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https://openalex.org/W2110143873	https://authors.library.caltech.edu/34334/1/Aad_EurPhysJournC2012p2056.pdf	English	null	Search for heavy neutrinos and right-handed W bosons in events with two leptons and jets in pp collisions at $\sqrt{s} = 7~\mathrm{TeV}$ with the ATLAS detector	European physical journal. C, Particles and fields	2,012	cc-by	21,271	Search for heavy neutrinos and right-handed W bosons in events with two leptons and jets in pp collisions at √s = 7 TeV with the ATLAS detector The ATLAS Collaboration⋆ CERN, 1211 Geneva 23, Switzerland Received: 24 March 2012 / Revised: 17 May 2012 / Published online: 3 July 2012 © CERN for the beneﬁt of the ATLAS collaboration 2012. This article is published with open access at Springerlink.com Received: 24 March 2012 / Revised: 17 May 2012 / Published online: 3 July 2012 © CERN for the beneﬁt of the ATLAS collaboration 2012. This article is published Abstract This letter reports on a search for hypothetical heavy neutrinos, N, and right-handed gauge bosons, WR, in events with high transverse momentum objects which in- clude two reconstructed leptons and at least one hadronic jet. The results were obtained from data corresponding to an integrated luminosity of 2.1 fb−1 collected in proton–proton collisions at √s = 7 TeV with the ATLAS detector at the CERN Large Hadron Collider. No excess above the Stan- dard Model background expectation is observed. Excluded mass regions for Majorana and Dirac neutrinos are presented using two approaches for interactions that violate lepton and lepton-ﬂavor numbers. One approach uses an effective oper- ator framework, the other approach is guided by the Left– Right Symmetric Model. The results described in this letter represent the most stringent limits to date on the masses of heavy neutrinos and WR bosons obtained in direct searches. same generation, and mN is the mass of a new heavy neu- trino, N. If the see-saw mechanism were to explain the mas- ses of the known neutrinos, both the light and the heavy neutrinos would have to be Majorana particles. This would violate lepton number conservation, and yield a striking sig- nature of two leptons with the same charge at the LHC [7]. p g This letter reports on a search for new heavy neutrinos of either Majorana or Dirac type, with data corresponding to an integrated luminosity of 2.1 fb−1 recorded with the ATLAS detector at the LHC. Two approaches are employed. The ﬁrst approach aims at exploring possible sources of new physics predicting heavy neutrinos using a Lagrangian of effective operators (referred to as HNEO hereafter) [8]. The theory is built on effective four-fermion operators (q ¯q′ →Nℓ) with the N decaying promptly via a three-body decay, N →ℓjj. The second approach is based on the concept of Left–Right Symmetry [9–11] which extends the electroweak part of the SM by a new gauge group. Eur. Phys. J. C (2012) 72:2056 DOI 10.1140/epjc/s10052-012-2056-4 Letter Letter Search for heavy neutrinos and right-handed W bosons in events with two leptons and jets in pp collisions at √s = 7 TeV with the ATLAS detector Its force particles (WR and Z′ bosons) could be produced at LHC energies. A particular implementation of left–right symmetry breaking [12], the Left–Right Symmetric Model (LRSM) with doubly charged Higgs bosons [13, 14] is used in the present analysis. Ac- cording to this model, the heavy neutrinos would be pro- duced in the decays of a WR boson via q ¯q′ →WR →ℓN, with N decaying subsequently via N →ℓW ∗ R →ℓjj. Thus, the ﬁnal state signature for both models consists of two leptons and two jets with high transverse momenta (pT). Only electrons and muons are considered in this analy- sis. ⋆e-mail: atlas.publications@cern.ch 1ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point in the center of the detector and the z-axis coinciding with the axis of the beam pipe. The x-axis points from the interaction point to the center of the LHC ring, and the y-axis points upward. Cylindrical coordinates (r,φ) are used in the transverse plane, φ being the azimuthal angle around the beam pipe. The pseudorapidity is deﬁned in terms of the polar angle θ as η = lntan(θ/2). 3 Trigger and data The data used in this analysis were recorded between March and August 2011 at a center-of-mass energy of 7 TeV. The application of beam, detector, and data quality requirements results in a total integrated luminosity of 2.1 fb−1 with an estimated uncertainty of ±3.7 % [25, 26]. The data were recorded with single lepton (e or μ) triggers [27]. At the last stage of the trigger decision, the electron trigger selects candidate electrons with transverse energy ET > 20 GeV, satisfying shower-shape requirements and matching an ID track. For the last part of the dataset, corresponding to an integrated luminosity of 0.5 fb−1, the threshold was raised to 22 GeV. The muon trigger selects candidate muons with pT > 18 GeV and \|η\| < 2.4. These triggers reach full ef- ﬁciency for electrons with pT > 25 GeV and muons with pT > 20 GeV. The typical trigger efﬁciencies measured from data for leptons selected for ofﬂine analysis are 99 ± 1 % for electrons, and 74 % and 91 % for muons in the barrel (\|η\| < 1.05) and end-cap (1.05 < \|η\| < 2.4) regions, respectively, with an uncertainty of about ±1 %. Heavy neutrinos were previously searched for at LEP and excluded for masses up to ≈100 GeV [17–20]. The most stringent direct limits on WR bosons [21, 22] come from the Tevatron, where WR →tb decays were searched for. As- suming a branching ratio of 100 %, WR boson masses be- low 825 GeV are excluded at 95 % conﬁdence level (C.L.). Recently, the ATLAS collaboration published an inclusive search for new physics in the same-sign dilepton signature for an integrated luminosity of 34 pb−1 [23]. The 95 % C.L. limits presented exclude WR masses up to about 1 TeV for the LRSM model and Majorana neutrino masses around 460 GeV for the HNEO model. 2 The ATLAS detector The ATLAS detector [24] is a multipurpose particle physics apparatus with a forward-backward symmetric cylindrical geometry and nearly 4π coverage in solid angle.1 The in- ner tracking detector (ID) covers the pseudorapidity range \|η\| < 2.5 and consists of: a silicon pixel detector, provid- ing typically three measurements per track; a silicon mi- crostrip detector (SCT), which provides typically four to ﬁve measurements; and, for \|η\| < 2.0, a transition radi- ation tracker (TRT), giving typically 30 straw-tube mea- surements per track. The ID is surrounded by a thin su- perconducting solenoid providing a 2 T magnetic ﬁeld. A high-granularity liquid-argon (LAr) sampling electromag- netic calorimeter covers the region \|η\| < 3.2. An iron-scinti- llator tile calorimeter provides hadronic coverage in the cen- tral rapidity range of \|η\| < 1.7. The end-cap and forward regions, spanning 1.5 < \|η\| < 4.9, are instrumented with LAr calorimeters for both electromagnetic and hadronic measurements. The muon spectrometer (MS) surrounds the calorimeters and consists of a system of air-core super- conducting toroid coils, precision tracking chambers up 1 Introduction The discovery of neutrino oscillations [1, 2] unambiguously establishes that neutrinos have non-zero mass and provides clear evidence for physics beyond the Standard Model (SM). One possible explanation for the mass of light neutrinos is provided by theoretical models based on a Grand Uniﬁed Theory (GUT). Such models often introduce one or more additional neutrino ﬁelds, which manifest themselves as new heavy particles that could be directly observable at the Large Hadron Collider (LHC). In the framework of GUT mod- els, the mass of the light neutrinos could be explained via the see-saw mechanism [3–6]. This predicts mν ≈m2 D/mN, where, for each generation, mν is the mass of a known light neutrino, mD is the Dirac mass for charged fermions of the The N invariant mass can be fully reconstructed from the decay products in both approaches. Given the s-channel production in the LRSM, the WR mass, mWR, can also be reconstructed in this model. The reconstructed WR boson and N masses are used to perform the search in the con- text of the HNEO and LRSM models, respectively. Like the SM neutrinos, heavy neutrinos can mix if their masses are Page 2 of 22 Eur. Phys. J. C (2012) 72:2056 different. Both the scenarios of no mixing [15] and maxi- mal mixing [16] between two generations of lepton ﬂavors (electron and muon) are investigated assuming that the mass difference between the heavy neutrinos is much smaller than the experimental resolution of their reconstructed invariant mass. In the case of maximal mixing, a mass difference of 2 GeV is assumed. If the heavy neutrinos are of Majorana type, they would contribute to both the same-sign (SS) and opposite-sign (OS) channels, while heavy Dirac neutrinos would contribute solely to the OS channel. to \|η\| < 2.7, and detectors for triggering in the region of \|η\| < 2.4. to \|η\| < 2.7, and detectors for triggering in the region of \|η\| < 2.4. 5 Object reconstruction and event selection The tree-level-generated dimension-6 operator OV corresponds to duNe, while Os1 and Os2 correspond to QuNL and LNQd, respectively, and Os3 corresponds to QNLd (e, u, d and L, where n is the operator dimension, Λ is the scale of LNV interactions, αi are the coupling constants between the neu- trino N and the leptons, and Oi are the effective opera- tors [8]. The signal samples are produced for four effective operator hypotheses: the four-fermion vector operator, OV , and four-fermion scalar operators, Os1, Os2, and Os3. The tree-level-generated dimension-6 operator OV corresponds to duNe, while Os1 and Os2 correspond to QuNL and LNQd, respectively, and Os3 corresponds to QNLd (e, u, d and L, Q denote the right-handed SU(2) singlets and left-handed SU(2) doublets, respectively). The production via the effec- tive operators Os1 and Os2 have the same cross section and lead to identical event kinematics, which makes them indis- tinguishable. The production cross sections for the Majorana and Dirac neutrinos in the framework of the effective La- grangian are related to the energy scale of new physics and the coupling constant σ ≈α2/Λ4, such that the coupling can be varied to scan for new physics at different Λ scales. Muons are required to be identiﬁed in both the ID and the MS systems. The ID track is required to have at least one pixel hit, more than ﬁve SCT hits, and a number of TRT hits that varies with η. Muon tracks that pass through an ac- tive region of the innermost pixel detector are required to have a measurement in that layer. The curvatures, as mea- sured by the ID and MS systems, must have the same sign. Only muons with pT > 25 GeV and \|η\| < 2.4 are consid- ered. Selection criteria on the displacement of the muon rel- ative to the primary vertex, selected as the one with the high- est p2 T of associated tracks, are required. The longitudi- nal (z0) and transverse (d0) impact parameters must satisfy \|z0\| < 5 mm, \|d0\| < 0.2 mm, and \|d0/σd0\| < 5, where σd0 is the uncertainty on d0. These cuts reduce the cosmic ray muon background to a negligible level and also reduce the background from non-prompt muons.2 The LRSM signal MC samples are generated using an implementation of this model [14] in PYTHIA, with modiﬁed leading-order parton distribution functions MRST2008LO* [41]. 2Leptons from W, Z and τ decays are classiﬁed as prompt leptons, while leptons any hadron decays are classiﬁed as non-prompt leptons. 4 Monte Carlo simulation Fully simulated Monte Carlo (MC) event samples are used to develop and validate the analysis procedure, es- timate the detector acceptance and reconstruction efﬁ- ciency, and aid in the background determination. The sim- ulation of background processes is described in detail in Ref. [28]. For the major backgrounds, Z/γ ∗+ jets pro- duction and top quark pair production, ALPGEN [29] and MC@NLO [30–32] are used, respectively. The leading- order parametrization CTEQ6L1 [33] of the parton density functions (PDF) is used for the ALPGEN simulation, while the next-to-leading order parametrization CTEQ6.6 [33] is used for the MC@NLO simulation. Fragmentation and hadronization are performed in both cases with HERWIG [34–36], using JIMMY [37] for the underlying event mod- elling. Diboson (WW, WZ, and ZZ) event samples are gen- erated using HERWIG, while MADGRAPH [38] interfaced to PYTHIA [39] is used for Wγ and Zγ production. Single top- quark production is generated with MC@NLO. The produc- tion of W +W + arising from a t-channel gluon exchange, resulting in two jets in the ﬁnal state and two same-sign W bosons, are generated with MADGRAPH interfaced to PYTHIA. The associated production of a vector boson with a t ¯t pair (t ¯tW, t ¯tZ, t ¯tγ ) is simulated with MADGRAPH interfaced with PYTHIA. Eur. Phys. J. C (2012) 72:2056 Eur. Phys. J. C (2012) 72:2056 Page 3 of 22 taken into account by reweighting MC events to match the pile-up conditions measured in data. The HNEO signal MC samples are generated using CALCHEP [40] and the leading-order PDF CTEQ6L [33], and hadronization simulated with PYTHIA. All lepton com- binations of e, μ or τ leading to lepton number violating (LNV) signatures, which produce SS or OS dilepton events, are included. The model is implemented via a Lagrangian of effective operators deﬁned as 5 Object reconstruction and event selection The criteria for electron and muon identiﬁcation closely fol- low those described in Ref. [46]. Electrons are required to pass the “medium” selection criteria, with pT > 25 GeV and \|η\| < 2.47, excluding the electromagnetic calorimeter tran- sition region, 1.37 < \|η\| < 1.52 [47]. To improve the back- ground rejection for \|η\| > 2.0, more stringent requirements are placed on the track-cluster matching in η and shower shape. Electron tracks that pass through an active region of the innermost pixel detector are required to have a measure- ment in that layer in order to suppress electrons from photon conversions. Additionally, an electron whose track matches the ID track of a muon candidate is rejected. L = ∞ n=5 1 Λn−4 · i αiOi(n), (1) (1) where n is the operator dimension, Λ is the scale of LNV interactions, αi are the coupling constants between the neu- trino N and the leptons, and Oi are the effective opera- tors [8]. The signal samples are produced for four effective operator hypotheses: the four-fermion vector operator, OV , and four-fermion scalar operators, Os1, Os2, and Os3. The tree-level-generated dimension-6 operator OV corresponds to duNe, while Os1 and Os2 correspond to QuNL and LNQd, respectively, and Os3 corresponds to QNLd (e, u, d and L, Q denote the right-handed SU(2) singlets and left-handed SU(2) doublets, respectively). The production via the effec- tive operators Os1 and Os2 have the same cross section and lead to identical event kinematics, which makes them indis- tinguishable. The production cross sections for the Majorana and Dirac neutrinos in the framework of the effective La- grangian are related to the energy scale of new physics and the coupling constant σ ≈α2/Λ4, such that the coupling can be varied to scan for new physics at different Λ scales. where n is the operator dimension, Λ is the scale of LNV interactions, αi are the coupling constants between the neu- trino N and the leptons, and Oi are the effective opera- tors [8]. The signal samples are produced for four effective operator hypotheses: the four-fermion vector operator, OV , and four-fermion scalar operators, Os1, Os2, and Os3. 6 Background estimation Several processes have the potential to contaminate the sig- nal regions. The main background to the SS dilepton ﬁ- nal state, which is referred to as “fake lepton” background, arises from SM W + jets, t ¯t, and multi-jet production where one or more jets are misidentiﬁed as prompt isolated lep- tons. This background is measured using a data-driven tech- nique rather than using less accurate estimates from MC simulation. The other signiﬁcant background arises from charge misidentiﬁcation of a reconstructed electron as a re- sult of hard bremsstrahlung followed by asymmetric conver- sion (e± hard →e± softγhard →e± softe± softe∓ hard). This background is estimated with a combination of MC and data-driven tech- niques. Small contributions from diboson and single top- quark events are also accounted for using MC. For the e±e∓and μ±μ∓ﬁnal states, the dominant back- grounds are Z/γ ∗+jets and t ¯t events, with about equal con- tributions after all selection criteria are applied. The e±μ∓ ﬁnal state is dominated by t ¯t production. The backgrounds from t ¯t, single top-quark, and diboson production are es- timated from MC simulation, while the estimation of the Z/γ ∗+ jets background is extracted from a normalization to the data. The fake lepton background is estimated from data, using the same method as for the SS ﬁnal states. Events are preselected by requiring exactly two identiﬁed leptons with pT > 25 GeV originating from the primary ver- tex and at least one jet with pT > 20 GeV. At least one of the lepton candidates must match a triggered lepton at the last stage of the trigger selection. To reduce the number of background events from Drell–Yan production and misiden- tiﬁed leptons, the dilepton invariant mass, mℓℓ, is required to be greater than 110 GeV. The signal region is then sub- divided into SS and OS dilepton events. In the OS dilepton channels, further background reduction is achieved by re- quiring that the scalar sum of the transverse energies of the two leptons and the leading two jets with pT > 20 GeV, de- noted by ST, is greater than 400 GeV. This event selection is referred to hereafter as the baseline selection. As mentioned previously, the mass of the N can be reconstructed from its decay products of one lepton and two jets. In the case where A data-driven approach, similar to the one described in Refs. 5 Object reconstruction and event selection The coupling constants for the WR and left-handed W boson are assumed to be the same, including the CKM ma- trix for WR boson couplings to right-handed chiral quark components. It is assumed that there is no mixing between the WR boson and the SM W boson. The LRSM signal MC samples are generated constraining the decays to e or μ and with mN < mWR. The branching fractions used are the ones predicted by PYTHIA. When the mass difference between the WR and the N is large, the leptonic branching fractions are ≈8 %, and they decrease with decreasing mass differ- ence. To reduce the background due to leptons from decays of hadrons (including heavy-ﬂavor hadrons) produced in jets, requirements on the isolation of leptons are imposed. To evaluate the isolation energy for electrons, the transverse en- ergies deposited in the calorimeter towers in a cone in η–φ space of radius R = (φ)2 + (η)2 = 0.2 around the electron direction are summed and corrected for energy de- position from pile-up events. In addition, the transverse en- ergy of the electron, ET, corrected for energy leakage into the neighboring towers, is subtracted. The isolation trans- verse energy is required to be less than 15 % of the elec- tron ET. An equivalent quantity, ER=0.3 T , is calculated for the muon using a cone size of R = 0.3. If there is no jet with pT > 20 GeV within R < 0.4 of the muon, ER=0.3 T is required to be less than 15 % of the muon pT. Addition- ally, muons with pT < 80 GeV should have no other track Both Majorana and Dirac type heavy neutrinos are con- sidered, assuming that the total production cross section is the same for both cases. The leading-order theoretical cross sections are used. All signal and background samples are generated us- ing the ATLAS underlying event tunes [42, 43] and pro- cessed through the ATLAS detector simulation [44] based on GEANT4 [45]. The MC samples are produced includ- ing the simulation of multiple interactions per LHC bunch crossing (pile-up). Varying pile-up conditions and their de- pendence on the instantaneous luminosity of the LHC are Eur. Phys. J. C (2012) 72:2056 Page 4 of 22 the N is boosted, the hadronic decay products can be recon- structed as one jet due to their proximity to each other. 5 Object reconstruction and event selection For scenarios with a large mass splitting between the WR and N, up to half of the signal events have only one jet. The WR bo- son invariant mass is reconstructed from the leptons and the two highest pT jets in events with at least two jets, or a sin- gle jet in events with only one jet. Anti-kt jets are massive, and therefore, the jet four-momenta are used in calculating the invariant mass. For the LRSM, the WR boson invariant mass, mℓℓj(j), is required to be greater than 400 GeV for both SS and OS ﬁnal states. with pT > 1 GeV originating from the primary vertex within a cone of R = 0.3 around the muon. Otherwise, if the muon has a jet nearby, it must satisfy pT > 80 GeV and (ER=0.3 T /pT −3)/pT > −0.02 GeV−1. These isolation re- quirements are powerful in rejecting background muons and highly efﬁcient for selecting signal muons produced in the decays of heavy neutrinos and reconstructed near the signal jets in cases where the heavy neutrino is boosted. with pT > 1 GeV originating from the primary vertex within a cone of R = 0.3 around the muon. Otherwise, if the muon has a jet nearby, it must satisfy pT > 80 GeV and (ER=0.3 T /pT −3)/pT > −0.02 GeV−1. These isolation re- quirements are powerful in rejecting background muons and highly efﬁcient for selecting signal muons produced in the decays of heavy neutrinos and reconstructed near the signal jets in cases where the heavy neutrino is boosted. Jets are reconstructed using the anti-kt jet clustering al- gorithm [48, 49] with a radius parameter R = 0.4. The input to this algorithm is clusters of calorimeter cells seeded by cells with energies signiﬁcantly above the measured noise. to this algorithm is clusters of calorimeter cells seeded by cells with energies signiﬁcantly above the measured noise. The energies and momenta of jets are evaluated by perform- ing a four-vector sum over these clusters, treating each clus- ter as an (E, p) four-vector with zero mass. Jets are cor- rected for calorimeter non-compensation, upstream material and other effects using pT and η-dependent calibration fac- tors [50] obtained from MC simulation [51], and validated with test-beam and collision-data studies. Only jets with pT > 20 GeV and \|η\| < 2.8 are considered. 5 Object reconstruction and event selection To avoid dou- ble counting, the closest jet within R < 0.5 of an electron candidate is discarded. The selected jets must pass quality requirements based on their shower shape, and their calori- meter signal timing must be consistent with the timing of the beam crossings [52]. Events with any jet that fails the jet quality criteria are rejected. To suppress jets unrelated to the hard scattering of interest, at least 75 % of the summed pT of all reconstructed tracks associated with a jet with \|η\| < 2.8 must come from tracks originating from the selected pri- mary vertex. During a part of the data-taking period, cor- responding to an integrated luminosity of 0.9 fb−1, an elec- tronic failure in a small η–φ region of the LAr EM calorime- ter created a dead region. For this integrated luminosity, events in data and MC containing either an identiﬁed elec- tron or a jet, with pT > 40 GeV, satisfying −0.1 < η < 1.5 and −0.9 < φ < −0.5 are rejected, leading to a loss of sig- nal efﬁciency of about 10 % for this portion of the data. 6 Background estimation [23, 28], is used to estimate the fake lepton back- ground. The method uses “loose” leptons in addition to the candidate leptons. Loose muons are deﬁned using the same identiﬁcation criteria as the candidate muons, except for the isolation requirements, which are not applied. For the electrons, looser requirements on the shower shape vari- ables, track-cluster matching, and track quality are used, and the isolation requirement is not applied. The method uses the fractions of these loose fake leptons, Rfake, and loose prompt leptons, Rprompt, which also pass the can- didate lepton requirements. A 4 × 4 matrix is then em- ployed on the “loose–loose” and “loose–tight” dilepton sample to predict the total fake lepton background con- tributing to the SS and OS dilepton ﬁnal states. The Rfake Eur. Phys. J. C (2012) 72:2056 Page 5 of 22 fractions are measured using fake lepton enriched control regions containing a single loose lepton. Additional cri- teria are imposed to reduce the true lepton contamina- tion from electroweak processes to a negligible level. For events in the control regions, the transverse mass, mT = 2 · pℓ T · Emiss T · (1 −cosφ(ℓ,Emiss T )), is required to be less than 40 GeV. Emiss T is deﬁned as the missing trans- verse momentum based on the calorimeter information and the transverse momenta of muons within \|η\| < 2.7 [46], while φ(ℓ,Emiss T ) is the azimuthal angle separation be- tween the lepton and the Emiss T vectors. Additionally, the fol- lowing requirements are imposed: φ(jet or e,Emiss T ) < 0.1 for the electron control region and φ(μ,Emiss T ) < 0.5 for the muon control region. For the muon control region, an additional requirement of at least one jet is imposed. After these criteria are applied, the remaining background from electroweak processes is estimated to be less than 5 %. The Rprompt fractions are measured using Z boson events satis- fying 86 GeV < mℓℓ< 96 GeV via a method referred to as the “tag-and-probe” method. The “tag” lepton is required to satisfy all lepton selection criteria, while the unbiased op- posite charge “probe” lepton should satisfy the loose crite- ria. The Rprompt fractions are parametrized as a function of the lepton pT and range from 89 % to 98 % for muons and 96 % to 99 % for electrons. 6 Background estimation To improve the accuracy of the prediction, the fractions are parametrized as a function of kinematic variables separately for leptons that pass the anal- ysis trigger requirement and those that do not. The muon Rfake is measured separately for muons that originate from heavy ﬂavor jets and those that do not, where the jet ﬂavor is identiﬁed using a combination of the secondary vertex [53] and impact parameter-based [54] b-tagging algorithms. For muons, Rfake ranges from 5 % to 10 % (5 % to 40 %) for heavy ﬂavor (light ﬂavor) jets. For electrons, Rfake ranges from 45 % to 60 %. The fraction of reconstructed electrons with charge mis- identiﬁcation due to hard bremsstrahlung is measured from simulated Z/γ ∗+ jets events, by comparing the MC gener- ated charge of the electron originating from the Z to that of the reconstructed electron candidate. The fraction is parame- trized as a function of the electron ET and η and applied to Z/γ ∗→e+e−and t ¯t →e±ℓ∓b ¯b MC backgrounds to ob- tain their contributions to the SS dilepton ﬁnal state, thus beneﬁting from the large number of simulated OS events. Since the MC overestimates the charge misidentiﬁcation as observed in the Z/γ ∗→ee data sample, η-dependent scale factors between data and MC simulation are obtained us- ing Z/γ ∗→e±e± events with 80 GeV < mℓℓ< 100 GeV. Both electrons are required to be within the same η range and with the same charge. These factors are applied to the MC estimates. The rate of charge misidentiﬁcation due to tracking resolution is found to be negligible within the lep- ton transverse momentum range of interest and is well de- scribed by the MC simulation. All other backgrounds are estimated from MC simulation and found to be small, as shown in Table 2. In the SS ee and eμ channels, the dominant background arises from fake lep- tons. The next most signiﬁcant background is diboson pro- duction for the eμ channel and Z/γ ∗production for the ee channel. The SS μμ channel is dominated by the diboson background with a smaller contribution from fake leptons. 8 Results and interpretation The quoted uncertainties include statis- tical and systematic components, excluding the luminosity uncertainty of ±3.7 %. The latter is relevant for all backgrounds except for the fake lepton(s) background, which is measured using data Physics processes e±e± μ±μ± e±μ± Total Z/γ ∗+ jets 26.1 ± 5.6 0.0+1.6 −0 1.2 ± 0.7 27 ± 6 Diboson 12.7 ± 2.3 7.2 ± 1.7 18.8 ± 3.0 39 ± 6 Top 5.8 ± 1.3 0.7 ± 0.3 6.8 ± 1.6 13 ± 3 Fake lepton(s) 93.6 ± 35.7 3.1 ± 1.6 53.8 ± 20.3 151 ± 50 Total background 138.3 ± 36.5 11.0+2.9 −2.5 80.7 ± 20.8 230 ± 52 Observed events 155 14 99 268 mℓℓj(j) ≥400 GeV Total background 48.4 ± 16.1 4.4+2.1 −1.3 24.6 ± 7.6 77 ± 21 Ob d 59 8 39 106 Physics processes e±e∓ μ±μ∓ e±μ∓ Total Z/γ ∗+ jets 136.1 ± 12.5 173.2 ± 15.1 0.8 ± 0.8 310 ± 20 Diboson 4.3 ± 1.8 7.3 ± 1.9 5.9 ± 1.6 18 ± 3 Top 103.1 ± 12.3 100.9 ± 12.0 199.4 ± 23.3 403 ± 46 Fake lepton(s) 12.5 ± 8.1 −0.2 ± 0.7 6.1 ± 4.2 18 ± 9 Total background 256.0 ± 26.2 281.2 ± 27.9 212.3 ± 33.8 750 ± 78 Observed events 248 245 247 740 mℓℓj(j) ≥400 GeV Total background 254.8 ± 25.8 279.7 ± 27.6 210.9 ± 33.4 745 ± 77 Observed events 246 241 244 731 Table 2 Summary of the expected background yields and observed numbers of events for the SS dilepton channels. The top part of the table shows the numbers obtained for events with two leptons, ≥1 jet and mℓℓ> 110 GeV. The bottom part of the table shows the num- bers for the ﬁnal LRSM selection, where an additional requirement mℓℓj(j) ≥400 GeV is imposed. The quoted uncertainties include statis- tical and systematic components, excluding the luminosity uncertainty of ±3.7 %. The latter is relevant for all backgrounds except for the fake lepton(s) background, which is measured using data mℓℓj(j) ≥400 GeV is imposed. The quoted uncertainties include statis- tical and systematic components, excluding the luminosity uncertainty of ±3.7 %. 7 Systematic uncertainties The dominant contribution to the systematic uncertainties in the SS ee and eμ channels arises from the fake lep- ton background estimate. As a ﬁrst step in validating the parametrization of Rfake and Rprompt, a closure test is per- formed in data. Measurements of Rfake and Rprompt are ob- tained by randomly sampling half of the control regions. The predicted values are then compared with the values mea- sured in the other half of the data. The closure test yields an agreement for Rfake and Rprompt of, respectively, ±40 % and ±5 % for muons and, for electrons, ±5 % (±20 % for 1 < \|η\| < 1.9) and ±2 %, which are propagated to the fake lepton background estimate. To evaluate the uncertainty on the overall fake lepton background estimate, the robustness of the procedure is tested against variations across samples. The estimated fake lepton background is compared to the observed background in SS events passing the same selec- tion as events in the signal region but where the sub-leading lepton has a transverse momentum between 15 GeV and 25 GeV. The fake lepton background contributes between 65 % in e±e± to 87 % in e±μ± of the total background. This study tests the reliability of both the parametrization and the use of Rfake and Rprompt to extract the background predic- tion. In this sample, the total background prediction agrees A partially data-driven approach is adopted to estimate Z/γ ∗→ee and Z/γ ∗→μμ contributions to the OS dilepton channels. A control region is deﬁned requiring 80 GeV < mℓℓ< 100 GeV and ≥1 jets, where non-Z bo- son contributions are found to be negligible. Normaliza- tion factors between the observed number of events in data and the MC prediction are obtained as a function of jet multiplicity from this region and applied to the MC esti- mates in the signal region. All other backgrounds, including Z/γ ∗→ττ, are estimated from MC simulation. The con- tribution of Z/γ ∗→ττ is found to be negligible after all selection criteria are applied. Table 1 summarises the back- ground estimates for the OS channels. In the OS ee and μμ channels, Z/γ ∗+ jets and t ¯t backgrounds dominate, while the t ¯t production contributes more than 90 % in the eμ channel. Smaller contributions arise from diboson pro- duction and events with fake leptons. Eur. Phys. J. 7 Systematic uncertainties C (2012) 72:2056 Page 6 of 22 with the observed data within ±10 %. A ±30 % overall sys- tematic uncertainty is assigned to cover for the differences between the predicted and the observed mℓℓspectra. up in the 2011 dataset. MC modelling uncertainties for t ¯t production [28] are derived using different MC generators and varying, within their uncertainties, the parameters that control initial and ﬁnal state radiation. The resulting uncer- tainties are ±15 % and ±(5–7) % for t ¯t and diboson contri- butions, respectively. The uncertainties on the background due to the electron charge misidentiﬁcation arise from the limited number of MC events used to parametrize the rate and the scale factors used to correct the simulation for differences between data and MC and contribute ±13 % and ±12 %, respectively. Due to the limited knowledge of PDFs and αs, the uncer- tainties are evaluated using a range of current PDF sets with the procedure described in Ref. [57]. The ﬁnal uncertainty is taken from the outer bounds of the overall error bands. The PDF uncertainties are estimated to be ±9 % for the LRSM signal and ±12 % for the HNEO signals. The background and signal estimates derived from MC are affected by the jet energy scale (JES) calibration and the jet energy resolution (JER), theoretical and MC modelling uncertainties, and pT and η dependent uncertainties on the lepton identiﬁcation and reconstruction efﬁciencies (identi- ﬁcation ±(0.2–3.3) %, pT scale ±(0.2–2) % and resolution ±(0.4–10) %) [47, 55, 56]. The JES (±(2–6) %) and JER (±(5–12) %) uncertainties applied depend on jet pT and η and are measured from the 2010 dataset [52]. An additional contribution of ±(2–7) % to the JES uncertainty is added in quadrature to account for the effect of high luminosity pile- 8 Results and interpretation The expected and observed numbers of events in each dilep- ton ﬁnal state for the baseline selection and the LRSM se- lections are compared in Tables 1 and 2 for the OS and requirement mℓℓj(j) ≥400 GeV is imposed. The quoted uncertainties include statistical and systematic components, excluding the luminos- ity uncertainty of ±3.7 %. The latter is relevant for all backgrounds except for the fake lepton(s) background, which is measured using data requirement mℓℓj(j) ≥400 GeV is imposed. The quoted uncertainties include statistical and systematic components, excluding the luminos- ity uncertainty of ±3.7 %. The latter is relevant for all backgrounds except for the fake lepton(s) background, which is measured using data Table 1 Summary of the expected background yields and observed numbers of events for the OS dilepton channels. The top part of the table shows the numbers obtained for events with two leptons, ≥1 jet, mℓℓ> 110 GeV, and ST > 400 GeV. The bottom part of the table shows the numbers for the ﬁnal LRSM selection, where an additional Table 1 Summary of the expected background yields and observed numbers of events for the OS dilepton channels. The top part of the table shows the numbers obtained for events with two leptons, ≥1 jet, mℓℓ> 110 GeV, and ST > 400 GeV. The bottom part of the table shows the numbers for the ﬁnal LRSM selection, where an additional Physics processes e±e∓ μ±μ∓ e±μ∓ Total Z/γ ∗+ jets 136.1 ± 12.5 173.2 ± 15.1 0.8 ± 0.8 310 ± 20 Diboson 4.3 ± 1.8 7.3 ± 1.9 5.9 ± 1.6 18 ± 3 Top 103.1 ± 12.3 100.9 ± 12.0 199.4 ± 23.3 403 ± 46 Fake lepton(s) 12.5 ± 8.1 −0.2 ± 0.7 6.1 ± 4.2 18 ± 9 Total background 256.0 ± 26.2 281.2 ± 27.9 212.3 ± 33.8 750 ± 78 Observed events 248 245 247 740 mℓℓj(j) ≥400 GeV Total background 254.8 ± 25.8 279.7 ± 27.6 210.9 ± 33.4 745 ± 77 Observed events 246 241 244 731 Table 2 Summary of the expected background yields and observed numbers of events for the SS dilepton channels. The top part of the table shows the numbers obtained for events with two leptons, ≥1 jet and mℓℓ> 110 GeV. The bottom part of the table shows the num- bers for the ﬁnal LRSM selection, where an additional requirement mℓℓj(j) ≥400 GeV is imposed. 8 Results and interpretation The latter is relevant for all backgrounds except for the fake lepton(s) background, which is measured using data Physics processes e±e± μ±μ± e±μ± Total Z/γ ∗+ jets 26.1 ± 5.6 0.0+1.6 −0 1.2 ± 0.7 27 ± 6 Diboson 12.7 ± 2.3 7.2 ± 1.7 18.8 ± 3.0 39 ± 6 Top 5.8 ± 1.3 0.7 ± 0.3 6.8 ± 1.6 13 ± 3 Fake lepton(s) 93.6 ± 35.7 3.1 ± 1.6 53.8 ± 20.3 151 ± 50 Total background 138.3 ± 36.5 11.0+2.9 −2.5 80.7 ± 20.8 230 ± 52 Observed events 155 14 99 268 mℓℓj(j) ≥400 GeV Total background 48.4 ± 16.1 4.4+2.1 −1.3 24.6 ± 7.6 77 ± 21 Observed events 59 8 39 106 Page 7 of 22 Eur. Phys. J. C (2012) 72:2056 SS events, respectively. Additionally, the reconstructed ma- sses of the N and WR candidates, mℓj(j) and, mℓℓj(j) re- spectively, are examined in each dilepton channel. Figures 1 and 2 show those distributions for the OS and SS channels (ee, μμ, and eμ combined). the LRSM, Aϵ varies between 40 % and 65 % across the (mWR,mN) plane. The lowest Aϵ occurs for small mN. It should be noted that the difference in Aϵ between the two models is dominated by the fact the decays to τ leptons are included in generating the HNEO samples, while only de- cays to e and μ are included in the LRSM samples. Table 3 quotes the limits obtained for each channel, after the base- line selection. Given the good agreement between the data and the ex- pectations from SM processes, the results are used to set limits at 95 % C.L. on the visible cross section, σAϵ, where σ is the cross section for new phenomena, A is the accep- tance (i.e. the fraction of events passing geometric and kine- matic selection requirements at the particle level), and ϵ is the efﬁciency (i.e. the detector reconstruction and identiﬁca- tion efﬁciency). For the HNEO model, Aϵ is about 10 % for mN = 0.1 TeV and reaches a plateau value of about 28 % at around mN = 0.8 TeV, for all six dilepton channels. For The resulting limits for the interpretation of the data in terms of the HNEO and LRSM models are derived using as templates the reconstructed masses of the N and WR candi- dates in each dilepton channel. 8 Results and interpretation The baseline selection is used for the HNEO model, while the additional cut of mℓℓj(j) is applied for the LRSM model. Systematic uncertainties Fig. 1 Distributions of the reconstructed N invariant mass, mℓj(j), for OS (top) and SS (bottom) dilepton events with ≥1 jets and mℓℓ> 110 GeV. A selection criterion ST ≥400 GeV is used for the OS selection. The hypothetical signal distributions for mN = 0.3 TeV for OV and Λ/√α = 2 TeV are superimposed Fig. 2 Distributions of the reconstructed WR invariant mass, mℓℓj(j), for OS (top) and SS (bottom) dilepton events with ≥1 jets, mℓℓ> 110 GeV, and mℓℓj(j) ≥400 GeV. A selection criterion ST ≥400 GeV is used for the OS selection. The hypothetical signal distributions for mWR = 1.2 TeV and mN = 0.1 TeV (mWR = 1.5 TeV and mN = 0.8 TeV) for the case of maximal mixing, are superimposed to the OS (SS) distribution Fig. 2 Distributions of the reconstructed WR invariant mass, mℓℓj(j), for OS (top) and SS (bottom) dilepton events with ≥1 jets, mℓℓ> 110 GeV, and mℓℓj(j) ≥400 GeV. A selection criterion ST ≥400 GeV is used for the OS selection. The hypothetical signal distributions for mWR = 1.2 TeV and mN = 0.1 TeV (mWR = 1.5 TeV and mN = 0.8 TeV) for the case of maximal mixing, are superimposed to the OS (SS) distribution Fig. 1 Distributions of the reconstructed N invariant mass, mℓj(j), for OS (top) and SS (bottom) dilepton events with ≥1 jets and mℓℓ> 110 GeV. A selection criterion ST ≥400 GeV is used for the OS selection. The hypothetical signal distributions for mN = 0.3 TeV for OV and Λ/√α = 2 TeV are superimposed Eur. Phys. J. C (2012) 72:2056 Page 8 of 22 Fig. 4 Expected and observed 95 % C.L. upper limits on the heavy neutrino and WR masses for the Majorana (top) and Dirac (bottom) cases, in the no-mixing and maximal-mixing scenarios Table 3 Observed (obs) and expected (exp) 95 % C.L. upper limits on the visible cross section, ⟨σAϵ⟩95, for each OS and SS dilepton channel after the baseline selection , ⟨ ⟩ , p channel after the baseline selection Channels ⟨σAϵ⟩95 obs [fb] ⟨σAϵ⟩95 exp [fb] e±e∓ 28.6 31.0 μ±μ∓ 25.1 36.7 e±μ∓ 50.9 36.4 e±e± 37.6 29.6 μ±μ± 6.1 4.6 e±μ± 25.4 16.2 Fig. 3 Expected and observed 95 % C.L. 8 Results and interpretation upper limits on Λ/√α as a function of the mass of a heavy neutrino, for the operators OV , Os1/Os2, and Os3, using the formalism of Lagrangian of effective op- erators, for the Majorana (top) and Dirac (bottom) scenarios Channels ⟨σAϵ⟩95 obs [fb] ⟨σAϵ⟩95 exp [fb] e±e∓ 28.6 31.0 μ±μ∓ 25.1 36.7 e±μ∓ 50.9 36.4 e±e± 37.6 29.6 μ±μ± 6.1 4.6 e±μ± 25.4 16.2 Fig. 4 Expected and observed 95 % C.L. upper limits on the heavy neutrino and WR masses for the Majorana (top) and Dirac (bottom) cases, in the no-mixing and maximal-mixing scenarios on Λ/√α are shown in Fig. 3 for the Majorana and Dirac scenarios using various effective operator hypotheses. Fig- ure 4 shows the exclusion limits for the masses of heavy neutrinos and the WR boson in the LRSM interpretation, for the no-mixing and maximal-mixing scenarios between Ne and Nμ neutrinos, for both the Majorana and Dirac heavy neutrinos hypotheses. The above results are obtained with a Bayesian [58] ap- proach, where systematic uncertainties are treated as nui- sance parameters with a truncated Gaussian as a prior shape. The prior shape on the parameters of interest, σ× BR, is as- sumed to be ﬂat. Fig. 3 Expected and observed 95 % C.L. upper limits on Λ/√α as a function of the mass of a heavy neutrino, for the operators OV , Os1/Os2, and Os3, using the formalism of Lagrangian of effective op- erators, for the Majorana (top) and Dirac (bottom) scenarios from JES and JER are included as variations in the signal and background templates. The uncertainties on the mea- surement of Rfake and Rprompt are included as variations in the fake lepton background templates. All other uncertain- ties have no signiﬁcant kinematic dependence. Correlations of uncertainties between signal and background, as well as across channels, are taken into account. 9 Conclusions A dedicated search for hypothetical heavy Majorana and Dirac neutrinos, and WR bosons in ﬁnal states with two high-pT same-sign or opposite-sign leptons and hadronic jets has been presented. In a data sample corresponding to an integrated pp luminosity of 2.1 fb−1 at √s = 7 TeV, no signiﬁcant deviations from the SM expectations are ob- served, and 95 % C.L. limits are set on the contributions The 95 % C.L. exclusion limits on the mass of the heavy neutrino in the HNEO model and their dependence Page 9 of 22 Eur. Phys. J. C (2012) 72:2056 of new physics. Excluded mass regions for Majorana and Dirac neutrinos are presented for various operators of an ef- fective Lagrangian framework and for the LRSM. The latter interpretation was used to extract a lower limit on the mass of the gauge boson WR. 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The ATLAS Collaboration C (2012) 72:2056 1University at Albany, Albany NY, United States of America 2 1University at Albany, Albany NY, United States of America 2Department of Physics, University of Alberta, Edmonton AB, Canada b 3(a)Department of Physics, Ankara University, Ankara; (b)Department of Physics, Dumlupinar University, Kutahya; ( ) (d) of Physics, Gazi University, Ankara; (d)Division of Physics, TOBB University of Economics and ( ) c)Department of Physics, Gazi University, Ankara; (d)Division of Physics, TOBB University of Econ Technology, Ankara; (e)Turkish Atomic Energy Authority, Ankara, Turkey Technology, Ankara; (e)Turkish Atomic Energy Authority, Ankara, Turkey 4LAPP, CNRS/IN2P3 and Université de Savoie, Annecy-le-Vieux, France High Energy Physics Division, Argonne National Laboratory, Argonne IL, United States of America Department of Physics, University of Arizona, Tucson AZ, United States of America 7Department of Physics, The University of Texas at Arlington, Arlington TX, United States of America s Department, University of Athens, Athens, Gree 9Physics Department, National Technical University of Athens, Zografou, Greece 10Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan Azerbaijan Academy of Sciences, Baku, Azerbaij d’Altes Energies and Departament de Física de la Universitat Autònoma de Barcelona and ICREA, 11Institut de Física d’Altes Energies and Departament de Física de la Universitat Autònoma de Barcelona and ICREA, B l S i 11Institut de Física d’Altes Energies and Departament de Física de la Universitat Autònoma de Barcelona and ICREA, Barcelona, Spain Page 18 of 22 Eur. Phys. J. The ATLAS Collaboration C (2012) 72:2056 12(a)Institute of Physics, University of Belgrade, Belgrade; (b)Vinca Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia 12(a)Institute of Physics, University of Belgrade, Belgrade; (b)Vinca Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia 13Department for Physics and Technology, University of Bergen, Bergen, Norway 14 14Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley CA, United States of America 5 14Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley CA, United States of America 5 15Department of Physics, Humboldt University, Berlin, Germany 16Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland 18(a)Department of Physics, Bogazici University, Istanbul; (b)Division of Physics, Dogus University, Istanbul; (c)Department of Physics Engineering, Gaziantep University, Gaziantep; (d)Department of Physics, Istanbul Technical University, Istanbul, Turkey 19(a)INFN Sezione di Bologna; (b)Dipartimento di Fisica, Università di Bologna, Bologna, Italy 20Physikalisches Institut, University of Bonn, Bonn, Germany 21 20Physikalisches Institut, University of Bonn, Bonn, Germany 21 20Physikalisches Institut, University of Bonn, Bonn, Germany 21Department of Physics, Boston University, Boston MA, United States of America 21Department of Physics, Boston University, Boston MA, United States of America 22Department of Physics, Brandeis University, Waltham MA, United States of America 22Department of Physics, Brandeis University, Waltham MA, United States of America 23(a)Universidade Federal do Rio De Janeiro COPPE/EE/IF Rio de Janeiro; (b)Federal University of Juiz de Fora (UFJF) 22Department of Physics, Brandeis University, Waltham MA, United States of America 23(a)Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b)Federal University of Juiz de Fora (UFJF), ( ) (d) 23(a)Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b)Federal University of 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 sics Department, Brookhaven National Laboratory, Upton NY, United States of America 24Physics Department, Brookhaven National Laboratory, Upton NY, United States of America 25(a)N ti l I tit t f Ph i d N l E i i B h t (b)U i it P lit h i 24Physics Department, Brookhaven National Laboratory, Upton NY, United States of America 25(a)National Institute of Physics and Nuclear Engineering, Bucharest; (b)University Politehnica Bucharest, Bucharest; Physics Department, Brookhaven National Laboratory, Upton NY, United States of America 25(a)National Institute of Physics and Nuclear Engineering, Bucharest; (b)University Politehnica Bu Physics Department, Brookhaven National Laboratory, Upton NY, United States of America 25(a)National Institute of Physics and Nuclear Engineering, Bucharest; (b)University Politehnica Bucharest, Bucharest; ( ) a)National Institute of Physics and Nuclear Engineering, Bucharest; (b)University Politehnica Bucha (c)West University in Timisoara, Timisoara, Romania 26Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Argentina 27Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 28Department of Physics, Carleton University, Ottawa ON, Canada 30Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America 31(a)D t t d Fi i P tiﬁi U i id d C tóli d Chil S ti (b)D Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America 30Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America 30Enrico Fermi Institute, University of Chicago, Chicago IL, United States of America 31( ) (b) 31(a)Departamento de Fisica, Pontiﬁcia Universidad Católica de Chile, Santiago; (b)Departamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile p , U v C C , S g ; p , U v Técnica Federico Santa María, Valparaíso, Chile 32( ) (b) 32(a)Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b)Department of Mo ( ) University of Science and Technology of China, Anhui; (c)Department of Physics, Nanjing University, Jiangsu; (d)School of Physics, Shandong University, Shandong, China 33Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Aubiere Cedex, France 34Nevis Laboratory, Columbia University, Irvington NY, United States of America 35 34Nevis Laboratory, Columbia University, Irvington NY, United States of America 35Niels Bohr Institute University of Copenhagen Kobenhavn Denmark 34Nevis Laboratory, Columbia University, Irvington NY, United States of Am 35Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark 36(a)INFN Gruppo Collegato di Cosenza; (b)Dipartimento di Fisica, Università della Calabria, Arcavata di Rende, Italy 37AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow, Poland 38The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Poland 39Physics Department, Southern Methodist University, Dallas TX, United States of America iewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Poland 38The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Krakow 39 38The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Krak 39Physics Department, Southern Methodist University, Dallas TX, United States of America Physics Department, Southern Methodist University, Dallas TX, United States of America 40Physics Department, University of Texas at Dallas, Richardson TX, United States of America 41DESY, Hamburg and Zeuthen, Germany 42Institut für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 43 43Institut für Kern- und Teilchenphysik, Technical University Dresden, Dresden, Germany 44 44Department of Physics, Duke University, Durham NC, United States of America 45SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 46Fachhochschule Wiener Neustadt, Johannes Gutenbergstrasse 3, 2700 Wiener Neustadt, Austria 51(a)E.Andronikashvili Institute of Physics, Tbilisi State University, Tbilisi; (b)High Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia Page 19 of 22 Eur. The ATLAS Collaboration Phys. J. The ATLAS Collaboration C (2012) 72:2056 52II Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 53SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 54II Ph ik li h I tit t G A t U i ität Götti G 54II Physikalisches Institut, Georg-August-Universität, Göttingen, Germany 55Laboratoire de Physique Subatomique et de Cosmologie, Université Joseph Fourier and CNRS/IN2P3 and Institut National Polytechnique de Grenoble, Grenoble, France National Polytechnique de Grenoble, Grenoble, France 56Department of Physics, Hampton University, Hampton VA, United States of Ame 56Department of Physics, Hampton University, Hampton VA, United States of America 58(a)Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg 58(a)Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b)Physikalisches Institut, ( ) irchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b)Physikalische recht-Karls-Universität Heidelberg, Heidelberg; (c)ZITI Institut für technische Informatik, ( )Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; ( )Physikali Ruprecht-Karls-Universität Heidelberg, Heidelberg; (c)ZITI Institut für technische Informatik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (c)ZITI Institut für technische Informa Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany 59Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan 60Department of Physics, Indiana University, Bloomington IN, United States of America 61Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria 62University of Iowa, Iowa City IA, United States of America 63Department of Physics and Astronomy, Iowa State University, Ames IA, United States of Americ 63Department of Physics and Astronomy, Iowa State University, A 64Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 65KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 66Graduate School of Science, Kobe University, Kobe, Japan 67Faculty of Science, Kyoto University, Kyoto, Japan 68Kyoto University of Education, Kyoto, Japan 68Kyoto University of Education, Kyoto, Japan 69Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina 70 70Physics Department, Lancaster University, Lancaster, United Kingdom 71(a)INFN Sezione di Lecce; (b)Dipartimento di Fisica, Università del Salento, Lecce, Italy 72Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 73Department of Physics, Jožef Stefan Institute and University of Ljubljana, Ljubljana, Slove 75Department of Physics, Royal Holloway University of London, Surrey, United Kingdom 77Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, P France 78Fysiska institutionen, Lunds Universitet, Lund, Sweden 9 78Fysiska institutionen, Lunds Universitet, Lund, Sweden 79 79Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain 80Institut für Physik, Universität Mainz, Mainz, Germany 80Institut für Physik, Universität Mainz, Mainz, Germany 81School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 81School of Physics and Astronomy, University of Manchester, Mancheste 82CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France 82CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France 83Department of Physics, University of Massachusetts, Amherst MA, United States of America 83Department of Physics, University of Massachusetts, Amherst MA, Un 84Department of Physics, McGill University, Montreal QC, Canada 84Department of Physics, McGill University, Montreal QC, Canada 85School of Physics, University of Melbourne, Victoria, Australia 85School of Physics, University of Melbourne, Victoria, Australia 86Department of Physics, The University of Michigan, Ann Arbor MI, United States of America Department of Physics and Astronomy, Michigan State University, East Lansing MI, United States ) (b) 87Department of Physics and Astronomy, Michigan State University, East Lansing MI, United Stat 88(a)INFN Sezione di Milano; (b)Dipartimento di Fisica, Università di Milano, Milano, Italy p y y g y g a)INFN Sezione di Milano; (b)Dipartimento di Fisica, Università di Milano, Milano, Italy 89B.I. The ATLAS Collaboration Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Republic of 90National Scientiﬁc and Educational Centre for Particle and High Energy Physics, Minsk, Republi 91Department of Physics, Massachusetts Institute of Technology, Cambridge MA, United States of Group of Particle Physics, University of Montreal, Montreal QC, Canada P.N. Lebedev Institute of Physics, Academy of Sciences, Moscow, Russia 94Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia 95Moscow Engineering and Physics Institute (MEPhI), Moscow, Russia 97Fakultät für Physik, Ludwig-Maximilians-Universität München, München, G 98 99Nagasaki Institute of Applied Science, Nagasaki, Japan 100Graduate School of Science, Nagoya University, Nagoya, Japan 100Graduate School of Science, Nagoya University, Nagoya, Japan Page 20 of 22 Eur. Phys. J. C (2012) 72:2056 01(a)INFN Sezione di Napoli; (b)Dipartimento di Scienze Fisiche, Università di Napoli, Napoli, Italy p p p p y 102Department of Physics and Astronomy, University of New Mexico, Albuquerque NM, United States of America 103Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands 02Department of Physics and Astronomy, University of New Mexico, Albuquerque NM, United Sta Department of Physics and Astronomy, University of New Mexico, Albuquerque NM, United States of America 103Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands p y y y q q 103Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands ional Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands 04Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Neth 105Department of Physics, Northern Illinois University, DeKalb IL, United States of America 105Department of Physics, Northern Illinois University, DeKalb IL, United States of America 106Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 107Department of Physics, New York University, New York NY, United States of America 107Department of Physics, New York University, New York NY, United States of America 109Faculty of Science, Okayama University, Okayama, Japan 110 109Faculty of Science, Okayama University, Okayama, Japan 110Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman OK, United States of America 110Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman OK, United States of America 11Department of Physics, Oklahoma State University, Stillwater OK, United States of America 112Palacký University, RCPTM, Olomouc, Czech Republic 113Center for High Energy Physics University of Oregon Eugene OR United States of America 113Center for High Energy Physics, University of Oregon, Eugene OR, United States of America 114LAL, Univ. The ATLAS Collaboration Paris-Sud and CNRS/IN2P3, Orsay, France 115Graduate School of Science, Osaka University, Osaka, Japan 116Department of Physics, University of Oslo, Oslo, Norway 117Department of Physics, Oxford University, Oxford, United Kingdom 118(a)INFN Sezione di Pavia; (b)Dipartimento di Fisica Nucleare e Teorica, Università di Pavia, Pa INFN Sezione di Pavia; Dipartimento di Fisica Nucleare e Teorica, Università di Pavia, Pavia, 119Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America 19Department of Physics, University of Pennsylvania, Philadelphia PA, United States of America 120Petersburg Nuclear Physics Institute, Gatchina, Russia 121(a)INFN Sezione di Pisa; (b)Dipartimento di Fisica E. The ATLAS Collaboration Fermi, Università di Pisa, Pisa, Italy 122Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, United States of America 123(a)Laboratorio de Instrumentacao e Fisica Experimental de Particulas - LIP, Lisboa, Portugal; (b)Departamento de Fisi 22Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh PA, United States of A 23(a) b i d i i i l d i l i b l (b) p y y, y g , g , 123(a)Laboratorio de Instrumentacao e Fisica Experimental de Particulas - LIP, Lisboa, Portugal; (b)Departamento de Fisica 23(a)Laboratorio de Instrumentacao e Fisica Experimental de Particulas - LIP, Lisboa, Portugal; (b)D Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada, Spain 124 Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada, Spain 124Institute of Physics, Academy of Sciences of the Czech Republic, Praha, Czech Republic 124Institute of Physics, Academy of Sciences of the Czech Republic, Praha, Czech Republic 125Faculty of Mathematics and Physics, Charles University in Prague, Praha, Czech Republic 126Czech Technical University in Prague, Praha, Czech Republic 127State Research Center Institute for High Energy Physics, Protvino, Russia 128 128Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom ics Department, Rutherford Appleton Laboratory, 128Particle Physics Department, Rutherford Appleton Laboratory, D 129Physics Department, University of Regina, Regina SK, Canada 129Physics Department, University of Regina, Regina SK, Canada 30Ritsumeikan University, Kusatsu, Shiga, Japan 131(a)INFN Sezione di Roma I; (b)Dipartimento di Fisica, Università La Sapienza, Roma, Italy 132( ) (b) 132(a)INFN Sezione di Roma Tor Vergata; (b)Dipartimento di Fisica, Università di Roma Tor Vergata, Roma, Italy 133( ) (b) 133(a)INFN Sezione di Roma Tre; (b)Dipartimento di Fisica, Università Roma Tre, Roma, Italy 134(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 S l li U i ité C di A d LPHEA M k h (d)F lté d S i U i ité M h d P i d 134(a)Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies - U Casablanca; (b)Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat; (c)Fa 134(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 des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies - Université Hassan a; (b)Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat; (c)Faculté des Science , y q g , )Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat; (c)Faculté des Sciences Université Cadi Ayyad, LPHEA-Marrakech; (d)Faculté des Sciences, Université Mohamed Premier a ( ) Semlalia, Université Cadi Ayyad, LPHEA-Marrakech; (d)Faculté des Sciences, Université Mo LPTPM, Oujda; (e)Faculté des Sciences, Université Mohammed V- Agdal, Rabat, Morocco 135DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat a l’Energie Atomique), Gif-sur-Yvette, France 136Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz CA, 13 stitute for Particle Physics, University of California Santa Cruz, Santa Cruz CA, United States of Am 137Department of Physics, University of Washington, Seattle WA, United States of America 138 37Department of Physics, University of Washington, Seattle WA, United States of America 38 38Department of Physics and Astronomy, University of Shefﬁeld, Shefﬁeld, United Kingdom 138Department of Physics and Astronomy, University of Shefﬁeld, Shefﬁeld, United Kingd 139Department of Physics, Shinshu University, Nagano, Japan 139Department of Physics, Shinshu University, Nagano, Japan 140Fachbereich Physik, Universität Siegen, Siegen, Germany 140Fachbereich Physik, Universität Siegen, Siegen, Germany 41Department of Physics, Simon Fraser University, Burnaby BC, Canada 141Department of Physics, Simon Fraser University, Burnaby BC, Canada 142SLAC National Accelerator Laboratory, Stanford CA, United States of America 142SLAC National Accelerator Laboratory, Stanford CA, United States of America 143(a)Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b)Department of Subnuclear 143(a)Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b)Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 144(a)Department of Physics, University of Johannesburg, Johannesburg; (b)School of Physics, University of the Wi d J h b S h Af i y y y p Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 144(a)Department of Physics, University of Johannesburg, Johannesburg; (b)School of Physics, University of the Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 144(a)Department of Physics, University of Johannesburg, Johannesburg; (b)School of Physics, University of the Witwatersrand, Johannesburg, South Africa Witwatersrand, Johannesburg, South Africa Page 21 of 22 Eur. The ATLAS Collaboration Phys. J. C (2012) 72:2056 145(a)Department of Physics, Stockholm University; (b)The Oskar Klein Centre, Stockholm, Swed 146Physics Department, Royal Institute of Technology, Stockholm, Sweden 46Physics Department, Royal Institute of Technology, Stockholm, Sweden y p y gy 147Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook NY, United States of America 147Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook NY, United States of America 147Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook NY, United States of America 48Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 148Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 149School of Physics, University of Sydney, Sydney, Australia 150Institute of Physics, Academia Sinica, Taipei, Taiwan 151Department of Physics, Technion: Israel Inst. of Technology, Haifa 151Department of Physics, Technion: Israel Inst. The ATLAS Collaboration of Technology, Haifa, Israel Raymond and Beverly Sackler School of Physics a y y y y 153Department of Physics, Aristotle University of Thessaloniki, Thessalonik 153Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 154International Center for Elementary Particle Physics and Department of Physics, The Un 155Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 155Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 156Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 157Department of Physics, University of Toronto, Toronto ON, Canada 158(a)TRIUMF, Vancouver BC; (b)Department of Physics and Astronomy, York University, Toronto ON, Canada 157Department of Physics, University of Toronto, Toronto ON, Canada 158(a)TRIUMF, Vancouver BC; (b)Department of Physics and Astronomy, York University, Toron p y y y 159Institute of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571, Japan 159Institute of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibar 160Science and Technology Center, Tufts University, Medford MA, United States of America 161Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia 162 161Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia Department of Physics and Astronomy, University of California Irvine, Irvine CA, United States of A 162Department of Physics and Astronomy, University of California Irvine, Irvine 163(a)INFN Gruppo Collegato di Udine, Udine; (b)ICTP, Trieste; (c)Dipartimento di Chimica, Fisica e Ambiente, Università di Udine, Udine, Italy 163(a)INFN Gruppo Collegato di Udine, Udine; (b)ICTP, Trieste; (c)Dipartimento di Chimica, Fisica e Ambiente, Università di Udine, Udine, Italy artment of Physics, University of Illinois, Urbana IL, United States of America 164Department of Physics, University of Illinois, Urbana IL, United States of America 165 165Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 166 artment of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 165Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 166 166Instituto de Física Corpuscular (IFIC) and Departamento de Física Atómica, Molecular y Nuclear and Departamento de Ingeniería Electrónica and Instituto de Microelectrónica de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain 166Instituto de Física Corpuscular (IFIC) and Departamento de Física Atómica, Molecular y Nuclear and Departamento de Ingeniería Electrónica and Instituto de Microelectrónica de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain 67Department of Physics, University of British Columbia, Vancouver BC, Canada 167Department of Physics, University of British Columbia, Vancouver BC, Canada 68Department of Physics and Astronomy, University of Victoria, Victoria BC, Canada 69 168Department of Physics and Astronomy, University of Victoria, Victoria BC, Canada 169Waseda University, Tokyo, Japan 171Department of Physics, University of Wisconsin, Madison WI, United States of America 171Department of Physics, University of Wisconsin, Madison WI, United States of America 72Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany 172Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany 173Fachbereich C Physik, Bergische Universität Wuppertal, Wuppertal, Germany 173Fachbereich C Physik, Bergische Universität Wuppertal, Wuppertal, Germany 74Department of Physics, Yale University, New Haven CT, United States of America 5 174Department of Physics, Yale University, New Haven CT, United States of Ameri 175Yerevan Physics Institute, Yerevan, Armenia 176Domaine scientiﬁque de la Doua, Centre de Calcul CNRS/IN2P3, Villeurbanne Cedex, France 176Domaine scientiﬁque de la Doua, Centre de Calcul CNRS/IN2 177Faculty of Science, Hiroshima University, Hiroshima, Japan 177Faculty of Science, Hiroshima University, Hiroshima, Japan aAlso at Laboratorio de Instrumentacao e Fisica Experimental de Particulas - LIP, Lisboa, Portuga bAlso at Faculdade de Ciencias and CFNUL, Universidade de Lisboa, Lisboa, Portugal cAlso at Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom dAlso at TRIUMF, Vancouver BC, Canada dAlso at TRIUMF, Vancouver BC, Canada eAlso at Department of Physics, California State University, Fresno CA, United States of America eAlso at Department of Physics, California fAlso at Novosibirsk State University, Novosibirsk, Russia gAlso at Fermilab, Batavia IL, United States of America gAlso at Fermilab, Batavia IL, United States of America hAlso at Department of Physics, University of Coimbra, Coimbra, Portugal iAlso at Università di Napoli Parthenope, Napoli, Italy iAlso at Università di Napoli Parthenope, Napoli, Italy jAlso at Institute of Particle Physics (IPP), Canada jAlso at Institute of Particle Physics (IPP), Canada kAlso at Department of Physics, Middle East Technical University, Ankara, Turkey kAlso at Department of Physics, Middle East Technical University, Ankara, Turke ech University, Ruston LA, United States of Amer mAlso at Department of Physics and Astronomy, University College London, London, United K mAlso at Department of Physics and Astronomy, University College London, London, United Kingdom nAlso at Group of Particle Physics, University of Montreal, Montreal QC, Canada oAlso at Department of Physics, University of Cape Town, Cape Town, South Afri pAlso at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan Eur. The ATLAS Collaboration Paris-Sud and CNRS/IN2P3, Orsay, France aeAlso at Department of Physics and Astronomy, University of Shefﬁeld, Shefﬁeld, United Kingdom aeAlso at Department of Physics and Astronomy, University of She afAlso at Department of Physics, Oxford University, Oxford, United Kingdom agAlso at Institute of Physics, Academia Sinica, Taipei, Taiwan agAlso at Institute of Physics, Academia Sinica, Taipei, Taiwan ahAlso at Department of Physics, The University of Michigan, Ann Arbor MI, United States of America i Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and aiAlso at Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Di aiAlso at Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, France * The ATLAS Collaboration Paris-Sud and CNRS/IN2P3, Orsay, France aeAlso at Department of Physics and Astronomy, University of Shefﬁeld, Shefﬁeld, United Kingdom afAlso at Department of Physics, Oxford University, Oxford, United Kingdom agAlso at Institute of Physics, Academia Sinica, Taipei, Taiwan ahAlso at Department of Physics, The University of Michigan, Ann Arbor MI, United States of America aiAlso at Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, France Deceased qAlso at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany qAlso at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany rAlso at Manhattan College, New York NY, United States of America rAlso at Manhattan College, New York NY, United States of America sAlso at School of Physics, Shandong University, Shandong, China sAlso at School of Physics, Shandong University, Shandong, China tAlso at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France uAlso at School of Physics and Engineering, Sun Yat-sen University, Guanzhou, China vAlso at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan vAlso at Academia Sinica Grid Computing, Institute of Physics, Aca Also at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan wAlso at DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Sacla l’Energie Atomique), Gif-sur-Yvette, France l’Energie Atomique), Gif-sur-Yvette, France xAlso at Section de Physique, Université de Genève, Geneva, Switzerland xAlso at Section de Physique, Université de Genève, Geneva, Switzerland yAlso at Departamento de Fisica, Universidade de Minho, Braga, Portugal yAlso at Departamento de Fisica, Universidade de Minho, Braga, Portugal yAlso at Departamento de Fisica, Universidade de Minho, Braga, Portugal zAlso at Department of Physics and Astronomy, University of South Carolina, Columbia SC, Unit p y y y aaAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary aaAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budap abAlso at California Institute of Technology, Pasadena CA, United States of America acAlso at Institute of Physics, Jagiellonian University, Krakow, Poland acAlso at Institute of Physics, Jagiellonian University, Krakow, Poland adAlso at LAL, Univ. Paris-Sud and CNRS/IN2P3, Orsay, France adAlso at LAL, Univ. The ATLAS Collaboration Phys. J. C (2012) 72:2056 Page 22 of 22 qAlso at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany rAlso at Manhattan College, New York NY, United States of America sAlso at School of Physics, Shandong University, Shandong, China tAlso at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France uAlso at School of Physics and Engineering, Sun Yat-sen University, Guanzhou, China vAlso at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan wAlso at DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat a l’Energie Atomique), Gif-sur-Yvette, France xAlso at Section de Physique, Université de Genève, Geneva, Switzerland yAlso at Departamento de Fisica, Universidade de Minho, Braga, Portugal zAlso at Department of Physics and Astronomy, University of South Carolina, Columbia SC, United States of America aaAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary abAlso at California Institute of Technology, Pasadena CA, United States of America acAlso at Institute of Physics, Jagiellonian University, Krakow, Poland adAlso at LAL, Univ. CNRS/IN2P3, Paris, France Deceased *Deceased
https://openalex.org/W4315705035	https://research-information.bris.ac.uk/ws/files/366575054/Nursing_Open_2023_Percival_Hospital_practitioner_views_on_the_benefits_of_continence_education_and_best_ways_to.pdf	English	null	Hospital practitioner views on the benefits of continence education and best ways to provide training	Nursing open	2,023	cc-by	8,258	Percival, J., Abbott, K., Allain, T., Bradley, R., Cramp, F., Donovan, J., McCabe, C., Neubauer, K., Redwood, S., & Cotterill, N. (2023). Hospital practitioner views on the benefits of continence education and best ways to provide training. Nursing Open, 10(5), 3305-3313. https://doi.org/10.1002/nop2.1582 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1002/nop2.1582 Link to publication record on the Bristol Research Portal PDF-document This is the final published version of the article (version of record). It first appeared online via Wiley at https://doi.org/10.1002/nop2.1582 . Please refer to any applicable terms of use of the publisher. Percival, J., Abbott, K., Allain, T., Bradley, R., Cramp, F., Donovan, J., McCabe, C., Neubauer, K., Redwood, S., & Cotterill, N. (2023). Hospital practitioner views on the benefits of continence education and best ways to provide training. Nursing Open, 10(5), 3305-3313. https://doi.org/10.1002/nop2.1582 Percival, J., Abbott, K., Allain, T., Bradley, R., Cramp, F., Donovan, J., McCabe, C., Neubauer, K., Redwood, S., & Cotterill, N. (2023). Hospital practitioner views on the benefits of continence education and best ways to provide training. Nursing Open, 10(5), 3305-3313. https://doi.org/10.1002/nop2.1582 Funding information K E Y W O R D S catheters, continence care, education, hospital healthcare practitioner, incontinence, nurses, pads, product, training R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Hospital practitioner views on the benefits of continence education and best ways to provide training To ensure better take-up of, and engagement with, continence training, it must be authorized as essential and provided in ways that reflect professional preferences and pragmatic resource considerations. K E Y W O R D S catheters, continence care, education, hospital healthcare practitioner, incontinence, nurses, pads, product, training Hospital practitioner views on the benefits of continence education and best ways to provide training John Percival1 \| Katharine Abbott2 \| Theresa Allain3 \| Rachel Bradley4 \| Fiona Cramp1 \| Jenny Donovan5 \| Candy McCabe6 \| Kyra Neubauer2 \| Sabi Redwood5 \| Nikki Cotterill1 1Faculty of Health and Applied Sciences, University of the West of England, Bristol, UK 1Faculty of Health and Applied Sciences, University of the West of England, Bristol, UK 2Complex Assessment and Liaison Service, North Bristol NHS Trust, Bristol, UK 3Medicine for Older Persons, University Hospitals Bristol NHS Foundation Trust, Bristol, UK 4Geriatric & Orthogeriatric Medicine, University Hospitals Bristol NHS Foundation Trust, Bristol, UK 5Bristol Medical School, University of Bristol, Bristol, UK 6College of Health, Science and Society, University of the West of England, Bristol, UK Correspondence John Percival, Faculty of Health and Applied Sciences, University of the West of England, Bristol, UK. Email: john.percival@uwe.ac.uk Funding information University Hospitals Bristol and Weston NHS Foundation Trust, Research Capability Funding (RCF) stream., Grant/ Award Number: 2018-Aut-03] 1Faculty of Health and Applied Sciences, University of the West of England, Bristol, UK 2Complex Assessment and Liaison Service, North Bristol NHS Trust, Bristol, UK 3Medicine for Older Persons, University Hospitals Bristol NHS Foundation Trust, Bristol, UK 4Geriatric & Orthogeriatric Medicine, University Hospitals Bristol NHS Foundation Trust, Bristol, UK 5Bristol Medical School, University of Bristol, Bristol, UK 6College of Health, Science and Society, University of the West of England, Bristol, UK Correspondence John Percival, Faculty of Health and Applied Sciences, University of the West of England, Bristol, UK. Email: john.percival@uwe.ac.uk Funding information University Hospitals Bristol and Weston NHS Foundation Trust, Research Capability Funding (RCF) stream., Grant/ Award Number: 2018-Aut-03] Abstract Aim: The aim of the study was to explore practitioners' experiences and perspectives on continence training, in order to understand its relevance to practice and how take- up of, and engagement with, such training may be improved. Design: 27 qualitative interviews were conducted with nursing, medical and allied health practitioners in three hospitals. Methods: We analysed data thematically, both manually and with the aid of NVivo software. The research adheres to the consolidated criteria for reporting qualitative research checklist. Results: Practitioners asserted the likely benefits of evidence-based continence train- ing, including more judicious use of products, reduction in associated infection, better patient skin care and more facilitative communication with patients. Practitioners also identified preferred methods of continence training, according to their role and work- load. © 2023 The Authors. Nursing Open published by John Wiley & Sons Ltd. Abstract Aim: The aim of the study was to explore practitioners' experiences and perspectives on continence training, in order to understand its relevance to practice and how take- up of, and engagement with, such training may be improved. Design: 27 qualitative interviews were conducted with nursing, medical and allied health practitioners in three hospitals. Methods: We analysed data thematically, both manually and with the aid of NVivo software. The research adheres to the consolidated criteria for reporting qualitative research checklist. Results: Practitioners asserted the likely benefits of evidence-based continence train- ing, including more judicious use of products, reduction in associated infection, better patient skin care and more facilitative communication with patients. Practitioners also identified preferred methods of continence training, according to their role and work- load. To ensure better take-up of, and engagement with, continence training, it must be authorized as essential and provided in ways that reflect professional preferences and pragmatic resource considerations. Correspondence John Percival, Faculty of Health and Applied Sciences, University of the West of England, Bristol, UK. Email: john.percival@uwe.ac.uk Funding information University Hospitals Bristol and Weston NHS Foundation Trust, Research Capability Funding (RCF) stream., Grant/ Award Number: 2018-Aut-03] Funding information University Hospitals Bristol and Weston NHS Foundation Trust, Research Capability Funding (RCF) stream., Grant/ Award Number: 2018-Aut-03] Nursing Open. 2023;10:3305–3313. University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ Received: 28 January 2022 \| Revised: 16 June 2022 \| Accepted: 15 December 2022 Received: 28 January 2022 \| Revised: 16 June 2022 \| Accepted: 15 December 2022 DOI: 10.1002/nop2.1582 2 \| BACKGROUND The interview schedule was not formally piloted but informed by previous audit data and existing evidence (Bowling, 2014), as well as by consensus input by experts in the clinical area, and reviewed by all authors. The interview schedule (see Appendix 1) included questions on how participants had learned about continence care, whether continence training had been made available, whether participants knew how to access such training, preferences regard- ing style of training and participants' awareness of continence care guidelines. As such, the study design was deliberately exploratory, offering opportunity for participants to reflect on relevant experi- ences and ideas about a health topic that has received relatively little attention (Hunter et al., 2018). Studies regularly highlight the emotional, social and physical impact of incontinence on patients, including anxiety, loss of self-esteem, decreased independence and isolation (Abrams et al., 2015; Holt- Lunstad et al., 2015; Ramage-Morin & Gilmour, 2013). Furthermore, incontinence substantially increases the risks of lengthy hospi- talization and nursing home admission in older adults (All Party Parliamentary Group Report, 2011) These wide-ranging effects indicate that appropriate management of incontinence symptoms is therefore essential and pro-active continence care can make de- monstrable improvements (Abrams et al., 2017; NHS England, 2014; Wagg et al., 2017). Treatment of incontinence, however, has often been reported as inadequate (Healthcare Quality Improvement Partnership, 2010; Taylor & Cahill, 2018), particularly for older adults (Harari et al., 2014; Vethanayagam et al., 2017). Indeed, the Francis public inquiry re- ported that the quality of continence care was the area of practice most frequently subject to complaint (Francis, 2013). Falls, pressure ulcers and moisture lesions are directly linked to poor continence care in older adults (DoH, 2014; Yates, 2017). Studies have also found that healthcare professionals in acute care settings incon- sistently identify, assess and conservatively manage incontinence, citing evidence of inappropriate use of catheters and pads, weak care planning and insufficient staff support and training (Age UK et al., 2018; Orrell et al., 2013; Wagg et al., 2008). 3.3 \| Participants Research participants were recruited in acute inpatient wards (mostly, though not exclusively, care of older people wards) at three hospitals in England: two large, city, teaching hospitals (one, a tertiary centre for Urology) and one a smaller, town-based non-teaching cen- tre. The main inclusion criterion was that research participants have responsibility for patients aged 65 and over. Twenty-seven partici- pants were recruited, including nursing staff, allied healthcare pro- fessionals, medical staff and healthcare assistants. All participants answered the questions put to them. Practitioner education is recognized as a crucial component of moves to improve the quality of continence care (All Party Parliamentary Group Report, 2011; NICE, 2019; Orrell et al., 2013). However, the availability of continence care training, and healthcare practitioners' engagement with it, are reported to be limited (Harari et al., 2014; United Kingdom Continence Society, 2015). There has been little exploration of why this is the case, beyond noting that continence care is sometimes seen by practitioners as unglamor- ous and a sensitive subject to discuss with patients (Read, 2018). A recent paper, also based on the study reported here, found that hospital practitioners believe continence care can be improved in patient-centred, time-efficient ways, if they had appropriate conti- nence training (Percival et al., 2021). In this current paper, we closely examine hospital practitioners' experiences of relevant training, their perspectives on its benefits to practice, and their preferences for the ways in which it is delivered, so as to understand how conti- nence education can best be provided, taken up and engaged with by more staff in the hospital setting. Recruitment was carried out through purposive sampling of staff groups to achieve representation across professional roles (Bowling, 2014). The lead researcher (JP) met with ward managers at each hospital to discuss the research and provide participant in- formation materials for display and distribution among staff. Once representation was achieved in respect of each staff group, nursing, medical and allied health, the sample size was determined during on- going analysis and interpretation of emerging themes, ensuring data collection parameters in line with the study's objectives (Johnson et al., 2020). One-off Individual interviews took place in the hospital settings and were carried out by JP. 3.4 \| Data collection Semi-structured exploratory interviews were carried out with hospital-based nursing, medical and allied health professional staff, to probe perspectives on continence care practice and ways to optimize such care. Interviews were audio-recorded, transcribed verbatim and then analysed using thematic analysis (Silverman, 2015). Initial data analysis involved close and repeated reading of each transcript by JP, establishing an opportunity for the development and refining of coding strategies in relation to emerging issues and insights. Transcript data were then imported into the qualitative data management software package NVivo 12 (King, 2004) with coding further enhanced by JP and the chief 3306 \| 2 \| BACKGROUND 3306 1 \| INTRODUCTION setting, is often lacking or not taken up (Harari et al., 2014). Lack of continence training among healthcare professionals is a worldwide issue (International Continence Society, 2009; Read, 2018). There is a clear need for improved continence care and training, together with a better understanding of healthcare workers' perspectives, as evidence is limited in these respects. (Harari et al., 2014; Wagg et al., 2017) As a result of these concerns, our study set out to inves- tigate the likely merits of continence training in hospitals and how education opportunities may be engaged with by more practitioners in that setting. Over 14 million UK citizens experience bladder control problems and 6.5 million suffer bowel control difficulties (NHS England, 2018); globally, the World Health Organization report that urinary incon- tinence is a highly prevalent condition in older people aged 60 years and over (WHO, 2017). Many who experience incontinence receive poor quality care and/or insufficient support (Abrams et al., 2017; NHS England, 2018). Continence education is key to improvements in care, but relevant training provision, particularly in the hospital This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2023 The Authors. Nursing Open published by John Wiley & Sons Ltd. wileyonlinelibrary.com/journal/nop2 Nursing Open. 2023;10:3305–3313. PERCIVAL et al. 3.2 \| Design 3.2 \| Design 2 \| BACKGROUND 4 \| RESULTS The study's total of 27 participants was evenly distributed across the three hospital sites, as shown in Table 1. This total included 17 nursing staff, 5 allied healthcare professionals, 3 medical staff and 2 healthcare assistants. Participants represented all age groups, were mainly female (as is usual in the healthcare workforce) and were pre- dominantly white British, reflecting the regional demographic. A ward sister observed that lack of monitoring regarding prod- ucts such as catheters showed how continence care was sometimes not “thought through,” with the result that aspects of conservative management, such as dietary advice, bladder retraining or pelvic floor muscle exercises, were not adequately considered. A nursing assistant suggested that lack of training had a particular bearing on this issue: In order to safeguard confidentiality, the source of interview excerpts included in this paper is identified using a letter signifying each hospital site, as shown in Table 1, followed by the practitioner's study identification number. When [staff]… have just been in the profession for a long time, pads just go on [patients]… A lot of people have not had training and just see incontinence pads and they are almost saying, “[I use them] just in case.” When really we should be moving away from that. [B47, nursing assistant]. The questions on training put to practitioners elicited opinions and experiences regarding four themes: challenges to understanding and managing continence care; availability of continence care edu- cation; perceived benefits of continence care education; and pre- ferred methods of training. The relevance of continence care training to meeting the chal- lenges outlined above was apparent throughout interviews. The availability of training made available to practitioners was therefore explored further. 4.1 \| Challenges to understanding, monitoring and managing continence care needs Although incontinence was rarely the principal reason for a patient's admission, practitioners' accounts suggested that it featured promi- nently in their everyday practice. Certainly, staff on ‘care of elderly’ wards across the three sites refer to numbers as high as 75–80 per cent of patients who “struggle in some way with their urinary or fae- cal output” [A02, apprentice healthcare support worker], leading 3.1 \| Aims The study aspired to understand hospital healthcare practitioners' views on the efficacy of continence education and the ways in which take-up and engagement with continence training can be improved. PERCIVAL et al. 3307 investigator (NC) using a coding frame devised to highlight theme connectivity and relevance to the primary areas of investigation (Silverman, 2015). one consultant physician to describe incontinence as “one of the geriatric giants” [B46]. Despite the prevalence of older hospital patients with incon- tinence, it was clear from interviews across the three sites that pro-forma hospital admission assessment procedures insufficiently captured continence care needs, due to “variable” or sometimes “in- complete” detail. Accurate record-keeping by ward staff, following routine checks of patients' skin condition, temperature and urine/ faecal output, was therefore seen as of compensatory importance to building a better understanding of the patient's continence care needs. However, participants disclosed that monitoring and record- keeping were not always carried out efficiently: 3.5 \| Ethics All interviews with healthcare practitioners were carried out in ac- cordance with research governance ethics protocols and Health Research Authority (HRA) approval (Research Ethics Committee reference: HAS.19.07.221, September 2019). Written consent was provided by each participant prior to interview. This morning a lady was TWOC'd [attempted a Trial WithOut Catheter]. she is mobile, gone to the toilet a couple of times last night but nobody has docu- mented to see if she has passed urine or not or how much. [A04; senior nursing assistant] 3.6 \| Rigour In order to build trustworthiness and quality about data analysis, the conduct and thematic outcomes of analysis were considered and agreed on by all authors. Furthermore, regular meetings of the researchers took place to discuss emerging themes and ratify their origin in the data. In addition, researcher reflexivity and peer review regularly took place during stages of data gathering. The research adheres to the COREQ reporting checklist (Tong et al., 2007). Lack of oversight and erroneous assumptions could result in pa- tients using inappropriate products that jeopardized independence: I have seen situations where people have been put in incontinence pads and then you ask them or you ask their relative and [are told] they do not use them at home. And it's kind of, I think we sometimes get in the habit of presuming that somebody needs that when they do not. [C23, staff nurse] 4.2 \| Availability of continence care education The majority of nursing staff interviewees told us they had re- ceived no “specific,” “formal,” hospital-based training in continence 20541058, 2023, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/nop2.1582 by Test, Wiley Online Library on [02/06/20 PERCIVAL et al. 3308 1058, 2023, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/nop2.1582 by Test, Wiley Online Library on [02/06/2023]. See the Terms and Conditions (ht TA B LE 1 Hospital healthcare practitioner characteristics Setting Prof title Age band Gender Practice years Band Ethnicity A Staff nurse 21–25 F 1.5 5 Black British A Staff nurse 21–25 F <1 5 White & Black Caribbean A Ward sister 26–30 F 6 7 White British A Senior staff nurse 26–30 F 6 6 White British A Senior staff nurse 51–55 F 9 6 White British A Senior staff nurse 26–30 F 5 6 White British A Apprentice healthcare support worker 18–20 F <1 2 White British A Therapy technician 31–35 F 6.5 4 White British A Senior nursing assistant 46–50 F 13 3 White British B Staff nurse 56–60 M 29 5 White British B Staff nurse 56–60 F 40 5 White British B Student nurse 31–35 F <1 N/A White British B Physiotherapist 21–25 F 1.5 5 White British B Therapy technician 46–50 F 6 3 White British B Consultant physician 36–40 M 14 N/A Indian B Nursing assistant 36–40 F 2 2 White British B Staff nurse 46–50 F 2 5 White British B Junior sister 31–35 F 9 6 White British C Sister 21–25 F 4 6 White British C Assistant nursing practitioner 31–35 F 5 4 Black South African C Staff nurse 26–30 F 1 5 White British C Consultant geriatrician 36–40 M 17 N/A White British C Physiotherapist 41–45 F 15 6 White British C Dementia specialist practitioner 51–55 F 35 7 White British C Occupational therapist 41–45 F 11 6 White British C Healthcare assistant 51–55 F 3 3 White British C Junior doctor 21–25 F <1 F1 White British Note: Table shows participants' setting (anonymized), role, age range, gender, length of time in practice, salary band where appropriate and ethnicity. I think there needs to be training to avoid seeing con- tinence just in terms of incontinence and needing a pad. [C28, occupational therapist] Practitioners suggested that dedicated continence care educa- tion would benefit their practice by getting people “thinking” more consciously about the subject, so “it would be at the forefront of our mind more” [A05, staff nurse]. Similarly, a senior staff nurse advo- cated raising the profile of continence care training when she said: Practitioners repeatedly said that continence training would help ensure safe use of continence care products and “training with awareness of why we are putting a pad on” [C27] as well as guid- ance on “how to make sure [staff] have got the right size pad” [C29] and “how often you change pads… we don't know” [B42]. As regards catheters, one therapy technician suggested staff would benefit from “teaching around… weighing up the effects of long term cath- eter and short term catheter [use]” [A03]. Provision of such teach- ing, it was said, would increase staff vigilance and caution regarding catheter use. We are very much aware of dementia, we are very much aware of Parkinson's. I think continence needs to be brought up to the forefront as well. [A09, senior staff nurse] 4.2 \| Availability of continence care education According to practitioners, improved access to relevant educa- tion would also facilitate more efficacious use of incontinence pads: 4.2 \| Availability of continence care education The management and support of patients in respect of their skin integrity was a particular concern for practitioners, as the fol- lowing excerpt exemplifies: provide much needed information about “how we manage it with- out going for the easy option [automatic use of pads and catheters]” [A05]. The management and support of patients in respect of their skin integrity was a particular concern for practitioners, as the fol- lowing excerpt exemplifies: A minority of practitioners referred to occasional episodes of training on aspects of care connected with incontinence, such as skin care. Some practitioners referred to industry “reps” who came on to the ward to provide updates on products, such as new incon- tinence pads and their correct use, or catheters and new discharge packs. Although most interviewees said they were aware of good practice guidelines on continence care in hospitals, few confirmed they had actually read them or been asked to do so. A number of interviewees referred to the availability of urology nurses, or senior staff, should they need advice on a continence care issue. However, none of our interviewees knew of, or any longer had access to, a designated, specialist, continence care advisor in their hospital. According to one ward sister, the lack of specialist advice contributes to “poor” continence care practice. We are constantly managing patients who have really poor skin from continence issues. I do not really know myself if there are other things we could be doing… We do our best but if we had kind of more education and understanding then maybe our care would be better. [C23, staff nurse] In this connection, practitioners predicted that continence care education could play a part in helping staff “get rid of” incontinence sheets, which were in some cases still used inappropriately and “causes their skin to break down” [A01]. Practitioners also believed that conti- nence care education would help improve their knowledge of relevant best practice guidelines, “what we should be doing, what is sort of the baseline that we should all be meeting for our patients” [B47]. Given these experiences of training availability, Figure 1, below, is presented to graphically depict the lesser availability of formal training in comparison with other sources of continence knowledge. Sources of education are shown in decreasing proportions, as de- scribed by practitioners. 4.2 \| Availability of continence care education TA B LE 1 Hospital healthcare practitioner characteristics y.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License care, not even as nursing assistants, but had learned about it from industry representatives or simply by “just doing it,” learning “on the job”: interviewees recalled incontinence being a noteworthy component of their student education and a few said their Hospital Trust “induction” had included a session on the management of patients with inconti- nence; otherwise, interviewees had gained knowledge through self- directed reading, mostly online. Staff indicated that although online learning had been useful, they were more likely to engage with training on continence care, and its complexities, if it was presented in person by knowledgeable and experienced practitioners. We have had people come in and talk to us about in- continence products like different types of catheters and different pads but not any specific training about incontinence as a whole. [C21, staff nurse] This lack of concerted continence training was a concern for many interviewees. Some spoke of the pressure on new staff, who may have “no background in care” [C22, nurse assistant], or the pressure on mental health nurses who, according to a junior doctor, “are not ‘physically’ trained nurses” but often deal with incontinence issues [C29]. Healthcare assistants, too, were said to be disadvan- taged, given that they work with “high continence needs but haven't really had much training in it” and so may be unaware, for example, of “the link between skin [integrity] and continence” [C23]. Doctors I have been here six-and-a-half years and I have never had any official continence, incontinence teaching. [A03, therapy technician] I have been here six-and-a-half years and I have never had any official continence, incontinence teaching. [A03, therapy technician] Nursing staff referred to study days that had been organized on “pain,” “mouth care” or other aspects of fundamental practice, with nothing comparable in respect of continence care. A small number of PERCIVAL et al. 3309 also conceded “we get very little [continence care] training… it's not high enough on our curriculum” [C24], and “if we did have [conti- nence care] training we might be able to stop these situations where we have forgotten to TWOC the patient” [C29]. provide much needed information about “how we manage it with- out going for the easy option [automatic use of pads and catheters]” [A05]. Little short bursts [of training] happening on the ward area I think is ideal. Because everybody is getting that same opportunity. Because study days, you can only release so many people at a time, if you are lucky. [A09, senior staff nurse] Little short bursts [of training] happening on the ward area I think is ideal. Because everybody is getting that same opportunity. Because study days, you can only release so many people at a time, if you are lucky. [A09, senior staff nurse] You can go over to somebody and say its fine, do not worry. When actually they are like, “I am worrying and it's not fine, it's not nice.” Sometimes it would maybe be nice to know how they would want us to approach it as opposed to [us] just batting it off. [A08, senior staff nurse] In addition, the advantage of “localised training coming in” was mentioned by practitioners who welcomed gaining greater knowl- edge, in this way, of relevant continence services that support people at home, enabling them to advise patients of “what is available [in the community] for people that are incontinent” [B45, therapy technician]. Lengthier, off-site, training workshops was a format favoured by one consultant [C24] as a more practical option for medical staff in hospitals, given ward-based demands on doctors: In addition, the advantage of “localised training coming in” was mentioned by practitioners who welcomed gaining greater knowl- edge, in this way, of relevant continence services that support people at home, enabling them to advise patients of “what is available [in the community] for people that are incontinent” [B45, therapy technician]. For all the reasons outlined above, participants often spoke of con- tinence care training with conviction and in the spirit of broadening professional horizons: Lengthier, off-site, training workshops was a format favoured by one consultant [C24] as a more practical option for medical staff in hospitals, given ward-based demands on doctors: I would love to have some [continence care] training… we had extra [mouth care] training and I learnt things that I never knew about mouth care… without that extra training, sometimes you do not get to know. So I think we could all learn something new [about con- tinence care] if we just got given a little bit of extra training. [B49, junior ward sister] For doctors, one-day training works well because the chance of getting us back to [short, follow-up] things is low. For nurses I think it's the other way round. In the past when I have tried to do training for nurses it's almost impossible for them because they cannot get off the wards. Little short bursts [of training] happening on the ward area I think is ideal. Because everybody is getting that same opportunity. Because study days, you can only release so many people at a time, if you are lucky. [A09, senior staff nurse] [C24, consultant geriatrician] What form this potential training should adopt was a question we put to practitioners, in order to develop our understanding of provision that takes account of needs and preferences and is, therefore, likely to be approved and supported. Another consultant endorsed this view and added that he preferred this workshop-type training as it offered sufficient time for audience participation and the generation, by speakers, of ideas and “insights.” A relatively small number of nursing and therapy staff elected the off-site, workshop training option, on the grounds that it offered the opportunity to hear from a number of different speakers and practi- tioners about various services and aspects of practice, “away from the practice area [where] you're not being pulled in all directions” [C26; de- mentia specialist practitioner]. One or two interviewees were open to “mixed methods” training. A physiotherapist said she likes power-point presentations supplemented by “learning through doing, otherwise it doesn't stick” [B44]. 4.4 \| Preferred methods of training When asked to consider their preferences regarding methods of training, opinions were mixed as to the advantages of regular, short duration, ward based, sessions or less frequent, half/full day, off- site, study sessions. No practitioner elected online e-learning as a preferred source of training. Generally, nursing staff favoured shorter, regular, “hands-on” training sessions and medical and allied health professionals intimated a preference for longer, occasional workshop-type provision. It is worth briefly setting out the reasons given by interviewees for their preferences, as these provide fur- ther insight into the likely interest in, and take up of, continence care training for hospital-based staff. Whatever the format, regularity of training was advocated as en- tirely appropriate, to ensure all staff are included regardless of shift patterns. One ward sister cautioned, however, that time pressures may make commitment to any format of training vulnerable to dis- ruption and could only be guaranteed to take place if authorized as “essential training” [A07, ward sister]. Staff speaking in favour of regular, short duration, ward-based training said they normally found it easier to focus and retain infor- mation with this format and, additionally, it offered opportunity for hands-on, practice-oriented learning: This format was also said to be practical, given the pressures on staff time and the difficulty releasing staff for lengthier periods of off- site training. A staff nurse amplified this last point when she said: confident “to ask those questions and not everyone is confident to do that” [B49], a situation that prompted participants to reflect on com- munication shortcomings: 4.3 \| Perceived benefits of continence care education In addition, practitioners spoke of the potential impact of training in respect of increasing their knowledge of local community continence care services and sources of “community support” [A03], to facilitate discharge planning and “signpost [patients] in the right way” [A08]. This better co-ordination of continence care was thought likely to further raise its profile and also help support patients' independence: Practitioners' accounts indicate that continence care education would help improve their “management of incontinence” [C21] and FI G U R E 1 Sources of education about continence care in decreasing proportions. Learning on the job Industry led Formal training I think staff knowledge [needs improving]… with knowledge we can then give patients the confidence that they can manage their own continence, whether its catheter, pads, whatever. [B45, therapy technician] A number of practitioners also advocated more attentive listen- ing to patients and their family members, in order to “get their per- spectives” in regard to continence care and planning. In this context, a junior ward sister acknowledged that staff had to be sufficiently FI G U R E 1 Sources of education about continence care in decreasing proportions. 3310 PERCIVAL et al. healthcare practitioners; the benefits to continence care practice of an increase in practitioner education; and ways in which continence care training might be promoted and delivered to maximize take-up by practitioners. Our study finds that relevant training is negligible, that practitioners want more continence education and that they have clear ideas on how such education can best be provided. Additionally, findings from our study show that practitioners see the benefit of training that would assist them in confidently facilitating conversations with patients, to help patients overcome embarrassment and more readily impart relevant information and experience. Training that achieves these outcomes is important when providing intimate care (Redwood et al., 2020) and is highly valued by patients, who are reassured when healthcare practitioners acknowledge that incontinence is not easy to talk about and encour- age a shared approach to care planning (Brett, 2021). Studies have drawn attention to the availability of formal con- tinence care training to healthcare practitioners and its occurrence in less than half of acute hospitals (United Kingdom Continence Society, 2015). Our study bears out these concerns and indicates the relative prominence of industry-led, product-focused instruction and the lack of service-led, broad, evidence-based training. Industry- led training, we suggest, may exacerbate practitioners' over-reliance on catheters and pads, while independent, comprehensive training is less influenced by marketing and likely to help practitioners ques- tion, and learn more from, their practice. Provision of training driving continence promotion is key. Recommendations have been made for continence education to be provided in ways that offer more structure, for example through modular delivery. Furthermore, the provision of continence educa- tion in different formats is key to ensuring that training is accessi- ble, given the range of staff involved and the constraints on their time (United Kingdom Continence Society, 2015). Our study iden- tified staff interest in various options for delivery of face-to-face training; in addition, education providers should consider e-learning packages to supplement in-person continence education sessions (Gourlay, 2011; McClurg et al., 2013). Whatever format of training delivery, practitioners need to be released from other work pres- sures and to have sufficient, dedicated time made available, for con- tinence education to be viable and effective (Ferdinand, 2018). Previous survey findings have also exposed weaknesses in the quality of student continence education. A survey of undergraduate programmes including medicine, nursing, physiotherapy and occu- pational therapy, in 84 UK universities, found significant lack of time allocated to continence education (an average of 7.3 hours for stu- dent nurses, 4.9 hours for medical students, 3.8 hours for student physiotherapists and 3.5 hours for occupational therapy students). The survey concluded that continence education was insufficiently supported as a stand-alone topic to approved standards within un- dergraduate curricula (McClurg et al., 2013). Studies have found evidence for continence education to be given greater emphasis in student training and for its content to be more fully developed (Ferdinand, 2018; Gourlay, 2011; Holroyd, 2015). Continence education should also feature in continuous profes- sional development for post-graduates if they care for patients with continence care needs (Gourlay, 2011; United Kingdom Continence Society, 2015). However, for designated training to be recognized as a necessary component within healthcare curricula, continence care has to assume a higher status and profile (Harari et al., 2014; Ferdinand, 2018). In this respect, and as our study infers, an in- crease in continence care leads and specialist nurses would play a significant role in raising awareness of good practice and organis- ing and developing relevant training (Harari et al., 2014; Unplanned Admissions Consensus Committee, 2019). Of relevance here are the recently updated standards of proficiency for Registered Nurses, which now stipulates that practitioners should use evidence-based approaches to meet continence needs (Nursing and Midwifery Council, 2018). In the literature search carried out for our study, there was no similar reference to continence education in the proficiency protocols pertaining to medical or allied health practi- tioner registration. Furthermore, over half of qualified nurses do not receive any post-registration continence education (Ferdinand, 2018). To help ensure continence care training gains traction and be- comes routine, there are recommendations for education pro- grammes to be ongoing and mandatory, helping cement its status as required and necessary learning (NHS England, 2018; Orrell et al., 2013). Indeed, a recent report highlighting the plight of pel- vic floor services recommends that nursing and medical education should include a sufficient emphasis on continence throughout the healthcare professional's career (The Pelvic Floor Society, 2021). Our study adds weight to this argument, given participants' view that continence care training would have to be authorized as essen- tial if it is to take place at all. Increased continence care education helps build a more knowl- edgeable workforce, capable of routinely carrying out timely, person- centred assessments, enabling greater patient self-management and more cost-effective treatment (Holroyd, 2015; Nursing and Midwifery Council, 2018). Indeed, relevant training would help raise healthcare practitioners' awareness and consideration of al- ternatives to, or more restricted use of, catheters and incontinence pads, yielding health and social care cost savings and improvements to patients and carer quality of life (Dealey et al., 2012; Murphy et al., 2015; Unplanned Admissions Consensus Committee, 2019). Our study has revealed the pertinence of such benefits to current continence care practice in hospital settings, which practitioners have told us is sometimes characterized by limited understanding, or awareness, of best practice regarding continence management, skin integrity issues and judicious use of products. 5 \| DISCUSSION We do have this teaching scheme where different people will come in and talk about different things… every week… it's short and sweet, I think that seems to work well. [A01, senior staff nurse] Our research findings highlight three key topics that merit discus- sion: adequacy of the continence care training available to hospital PERCIVAL et al. 3311 6 \| CONCLUSION Bowling, A. (2014). Research methods in health: Investigating health and health services. Open University Press. Brett, L. (2021). Incontinence is lonely and hard to talk about. BMJ, 24(372), n207. https://doi.org/10.1136/bmj.n207 Obstacles to good continence care include insufficient understanding and monitoring of patients' continence needs, leading to over-reliance on, or inappropriate use of, catheters and pads. However, the avail- ability of broad, structured training that might address such issues is negligible and most practitioners also lack recourse to specialist ad- vice, other than product-focused training by industry reps. Findings indicate that concerted continence care training would help improve practitioners' management and communication of continence issues and develop safer practice. The delivery style of training needs to be flexible to reflect different practitioner preferences and pragmatic resource considerations. Most importantly, the study's findings offer insights that should help health service and training providers engage more practitioners in continence education experienced as accessible, worthwhile and effective. Dealey, C., Posnett, J., & Walker, A. (2012). The cost of pressure ulcers in the United Kingdom. Journal of Wound Care, 21(6), 261–266. Department of Health. (2014). Annual report of the chief medical officer, Department of Health. (2014). Annual report of the chief medical officer, 2014. The health of the 51%: women. Department of Health. https:// www.networks.nhs.uk/nhs-networks/staffordshire-shropshire -and-black-country-newborn/documents/documents/chief-medic al-officers-report-2014 Ferdinand, S. (2018). Continence care education: Views of students and registered nurses. The British Journal of Nursing, 27(15), 852–859. https://doi.org/10.12968/bjon.2018.27.15.852 https://doi.org/10.12968/bjon.2018.27.15.852 Francis, R. (2013). Report of the mid staffordshire NHS Foundation Trust public inquiry. Stationery Office. http://webarchive.nationalar chives.gov.uk/20150407084003/http://www.midstaffspublic inquiry.com/ Gourlay, K. (2011). Continence care education for community pharmacy staff – Evaluation of an e-learning facility compared with other modes of training. Australia and New Zealand Continence Journal, 17(4), 115. CONFLICT OF INTEREST The authors declare that there is no conflict of interest. The authors declare that there is no conflict of interest. 5.1 \| Strengths and limitations Key strengths of the study include its targeted focus on continence care training, a reportedly important but under-researched aspect of healthcare. Practitioners' preferences for regular ward-based train- ing or off-site workshops help inform continence training that is fit for purpose and with improved take-up by staff. In addition, the data 3312 PERCIVAL et al. France, Germany, the UK and the USA. BJU International, 115(1), 143–152. we gathered helps strengthen the health policy case to reduce the unnecessary use of products known to have adverse health conse- quences. A limitation of the study is an imbalance in research par- ticipants, given the higher representation of nursing staff in relation to medical and allied health practitioners. A more even spread of practitioners would potentially have helped further refine interpre- tations of our research data. Age UK, The Urology Foundation, Marie Curie, Parkinson's UK, Alzheimer's Society, NIHR, D4D, JLA, & Core BR. (2018). “My bladder and bowel own my life” – Continence Research: Exploring the themes, needs and recommendations raised at a collaborative research workshop. https://www.alzheimers.org.uk/sites/defau lt/files/2018-08/Incontinence%20needs%20-%202018%20rep ort%20v9.pdf All Party Parliamentary Group Report. (2011). Cost-effective commission- ing for continence care. RCP. https://www.rcplondon.ac.uk/proje cts/outputs/cost-effective-commissioning-continence-care ACKNOWLEDGEMENTS We would like to thank the study participants for their valuable con- tributions and for sharing their views so openly. We are also grateful for the valuable patients and public contributions to the develop- ment of this study. Harari, D., Husk, J., Lowe, D., & Wagg, A. (2014). National audit of conti- nence care: Adherence to National Institute for Health and Clinical Excellence (NICE) guidance in older versus younger adults with fae- cal incontinence. Age and Ageing, 43(6), 785–793. Healthcare Quality Improvement Partnership (HQIP) & Royal College of Physicians. (2010). National audit of continence care: Combined organisational and clinical report. https://www.hqip.org.uk/wp- content/uploads/2010/01/2010-NACC-Annual-Repot pdf John Percival https://orcid.org/0000-0002-0608-997X John Percival https://orcid.org/0000-0002-0608-997X International Continence Society. (2009). Continence Promotion, Education & Primary Prevention. https://www.ics.org/Publicatio ns/ICI_4/files-book/comite-21.pdf DATA AVAILABILITY STATEMENT Hunter, D. J., McCallum, J., & Howes, D. (2018). Defining exploratory- descriptive qualitative (EDQ) research and considering its applica- tion to healthcare. In Proceedings of worldwide nursing conference 2018. 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https://openalex.org/W2987510459	https://research-information.bris.ac.uk/ws/files/143360550/Full_text_PDF_final_published_version_.pdf	English	null	THE CONCISE GUIDE TO PHARMACOLOGY 2019/20: Nuclear hormone receptors	British journal of pharmacology	2,019	cc-by	11,672	Alexander, S. P. H., Cidlowski, J. A., Kelly, E., Marrion, N. V., Peters, J. A., Faccenda, E., Harding, S. D., Pawson, A. J., Sharman, J. L., Southan, C., Davies, J. A. (2017). THE CONCISE GUIDE TO PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology, 174, S208-S224. https://doi.org/10.1111/bph.13880 Alexander, S. P. H., Cidlowski, J. A., Kelly, E., Marrion, N. V., Peters, J. A., Faccenda, E., Harding, S. D., Pawson, A. J., Sharman, J. L., Southan, C., Davies, J. A. (2017). THE CONCISE GUIDE TO PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology, 174, S208-S224. https://doi.org/10.1111/bph.13880 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1111/bph.13880 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.1111/bph.13880 Link to publication record on the Bristol Research Portal PDF-document This is the final published version of the article (version of record). It first appeared online via Wiley at https://doi.org/10.1111/bph.13880 . Please refer to any applicable terms of use of the publisher. The authors state that there are no conﬂicts of interest to declare. © 2017 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2017 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in a in the nucleus in the unliganded state and interact with other nuclear receptors to form heterodimers, as well as with other reg- ulators of gene transcription, leading to changes in gene tran- scription upon agonist binding. Two major subclasses of nuclear receptors with identiﬁed endoge- nous agonists can be identiﬁed: steroid and non-steroid hor- mone receptors. Steroid hormone receptors function typically as dimeric entities and are thought to be resident outside the nucleus in the unliganded state in a complex with chaperone proteins, which are liberated upon agonist binding. Migration to the nucleus and interaction with other regulators of gene transcription, including RNA polymerase, acetyltransferases and deacetylases, allows gene transcription to be regulated. Non- steroid hormone receptors typically exhibit a greater distribution Two major subclasses of nuclear receptors with identiﬁed endoge- nous agonists can be identiﬁed: steroid and non-steroid hor- mone receptors. Steroid hormone receptors function typically as dimeric entities and are thought to be resident outside the nucleus in the unliganded state in a complex with chaperone proteins, which are liberated upon agonist binding. Migration to the nucleus and interaction with other regulators of gene transcription, including RNA polymerase, acetyltransferases and deacetylases, allows gene transcription to be regulated. Non- steroid hormone receptors typically exhibit a greater distribution Overview: Nuclear receptors are specialised transcription fac- tors with commonalities of sequence and structure, which bind as homo- or heterodimers to speciﬁc consensus sequences of DNA (response elements) in the promoter region of particular target genes. They regulate (either promoting or repressing) transcrip- tion of these target genes in response to a variety of endogenous ligands. Endogenous agonists are hydrophobic entities which, when bound to the receptor promote conformational changes in the receptor to allow recruitment (or dissociation) of protein partners, generating a large multiprotein complex. Abstract The Concise Guide to PHARMACOLOGY 2017/18 provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full. Nuclear hormone receptors are one of the eight major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ligand-gated ion channels, voltage-gated ion channels, other ion channels, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2017, and supersedes data presented in the 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature Committee of the Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing ofﬁcial IUPHAR classiﬁcation and nomenclature for human drug targets, where appropriate. University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ MACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British J S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S THE CONCISE GUIDE TO PHARMACOLOGY 2017/18: Nuclear hormone receptors Stephen PH Alexander1, John A Cidlowski2, Eamonn Kelly3, Neil V Marrion3, John A Peters4, Elena Faccenda5, Simon D Harding5, Adam J Pawson5, Joanna L Sharman5, Christopher Southan5, Jamie A Davies5 and CGTP Collaborators 1School of Life Sciences, University of Nottingham Medical School, Nottingham, NG7 2UH, UK 2National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA g , , 3School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, BS8 1TD, UK 4N i Di i i M di l Ed i I i Ni ll H i l d M di l S h l U i , , p 5Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK 5Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD Conﬂict of interest The authors state that there are no conﬂicts of interest to declare. 1A. Thyroid hormone receptors erodimer with members of the retinoid X receptor family. NH-3 has been described as an antagonist at TRs with modest selectivity for TRβ [110]. erodimer with members of the retinoid X receptor family. NH-3 has been described as an antagonist at TRs with modest selectivity for TRβ [110]. macronutrient metabolism, cognition and cardiovascular home- ostasis. TRs are activated by thyroxine (T4) and thyroid hormone (triiodothyronine). Once activated by a ligand, the receptor acts as a transcription factor either as a monomer, homodimer or het- Overview: Thyroid hormone receptors (TRs, nomencla- ture as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [41]) are nuclear hormone receptors of the NR1A family, with diverse roles regulating Nomenclature Thyroid hormone receptor-α Thyroid ho Systematic nomenclature NR1A1 NR1A2 HGNC, UniProt THRA, P10827 THRB, P10 Rank order of potency triiodothyronine > T4 triiodothy Agonists dextrothyroxine [19] dextrothy Selective agonists – sobetirom Thyroid hormone receptor-α Thyroid hormone receptor-β NR1A1 NR1A2 THRA, P10827 THRB, P10828 triiodothyronine > T4 triiodothyronine > T4 dextrothyroxine [19] dextrothyroxine [19] – sobetirome [26, 130] Nomenclature Systematic nomenclature HGNC, UniProt Rank order of potency Agonists Selective agonists Comments: An interaction with integrin αVβ3 has been suggested to underlie plasma membrane localization o binding domain and appears to act as a transcription suppressor. g pp p pp Although radioligand binding assays have been described for these receptors, the radioligands are not commercially available. g pp p pp Although radioligand binding assays have been described for these receptors, the radioligands are not commercia g pp p pp Although radioligand binding assays have been described for these receptors, the radioligand The authors state that there are no conﬂicts of interest to declare. Selectivity of gene regulation is brought about through interac- tion of nuclear receptors with particular consensus sequences of DNA, which are arranged typically as repeats or inverted palin- dromes to allow accumulation of multiple transcription factors in the promoter regions of genes. Nuclear hormone receptors Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full S208 MACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British J S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 Family structure S209 1A. Thyroid hormone receptors S210 1B. Retinoic acid receptors S210 1C. Peroxisome proliferator-activated receptors S211 1D. Rev-Erb receptors S212 1F. Retinoic acid-related orphans S212 1H. Liver X receptor-like receptors S213 1I. Vitamin D receptor-like receptors S214 2A. Hepatocyte nuclear factor-4 receptors S215 2B. Retinoid X receptors S216 2C. Testicular receptors S216 2E. Tailless-like receptors S217 2F. COUP-TF-like receptors S217 3B. Estrogen-related receptors S218 4A. Nerve growth factor IB-like receptors S219 5A. Fushi tarazu F1-like receptors S219 6A. Germ cell nuclear factor receptors S220 0B. DAX-like receptors S221 Steroid hormone receptors S221 3A. Estrogen receptors S222 3C. 3-Ketosteroid receptors Further reading on 1A. Thyroid hormone receptors Flamant F et al. (2006) International Union of Pharmacology. LIX. The pharmacology and classi- ﬁcation of the nuclear receptor superfamily: thyroid hormone receptors. Pharmacol. Rev. 58: 705-11 [PMID:17132849] Mendoza A et al. (2017) New insights into thyroid hormone action. Pharmacol Ther 173: 135-145 [PMID:28174093] Davis PJ et al. (2016) Nongenomic actions of thyroid hormone. Nat Rev Endocrinol 12: 111-21 [PMID:26668118] Elbers LP et al. (2016) Thyroid Hormone Mimetics: the Past, Current Status and Future Challenges. Curr Atheroscler Rep 18: 14 [PMID:26886134] 1A. Thyroid hormone receptors S209 Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full 1A. Thyroid hormone receptors S209 1A. Thyroid hormone receptors S209 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 MACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 Further reading on 1B. Retinoic acid receptors Further reading on 1B. Retinoic acid receptors Larange A et al. (2016) Retinoic Acid and Retinoic Acid Receptors as Pleiotropic Modulators of the Immune System. Annu Rev Immunol 34: 369-94 [PMID:27168242] Saeed A et al. (2017) The interrelationship between bile acid and vitamin A homeostasis. Biochim Biophys Acta 1862: 496-512 [PMID:28111285] Larange A et al. (2016) Retinoic Acid and Retinoic Acid Receptors as Pleiotropic Modulators of the Immune System. Annu Rev Immunol 34: 369-94 [PMID:27168242] Duong V et al. (2011) The molecular physiology of nuclear retinoic acid receptors. From health to disease. Biochim. Biophys. Acta 1812: 1023-31 [PMID:20970498] G i P t l (2006) I t ti l U i f Ph l LX R ti i id t Ph y [ ] Saeed A et al. (2017) The interrelationship between bile acid and vitamin A homeostasis. Biochim Biophys Acta 1862: 496-512 [PMID:28111285] Germain P et al. (2006) International Union of Pharmacology. LX. Retinoic acid receptors. Phar- macol. Rev. 58: 712-25 [PMID:17132850] Nuclear hormone receptors →1B. Retinoic acid receptors ceptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [46]) are nuclear hormone receptors of the NR1B family erived agonists tretinoin (ATRA) and alitretinoin, and the RAR-selective synthetic agonists TTNPB and adapalene. BMS493 is a family-selective antagonist [47]. Overview: Retinoic acid receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nu activated by the vitamin A-derived agonists tretinoin (ATRA) and alitretinoin, and the RAR-selective synthetic ag Nomenclature Retinoic acid receptor-α Retinoic acid receptor-β Retinoic acid receptor-γ Systematic nomenclature NR1B1 NR1B2 NR1B3 HGNC, UniProt RARA, P10276 RARB, P10826 RARG, P13631 Agonists tretinoin [25] tretinoin [25] tretinoin [25] Sub/family-selective agonists tazarotene [25] tazarotene [25], adapalene [24] tazarotene [25], adapalene [24] Selective agonists BMS753 [53], tamibarotene [146], Ro 40-6055 [33] AC261066 [89], AC55649 [88, 89] AHPN [24] Selective antagonists Ro 41-5253 (pIC50 6.3–7.2) [2, 69] – MM 11253 [76] Comments: Ro 41-5253 has been suggested to be a PPARγ agonist [129]. LE135 is an antagonist with selectivity for RARα and RARβ compared with RARγ [84]. Comments: Ro 41-5253 has been suggested to be a PPARγ agonist [129]. LE135 is an antagonist with selectivity for RARα and RARβ compared with RARγ [84]. Comments: Ro 41-5253 has been suggested to be a PPARγ agonist [129]. LE135 is an antagonist with selectivity for RARα and RARβ compared with RARγ [84]. Comments: Ro 41-5253 has been suggested to be a PPARγ agonist [129]. LE135 is an antagonist with selectiv 1C. Peroxisome proliferator-activated receptors Nuclear hormone receptors →1C. Peroxisome proliferator-activated receptors British Journal of Pharmacology (2017) 174, S208–S224 omenclature Peroxisome proliferator-activated receptor-α Peroxisome proliferator-activated receptor-β/δ Peroxisome proliferator-activated receptor-γ ystematic omenclature NR1C1 NR1C2 NR1C3 GNC, UniProt PPARA, Q07869 PPARD, Q03181 PPARG, P37231 elective gonists GW7647 [17, 18], CP-775146 [67], pirinixic acid [155], gemﬁbrozil [31] GW0742X [50, 148], GW501516 [112] GW1929 [17], bardoxolone (Partial agonist) [149], rosiglitazone [59, 80, 86, 161], troglitazone [59, 161], pioglitazone [7, 59, 127, 161], ciglitazone [59] elective ntagonists GW6471 (pIC50 6.6) [158] GSK0660 (pIC50 6.5) [131] T0070907 (pKi 9) [77], GW9662 (Irreversible inhibition) (pIC50 8.1) [78], CDDO-Me (pKi 6.9) [149] ents: As with the estrogen receptor antagonists, many agents show tissue-selective efﬁcacy (e.g. [12, 109, 124]). Agonists with mixed activity at PPARα and PPARγ have also been described (e.g [35, ]). er reading on 1C. Peroxisome proliferator-activated receptors WS et al. (2015) The peroxisome proliferator-activated receptors in cardiovascular diseases: erimental beneﬁts and clinical challenges. Br J Pharmacol 172: 5512-22 [PMID:25438608] et al. (2017) PPARs in obesity-induced T2DM, dyslipidaemia and NAFLD. Nat Rev Endocrinol 36-49 [PMID:27636730] b P t l (2016) Th l i d di i PPAR i t Li i th Michalik L et al. (2006) International Union of Pharmacology. LXI. Peroxisome proliferator- activated receptors. Pharmacol. Rev. 58: 726-41 [PMID:17132851] Sauer S. (2015) Ligands for the Nuclear Peroxisome Proliferator-Activated Receptor Gamma. Trends Pharmacol Sci 36: 688-704 [PMID:26435213] Nomenclature Peroxisome proliferator-activated receptor-α Systematic nomenclature NR1C1 HGNC, UniProt PPARA, Q07869 Selective agonists GW7647 [17, 18], CP-775146 [67], pirinixic acid [155], gemﬁbrozil [31] Selective antagonists GW6471 (pIC50 6.6) [158] T0070907 (pKi 9) [77], GW9662 (Irreversible inhibition) (pIC50 8.1) [78], CDDO-Me (pKi 6.9) [149] Comments: As with the estrogen receptor antagonists, many agents show tissue-selective efﬁcacy (e.g. [12, 109, 1 52, 159]). Comments: As with the estrogen receptor antagonists, many agents show tissue-selective efﬁcacy (e.g. [12, 109, 124]). Agonists with mixed activity at PPARα and PPARγ 52, 159]). Michalik L et al. (2006) International Union of Pharmacology. LXI. Peroxisome proliferator- activated receptors. Pharmacol. Rev. 58: 726-41 [PMID:17132851] Sauer S. (2015) Ligands for the Nuclear Peroxisome Proliferator-Activated Receptor Gamma. Trends Pharmacol Sci 36: 688-704 [PMID:26435213] Further reading on 1C. Peroxisome proliferator-activated receptors Cheang WS et al. (2015) The peroxisome proliferator-activated receptors in cardiovascular diseases: experimental beneﬁts and clinical challenges. Br J Pharmacol 172: 5512-22 [PMID:25438608] Gross B et al. (2017) PPARs in obesity-induced T2DM, dyslipidaemia and NAFLD. Nat Rev Endocrinol 13: 36-49 [PMID:27636730] [ ] Hallenborg P et al. (2016) The elusive endogenous adipogenic PPARgamma agonists: Lining up the suspects. Prog Lipid Res 61: 149-62 [PMID:26703188] 1D. Rev-Erb receptors Nuclear hormone receptors →1D. Rev-Erb receptors 1C. Peroxisome proliferator-activated receptors Nuclear hormone receptors →1C. Peroxisome proliferator-activated receptors cyclin (PGI2), many fatty acids and their oxidation products, lysophosphatidic acid (LPA) [98], 13-HODE, 15S-HETE, Paz-PC, azelaoyl-PAF and leukotriene B4 (LTB4). Bezaﬁbrate acts as a non-selective agonist for the PPAR family [155]. These recep- tors also bind hypolipidaemic drugs (PPARα) and anti-diabetic thiazolidinediones (PPARγ), as well as many non-steroidal anti- inﬂammatory drugs, such as sulindac and indomethacin. Once Overview: Peroxisome proliferator-activated receptors (PPARs, nomenclature as agreed by the NC-IUPHAR Subcom- mittee on Nuclear Hormone Receptors [101]) are nuclear hormone receptors of the NR1C family, with diverse roles reg- ulating lipid homeostasis, cellular differentiation, proliferation and the immune response. PPARs have many potential endoge- nous agonists [13, 101], including 15-deoxy-12,14-PGJ2, prosta- activated by a ligand, the receptor forms a heterodimer with members of the retinoid X receptor family and can act as a tran- scription factor. Although radioligand binding assays have been described for all three receptors, the radioligands are not com- mercially available. Commonly, receptor occupancy studies are conducted using ﬂuorescent ligands and truncated forms of the receptor limited to the ligand binding domain. cyclin (PGI2), many fatty acids and their oxidation products, lysophosphatidic acid (LPA) [98], 13-HODE, 15S-HETE, Paz-PC, azelaoyl-PAF and leukotriene B4 (LTB4). Bezaﬁbrate acts as a cyclin (PGI2), many fatty acids and their oxidation products, lysophosphatidic acid (LPA) [98], 13-HODE, 15S-HETE, Paz-PC, azelaoyl-PAF and leukotriene B4 (LTB4). Bezaﬁbrate acts as a y ( 4) non-selective agonist for the PPAR family [155]. These recep- tors also bind hypolipidaemic drugs (PPARα) and anti-diabetic thiazolidinediones (PPARγ), as well as many non-steroidal anti- inﬂammatory drugs, such as sulindac and indomethacin. Once 1C. Peroxisome proliferator-activated receptors S210 Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full MACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British J S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 Further reading on 1D. Rev-Erb receptors Gustafson CL et al. (2015) Emerging models for the molecular basis of mammalian circadian tim- ing. Biochemistry 54: 134-49 [PMID:25303119] Sousa EH et al. (2017) Drug discovery targeting heme-based sensors and their coupled activities. J Inorg Biochem 167: 12-20 [PMID:27893989] Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] Gonzalez-Sanchez E et al. (2015) Nuclear receptors in acute and chronic cholestasis. Dig Dis 33: 357-66 [PMID:26045270] [ ] Gonzalez-Sanchez E et al. (2015) Nuclear receptors in acute and chronic cholestasis. Dig Dis 33: 357-66 [PMID:26045270] 1F. Retinoic acid-related orphans Nuclear hormone receptors →1F. Retinoic acid-related orphans eptor-related orphan receptors (ROR, nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be assigned a , although RORα may be synthesized with a ‘captured’ agonist such as cholesterol [65, 66]. Overview: Retinoic acid receptor-related orphan receptors (ROR, nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be assigned a deﬁnitive endogenous ligand, although RORα may be synthesized with a ‘captured’ agonist such as cholesterol [65, 66]. Overview: Retinoic acid receptor-related orphan receptors (ROR, nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Horm deﬁnitive endogenous ligand, although RORα may be synthesized with a ‘captured’ agonist such as cholesterol [65, 66]. Overview: Retinoic acid receptor-related orphan receptors (ROR, nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be assigned a deﬁnitive endogenous ligand, although RORα may be synthesized with a ‘captured’ agonist such as cholesterol [65, 66]. Nomenclature RAR-related orphan receptor-α RAR-related orphan receptor-β RAR-related orphan receptor-γ Systematic nomenclature NR1F1 NR1F2 NR1F3 HGNC, UniProt RORA, P35398 RORB, Q92753 RORC, P51449 Endogenous agonists cholesterol [66, 114] – – Selective agonists 7-hydroxycholesterol [14], cholesterol sulphate [14, 66] – – Comments: tretinoin shows selectivity for RORβ within the ROR family [136] RORα has been suggested to be a nuclear receptor responding to melatonin [154] Nomenclature RAR-related orphan receptor-α RAR-related orphan receptor-β RAR-related orphan rece Systematic nomenclature NR1F1 NR1F2 NR1F3 HGNC, UniProt RORA, P35398 RORB, Q92753 RORC, P51449 Endogenous agonists cholesterol [66, 114] – – Selective agonists 7-hydroxycholesterol [14], cholesterol sulphate [14, 66] – – Comments: tretinoin shows selectivity for RORβ within the ROR family [136]. RORα has been suggested to be a nuclear receptor responding to melatonin [154]. β within the ROR family [136]. RORα has been suggested to be a nuclear receptor responding to melatonin [154]. Comments: tretinoin shows selectivity for RORβ within the ROR family [136]. RORα has been suggested to be a 1F. Retinoic acid-related orphans S212 Gustafson CL et al. (2015) Emerging models for the molecular basis of mammalian circadian tim- ing. Biochemistry 54: 134-49 [PMID:25303119] Sousa EH et al. (2017) Drug discovery targeting heme-based sensors and their coupled activities. J Inorg Biochem 167: 12-20 [PMID:27893989] Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full 1D. Rev-Erb receptors Nuclear hormone receptors →1D. Rev-Erb receptors p Nuclear hormone receptors →1D. Rev-Erb receptors Overview: Rev-erb receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be ofﬁcially paired with an endogenous ligand, but are thought to be activated by heme. Nomenclature Rev-Erb-α Rev-Erb-β Systematic nomenclature NR1D1 NR1D2 HGNC, UniProt NR1D1, P20393 NR1D2, Q14995 Endogenous agonists heme [121, 160] heme [97, 121, 160] Selective agonists GSK4112 [51], GSK4112 [70] – Selective antagonists SR8278 (pIC50 6.5) [70] – Searchable database: http://www.guidetopharmacology.org/index.jsp 1D. Rev-Erb receptors S211 Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full Overview: Rev-erb receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be ofﬁcially paired with an endogenous ligand, but are thought to be activated by heme. Overview: Rev-erb receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear but are thought to be activated by heme. 1D. Rev-Erb receptors S211 Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 Further reading on 1F. Retinoic acid-related orphans Guillemot-Legris O et al. (2016) Oxysterols in Metabolic Syndrome: From Bystander Molecules to Bioactive Lipids. Trends Mol Med 22: 594-614 [PMID:27286741] Mutemberezi V et al. (2016) Oxysterols: From cholesterol metabolites to key mediators. Prog Lipid Res 64: 152-169 [PMID:27687912] Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] Cyr P et al. (2016) Recent progress on nuclear receptor RORgamma modulators. Bioorg Med Chem Lett 26: 4387-93 [PMID:27542308] Dahlman-Wright K et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol Rev 58: 685-704 [PMID:17132848] Guillemot-Legris O et al. (2016) Oxysterols in Metabolic Syndrome: From Bystander Molecules to Bioactive Lipids. Trends Mol Med 22: 594-614 [PMID:27286741] Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] Cyr P et al. (2016) Recent progress on nuclear receptor RORgamma modulators. Bioorg Med Chem Lett 26: 4387-93 [PMID:27542308] Dahlman-Wright K et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol Rev 58: 685-704 [PMID:17132848] p [ ] Mutemberezi V et al. (2016) Oxysterols: From cholesterol metabolites to key mediators. Prog Lipid Res 64: 152-169 [PMID:27687912] Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full 1F. Retinoic acid-related orphans S212 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 1H. Liver X receptor-like receptors S213 1H. Liver X receptor-like receptors Nuclear hormone receptors →1H. Liver X receptor-like receptors Overview: Liver X and farnesoid X receptors (LXR and FXR, nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors analogue-activated nuclear receptor subfamily, which form heterodimers with members of the retinoid X receptor family. Endogenous ligands for LXRs include hydrox appear to be activated by bile acids. iew: Liver X and farnesoid X receptors (LXR and FXR, nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [105]) are members of a steroid ue-activated nuclear receptor subfamily, which form heterodimers with members of the retinoid X receptor family. Endogenous ligands for LXRs include hydroxycholesterols (OHC), while FXRs to be activated by bile acids Nomenclature Farnesoid X receptor Farnesoid X receptor-β Liver X receptor-α Liver X receptor-β Systematic nomenclature NR1H4 NR1H5 NR1H3 NR1H2 HGNC, UniProt NR1H4, Q96RI1 NR1H5P, – NR1H3, Q13133 NR1H2, P55055 Potency order chenodeoxycholic acid > lithocholic acid, deoxycholic acid [92, 115] – 20S-hydroxycholesterol, 22R-hydroxycholesterol, 24(S)-hydroxycholesterol > 25-hydroxycholesterol, 27-hydroxycholesterol [79] 20S-hydroxycholesterol, 22R-hydroxycholesterol, 24(S)-hydroxycholesterol > 25-hydroxycholesterol, 27-hydroxycholesterol [79] Endogenous agonists – lanosterol [113] – Mouse – – Selective agonists GW4064 [94], obeticholic acid [116], fexaramine [36] – – – Selective antagonists guggulsterone (pIC50 5.7–6) [157] – – – Comments: T0901317 [122] and GW3965 [27] are synthetic agonists acting at both LXRα and LXRβ with less than 10-fold selectivity. omenclature Farnesoid X receptor Farnesoid X receptor-β Liver X receptor-α Liver X receptor-β stematic menclature NR1H4 NR1H5 NR1H3 NR1H2 GNC, UniProt NR1H4, Q96RI1 NR1H5P, – NR1H3, Q13133 NR1H2, P55055 tency order chenodeoxycholic acid > lithocholic acid, deoxycholic acid [92, 115] – 20S-hydroxycholesterol, 22R-hydroxycholesterol, 24(S)-hydroxycholesterol > 25-hydroxycholesterol, 27-hydroxycholesterol [79] 20S-hydroxycholesterol, 22R-hydroxycholesterol, 24(S)-hydroxycholesterol > 25-hydroxycholesterol, 27-hydroxycholesterol [79] dogenous agonists – lanosterol [113] – Mouse – – ective agonists GW4064 [94], obeticholic acid [116], fexaramine [36] – – – ective antagonists guggulsterone (pIC50 5.7–6) [157] – – – Comments: T0901317 [122] and GW3965 [27] are synthetic agonists acting at both LXRα and LXRβ with less th Further reading on 1H. Liver X receptor-like receptors Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full Further reading on 1H. Liver X receptor-like receptors Courtney R et al. (2016) LXR Regulation of Brain Cholesterol: From Development to Disease. Trends Endocrinol Metab 27: 404-14 [PMID:27113081] [ ] Gadaleta RM et al. (2017) Bile acids and colon cancer: Is FXR the solution of the conundrum? Mol Aspects Med [PMID:28400119] Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full 1H. Liver X receptor-like receptors S213 1H. Liver X receptor-like receptors S213 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 p Nuclear hormone receptors →1I. Vitamin D receptor-like receptors ), Pregnane X (PXR) and Constitutive Androstane (CAR) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors R1I family of nuclear receptors, which form heterodimers with members of the retinoid X receptor family. PXR and CAR are activated by a range of exogenous compounds, ous physiological agonists, although high concentrations of bile acids and bile pigments activate PXR and CAR [105]. Overview: Vitamin D (VDR), Pregnane X (PXR) and Constitutive Androstane (CAR) receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [105]) are members of the NR1I family of nuclear receptors, which form heterodimers with members of the retinoid X receptor family. PXR and CAR are activated by a range of exogenous compounds, with no established endogenous physiological agonists, although high concentrations of bile acids and bile pigments activate PXR and CAR [105]. Vitamin D receptor NR1I1 VDR, P11473 1,25-dihydroxyvitamin D3 [11, 39] seocalcitol [28, 153], doxercalciferol TEI-9647 (pIC50 8.2) [126] – Chicken, ZK159222 (pIC50 7.5) [42, 60] Comments Further reading on 1I. Vitamin D receptor-like receptors Moore DD et al. (2006) International Union of Pharmacology. LXII. The NR1H and NR1I receptors: constitutive androstane receptor, pregnene X receptor, farnesoid X receptor alpha, farnesoid X receptor beta, liver X receptor alpha, liver X receptor beta, and vitamin D receptor. Pharmacol. Rev. 58: 742-59 [PMID:17132852] 1I. Vitamin D receptor-like receptors p Nuclear hormone receptors →1I. Vitamin D receptor-like receptors 2A. Hepatocyte nuclear factor-4 receptors S214 Overview: The nomenclature of hepatocyte nuclear factor-4 receptors is agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]. While linoleic acid has been identiﬁed as the endogenous ligand for HNF4α its function remains ambiguous [163]. HNF4γ has yet to be paired with an endogenous ligand. Searchable database: http://www.guidetopharmacology.org/index.jsp 2A. Hepatocyte nuclear factor-4 receptors S214 Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full nuclear factor-4 receptors is agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]. While linoleic acid has been identiﬁed as n remains ambiguous [163]. HNF4γ has yet to be paired with an endogenous ligand. Overview: The nomenclature of hepatocyte nuclear factor-4 receptors is agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]. While linoleic acid has the endogenous ligand for HNF4α its function remains ambiguous [163]. HNF4γ has yet to be paired with an endogenous ligand. he nomenclature of hepatocyte nuclear factor-4 receptors is agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]. While linoleic acid has been identiﬁed as us ligand for HNF4α its function remains ambiguous [163]. HNF4γ has yet to be paired with an endogenous ligand. Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full MACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British J Hepatocyte nuclear factor-4-α Hepatocyte nuclear factor-4-γ nclature NR2A1 NR2A2 HNF4A, P41235 HNF4G, Q14541 nists linoleic acid [163] – nists BI6015 [71] – HNF4α has constitutive transactivation activity [163] and binds DNA as a homodimer [63]. – Further reading on 2A. Hepatocyte nuclear factor-4 receptors Further reading on 2A. Hepatocyte nuclear factor-4 receptors Lu H. (2016) Crosstalk of HNF4alpha with extracellular and intracellular signaling pathways in the regulation of hepatic metabolism of drugs and lipids. Acta Pharm Sin B 6: 393-408 [PMID:27709008] Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] [ ] Germain P et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol. Rev. 58: 685-704 [PMID:17132848] 685 704 [PMID:17132848] Garattini E et al. (2016) Lipid-sensors, enigmatic-orphan and orphan nuclear receptors as thera- peutic targets in breast-cancer. Oncotarget 7: 42661-42682 [PMID:26894976] Lu H. (2016) Crosstalk of HNF4alpha with extracellular and intracellular signaling pathways in the regulation of hepatic metabolism of drugs and lipids. Acta Pharm Sin B 6: 393-408 [PMID:27709008] Walesky CE et al. (2015) Role of hepatocyte nuclear factor 4alpha (HNF4alpha) in cell proliferation and cancer. Gene Expr 16: 101-8[PMID:25700366] 2B. Retinoid X receptors S215 Further reading on 2B. Retinoid X receptors Germain P et al. (2006) International Union of Pharmacology. LXIII. Retinoid X receptors. Phar- macol. Rev. 58: 760-72 [PMID:17132853] Long MD et al. (2015) Vitamin D receptor and RXR in the post-genomic era. J Cell Physiol 230: 758-66 [PMID:25335912] Menendez-Gutierrez MP et al. (2017) The multi-faceted role of retinoid X receptor in bone remod- eling. Cell Mol Life Sci 74: 2135-2149 [PMID:28105491] S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 der et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 Overview: Testicular receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be ofﬁcially paired with an endogenous ligand, although testicular receptor 4 has been reported to respond to retinoids. Nomenclature Testicular receptor 2 Testicular receptor 4 Systematic nomenclature NR2C1 NR2C2 HGNC, UniProt NR2C1, P13056 NR2C2, P49116 Endogenous agonists – retinol [169], tretinoin [169] Comments Forms a heterodimer with TR4; gene disruption appears without effect on testicular development or function [132]. Forms a heterodimer with TR2. Further reading on 2C. Testicular receptors Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] Dahlman-Wright K et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol Rev 58: 685-704 [PMID:17132848] Safe S et al. (2014) Minireview: role of orphan nuclear receptors in cancer and potential as drug targets. Mol Endocrinol 28: 157-72 [PMID:24295738] Wu D et al. (2016) The emerging roles of orphan nuclear receptors in prostate cancer. Biochim. Biophys. Acta 1866: 23-36 [PMID:27264242] 2E. Tailless-like receptors Nuclear hormone receptors →2E. Tailless-like receptors Overview: Tailless-like receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be ofﬁcially paired with an endogenous ligand. Searchable database: http://www.guidetopharmacology.org/index.jsp 2E. Tailless-like receptors S216 Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full Overview: Testicular receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be ofﬁcially paired with an endogenous ligand, although testicular receptor 4 has been reported to respond to retinoids. Nomenclature Testicular receptor 2 Testicular receptor 4 Systematic nomenclature NR2C1 NR2C2 HGNC, UniProt NR2C1, P13056 NR2C2, P49116 Endogenous agonists – retinol [169], tretinoin [169] Comments Forms a heterodimer with TR4; gene disruption appears without effect on testicular development or function [132]. Forms a heterodimer with TR2. Further reading on 2C. Testicular receptors Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] Dahlman-Wright K et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol Rev 58: 685-704 [PMID:17132848] Safe S et al. (2014) Minireview: role of orphan nuclear receptors in cancer and potential as drug targets. Mol Endocrinol 28: 157-72 [PMID:24295738] Wu D et al. (2016) The emerging roles of orphan nuclear receptors in prostate cancer. Biochim. Biophys. Acta 1866: 23-36 [PMID:27264242] 2E. Tailless-like receptors Nuclear hormone receptors →2E. Tailless-like receptors Overview: Tailless-like receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be ofﬁcially paired with an endogenous ligand. Searchable database: http://www.guidetopharmacology.org/index.jsp 2E. Tailless-like receptors S216 Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full Overview: Testicular receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be ofﬁcially pa although testicular receptor 4 has been reported to respond to retinoids. Further reading on 2C. Testicular receptors 2E. Tailless-like receptors S216 2B. Retinoid X receptors Nuclear hormone receptors →2B. Retinoid X receptors tinoid X receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [45]) are NR2B family members activated by alitretinoin and ive agonists bexarotene and LG100268, sometimes referred to as rexinoids. UVI3003 [108] and HX 531 [37] have been described as a pan-RXR antagonists. These receptors form RXR-RAR nd RXR RXR homodimers [22 96] Overview: Retinoid X receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [45]) are NR2B family members activated by alitretinoin and the RXR-selective agonists bexarotene and LG100268, sometimes referred to as rexinoids. UVI3003 [108] and HX 531 [37] have been described as a pan-RXR antagonists. These receptors form RXR-RAR heterodimers and RXR-RXR homodimers [22, 96]. Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full 2B. Retinoid X receptors S215 Further reading on 2C. Testicular receptors Safe S et al. (2014) Minireview: role of orphan nuclear receptors in cancer and potential as drug targets. Mol Endocrinol 28: 157-72 [PMID:24295738] Wu D et al. (2016) The emerging roles of orphan nuclear receptors in prostate cancer. Biochim. Biophys. Acta 1866: 23-36 [PMID:27264242] Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] [ ] Dahlman-Wright K et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol Rev 58: 685-704 [PMID:17132848] 2E. Tailless-like receptors Nuclear hormone receptors →2E. Tailless-like receptors Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full 2E. Tailless-like receptors S216 ACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British J S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 Nomenclature TLX PNR Systematic nomenclature NR2E1 NR2E3 HGNC, UniProt NR2E1, Q9Y466 NR2E3, Q9Y5X4 Comments Gene disruption is associated with abnormal brain development [75, 104]. – Further reading on 2E. Tailless-like receptors Benod C et al. (2016) TLX: An elusive receptor. J. Steroid Biochem. Mol. Biol. 157: 41-7 [PMID:26554934] Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] Germain P et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol. Rev. 58: 685- 704 [PMID:17132848] 2F. COUP-TF-like receptors Nuclear hormone receptors →2F. COUP-TF-like receptors Overview: COUP-TF-like receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be ofﬁcially paired with an endogenous ligand. TLX PNR nclature NR2E1 NR2E3 NR2E1, Q9Y466 NR2E3, Q9Y5X4 Gene disruption is associated with abnormal brain development [75, 104]. – Nomenclature TLX PNR Systematic nomenclature NR2E1 NR2E3 HGNC, UniProt NR2E1, Q9Y466 NR2E3, Q9Y5X4 Comments Gene disruption is associated with abnormal brain development [75, 104]. – Further reading on 2E. Tailless-like receptors Germain P et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol. Rev. 58: 685- 704 [PMID:17132848] [ ] Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] p Nuclear hormone receptors →2F. COUP-TF-like receptors c e e ecep → e ecep Overview: COUP-TF-like receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be ofﬁcially paired with an endogenous li d p p iew: COUP-TF-like receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be ofﬁcially paired with an endogenous omenclature COUP-TF1 COUP-TF2 V-erbA-related gene ystematic nomenclature NR2F1 NR2F2 NR2F6 GNC, UniProt NR2F1, P10589 NR2F2, P24468 NR2F6, P10588 omments Gene disruption is perinatally lethal [120]. Gene disruption is embryonically lethal [117]. Gene disruption impairs CNS development [151]. er reading on 2F. COUP-TF-like receptors G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. armacol. Rev. 58: 798-836 [PMID:17132856] n P et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol. Rev. 58: 685- 4 [PMID:17132848] Wu D et al. (2016) The emerging roles of orphan nuclear receptors in prostate cancer. Biochim. Biophys. Acta 1866: 23-36 [PMID:27264242] Wu SP et al. (2016) Choose your destiny: Make a cell fate decision with COUP-TFII. J Steroid Biochem Mol Biol 157: 7-12 [PMID:26658017] p p OUP-TF-like receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be ofﬁcially paired with an endogenous p p Overview: COUP-TF-like receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be ofﬁcially paired with an endogenous ligand. Nomenclature COUP-TF1 COUP-TF2 V-erbA-related gene Systematic nomenclature NR2F1 NR2F2 NR2F6 HGNC, UniProt NR2F1, P10589 NR2F2, P24468 NR2F6, P10588 Comments Gene disruption is perinatally lethal [120]. Gene disruption is embryonically lethal [117]. Gene disruption impairs CNS development [151]. Further reading on 2F. COUP-TF-like receptors Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] Germain P et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol. Rev. 58: 685- 704 [PMID:17132848] Wu D et al. (2016) The emerging roles of orphan nuclear receptors in prostate cancer. Biochim. Biophys. Acta 1866: 23-36 [PMID:27264242] Wu SP et al. (2016) Choose your destiny: Make a cell fate decision with COUP-TFII. J Steroid Biochem Mol Biol 157: 7-12 [PMID:26658017] Searchable database: http://www.guidetopharmacology.org/index.jsp 2F. COUP-TF-like receptors S217 Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full Overview: COUP-TF-like receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be ofﬁc ligand 2F. COUP-TF-like receptors p Nuclear hormone receptors →2F. COUP-TF-like receptors Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full Further reading on 2F. COUP-TF-like receptors Further reading on 2F. COUP-TF-like receptors Wu D et al. (2016) The emerging roles of orphan nuclear receptors in prostate cancer. Biochim. Biophys. Acta 1866: 23-36 [PMID:27264242] Wu SP et al. (2016) Choose your destiny: Make a cell fate decision with COUP-TFII. J Steroid Biochem Mol Biol 157: 7-12 [PMID:26658017] 2F. COUP-TF-like receptors S217 Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full 2F. COUP-TF-like receptors S217 der et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 Further reading on 3B. Estrogen-related receptors Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] Divekar SD et al. (2016) Estrogen-related receptor β (ERRβ) - renaissance receptor or receptor re- naissance? Nucl Recept Signal 14: e002 [PMID:27507929] Germain P et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol. Rev. 58: 685- 704 [PMID:17132848] Tam IS et al. (2016) There and back again: The journey of the estrogen-related receptors in the cancer realm. J Steroid Biochem Mol Biol 157: 13-9[PMID:26151739] Wu D et al. (2016) The emerging roles of orphan nuclear receptors in prostate cancer. Biochim. Biophys. Acta 1866: 23-36 [PMID:27264242] Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] [ ] Divekar SD et al. (2016) Estrogen-related receptor β (ERRβ) - renaissance receptor or receptor re- naissance? Nucl Recept Signal 14: e002 [PMID:27507929] p g [ ] Germain P et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol. Rev. 58: 685- 704 [PMID:17132848] 3B. Estrogen-related receptors g Nuclear hormone receptors →3B. Estrogen-related receptors Overview: Estrogen-related receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee o ligand. Further reading on 3B. Estrogen-related receptors Further reading on 4A. Nerve growth factor IB-like receptors Rodriguez-Calvo R et al. (2017) The NR4A subfamily of nuclear receptors: potential new therapeu- tic targets for the treatment of inﬂammatory diseases. Expert Opin Ther Targets 21: 291-304 [PMID:28055275] Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] Germain P et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol. Rev. 58: 685- 704 [PMID:17132848] Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] Germain P et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol. Rev. 58: 685- 704 [PMID:17132848] Pharmacol. Rev. 58: 798 836 [PMID:17132856] Germain P et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol. Rev. 58: 685- 704 [PMID:17132848] [PMID:28055275] Safe S et al. (2016) Nuclear receptor 4A (NR4A) family - orphans no more. J Steroid Biochem Mol Biol 157: 48-60 [PMID:25917081] [ ] Ranhotra HS. (2015) The NR4A orphan nuclear receptors: mediators in metabolism and diseases. J Recept Signal Transduct Res 35: 184-8 [PMID:25089663] S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British J S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 Rodriguez-Calvo R et al. (2017) The NR4A subfamily of nuclear receptors: potential new therapeu- tic targets for the treatment of inﬂammatory diseases. Expert Opin Ther Targets 21: 291-304 [PMID:28055275] Safe S et al. (2016) Nuclear receptor 4A (NR4A) family - orphans no more. J Steroid Biochem Mol Biol 157: 48-60 [PMID:25917081] 5A. Fushi tarazu F1-like receptors S219 Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full 4A. Nerve growth factor IB-like receptors Nuclear hormone receptors →4A. Nerve growth factor IB-like receptors Overview: Nerve growth factor IB-like receptors (nomenclature as agreed by the NC-IUPHAR Subcomm endogenous ligand. An endogenous agonist, cytosporone B, has been described [164], although structural analysis and molecular modelling has not identiﬁed a ligand binding site [4, 40, 150]. 4A. Nerve growth factor IB-like receptors S218 Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full 5A. Fushi tarazu F1-like receptors Nuclear hormone receptors →5A. Fushi tarazu F1-like receptors The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 6A. Germ cell nuclear factor receptors Nuclear hormone receptors →6A Germ cell nuclear factor receptors Nuclear hormone receptors →6A. Germ cell nuclear factor receptors Overview: Germ cell nuclear factor receptors (nomenclature as agreed by the NC-IUPHAR Subcomm endogenous ligand. Germ cell nuclear factor NR6A1 NR6A1, Q15406 Nomenclature Systematic nomenclature HGNC, UniProt 5A. Fushi tarazu F1-like receptors Nuclear hormone receptors →5A. Fushi tarazu F1-like receptors Overview: Fushi tarazu F1-like receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be ofﬁcially paired with an endogenous ligand. Nomenclature Steroidogenic factor 1 Liver receptor homolog-1 Systematic nomenclature NR5A1 NR5A2 HGNC, UniProt NR5A1, Q13285 NR5A2, O00482 Comments Reported to be inhibited by AC45594 [32] and SID7969543 [90]. – Further reading on 5A. Fushi tarazu F1-like receptors Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] Garattini E et al. (2016) Lipid-sensors, enigmatic-orphan and orphan nuclear receptors as thera- peutic targets in breast-cancer. Oncotarget. 7: 42661-42682 [PMID:26894976] Germain P et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol. Rev. 58: 685-704 [PMID:17132848] Zhi X et al. (2016) Structures and regulation of non-X orphan nuclear receptors: A retinoid hy- pothesis. J Steroid Biochem Mol Biol. 157: 27-40 [PMID:26159912] Zimmer V et al. (2015) Nuclear receptor variants in liver disease. Dig Dis. 33: 415-9 [PMID:26045277] hi tarazu F1-like receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be ofﬁcially paired with an endogenous ture Steroidogenic factor 1 Liver receptor homolog-1 nomenclature NR5A1 NR5A2 iProt NR5A1, Q13285 NR5A2, O00482 s Reported to be inhibited by AC45594 [32] and SID7969543 [90]. – ing on 5A. Fushi tarazu F1-like receptors (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Rev. 58: 798-836 [PMID:17132856] l. (2016) Lipid-sensors, enigmatic-orphan and orphan nuclear receptors as thera- ets in breast-cancer. Oncotarget. 7: 42661-42682 [PMID:26894976] l. (2006) Overview of nomenclature of nuclear receptors. Pharmacol. Rev. 58: MID:17132848] Zhi X et al. (2016) Structures and regulation of non-X orphan nuclear receptors: A retinoid hy- pothesis. J Steroid Biochem Mol Biol. 157: 27-40 [PMID:26159912] Zimmer V et al. (2015) Nuclear receptor variants in liver disease. Dig Dis. 33: 415-9 [PMID:26045277] g NR5A1 g NR5A1 her reading on 5A. Fushi tarazu F1-like receptors Zhi X et al. (2016) Structures and regulation of non-X orphan nuclear receptors: A retinoid hy- pothesis. J Steroid Biochem Mol Biol. 157: 27-40 [PMID:26159912] Zimmer V et al. (2015) Nuclear receptor variants in liver disease. Dig Dis. 33: 415-9 [PMID:26045277] 5A. Fushi tarazu F1-like receptors S219 5A. Fushi tarazu F1-like receptors S219 Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 S.P.H. Alexander et al. Safe S et al. ((2014) Minireview: role of orphan nuclear receptors in cancer and potential as drug targets. Mol Endocrinol. 28: 157-72 [PMID:24295738] Zhi X et al. (2016) Structures and regulation of non-X orphan nuclear receptors: A retinoid hy- pothesis. J Steroid Biochem Mol Biol. 157: 27-40 [PMID:26159912] Further reading on 6A. Germ cell nuclear factor receptors Further reading on 6A. Germ cell nuclear factor receptors Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] Further reading on 0B. DAX-like receptors Benoit G et al. (2006) International Union of Pharmacology. LXVI. Orphan nuclear receptors. Pharmacol. Rev. 58: 798-836 [PMID:17132856] [ ] Garattini E et al. (2016) Lipid-sensors, enigmatic-orphan and orphan nuclear receptors as thera- peutic targets in breast-cancer. Oncotarget 7: 42661-42682 [PMID:26894976] p g g [ ] Germain P et al. (2006) Overview of nomenclature of nuclear receptors. Pharmacol. Rev. 58: 685-704 [PMID:17132848] S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 er et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 Safe S et al. (2014) Minireview: role of orphan nuclear receptors in cancer and potential as drug targets. Mol Endocrinol 28: 157-72 [PMID:24295738] Wu D et al. (2016) The emerging roles of orphan nuclear receptors in prostate cancer. Biochim. Biophys. Acta 1866: 23-36 [PMID:27264242] [ ] A 7TM receptor responsive to estrogen (GPER1, Q99527, also known as GPR30, see [118]) has been described. Human ortho- logues of 7TM ’membrane progestin receptors’ (PAQR7, PAQR8 and PAQR5), initially discovered in ﬁsh [170, 171], appear to lo- calize to intracellular membranes and respond to ’non-genomic’ progesterone analogues independently of G proteins [134]. Nuclear hormone receptors →0B. DAX-like receptors Overview: Dax-like receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [6]) have yet to be ofﬁcially paired with an endogenous ligand. Nomenclature DAX1 SHP Systematic nomenclature NR0B1 NR0B2 HGNC, UniProt NR0B1, P51843 NR0B2, Q15466 Searchable database: http://www.guidetopharmacology.org/index.jsp 0B. DAX-like receptors S220 Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full 0B. DAX-like receptors S220 Steroid hormone receptors Nuclear hormone receptors →Steroid hormone receptors Overview: Steroid hormone receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [30, 87]) are nuclear hormone recep- tors of the NR3 class, with endogenous agonists that may be divided into 3-hydroxysteroids (estrone and 17β-estradiol) and 3-ketosteroids (dihydrotestosterone [DHT], aldosterone, cortisol, corticosterone, progesterone and testosterone). These recep- tors exist as dimers coupled with chaperone molecules (such as hsp90β (HSP90AB1, P08238) and immunophilin FKBP52:FKBP4, Q02790), which are shed on binding the steroid hormone. Al- though rapid signalling phenomena are observed [83, 119], the principal signalling cascade appears to involve binding of the activated receptors to nuclear hormone response elements of the genome, with a 15-nucleotide consensus sequence AGAA- CAnnnTGTTCT (i.e. an inverted palindrome) as homo- or het- erodimers. They also affect transcription by protein-protein interactions with other transcription factors, such as activator protein 1 (AP-1) and nuclear factor κB (NF-κB). Splice vari- ants of each of these receptors can form functional or non- functional monomers that can dimerize to form functional or non-functional receptors. For example, alternative splicing of PR mRNA produces A and B monomers that combine to produce functional AA, AB and BB receptors with distinct characteristics [145]. [ ] A 7TM receptor responsive to estrogen (GPER1, Q99527, also known as GPR30, see [118]) has been described. Human ortho- logues of 7TM ’membrane progestin receptors’ (PAQR7, PAQR8 and PAQR5), initially discovered in ﬁsh [170, 171], appear to lo- calize to intracellular membranes and respond to ’non-genomic’ progesterone analogues independently of G proteins [134]. Further reading on 3A. Estrogen receptors Further reading on 3A. Estrogen receptors Hewitt SC et al. (2016) What’s new in estrogen receptor action in the female reproductive tract. J. Mol. Endocrinol. 56: R55-71 [PMID:26826253] Jameera Begam A et al. (2017) Estrogen receptor agonists/antagonists in breast cancer therapy: A critical review. Bioorg Chem 71: 257-274 [PMID:28274582] Warner M et al. (2017) Estrogen Receptor β as a Pharmaceutical Target. Trends Pharmacol. Sci. 38: 92-99 [PMID:27979317] Hewitt SC et al. (2016) What’s new in estrogen receptor action in the female reproductive tract. J. Mol. Endocrinol. 56: R55-71 [PMID:26826253] Jameera Begam A et al. (2017) Estrogen receptor agonists/antagonists in breast cancer therapy: A critical review. Bioorg Chem 71: 257-274 [PMID:28274582] Warner M et al. (2017) Estrogen Receptor β as a Pharmaceutical Target. Trends Pharmacol. Sci. 38: 92-99 [PMID:27979317] Dahlman-Wright K et al. (2016) Estrogen Receptor Ligands: A Review (2013-2015). Sci Pharm 84: 409-427 [PMID:28117309] [ ] Gonzalez-Sanchez E et al. (2015) Nuclear receptors in acute and chronic cholestasis. Dig Dis 33: 357-66 [PMID:26045270] 357 66 [PMID:26045270] Gustafsson (2006) International Union of Pharmacology. LXIV. Estrogen receptors. Pharmacol Rev 58: 773-81 [PMID:17132854] g p Nuclear hormone receptors →Steroid hormone receptors →3A. Estrogen receptors Estrogen receptors may be blocked non-selectively by tamoxifen and raloxifene and labelled by [3H]17β-estradiol and [3H]tamoxifen. strogen receptors appear to have tissue-speciﬁc efﬁcacy (e.g. Tamoxifen is an antagonist at estrogen receptors in the breast, but is an agonist at estrogen M (selective estrogen receptor modulator) or SnuRM (selective nuclear receptor modulator). Y134 has been suggested to be an ERα-selective estrogen g p Nuclear hormone receptors →Steroid hormone receptors →3A. Estrogen receptors r (ER) activity regulates diverse physiological processes via transcriptional modulation of target genes. The selection of target genes and the magnitude of the response, be it etermined by many factors, including the effect of the hormone ligand and DNA binding on ER structural conformation, and the local cellular regulatory environment. The the speciﬁc complement of DNA enhancer and promoter elements present and the availability of coregulators to form functional transcription complexes. Together, these Overview: Estrogen receptor (ER) activity regulates diverse physiological processes via transcriptional modulation of target genes. The selection of target genes and the magnitude of the response, be it induction or repression, are determined by many factors, including the effect of the hormone ligand and DNA binding on ER structural conformation, and the local cellular regulatory environment. The cellular environment deﬁnes the speciﬁc complement of DNA enhancer and promoter elements present and the availability of coregulators to form functional transcription complexes. Together, these determinants control the resulting biological response. 3A. Estrogen receptors S221 Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full MACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. Britis d) ture Estrogen receptor-α Estrogen receptor-β agonists propylpyrazoletriol [73, 135], ethinylestradiol [62] WAY200070 [93], diarylpropionitrile [100, 135], prinaberel [29, 93] y-selective antagonists bazedoxifene (pIC50 7.6) [103] bazedoxifene (pIC50 7.1) [103] antagonists clomiphene (pKi 8.9) [3], methyl-piperidino-pyrazole (pKi 8.6) [139] R,R-THC (pKi 8.4) [99, 140], PHTPP (pKi 6.9) [168] Estrogen receptor-β WAY200070 [93], diarylpropionitrile [100, 135], prinaberel [29, 93] bazedoxifene (pIC50 7.1) [103] R,R-THC (pKi 8.4) [99, 140], PHTPP (pKi 6.9) [168] Estrogen receptor-α propylpyrazoletriol [73, 135], ethinylestradiol [62] bazedoxifene (pIC50 7.6) [103] clomiphene (pKi 8.9) [3], methyl-piperidino-pyrazol Comments: R,R-THC exhibits partial agonist activity at ERα [99, 140]. Estrogen receptors may be blocked non-selectively by tamoxifen and raloxifene and labelled by [3H]17β-estradiol and [3H]tamoxifen. Many agents thought initially to be antagonists at estrogen receptors appear to have tissue-speciﬁc efﬁcacy (e.g. Tamoxifen is an antagonist at estrogen receptors in the breast, but is an agonist at estrogen receptors in the uterus), hence the descriptor SERM (selective estrogen receptor modulator) or SnuRM (selective nuclear receptor modulator). Y134 has been suggested to be an ERα-selective estrogen receptor modulator [111]. ty at ERα [99, 140]. Lu NZ et al. (2006) International Union of Pharmacology. LXV. The pharmacology and classiﬁca- tion of the nuclear receptor superfamily: glucocorticoid, mineralocorticoid, progesterone, and androgen receptors. Pharmacol Rev 58: 782-97 [PMID:17132855] Lucas-Herald AK et al. (2017) Genomic and non-genomic effects of androgens in the cardiovascular system: clinical implications. Clin Sci (Lond) 131: 1405-1418 [PMID:28645930] Wadosky KM et al. (2017) Androgen receptor splice variants and prostate cancer: From bench to bedside. Oncotarget 8: 18550-18576 [PMID:28077788] Weikum ER et al. (2017) Glucocorticoid receptor control of transcription: precision and plasticity via allostery. Nat Rev Mol Cell Biol 18: 159-174 [PMID:28053348] 3C. 3-Ketosteroid receptors Nuclear hormone receptors →Steroid hormone receptors →3C. 3-Ketosteroid receptors 3C. 3-Ketosteroid receptors Nuclear hormone receptors →Steroid hormone receptors →3C. 3-Ketosteroid p Nuclear hormone receptors →Steroid hormone receptors →3C. 3-Ketosteroid receptors Nomenclature Androgen receptor Glucocorticoid receptor Systematic nomenclature NR3C4 NR3C1 HGNC, UniProt AR, P10275 NR3C1, P04150 Rank order of potency dihydrotestosterone [142]> testosterone cortisol, corticosterone ≫aldosterone, deoxycortisone [125] Selective agonists testosterone propionate [95], mibolerone [49], ﬂuoxymesterone [61], methyltrienolone [148], dromostanolone propionate ﬂuticasone propionate [10], beclometasone [3], methylprednisolone [3], ﬂuocinonide [3], betamethasone [3], budesonide [102] Selective antagonists bicalutamide (pKi 7.7) [70], PF0998425 (pIC50 7.1–7.5) [85], enzalutamide (pIC50 7.4) [143], nilutamide (pIC50 7.1–7.1) [133], hydroxyﬂutamide (pEC50 6.6) [148], galeterone (pIC50 6.4) [56], ﬂutamide (pKi 5.4) [147] onapristone (pIC50 7.6) [165], ZK112993 Labelled ligands [3H]dihydrotestosterone (Selective Agonist), [3H]methyltrienolone (Selective Agonist), [3H]mibolerone (Agonist) [3H]dexamethasone (Agonist) Searchable database: http://www.guidetopharmacology.org/index.jsp 3C. 3-Ketosteroid receptors S222 Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full [3H]dexamethasone (Agonist) 3C. 3-Ketosteroid receptors S222 Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full MACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British J Nomenclature Mineralocorticoid receptor Progesterone receptor Systematic nomenclature NR3C2 NR3C3 HGNC, UniProt NR3C2, P08235 PGR, P06401 Rank order of potency corticosterone, cortisol, aldosterone [58, 125], progesterone [125] progesterone [38] Selective agonists – medroxyprogesterone (Afﬁnity at human PR-A) [166], ORG2058, levonorgestrel [9, 128] Selective antagonists ﬁnerenone (pIC50 7.7) [20], eplerenone (pKi 6.9) [5], onapristone (pIC50 6.3) [165], RU28318, ZK112993 ulipristal acetate (pIC50 9.7) [123], mifepristone (Mixed) (pKi 9) [167], onapristone (pKi 7.7) [54], ZK112993 Labelled ligands [3H]aldosterone (Selective Agonist) [44, 137] – Rat [3H]ORG2058 (Selective Agonist) Comments: [3H]dexamethasone also binds to MR in vitro. PR antagonists have been suggested to subdivide into Type I (e.g. onapristone) and Type II (e.g. ZK112993) groups. These groups appear to promote binding of PR to DNA with different efﬁcacies and evoke distinct conformational changes in the receptor leading to a transcription-neutral complex [43 82] Mutations in AR underlie testicular Comments: [3H]dexamethasone also binds to MR in vitro. PR antagonists have been suggested to subdivide into Type I (e.g. onapristone) and Type II (e.g. ZK112993) groups. These groups appear to promote binding of PR to DNA with different efﬁcacies and evoke distinct conformational changes in the receptor, leading to a transcription-neutral complex [43, 82]. 3C. 3-Ketosteroid receptors Nuclear hormone receptors →Steroid hormone receptors →3C. 3-Ketosteroid receptors Mutations in AR underlie testicular feminization and androgen insensitivity syndromes, spinal and bulbar muscular atrophy (Kennedy’s disease). 3C. 3-Ketosteroid receptors S223 Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full Further reading on 3C. 3-Ketosteroid receptors Further reading on 3C. 3-Ketosteroid receptors Baker ME et al. (2017) 30 YEARS OF THE MINERALOCORTICOID RECEPTOR: Evolution of the mineralocorticoid receptor: sequence, structure and function. J Endocrinol 234: T1-T16 [PMID:28468932] Baker ME et al. (2017) 30 YEARS OF THE MINERALOCORTICOID RECEPTOR: Evolution of the mineralocorticoid receptor: sequence, structure and function. J Endocrinol 234: T1-T16 [PMID:28468932] [PMID:28468932] Carroll JS et al. (2017) Deciphering the divergent roles of progestogens in breast cancer. Nat Rev Cancer 17: 54-64 [PMID:27885264] [ ] Carroll JS et al. (2017) Deciphering the divergent roles of progestogens in breast cancer. Nat Rev Cancer 17: 54-64 [PMID:27885264] [ ] Cohen DM et al. (2017) Nuclear Receptor Function through Genomics: Lessons from the Gluco- corticoid Receptor. Trends Endocrinol Metab 28: 531-540 [PMID:28495406] [ ] Cohen DM et al. (2017) Nuclear Receptor Function through Genomics: Lessons from the Gluco- corticoid Receptor. Trends Endocrinol Metab 28: 531-540 [PMID:28495406] p [ ] de Kloet ER et al. (2017) Brain mineralocorticoid receptor function in control of salt balance and stress-adaptation. Physiol. Behav. [PMID:28089704] p [ ] de Kloet ER et al. (2017) Brain mineralocorticoid receptor function in control of salt balance and stress-adaptation. Physiol. Behav. [PMID:28089704] p y [ ] Garg D, et al. (2017) Progesterone-Mediated Non-Classical Signaling. Trends Endocrinol Metab [PMID:28651856] p y [ ] Garg D, et al. (2017) Progesterone-Mediated Non-Classical Signaling. Trends Endocrinol Metab [PMID:28651856] 3C. 3-Ketosteroid receptors S223 Searchable database: http://www.guidetopharmacology.org/index.jsp Full Contents of ConciseGuide: http://onlinelibrary.wiley.com/doi/10.1111/bph.13880/full 3C. 3-Ketosteroid receptors S223 MACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British J S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2017/18: Nuclear hormone receptors. British Journal of Pharmacology (2017) 174, S208–S224 gg ( ) [ 11. Bishop JE et al. (1994) [7976510] y 143. 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https://openalex.org/W4205595133	https://digital.csic.es/bitstream/10261/259199/1/Pineda%202022.pdf	English	null	Local hydrological conditions and spatial connectivity shape invertebrate communities after rewetting in temporary rivers	Hydrobiologia	2,022	cc-by	11,728	Hydrobiologia https://doi.org/10.1007/s10750-022-04799-8 (0123456789().,-volV)( 01234567 89().,-volV) https://doi.org/10.1007/s10750-022-04799-8 (0123456789().,-volV)( 01234567 89().,-volV) PRIMARY RESEARCH PAPER Local hydrological conditions and spatial connectivity shape invertebrate communities after rewetting in temporary rivers David Pineda-Morante . Jose´ Marı´a Ferna´ndez-Calero . Sebastian Po¨lsterl . David Cunillera-Montcusı´ . Nu´ria Bonada . Miguel Can˜edo-Argu¨elles David Pineda-Morante . Jose´ Marı´a Ferna´ndez-Calero . Sebastian Po¨lsterl . David Cunillera-Montcusı´ . Nu´ria Bonada . Miguel Can˜edo-Argu¨elles Received: 10 March 2021 / Revised: 22 December 2021 / Accepted: 8 January 2022 The Author(s) 2022 invertebrate communities of seven not impacted Mediterranean TRs after rewetting. We quantiﬁed the frequency and duration of drying events and the time since ﬂow resumed. We also quantiﬁed spatial connectivity based on each site’s position in the river network (i.e. network connectivity) and the presence of nearby disconnected streams. Overall, we found that both hydrological conditions and network con- nectivity played a signiﬁcant role in structuring aquatic invertebrate communities after rewetting. Taxonomic richness, functional richness and func- tional redundancy decreased with the frequency and duration of drying events and increased with time since the most recent rewetting. Network connectivity showed a signiﬁcant unimodal relationship with Abstract Temporary rivers (TRs) dominate global river networks and are increasing in occurrence and spatiotemporal extent. However, few studies have investigated the communities that establish after rewetting events (i.e. the end of the dry phase), when local hydrological conditions can shape the commu- nities through species sorting, and the spatial connec- tivity of sites can also inﬂuence colonisation. Here, we analysed the relative importance of both local hydro- logical conditions and spatial connectivity on the Handling editor: Dani Boix Handling editor: Dani Boix Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/ s10750-022-04799-8. Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/ s10750-022-04799-8. D. Pineda-Morante FEHM-Lab (Freshwater Ecology, Hydrology and Management), Departament de Biologia Evolutiva, Ecologia i Cie`ncies Ambientals, Universitat de Barcelona, Barcelona, Spain S. Po¨lsterl Artiﬁcial Intelligence in Medical Imaging (AI-Med), Department of Child and Adolescent Psychiatry, Ludwig- Maximilians-Universita¨t, Munich, Germany D. Cunillera-Montcusı´ WasserCluster Lunz, Biologische Station GmbH, Dr. Carl Kupelwieser Promenade 5, 3293 Lunz am See, Austria D. Pineda-Morante Integrative Freshwater Ecology Group, Centre d’Estudis Avanc¸ats de Blanes (CEAB-CSIC), Blanes, Spain e-mail: mcanedo.fem@gmail.com 123 123 Hydrobiologia by surface runoff, ﬂow resumption is typically triggered by increased precipitation and runoff in the headwaters, which is sometimes intense enough to cause ﬂash ﬂoods that swiftly transform channels from completely dry to ﬂowing strongly (Jacobson et al., 2000; Cohen & Laronne, 2005). taxonomic and functional metrics. In contrast, the presence of nearby disconnected streams was nega- tively related to functional richness and functional dispersion. Given that ﬂow intermittence in Mediter- ranean areas is expected to intensify under future global change scenarios, our results can be helpful to guide future conservation and management actions. David Pineda-Morante . Jose´ Marı´a Ferna´ndez-Calero . Sebastian Po¨lsterl . David Cunillera-Montcusı´ . Nu´ria Bonada . Miguel Can˜edo-Argu¨elles Invertebrate community recovery upon rewetting depends on the temporal dynamics of drying events (including frequency and duration) and the river network connectivity (Larned et al., 2010; Bogan et al., 2015; Crabot et al., 2020). Within this context, resistance and resilience strategies play a key role in determining community assembly (Hershkovitz & Gasith, 2013; Bogan et al., 2017). Resistance strate- gies relate to the persistence of organisms in situ in dry habitats such as in the seedbank (Bohonak & Jenkins, 2003; Stubbington & Datry, 2013), and wet refuges such as isolated pools (Cid et al., 2020), whereas resilience strategies relate to the persistence of organ- isms ex situ and their subsequent colonisation via dispersal (Datry et al., 2014). The relative contribution of both strategies to community recovery upon rewetting is highly variable across regions and among river and disturbance types (Bonada et al., 2007a). Certain resistance strategies, such as the persistence of organisms in the hyporheic zone and its contribution to community recovery, may vary considerably depend- ing on the river type (Dole-Olivier, 2011; Stubbington, 2012). Previous studies of aquatic invertebrate com- munities after rewetting in TRs across multiple climate types (e.g. arid: Stanley et al., 1994; temper- ate: Stubbington et al., 2009; Chester & Robson, 2011; Datry et al., 2012; tropical: Paltridge et al., 1997) have identiﬁed high richness of desiccation-tolerant taxa. In contrast, sediment rewetting experiments indicate that resistance strategies may play a negligible role in our study area (Folch, 2020). Resilience strategies can also be the main strategy for surviving the dry period in many regions (Fritz & Dodds, 2004; Acun˜a et al., 2005; Datry et al., 2014). This recolonisation via spatial dispersal can occur from perennial refuges including isolated water bodies as well as upstream and downstream reaches, by overland aerial dispersal (Bogan et al., 2015; Can˜edo-Argu¨elles et al., 2015) or drift and active migration, respectively (Eveleens et al., 2019; Parˇil et al., 2019). The fragmentation of TR river networks by ﬂow Keywords Flow intermittence Aquatic insects Functional traits Spatial connectivity Network analysis Dispersal Keywords Flow intermittence Aquatic insects Functional traits Spatial connectivity Network analysis Dispersal Introduction River ecosystems that recurrently cease to ﬂow or dry up at some point in time and space (i.e. temporary rivers, TRs) occupy more than 50% of the global river network (Messager et al., 2021) and they are becoming more abundant due to global change (Do¨ll & Schmied, 2012; Sauquet et al., 2021). TRs are common in Mediterranean climates (Bonada & Resh, 2013; Cid et al., 2017) where they experience seasonal and predictable ﬂoods and droughts (McElravy et al., 1989; Gasith & Resh, 1999). These ecosystems ﬂuctuate between two main periods: dry and wet, in which TRs experience different hydrological condi- tions (Gallart et al., 2012; Bonada et al., 2020). During the dry period, TRs dry up or have disconnected pools that can persist throughout the summer, whereas during the wet period TRs ﬂow and instream habitats include both rifﬂes and pools. These changing instream conditions impose a strong environmental ﬁlter that directly controls the taxonomic and func- tional composition of invertebrate communities (Wil- liams, 2006; Gallart et al., 2012). In Mediterranean TRs, drying usually starts in late spring and early summer, when an increase in water temperature and a decrease in oxygen concentrations eliminate many lotic taxa, such as mayﬂies (Ephemeroptera), stone- ﬂies (Plecoptera) and caddisﬂies (Trichoptera), i.e. EPT (Boulton & Lake, 2008; Bonada & Resh, 2013). During the dry period, many EPT insects cannot tolerate the conditions in pools, whereas lentic taxa as dragonﬂies (Odonata), beetles (Coleoptera) and true bugs (Hemiptera), i.e. OCH, increase in relative abundance (Acun˜a et al., 2005; Bonada et al., 2020). Later, in Mediterranean river catchments that are fed The fragmentation of TR river networks by ﬂow intermittence has different effects on the recolonisa- tion of aquatic invertebrates depending on their 12 123 Hydrobiologia in seven not impacted TRs in a Mediterranean river network. This time period is sufﬁcient for aquatic invertebrate communities to assemble after ﬂow resumption (Fowler, 2004; Vander Vorste et al., 2016), which usually occurs as ﬂoods in Mediter- ranean rivers (Cid et al., 2017). We used temperature data recorded continuously over 1.5 years to assess the temporal dynamics of drying events. Additionally, we analysed spatial connectivity using two metrics, one based on each site’s position in the river network (i.e. network connectivity), which inﬂuences recolonisa- tion by drift, and one based on its proximity to nearby disconnected streams (i.e. Introduction streams that did not ﬂow directly into each studied subcatchment), which affects overland dispersal. Our ﬁrst hypothesis (H1) was that taxonomic and EPT richness and functional metrics would decrease linearly with frequency and duration of drying events, and increase over time after the resumption of ﬂow. We expected OCH richness to show the opposite response to hydrological metrics since OCH could be more common in pool conditions. Secondly, we hypothesised (H2) a unimodal relation- ship between both taxonomic and functional metrics and spatial connectivity metrics. Our third hypothesis (H3) was that aquatic passive dispersal would be more important than aerial active dispersal for colonising rewetted habitats, because high river ﬂows during rewetting could favour drift of organisms from upstream reaches (McArthur & Barnes, 1985; Pal- tridge et al., 1997). dispersal abilities (Can˜edo-Argu¨elles et al., 2015; Phillipsen et al., 2015; Sarremejane et al., 2017a; Crabot et al., 2020). Strong drifters show local dispersal that is highly dependent on network connec- tivity (Bogan & Boersma, 2012; Sarremejane et al., 2020a), whereas insects with a winged adult phase might rapidly recolonise rewetted habitats even if they are relatively isolated from the river network (Can˜edo- Argu¨elles et al., 2015; Sarremejane et al., 2017b). Overall, both local hydrological conditions and spatial connectivity can determine the taxonomic richness and functional diversity of aquatic inverte- brate communities upon rewetting. Firstly, local hydrological conditions can constrain the number of taxa that inhabit a given site and ﬁlter certain ecological traits, therefore, affecting taxonomic and functional metrics. Several studies have shown that taxonomic and functional richness decline along a gradient of ﬂow permanence (Bonada et al., 2007b; Schriever et al., 2015). Thus, taxonomic and func- tional richness may decrease in rewetted habitats as the length of the preceding dry phase increases. Also, rheophilic taxa such as EPT can be more common in TRs with greater connectivity to perennial reaches, where they are favoured by continuous ﬂow (Arscott et al., 2010; Datry et al., 2014), whereas OCH can be more common in TRs with higher dry period durations because of their high afﬁnity for pool habitats (Williams, 1996; Bonada et al., 2007b). Secondly, spatial connectivity can determine community assem- bly by affecting the capacity of dispersing inverte- brates to reach suitable habitats (Heino et al., 2015; Can˜edo-Argu¨elles et al., 2015; Sarremejane et al., 2017b). Introduction At high levels of spatial connectivity, species with strong dispersal abilities can colonise suit- able habitats via mass effects and maintain viable populations throughout the river network, promoting their widespread dominance (Heino et al., 2015). Conversely, at highly isolated sites, dispersal limita- tion can lead stochastic processes to be the dominant inﬂuence on species distributions and thus community composition (Mykra¨ et al., 2007). Therefore, taxo- nomic richness can peak at intermediate levels of spatial connectivity (Mouquet & Loreau, 2003; Alter- matt et al., 2013; Vanschoenwinkel et al., 2013). Study area The study was carried out in Sant Llorenc¸ del Munt i l’Obac Natural Park, a protected area in the Valle`s Occidental region (Catalonia, NE Spain) (Fig. 1a). The area has a Mediterranean climate, with irregular and heavy rainfall, mostly in winter, some rainfall in spring and autumn, and dry summers. The underlying geology is dominated by karst limestone, with highly permeable substrates. Therefore, during summer, surface ﬂow in TRs persists for hours to days after rainfall. Almost all TRs within the park dry up during summer, either with disconnected pools or completely dry riverbeds. There are also several permanent streams in areas where discharge from karstic aquifers In this study, we analysed the effects of local hydrological conditions and the relative importance of spatial connectivity on the composition of invertebrate communities approximately one month after rewetting 12 3 3 Hydrobiologia exists with very low ﬂows during summer The TRs gradient Twenty-ﬁve sites were sampled approxi- Fig. 1 Map of the sites in Sant Llorenc¸ del Munt i l’Obac Natural Park, Spain (a). Site locations within each subcatchment, indicating the % ﬂow intermittence for each site (b). Site codes are deﬁned in the Fig. 2 caption Fig. 1 Map of the sites in Sant Llorenc¸ del Munt i l’Obac Natural Park, Spain (a). Site locations within each subcatchment, indicating the % ﬂow intermittence for each site (b). Site codes are deﬁned in the Fig. 2 caption Fig. 1 Map of the sites in Sant Llorenc¸ del Munt i l’Obac Natural Park, Spain (a). Site locations within each subcatchment, indicating the % ﬂow intermittence for each site (b). Site codes are deﬁned in the Fig. 2 caption exists, with very low ﬂows during summer. The TRs sampled belong to two main catchments that discharge to the Mediterranean Sea: the Beso`s and the Llobregat. gradient. Twenty-ﬁve sites were sampled approxi- mately one month after rewetting, from the 18–21 November 2019, to ensure enough time for assembly of post-rewetting communities. A few weeks before sampling (from 21 to 23 October) intense rainfall and ﬂash ﬂoods affected all of the sampled TRs (Supple- mentary Figs. 1, 2). Fig. 2 Synthetic network of the identiﬁed subcatchments, indicating sampled reaches (red nodes) within seven TRs and their corresponding values of closeness: a Sanana (SA), b Rellinars (R), c Vall d’Horta (H), d Castello´ (CA), e Talamanca (T), f Mura (MU), and g Santa Creu (SC). River conﬂuences are represented as network nodes and their links correspond to the directed network structure (upstream to downstream). Node size and colour represent closeness values Invertebrate sampling and processing Invertebrate sampling and processing We sampled seven TRs with similar instream condi- tions (Supplementary Table S1). Within them, we selected 32 sites which, based on previous studies (Rieradevall et al., 1999; Bonada et al., 2007b) and ﬁeld observations, encompassed a wide hydrological Each site consisted of a 100-m-long reach and was located 100–500 m from its nearest site. Invertebrates were collected by kick sampling with a 250-lm mesh net, covering all habitats in proportion to their 12 123 Hydrobiologia Fig. 2 Synthetic network of the identiﬁed subcatchments, indicating sampled reaches (red nodes) within seven TRs and their corresponding values of closeness: a Sanana (SA), b Rellinars (R), c Vall d’Horta (H), d Castello´ (CA), e Talamanca (T), f Mura (MU), and g Santa Creu (SC). River conﬂuences are represented as network nodes and their links correspond to the directed network structure (upstream to downstream). Node size and colour represent closeness values 123 3 Hydrobiologia resulting community-level matrix representing the mean trait proﬁle for each site (i.e. sites 9 traits). occurrence. Samples were preserved in 96% ethanol. Invertebrates were counted and identiﬁed in the laboratory to the lowest practical taxonomic level, usually genus except for some Diptera (Supplemen- tary Table S2). Three taxonomic metrics were calcu- lated: total taxa richness (TRic), EPT richness and OCH richness. We deﬁned the functional space as a multidimen- sional Euclidean space, and we performed a Principal Component Analysis (PCA) using the taxa 9 traits matrix to obtain a set of uncorrelated axes that represented the variability of the trait categories contained in that matrix (Gutie´rrez-Ca´novas et al., 2015). We retained the ﬁrst three principal compo- nents, which were the signiﬁcant ones resulting from the application of the broken stick rule (Jackson, 1993). These principal components constituted the dimensions of the functional space in which we quantiﬁed functional metrics, and we checked their homogeneity following the method described in Mu´rria et al. (2020). Then, we built several simulated binary matrices with one category per trait, following a random sampling based on the probability that each category was present in a randomly generated indi- vidual belonging to a genus. These matrices were projected into the generated functional space to create clouds of points that simulate the suite of potential trait combinations that comprise inter-and intra-genus functional variability. Invertebrate sampling and processing We then calculated three com- ponents of functional diversity at the whole-commu- nity level: functional richness (FRic), functional dispersion (FDis), and functional redundancy (FR), using the R packages ‘vegan’ (Oksanen et al., 2019) and ‘ade4’ (Dray & Dufour, 2007). FRic quantiﬁes the ﬁlling of the functional space and was estimated as the hypervolume enclosing the functional space ﬁlled by the community (Ville´ger et al., 2008). FDis describes the distribution of taxa in functional space and was calculated by averaging the Euclidean distance from each simulated point to the community centroid (Laliberte´ & Legendre, 2010). FR quantiﬁes the degree of functional space overlap among taxa in the functional space (Rosenfeld, 2002) and was calculated by summing the total overlap in the functional space between each taxon pair in a community (Gutie´rrez- Canovas et al., 2015). FRic reaches higher values when taxa have contrasting trait proﬁles, FDis when there are taxa with trait proﬁles that differ greatly from the community mean values, and FR when many taxa are functionally similar. Finally, each functional metric was standardised by its maximum from all communities, so that index values ranged between 0 and 1 (Mouchet et al., 2010; Bruno et al., 2016). Functional traits We used eight biological traits (and 39 categories) to characterise the functional features of invertebrate communities: life-cycle features, reproduction, aqua- tic stages, dispersal mode, resistance forms, respira- tion, and locomotion and substrate relation (Tachet et al., 2010, Supplementary Table S3). These traits can be related to resistance and resilience to drying (e.g. Bonada et al., 2007b; Aspin et al., 2019; Belmar et al., 2019). Feeding habits and food types were excluded because they have no direct relationship to resistance or resilience to drying (Sarremejane et al., 2020a; Crabot et al., 2020), and body size was excluded because trait databases may show limited concordance with the true size distribution of specimens (Orlofske & Baird, 2014). Each genus was coded according to its afﬁnity to each trait category using a fuzzy coding approach (Chevenet et al., 1994), from 0 for no afﬁnity, to 3 for the strongest afﬁnity, except for one trait, locomotion and substrate relation, which was coded from 0 to 5 (Supplementary Table S4). Trait information for Oligochaeta and Hydrachnidia was unavailable and, therefore, these taxa were excluded from trait analyses. We quantiﬁed the relative proportion of the four major dispersal modes considered in invertebrates from freshwater ecosystems, i.e. aquatic passive, aquatic active, aerial passive and aerial active (Sar- remejane et al., 2020b), using the database of Tachet et al. (2010). The proportion of aerial active and aquatic passive dispersers served to test H3. Spatial connectivity We delimited the entire river network of the Sant Llorenc¸ del Munt i l’Obac Natural Park using geospatial data. Then, the network was divided into subcatchments, which represented the most coherent river networks in the dataset, and each containing one or more TR. Using these subcatchments, we built a synthetic network taking the river conﬂuences as nodes (Fig. 2) using the R package ‘igraph’ (Csardi & Nepusz, 2006), with each site assigned to its nearest node using the function ‘nn2’ in the R package ‘RANN’ (Arya et al., 2019). We calculated each node’s closeness centrality, to indicate the capacity of their position within the network to connect to other nodes (Freeman, 1979). We deﬁned our TRs as directed networks with an upstream to downstream direction (i.e. dendritic network; Fig. 2). Therefore, we categorised closeness centrality by calculating the ‘out-closeness’ using the function ‘closeness’ in the package ‘igraph’. By considering the ‘out-closeness’ as our centrality metric we assume that a higher closeness value for a given site implies higher isolation (i.e. upstream reaches) within a network (Borthagaray et al., 2020). Closeness provides a solid proxy of dendritic network structure and can relate signiﬁcantly to biodiversity in river networks at theoretical, experimental, observational and manage- ment levels (Estrada & Bodin, 2008; Economo & Keitt, 2010; Altermatt, 2013; Borthagaray et al., 2020). We used closeness to characterise the potential for recolonisation through the river network by small- to large-scale drift. Due to data logger malfunction on very cold days and the loss of loggers washed out by ﬂoods, 44 of 69 loggers placed within the river had more missing values than recorded values. We ﬁlled in missing information about each site’s status (wet/dry) using a logistic regression model based on the channel slope, altitude, geology, as well as antecedent humidity, precipitation and temperature. These variables have a major inﬂuence on the hydrology of TRs (Cid et al., 2017; Datry et al., 2017). For humidity, precipitation and temperature, we computed summary measures for 14, 30, and 90-day periods. For each period, we summarised the daily measurements as the median, mean and slope of a linear regression model across all measurements recorded during that period. We excluded days with missing measurements in this aggregation step. Hence, for each data logger and day, we obtained a total of 27 statistics summarising changes in humidity, precipitation, and temperature (Supplementary Fig. Local hydrological conditions evaluation of its predictive performance are provided in Supplementary Appendix 1. We used the entire 1.5- year data period to calculate two metrics to charac- terise the temporal dynamics of drying events for each sample: the total duration of drying events (TotDur), which represents the total number of zero-ﬂow days, and the frequency of drying events (TotNum), which represents the number of drying events. In addition, we calculated the number of days between the most recent rewetting and each sampling event (i.e. days since rewetting). Two temperature data loggers (HOBO Pendant Temperature/Light logger) were installed from July 2018 to December 2019 in each site, one in a rifﬂe and one in a pool, to monitor the ﬂow intermittence, i.e. the proportion of zero-ﬂow days for the entire data period. Loggers were set to log temperature at hourly inter- vals. We compared the diurnal temperature variation of loggers placed within the river with loggers hung from nearby trees to infer when a site was wet or dry (Gungle, 2006). First, we calculated the daily temper- ature range as the difference between the maximum and the minimum temperature. Then we ﬁtted linear and sinusoidal regression models, being the former typical of loggers placed in perennial rivers and the latter typical of those installed in TRs (Supplementary Fig. 3). For each daily temperature range value, we calculated if it was closer to those predicted by the linear or the sinusoidal model. In the ﬁrst case, we considered the site as wet, i.e. surface water was present, whereas in the latter, we considered the site as dry, i.e. surface water was absent or restricted to disconnected pools. We validated this approach using ﬁeld observations and time-lapse photographs and found a prediction accuracy of 76%. Functional metrics We used three matrices to characterise the functional space and to estimate the functional metrics of aquatic invertebrates: a taxon counts by site matrix (i.e. sites 9 taxa), a matrix containing the fuzzy-coded trait proﬁle for each taxon (i.e. taxa 9 traits), and the 12 123 Hydrobiologia Spatial connectivity 4), and combined these statistics with information on the river’s slope, altitude, and geology to yield a set of 30 variables used to predict a site’s status. The logistic regression model and the We calculated a spatial connectivity metric, ‘sur- rounding water’, to capture overland dispersal by ﬂying adult insects. For each site, surrounding water represents the total length of streams that did not ﬂow 12 3 3 3 Hydrobiologia chance, and thus to indicate whether observed func- tional metric responses were driven by taxonomic richness or predictor variables (Ville´ger et al., 2008). Basing on the sites 9 taxa and taxa 9 trait matrices used to construct the functional space, we randomly reassigned functional traits to each taxon (99 simula- tions) to recalculate their relationships with predictor variables (Bruno et al., 2016). For each simulation, we used the same procedure as used for the data models: we calculated FRic, FR, and FDis and re-examined their relationship with the same predictors using LMM regressions to obtain the simulated intercepts and slopes for each relationship. All analyses were conducted in R version 4.1.0 (R Development Core Team, 2021). directly into the subcatchment where the site was located within a Euclidean distance of 1 km. We chose a 1 km distance because it can capture most inverte- brate colonisation events (Tonkin et al., 2014). This metric incorporates streams that are not connected to each other through the river network, and therefore indicates dispersal across a landscape (Supplementary Fig. 5). We used ArcMap 10.0 (ESRI, 1999) to generate the corresponding buffers and to quantify the total length of nearby disconnected streams. In spite of its ecological relevance (Can˜edo-Argu¨elles et al., 2015), we did not calculate the distance from each site to the nearest perennial site, due to a lack of detailed hydrological data for the stream network. Modelling The relationships between response variables (i.e. TRic, EPT and OCH richness, FRic, FR, and FDis) and predictor variables (days since rewetting, TotDur, TotNum, closeness, and surrounding water) were tested using linear mixed-effect models (LMM) by applying the function ‘lme’ included in the R package ‘nlme’ (Pinheiro et al., 2021), after validating the Gaussian distribution of the response variables. Each model included predictor variables as ﬁxed effects and the river as a random effect to control for the potential non-independence of sites within TRs. We used the function ‘r.squaredGLMM’ in the R package ‘MuMIn’ (Barton´, 2020) to calculate two goodness- of-ﬁt measures: the marginal R2 (R2m), which indi- cates the variance explained by ﬁxed effects, and the conditional R2 (R2c), which represents that explained by both ﬁxed and random effects. Based on explora- tory analyses to identify the best-ﬁtting models, unimodal relationships were used between community metrics and closeness. Closeness was log-transformed to improve the ﬁt with response variables and close- ness values were represented inverted so that its graphical representation is more intuitive (higher values correspond to higher centrality). To test the signiﬁcance of unimodal relationships, each model was ﬁtted using the maximum likelihood (ML) to compare it to the associated null model with likelihood ratio tests (Luke, 2017), using the function ‘lrtest’ in the R package ‘lmtest’ (Zeileis & Hothorn, 2002). Community composition We collected a total of 8334 organisms belonging to 86 taxa (Supplementary Table S2). Invertebrate com- munities varied in their TRic (mean ± SD: 21 ± 12 taxa; range: 36 taxa) and their total abundance (333 ± 308 organisms; range: 1081 organisms). Overall, the three most abundant orders were Diptera, Ephemeroptera and Plecoptera, representing 61.5, 21.6 and 8.5% of the total abundance, respectively. Diptera was also the most taxon-rich order (22 taxa), followed by Coleoptera (21 taxa; 2.5% abundance), Odonata (10 taxa; 1.3% abundance), Trichoptera (9 taxa; 1.4% abundance), Ephemeroptera (7 taxa) and Hemiptera (6 taxa; 1.3% abundance). Local hydrological conditions Flow intermittence varied along a gradient between \ 1% to 66% across the seven TRs (Fig. 1b), being highest at SC2 (66%; TotDur: 339 days; TotNum: 25 events), and lowest at MU4 (0.2%; TotDur: 1 day; TotNum: 1 event). TotDur (mean ± SD) was 113 ± 105 days and TotNum was 11 ± 10 events (Table 1). The maximum period of consecutive dry days occurred at H5 (195 days), whereas T3 experi- enced the highest number of drying events (35) (Table 1). After the ﬂood events (in late October 2019), all sites were wet. Some TRs, including H and SA, had already been ﬂowing for approx. Local hydrological conditions 50 days We used a null model procedure to determine whether the relationships between taxonomic and functional richness differed from those expected by 123 123 Hydrobiologia Table 1 Number of sites (N), the mean, standard deviation (SD), maximum (Max) and minimum (Min) number of days since rewetting, duration of drying events (TotDur), frequency of drying events (TotNum), closeness, and surrounding water River All Castello´ Vall d’Horta Mura Santa Creu Talamanca Rellinars Sanana Code CA H MU SC T R SA N 25 4 4 3 4 4 1 5 Days since rewetting Mean 70 44 68 60 26 70 69 137 SD 67 25 15 27 8 69 – 124 Max 329 71 76 75 36 171 – 329 Min 12 12 46 29 16 22 – 52 TotDur (days) Mean 113 108 38 38 285 160 8 57 SD 105 76 73 62 60 85 – 45 Max 339 205 147 109 339 249 – 103 Min 1 30 1 2 227 76 – 10 TotNum (events) Mean 11 10 3 7 19 11 5 8 SD 10 7 4 11 10 10 – 7 Max 35 19 9 20 29 35 – 21 Min 1 4 1 1 10 1 – 1 Closeness Mean 2.02 9 10–5 1.00 9 10–5 1.05 9 10–5 1.31 9 10–6 1.27 9 10–6 1.30 9 10–6 9.91 9 10–5 6.18 9 10–5 SD 2.86 9 10–5 1.52 9 10–8 5.50 9 10–8 0 7.05 9 10–10 7.30 9 10–10 – 2.07 9 10–7 Max 9.91 9 10–5 1.00 9 10–5 1.05 9 10–5 1.31 9 10–6 1.27 9 10–6 1.30 9 10–6 – 6.19 9 10–5 Min 1.27 9 10–6 1.00 9 10–5 1.04 9 10–5 1.31 9 10–6 1.27 9 10–6 1.30 9 10–6 – 6.14 9 10–5 Surrounding water (km) Mean 0.47 0.36 0.23 0.10 1.08 0.57 2.08 0.07 SD 0.53 0.06 0.29 0.10 0.53 0.10 – 0.09 Max 2.08 0.42 0.59 0.21 1.53 0.72 – 0.21 Min 0.00 0.29 0.00 0.00 0.46 0.48 – 0.00 Table 1 Number of sites (N), the mean, standard deviation (SD), maximum (Max) and minimum (Min) number of days since rewetting, duration of drying events (TotDur), frequency of drying events (TotNum), closeness, and surrounding water before the ﬂash ﬂoods (Fig. 1b), whereas in SC ﬂow resumed with the ﬂoods (days since rewetting: 26 ± 8 days) (Table 1). Local hydrological conditions Days since rewetting ranged from 12 to 329 (70 ± 67 days), and although sites within the same TR were within \ 500 m of each other (Fig. 1b), they experienced contrasting hydro- logical conditions, e.g. days since rewetting in T varied from 22 to 171 days at T3 and T1, respectively (Table 1 and Fig. 1b). TotDur was not signiﬁcant (P = 0.078). In contrast, FDis was not signiﬁcantly related to any hydrological metric (Table 2 and Fig. 3p, q, r). FR was strongly explained by hydrological metrics regardless of the river identity (R2m = 0.174–0.327) (Table 2). Spatial connectivity 0.003 0.149 0.785 - 0.020 0.153 0.691 - 0.002 0.173 0.761 EPT richness ? 0.010 0.185 0.596 - 0.078 0.120 0.493 - 0.004 0.217 0.625 OCH richness ? \ 0.001* 0.272 0.823 - 0.047* 0.141 0.558 - 0.010** 0.159 0.632 FRic ? 0.033* 0.125 0.583 - 0.044* 0.170 0.416 - 0.039* 0.132 0.475 FR ? 0.002** 0.327 0.552 - 0.041* 0.174 0.199 - 0.009 0.264 0.318 FDis ? 0.387 0.032 0.032 - 0.898 \ 0.001 \ 0.001 - 0.645 0.010 0.010 Aerial active ? 0.123 0.099 0.173 - 0.097 0.114 0.114 - 0.466 0.022 0.126 Aquatic passive - 0.989 \ 0.001 0.186 - 0.126 0.010 0.111 - 0.294 0.045 0.130 Abbreviated taxonomic, functional and hydrological metrics are deﬁned in the Figs. 3 and 4 caption. For clarity, only the sign of the estimate was represented: it indicates if the hydrological metrics had a positive or a negative effect on community metrics nodes (2.92 ± 1.45 km and 3.35 ± 1.89 km, respec- tively) and highest closeness values (6.18 9 10–5 and 9.91 9 10–5, respectively), indicating higher isolation (Table 1). Finally, H (Fig. 2c) and CA (Fig. 2d) had 328 nodes, a mean Euclidean distance between nodes of 5.10 ± 2.79 km, and intermediate closeness values (1.05 9 10–5 and 1.00 9 10–5, respectively) (Table 1). The total length of nearby disconnected streams (i.e. surrounding water) was highest at R (2.08 km) and SC (1.08 ± 0.53 km), and lowest at SA (0.07 ± 0.09 km) and MU (0.10 ± 0.10 km) (Table 1; Sup- plementary Fig. 5). nodes (2.92 ± 1.45 km and 3.35 ± 1.89 km, respec- tively) and highest closeness values (6.18 9 10–5 and 9.91 9 10–5, respectively), indicating higher isolation (Table 1). Finally, H (Fig. 2c) and CA (Fig. 2d) had 328 nodes, a mean Euclidean distance between nodes of 5.10 ± 2.79 km, and intermediate closeness values (1.05 9 10–5 and 1.00 9 10–5, respectively) (Table 1). The total length of nearby disconnected streams (i.e. surrounding water) was highest at R (2.08 km) and SC (1.08 ± 0.53 km), and lowest at SA (0.07 ± 0.09 km) and MU (0.10 ± 0.10 km) (Table 1; Sup- plementary Fig. 5). TRic, EPT and OCH richness showed signiﬁcant unimodal relationships with closeness (R2m = 0.414– 0.607; Fig. 4a, c, e), so did FRic and FR (R2m = 0.409 and 0.283, respectively; Fig. 4b, d) (Table 3). Spatial connectivity T, MU and SC had the largest number of nodes (895; Fig. 2e, f, g), highest mean Euclidean distance between nodes (mean ± SD: 8.82 ± 4.73 km) and lowest closeness values (1.27 9 10–6–1.31 9 10–6), indicating higher centrality and spatial connectivity (Table 1). In contrast, SA (Fig. 2a) and R (Fig. 2b) had the lowest number of nodes (133 and 110, respec- tively), lowest mean Euclidean distance between T, MU and SC had the largest number of nodes (895; Fig. 2e, f, g), highest mean Euclidean distance between nodes (mean ± SD: 8.82 ± 4.73 km) and lowest closeness values (1.27 9 10–6–1.31 9 10–6), indicating higher centrality and spatial connectivity (Table 1). In contrast, SA (Fig. 2a) and R (Fig. 2b) had the lowest number of nodes (133 and 110, respec- tively), lowest mean Euclidean distance between Taxonomic (i.e. TRic, EPT and OCH richness) and functional (i.e. FRic and FR) metrics were positively related to days since rewetting (R2m = 0.149–0.327; Fig. 3a, d, g, j, m) and negatively related to TotDur (R2m = 0.120–0.174; Fig. 3b, e, h, k, n) and TotNum (R2m = 0.159–0.264; Fig. 3c, f, i, l, o) (Table 2), although the decrease in EPT richness with increasing 12 3 3 Hydrobiologia Fig. 3 Relationships between both taxonomic (a–i) and func- tional (j–r) metrics and hydrological metrics (i.e. days since rewetting; duration of drying events, TotDur; frequency of drying events, TotNum). Solid lines represent the ﬁtted LMM values (see Table 2). a–c TRic taxonomic richness, d–f EPT Ephemeroptera, Plecoptera and Trichoptera richness, g–i OCH Odonata, Coleoptera and Hemiptera richness, j–l FRic func- tional richness, m–o FR functional redundancy, p–r FDis functional dispersion 1 Ephemeroptera, Plecoptera and Trichoptera richness, g–i OCH Odonata, Coleoptera and Hemiptera richness, j–l FRic func- tional richness, m–o FR functional redundancy, p–r FDis functional dispersion Fig. 3 Relationships between both taxonomic (a–i) and func- tional (j–r) metrics and hydrological metrics (i.e. days since rewetting; duration of drying events, TotDur; frequency of drying events, TotNum). Solid lines represent the ﬁtted LMM values (see Table 2). a–c TRic taxonomic richness, d–f EPT 12 3 Hydrobiologia Table 2 LMM results relating taxonomic and functional metrics to hydrological metrics as ﬁxed effects and river as a random effect Days since rewetting TotDur TotNum Estimate P R2m R2c Estimate P R2m R2c Estimate P R2m R2c TRic ? Spatial connectivity Close- ness explained more variance in both taxonomic and functional metrics (R2m = 0.283–0.607) than any hydrological metric (R2m = 0.120–0.272). Also, FRic and FDis experienced a signiﬁcant linear decrease with surrounding water (R2m = 0.434 and 0.165, Fig. 5b, f, respectively) (Table 3). Dispersal trait analysis The proportion of aerial active and aquatic passive dispersers was not related to any hydrological metric (Table 2 and Supplementary Fig. 6) nor surrounding water (Fig. 5g, h). Similarly, none of the disperser groups showed a strong unimodal relationship with closeness (Fig. 4g, h), although the relationship was marginally signiﬁcant for aerial dispersers (Table 3). Relationships between aerial passive and aquatic active dispersers and predictor variables are provided in Supplementary Figs. 6, 7 and Tables S5, S6. Null models For the signiﬁcant relationships observed between functional metrics and predictor variables (Table 2, 3), null models revealed that the intercepts and slopes for the FRic models including hydrological metrics, closeness and surrounding water differed from null distributions (P \ 0.05), suggesting non-randomness (Supplementary Fig. 8 and Table S7). In contrast, the intercepts and slopes for the FR and FDis models were comparable to the null distribution (P [ 0.05), sug- gesting that the signiﬁcant relationships found between FR and hydrological metrics and closeness, 12 3 3 Hydrobiologia and the signiﬁcant relationship between FDis and surrounding water were all driven by TRic (Supple- mentary Fig. 8 and Table S7). Discussion invertebrate community composition after rewetting. Similarly, Can˜edo-Argu¨elles et al. (2020) used a different but complementary approach (analysis of Bray–Curtis pairwise dissimilarities between commu- nities) and found that both local hydrological condi- tions and spatial connectivity shaped invertebrate metacommunities in Mediterranean TR networks Fig. 4 Relationships between taxonomic (a, c, e) and functional (b, d, f) metrics, dispersal trait categories (g, h) and closeness. Solid lines represent the ﬁtted LMM values using unimodal relationships (see Table 3). Log-transformed closeness values were inverted so that higher values corresponded to a higher centrality (i.e. connectivity). Abbreviations (a–f) are deﬁned in the Fig. 3 caption. g Aerial active = proportion of aerial active dispersers, and h Aquatic passive = proportion of aquatic passive dispersers invertebrate community composition after rewetting. Similarly, Can˜edo-Argu¨elles et al. (2020) used a different but complementary approach (analysis of Bray–Curtis pairwise dissimilarities between commu- nities) and found that both local hydrological condi- tions and spatial connectivity shaped invertebrate metacommunities in Mediterranean TR networks close to our study area. In addition, Crabot et al. (2020) found that local hydrological conditions invertebrate community composition after rewetting. Similarly, Can˜edo-Argu¨elles et al. (2020) used a different but complementary approach (analysis of Bray–Curtis pairwise dissimilarities between commu- nities) and found that both local hydrological condi- tions and spatial connectivity shaped invertebrate metacommunities in Mediterranean TR networks close to our study area. In addition, Crabot et al. (2020) found that local hydrological conditions and the signiﬁcant relationship between FDis and surrounding water were all driven by TRic (Supple- mentary Fig. 8 and Table S7). Discussion We found that both local hydrological conditions and network connectivity signiﬁcantly affected 12 Hydrobiologia Table 3 LMM results relating taxonomic and functional metrics to spatial connectivity metrics as ﬁxed effects and river as a random effect logCloseness Surrounding water Likelihood ratio test Chi2 P R2m R2c Estimate P R2m R2c TRic 15.318 \ 0.001* 0.607 0.619 - 0.096 0.124 0.603 EPT richness 10.812 0.004 0.414 0.421 - 0.238 0.068 0.390 OCH richness 12.379 0.002 0.500 0.523 - 0.100 0.127 0.517 FRic 9.986 0.007 0.409 0.469 - 0.001* 0.434 0.613 FR 8.822 0.012* 0.283 0.283 - 0.074 0.133 0.145 FDis 0.828 0.661 0.030 0.030 - 0.044* 0.165 0.165 Aerial active 5.817 0.055 0.202 0.202 - 0.193 0.073 0.098 Aquatic passive 0.700 0.705 0.035 0.342 - 0.776 0.004 0.176 Abbreviated taxonomic and functional metrics are deﬁned in the Figs. 3 and 4 caption. We used quadratic regressions for closeness and linear regressions for surrounding water. Estimate was deﬁned in the Table 2 caption Signiﬁcant results are denoted by asterisks (P \ 0.05, P \ 0.01, *P \ 0.001) Abbreviated taxonomic and functional metrics are deﬁned in the Figs. 3 and 4 caption. We used quadratic regressions for closeness and linear regressions for surrounding water. Estimate was deﬁned in the Table 2 caption Signiﬁcant results are denoted by asterisks (P \ 0.05, P \ 0.01, *P \ 0.001) K-strategists) from colonising and persisting (Schrie- ver et al., 2015; Stubbington et al., 2017). Time since rewetting also inﬂuenced taxonomic and functional metrics, suggesting that the relatively long-term characteristics of drying regimes (here, TotDur and TotNum) and recent instream conditions both inﬂu- ence invertebrate community composition in TRs. In contrast, Arias-Real et al. (2021) found that the time since the last rewetting event had a minor effect on density and diversity metrics compared with the annual duration and frequency of drying events. This difference between Arias-Real et al. (2021) and or study could be related with the sampling period. Arias- Real et al. (2021) collected invertebrate samples just after the rainy season (February), whereas we col- lected them after rewetting (November). It is very likely that our study better captured the effects of the rewetting in terms of dispersal and colonisation, that could be lost to some extent in February when communities are stabilised and density-dependent processes (e.g. competition, predation) operate (Closs & Lake, 1994; Godoy et al., 2016). Discussion These ﬁndings highlight the importance of considering both recent and longer-term antecedent hydrological conditions (Sa´nchez-Montoya et al., 2018), especially in TRs with highly variable hydrological regimes, such as those in our semi-arid study area (Gallart et al., 2012). Supporting our H1, EPT richness decreased with drying frequency (i.e. TotNum), likely because many inﬂuenced invertebrate beta diversity, but the effects depended on the spatial drying pattern (e.g. beta diversity was higher for basins with intermittent headwaters and perennial downstream reaches than for basins with perennial headwaters and intermittent downstream reaches). These patterns likely reﬂect a combination of strong abiotic ﬁlters associated with seasonal hydrological variability in TRs (Stubbington et al., 2017), and dispersal limitation associated with river network fragmentation by ﬂow intermittence (Can˜edo-Argu¨elles et al., 2015, 2020; Sarremejane et al., 2017a). We also found a reduction in taxonomic and functional metrics as the frequency and duration of drying events increased, supporting our H1. Our results are consistent with previous studies focusing on the rewetting phase (e.g. Acun˜a et al., 2005; Doretto et al., 2020) and other analyses of richness patterns in TRs (e.g. Datry et al., 2014; Schriever et al., 2015; Sarremejane et al., 2020a; Crabot et al., 2021). Sites with lower TotDur may support richer commu- nities including taxa with a wider range of trait proﬁles, which can colonise and persist in less stressful conditions, thus reducing the inﬂuence of environ- mental ﬁltering (Bonada et al., 2007b; Leigh & Datry, 2017; Sa´nchez-Montoya et al., 2018). Similarly, a high frequency of drying events (i.e. high TotNum) can limit the maximum taxonomic and functional richness that a community can attain, because a high disturbance frequency prevents some taxa (e.g. Supporting our H1, EPT richness decreased with drying frequency (i.e. TotNum), likely because many EPT taxa are rheophiles and ﬂow cessation and drying 12 3 3 Hydrobiologia ay prevent their persistence in TRs (Acun˜a et al., 005; Williams, 2006; Sa´nchez-Montoya et al., 2018; spin et al., 2018). However, OCH richness also to dry fast without retaining lentic habitats, and thus did not have OCH populations that persisted after ﬂow resumed. g. 5 Relationships tween taxonomic (a, c, and functional (b, d, metrics, dispersal trait tegories (g, h) and rrounding water (km). olid lines represent the ted LMM values (see able 3). Abbreviations are ﬁned in the Figs. Discussion 3 and 4 ptions may prevent their persistence in TRs (Acun˜a et al., 2005; Williams, 2006; Sa´nchez-Montoya et al., 2018; Aspin et al., 2018). However, OCH richness also decreased signiﬁcantly with TotDur and TotNum, partially rejecting our H1. After ﬂow resumes, water ﬂows through the river network, and lentic habitats which support high OCH richness in TRs (Williams, 1996; Bonada et al., 2007b) are less abundant. Moreover, sites with longer TotDur were more likely to dry fast without retaining lentic habitats, and thus did not have OCH populations that persisted after ﬂow resumed. Functional analyses revealed that invertebrate communities from TRs with lower TotDur and TotNum occupied a higher trait hypervolume (i.e. higher FRic) and had a higher number of taxa with similar traits (i.e. higher FR). These results suggest a lower impact of local species loss on functional Table 3). Abbreviations are deﬁned in the Figs. 3 and 4 captions to dry fast without retaining lentic habitats, and thus did not have OCH populations that persisted after ﬂow resumed. may prevent their persistence in TRs (Acun˜a et al., 2005; Williams, 2006; Sa´nchez-Montoya et al., 2018; Aspin et al., 2018). However, OCH richness also decreased signiﬁcantly with TotDur and TotNum, partially rejecting our H1. After ﬂow resumes, water ﬂows through the river network, and lentic habitats which support high OCH richness in TRs (Williams, 1996; Bonada et al., 2007b) are less abundant. Moreover, sites with longer TotDur were more likely may prevent their persistence in TRs (Acun˜a et al., 2005; Williams, 2006; Sa´nchez-Montoya et al., 2018; Aspin et al., 2018). However, OCH richness also decreased signiﬁcantly with TotDur and TotNum, partially rejecting our H1. After ﬂow resumes, water ﬂows through the river network, and lentic habitats which support high OCH richness in TRs (Williams, 1996; Bonada et al., 2007b) are less abundant. Moreover, sites with longer TotDur were more likely Functional analyses revealed that invertebrate communities from TRs with lower TotDur and TotNum occupied a higher trait hypervolume (i.e. higher FRic) and had a higher number of taxa with similar traits (i.e. higher FR). Discussion These results suggest a lower impact of local species loss on functional 12 123 Hydrobiologia (Heino et al., 2015) and empirical studies (Brown & Swan, 2010; Tolonen et al., 2017), and taking the dispersal abilities of the studied organisms into consideration (Sarremejane et al., 2020b), mass effects should be operating at the spatial scale that we assessed (i.e. differences within streams and between nearby streams). Contrarily, our results suggest that dispersal limitation could take place at small spatial scales in river and stream networks fragmented by droughts (i.e. TRs). However, this message needs to be taken with caution since we sampled the communities only one month after rewetting, when no sufﬁcient time might have elapsed to allow for mass effects. diversity in less intermittent rivers, potentially sup- porting recovery of ecosystem functioning after dis- turbances such as drying events (Schriever et al., 2015). In contrast, at the most intermittent sites the loss of taxonomic diversity might have an impact in ecosystem functioning through a reduction or the loss of key ecological traits (Crabot et al., 2021). However, the relationships between FR and hydrological metrics should be taken with caution since they were signif- icantly affected by differences in TRic, conﬁrming that null models are essential to assess changes in FR due to the intimate relationship between taxonomic and functional richness. Our surrounding water metric, which described the spatial connectivity based on the length of nearby disconnected streams, had a negligible effect on taxonomic richness and trait dispersal, except for a negative relationship with functional richness and functional dispersion that was signiﬁcantly affected by differences in TRic. Thus, overland aerial dispersal seems to have a minor importance for community assembly after rewetting in the studied river network. Although overland dispersal can play a key role for community assembly in river networks that are fragmented by droughts (Can˜edo-Argu¨elles et al., 2015; Razeng et al., 2016), the dispersal by drift and following the river network seems to be more impor- tant after the rewetting, when the whole network has been connected by ﬂoods. Accordingly, Doretto et al. (2020) reported that dispersal occurred mainly by drift through the river network in Alpine streams recover- ing from a dry period. Also, previous studies in perennial river networks showed that aquatic inverte- brates disperse mainly following the river channel (Hughes, 2007; Brown & Swan, 2010; Rouquette et al., 2013). Conclusion 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/. This study advances our understanding on how aquatic invertebrate communities respond to the resumption of surface ﬂow throughout river networks after periods of fragmentation by drying. We found that both hydrological conditions and spatial connectivity inﬂu- enced the taxonomic richness and functional diversity of aquatic invertebrate communities. The main path- way for invertebrate recolonisation after rewetting was through the river network from upstream to downstream reaches. By characterising how network connectivity inﬂuences community composition after ﬂow resumption in TRs, our results could inform conservation and management actions in these rivers, especially under future global change scenarios. For example, biodiversity conservation planning in TRs could be improved by quantifying the inﬂuence of spatial connectivity on recolonisation processes and identifying key refuges that serve as sources of colonists after rewetting. Discussion This could explain why we found no relationship between the spatial connectivity metrics and the relative abundance of aerial and aquatic dispersers (thereby rejecting our H3). The negative relationship between surrounding water with func- tional richness could be related with the fact that disconnected streams can promote the dispersal of strong ﬂyers (e.g. Heteroptera and Odonata) that could dominate the communities through mass effects (Can˜edo-Argu¨elles et al., 2015). However, additional studies covering a wider range of surrounding water are needed to validate this hypothesis. As hypothesised in H2, we found a unimodal relationship between taxonomic metrics and the longitudinal connectivity of each site to the rest of the river network (i.e. closeness) that was usually not dependent on the river identity (i.e. small differences between R2m and R2c). In this study, closeness centrality captured the upstream–downstream relative position of the sites, which after a rewetting event is of great importance (Doretto et al., 2020). As all TRs were ﬂooded during the same rainfall event, the identity of each TR lost relevance against the spatial isolation. The relevance of closeness has already been shown at larger landscape scales when assessing changes in species richness (Borthagaray et al., 2020). Interestingly, here this effect was detected at a smaller spatial scale, highlighting the potential of this metric for explaining diversity patterns in dendritic networks (Economo & Keitt, 2010; Carrara et al., 2012; Altermatt, 2013). Taxa richness may peak at interme- diate levels of network connectivity (Altermatt et al., 2013; Vanschoenwinkel et al., 2013) or species dispersal (Mouquet & Loreau, 2003), and this uni- modal relationship likely reﬂects the interplay between the effects of dispersal limitation at isolated sites and mass effects at well-connected sites (Heino et al., 2015; Brown et al., 2018). At intermediate connectivity levels, low dispersal limitation allows the species to track environmental gradients and, at the same time, communities are not homogenised by mass effects (Heino et al., 2015). Overall, this could lead to a higher niche occupancy and a higher number of taxa. 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https://openalex.org/W4390616704	https://ciencialatina.org/index.php/cienciala/article/download/9033/13466	es		Modelo de Gestión de Proyectos Inmobiliarios de Vivienda Para Empresas Medianas desde la Fase Conceptual Hasta la Fase de Posventa	Ciencia latina	2,024	cc-by	4,797	Ciencia Latina Revista Científica Multidisciplinar Noviembre-Diciembre, 2023, Volumen 7, Número 6 https://doi.org/10.37811/cl_rcm.v7i6.9033 Modelo de Gestión de Proyectos Inmobiliarios de Vivienda Para Empresas Medianas desde la Fase Conceptual Hasta la Fase de Posventa Mtra. Lizzet Ina Macedo Valladares1 lizzetmacedov@gmail.com https://orcid.org/ 0009-0009-0680-9672 Universidad Nacional de Ingeniería, Lima-Perú MBA Carlos Adolfo Noriega Niño de Guzmán canoriega@gmail.com https://orcid.org/0000-0001-5747-8038 Universidad Nacional de Ingeniería, Lima-Perú Dr. Johnny Félix Farfán-Pimentel felix13200@hotmail.com https://orcid.org/0000-0001-6109-4416 Universidad César Vallejo, Lima-Perú RESUMEN El presente trabajo de investigación desarrolla desde el planteamiento del problema, el cual radica en los problemas de diseño, construcción y operación que conllevan el seguir empleando procesos tradicionales en 2D y como estos afectan la calidad de los proyectos y en la rentabilidad de las empresas que lo ejecutan; se planteó emplear modelos de gestión basados en nuevas tecnologías que permitan optimizar los procesos y recursos en todas las etapas de un proyecto de construcción de viviendas maximizando su rentabilidad y calidad; teniendo como propósito lograr un edificio de alto rendimiento. Se justifica por los resultados de casos de éxitos y lecciones aprendidas de diferentes expertos y proyectos a nivel mundial. Los procesos constructivos impulsan a la aplicación de nuevas tecnologías que ayuden a tener una mejor comprensión y análisis de la información de campo y sobre esa base se diseñan los aspectos técnicos empleando: (i) BIM (Building Information Modeling,), definida como una representación digital de las características físicas y funcionales de una edificación, siendo fuente confiable de información desde su concepción hasta su demolición; (ii) Lean Construction, definida como una metodología basada en la mejora continua, en minimizar pérdidas y maximizar el valor del producto final; y (iii) VDC (Virtual Design and Construction). Palabras clave: proyectos; construcción; implementación; desarrollo; ejecución . 1 Autor Principal Correspondencia: lizzetmacedov@gmail.com pág. 4747 Management Model for Housing Real Estate Projects for Medium-Sized Companies from The Conceptual Phase to The Post-Sale Pase ABSTRACT This research work develops from the problem statement, which lies in the problems of design, construction and operation that involve the continued use of traditional processes in 2D and how these affect the quality of projects and the profitability of the companies that execute them; it was proposed to use management models based on new technologies to optimize processes and resources at all stages of a housing construction project maximizing its profitability and quality; aiming to achieve a high performance building. It is justified by the results of success stories and lessons learned from different experts and projects worldwide. The construction processes drive the application of new technologies that help to have a better understanding and analysis of field information and on that basis the technical aspects are designed using: (i) BIM (Building Information Modeling), defined as a digital representation of the physical and functional characteristics of a building, being a reliable source of information from its conception to its demolition; (ii) Lean Construction, defined as a methodology based on continuous improvement, minimizing losses and maximizing the value of the final product; and (iii) VDC (Virtual Design and Construction). Key words: projects; construction; implementation; development; execution Artículo recibido: 17 noviembre 2023 Aceptado para publicación: 26 diciembre 2023 pág. 4748 INTRODUCCIÓN Según Dantas et al. (2015) la industria de la construcción se considera una de las más antiguas del mundo, donde la coordinación se lleva a cabo en su mayoría comparando dibujos en 2D de las diferentes especialidades de un proyecto, donde identificar las interferencias y/o conflictos resulta ser una tarea ardua y muchos de estos errores pueden quedar el aire sin descubrirse, hasta el inicio de la construcción, conllevando a retrabajos que reducen la productividad de los procesos. También uno de los principales factores responsables de la reducción del rendimiento general y la eficiencia de los edificios es la mala gestión del proyecto. Siendo esta problemática la que genera el mayor número de retrasos, sobrecostos y bajos estándares de calidad en los proyectos. La aplicación de nuevos modelos y herramientas basados en nuevas tecnologías, aportan un valor significativo a los procesos de las distintas especialidades de diseño y apoyan directamente la constructibilidad; un concepto que surgió a finales de la década de 1970 y que se basa en el conocimiento y la capacidad de construir, tomando como base la experiencia de planificación, diseño, ingeniería y suministro; mejorando la calidad y la productividad, así como reduciendo el tiempo, desperdicio y costos. Actualmente, las limitaciones que presentan las herramientas de visualización virtual basadas en tecnologías de información (TI), son mínimas, respecto al alcance en tecnología que requiere el sector construcción. Estas novedosas herramientas, brindan capacidades avanzadas de visualización y simulación beneficiando el flujo anticipado de información y simulando la realidad (Eyzaguirre, 2015). Es así que empresas desarrolladoras como Autodesk, Bentley, Graphisoft, entre otras trabajan en softwares y plataformas que permiten desarrollar un trabajo colaborativo en tiempo real entre un número ilimitado de colaboradores ubicados en cualquier punto en todos los horarios permitidos (Autodesk, 2021). Así, como autores de distintas partes del mundo desarrollan información basada en plataformas y usos de softwares en entornos colaborativos, que pretenden establecer un manual de estándar universal, así es como lo plantea David Barco (2018) en su libro Diario de un BIM Manager. Por otro lado, existen proyectos e investigaciones con resultados positivos en el uso de tecnología y entornos colaborativos que sientan las bases para el desarrollo e implementación de estas buenas prácticas en los distintos proyectos del sector construcción, como por ejemplo el caso de éxito del UHS pág. 4749 Temecula Valley Hospital, proyecto construido por DPR Consrruction (USA), en tan solo 11 meses en el que se logró cumplir con el objetivo del cliente generando un tercio más de ganancias. Sheng,Wei y Faris (2016) demostraron que las metodologías colaborativas y modelos de gestión se pueden emplear en proyectos de cualquier magnitud, enfocando sus estudios en los modelos BIM en el tiempo (4D) y costo (5D). Asi también, Bassam (2017) aplicó los modelos BIM en una empresa de aceites naturales, en el cual se logró mejorar los flujos de trabajo y así reducir significativamente el costo de producción del producto final. Del mismo modo, Kraatz, Sànchez y Hampson (2014) lograron resultados positivos en el desarrollo de un proyecto de infraestructura de transporte, mediante el uso de modelo BIM, diseño y construcción virtual (VDC). Asimismo, Belsvik, Laadre y Hjelseth (2019) desarrollaron un estudio en el que demostraron la importancia de métricas para evaluar el éxito de un proyecto para obtener con estos una mejora continua en un entorno colaborativo. Tariq y Muneeb (2020) desarrollaron un estudio de la aplicación de Modelos BIM en la gestión de seguridad en obra y Dantas et al. (2015), realizaron una investigación sobre la coordinación de especialidades empleando modelos BIM enfocados en los retrasos en la construcción, incrementos en la carga administrativa, presupuesto y tiempo. Los estudios han encontrado que la integración entre los procesos de diseño y construcción se ha convertido en un requisito importante para mejorar el desempeño del proyecto. El mundo de hoy demanda ofrecer cambios y transiciones tecnológicas, orientados a incrementar la eficiencia, productividad, calidad, tiempo y costos de los proyectos; por lo tanto, es necesario implementar nuevos modelos de gestión, que involucren tecnologías de información, desde el inicio del proyecto teniendo como base la constructibilidad. En ese sentido, Fischer (2006) indica que el diseño y la construcción virtuales se definen como “el uso de modelos de desempeño multidisciplinario de proyectos de diseño-construcción, incluido el producto (arquitectura, estructura e instalaciones), organización del equipo y procesos de trabajo para apoyar a los objetivos comerciales explícitos y públicos”. Las nuevas herramientas y tecnologías de diseño digital han sido de reciente interés como medios para mejorar las prácticas en todos los proyectos de construcción a nivel mundial. Almonacid et al. (2015), mencionan en su investigación que una de las principales formas para lograr que el proyecto tenga un diseño óptimo y el mejor número de incompatibilidades, es el de incluir a los ingenieros, arquitectos, supervisores y todos los involucrados pág. 4750 en el proyecto en etapas tempranas, para así evaluar restricciones y solucionar antes de generar un impacto. Entonces, la industria de la construcción es un entorno de información intensa, abundante y única; donde el papel sigue siendo aún la manera más común de comunicar la información a los diferentes actores del proyecto. Debido al crecimiento de la demanda de viviendas, las empresas inmobiliarias y constructoras se han visto en la necesidad de incrementar la velocidad en el diseño, conllevando a proyectos incompletos, con falta de detalles e incompatibilidades; generando problemas en la construcción. Por ello implementar un nuevo modelo de gestión basado en tecnologías implica un cambio de visión en los involucrados en la industria de arquitectura, ingeniería, construcción y operaciones (AECO, por sus siglas en inglés) y el Perú, no es ajeno a este cambio progresivo inmerso en nuevas tecnologías y plataformas colaborativas, que después de la pandemia del COVID 19 va en crecimiento paulatino junto al PBI, que según los informes del BCR realizado en junio del 2021, proyectan un crecimiento del 5.0% al 9.0% (notas BCR Nº.46, 2021). Por otro lado, Bravo et al. (2019) aplicaron un método integrador en la etapa de diseño de un proyecto, en el que obtuvieron el 27.6% de reducción en los presupuestos adicionales, reflejándose en la reducción de órdenes de cambio, requerimientos de cambios y tiempos de respuestas en ambos. Finalmente, Chingay, A (2015) concluyó en su investigación que, el uso de las sesiones ICE en el proyecto cambiaron la estructura de las reuniones tradicionales de la contratista, hizo más proactiva la toma de decisiones entre los equipos multidisciplinario. El objetivo de la investigación fue establecer un modelo de gestión de proyectos inmobiliarios de vivienda para empresas medianas desde la fase conceptual hasta la fase de posventa. METODOLOGÍA La investigación se basa en el análisis de modelos de gestión de proyectos existentes y en el análisis de la percepción y aceptación de modelos de gestión basados en nuevas tecnologías y metodologías de trabajo como el BIM, Lean Construction y el VDC; acompañados de entrevistas a expertos involucrados en el proceso; para obtener como resultado un modelo de gestión acorde a las necesidades de las empresas inmobiliarias medianas de viviendas en el Perú. Dentro de estas nuevas tecnologías y metodologías de trabajo como: (i) BIM (Building Information Modeling,), definida como una pág. 4751 representación digital de las características físicas y funcionales de una edificación, siendo fuente confiable de información desde su concepción hasta su demolición; (ii) Lean Construction, definida como una metodología basada en la mejora continua, en minimizar pérdidas y maximizar el valor del producto final; y (iii) VDC (Virtual Design and Construction), que tiene como objetivo la eficiencia de la gestión de proyectos a través del uso de modelos multidisciplinarios, incluyendo el producto, procesos de trabajo y organización de equipos. RESULTADOS Y DISCUSIÓN Según lo expuesto, en la investigación se ha logrado desarrollar un modelo de gestión de proyectos que brindará a las empresas inmobiliarias de viviendas las herramientas y metodologías necesarias para lograr optimizaciones y mejoras en los resultados del tiempo, costo y calidad de los proyectos y así lograr incrementar su rentabilidad. En la investigación se ha logrado realizar un análisis comparativo de los principales modelos teóricos de gestión de proyectos, los cuales sirvieron de base para desarrollar el modelo de gestión propuesto en esta investigación. En el estudio se ha logrado realizar un análisis económico del sector construcción, tomando en cuenta las implicaciones del COVID- 19, pandemia que afecta la economía mundial del 2020, donde los expertos expresan que una de las vías para superar esta crisis es implementar modelos dinámicos, respaldados por la tecnología y la innovación. Estas referencias refuerzan el modelo de gestión propuesto, ya que se basa en el uso de metodologías colaborativas y tecnológicas. En este análisis se ha logrado evaluar la percepción de los modelos de gestión revisados y sus respectivos componentes, en las empresas inmobiliarias medianas y presentar un referente de sus usos y aplicaciones. Se logró desarrollar una propuesta económica referencial, que servirá de base para la implementación y aplicación del modelo de gestión en proyectos inmobiliarios de viviendas. En tal sentido, el inicio del uso de nuevas metodologías de trabajo, empleando la tecnología como aliado, tiene sus inicios casi una década atrás; sin un logro aún acertado, en la integración con los modelos de gestión; es por ello que, en la actualidad estos se han afianzado en mayor medida. Dentro de los modelos de gestión y metodologías colaborativas revisados, se ha evaluado las ventajas y desventajas, relacionándolas entre sí y en los procesos en los que participan. Además, se realizó un cuadro comparativo, donde se comprueba que no todas las metodologías aportan de igual forma a los pág. 4752 procesos, no lográndose cumplir completamente con los objetivos planteados. En relación al análisis del entorno económico del sector construcción. Según la Asociación de desarrolladores Inmobiliarios (ADI) existen cinco tendencias las cuales se está inclinando el sector inmobiliario en el Perú: precios en aumento, viviendas más pequeñas funcionales, atractivas áreas comunes, mayor altura (hasta 25 pisos) y vivienda verde (uso eficiente de los recursos). Según el análisis de las respuestas obtenidas de los expertos, la variabilidad del uso de metodologías colaborativas es amplía, siendo los más empleados BIM, VDC, IPD y el Lean Construction, utilizados, sobre todo, en los procesos de ejecución y control; como detección de interferencias, metrados y planificación del proyecto con la simulación 4D. De esta manera, se logra una alta aprobación y satisfacción de todos los actores del proyecto, especialmente la del cliente, quien fue el promotor, en términos de los expertos concluyeron en mejoras en la calidad del proyecto, costo y tiempo de ejecución. Del análisis del trabajo de campo se obtuvo como resultado que existe un gran interés por colaboradores de distintos sectores, como el estatal y privado; para emplear y recomendar el uso de estas nuevas plataformas de trabajo; colaboradores que van desde propietarios y jefes de proyecto a modeladores y prácticas de ingeniería. Por otro lado, el mayor uso de estas nuevas metodologías se presenta en proyectos de vivienda multifamiliares de 10 a pisos a más, debido al número de metros cuadrados involucrados y al valor que generaría el compatibilizar un piso y replicarlo en los restantes y así disminuir el tiempo de ejecución. Dentro de los usos más frecuentes se tiene a las visualizaciones, integración, reporte de interferencias y la posterior compatibilización; donde la demanda del cliente y la política organizacional, fueron las claves del éxito de la implementación, con un acertado apoyo de los colaboradores en los distintos procesos; obteniendo resultados satisfactorios en los diferentes proyectos inmobiliarios. El modelo propuesto plantea la integración de metodologías colaborativas como Integrated Project Delibery(IPD), Lean Constructión, Building Information Modeling (BIM) y el Virtual Design Construction (VDC), ubicándolas en distintas etapas dentro de los proceso para maximizar sus resultados. Es así que las premisas sobre principios de uso, sustentabilidad, constructibilidad y operabilidad del IPD exigen contar con una organización, procesos e información integrados; teniendo pág. 4753 como herramientas modelos de productos como el Building Information Modeling (BIM), reuniones concurrentes (ICE) y una adecuada gestión de producción; englobados a la vez en la Metodología del Virtual Design Construction (VDC). El modelo de gestión propuesto es posible emplearlo en todas las etapas y procesos de un proyecto, desde la factibilidad en el proceso de inicio hasta la gestión de posventa en el proceso de cierre. Para ello se debe emplear modelos tridimensionales, la gestión colaborativa en reuniones ICE, la simulación 4D de la planificación, la fluidez y centralización de la documentación para crear modelos de fabricación a detalle. Luego estos serán instalados o construidos en el proyecto y estas mismas herramientas permitirán hacer el seguimiento y control de la correcta realización en obra. Todos estas etapas y procesos pueden ser documentados y, su vez, obtener métricas para mayor control y una retroalimentación a través de lecciones aprendidas. Por último, se hace hincapié que la implementación de este modelo de gestión puede ser un proceso largo y los resultados no se verán en una primera instancia, por lo cual se recomienda contar con el apoyo permanente de la organización y llevar, sobre todo, un proceso de implementación ordenado, desde el análisis inicial de la organización para conocer los puntos a reforzar y/o modificar. pág. 4754 Ilustraciones, Tablas, Figuras. Figura 1. Tablero de control de mando: indicadores TABLERO DE CONTROL DE MANDO: INDICADORES PROCESO USOS Y APLICACIONES INDICADORES NOMBRE MÉTRICA Terreno Precio # interferencias precio/m2 DESCRIPCIÓN Factibilidad Evaluación del Terreno Búsqueda de Inversionistas INICIO PLANIFICACIÓN Lograr el mejor perfil urbanístico con el menor costo m2 /piso Lograr el equilibrio entre lo que se puede realizar y lo que el cliente desea. Optmización m2 vendibles m2 vendibles/piso Desarrollo diseño especialidades Tiempo Costo Tiempo/costo Elaboración Anteproyecto Elaboración proyecto Tiempo # revisiones /tiempo Detección de Interferencias interferencias # interferencias Colaboración concurrente (ICE) Tiempo Cronograma maestro y simulación 4D Tiempo Gestión de seguridad Incidencias Gestión de la producción Tiempo Costo Tiempo Costo Opciones de diseño que me permitan generar mayor área vendible Lograr los sistemas más óptimos y económicos en el menor tiempo posible Lograr la gestión y aprobación en el menor tiempo posible Identificar el mayor número de interferencias ayudará a tener un proyecto más compatibilizado. # Optimizar las reuniones de colaboración reuniones/tiempo # cronogramas/ Lograr una adecuada gestión de logística tiempo # incidencias Lograr el menor número de incidencias # partidas/tiempo Lograr mayor número de partidas construidas en el menor tiempo posible (Tiempo real/ tiempo planificado)/(u producidas/u planificadas) Diferenciales entre el tiempo y el costo Tiempos menores de entrega en la documentación requerida Documentación detallada Tiempo # entregas/ tiempo Esquemas y planos de trabajos Sectorizaciones Visualizaciónes 3D Tiempo Alcance # entregas/ tiempo Lograr el mayor alcance posible en el menor tiempo Gestión y centralización de la informacíón Tiempo CONTROL Control de calidad de instalaciones CIERRE # pisos/ área Desarrollo diseño Arquitectónico Gestión de la productividad EJECUCIÓN Rentabilidad Parámetros urbanísticos Diseño alternativas conceptuales Costo Desarrollo de diseño Necesidades del cliente Partida de diseño Análisis estudio de suelos y estudio de títulos Análisis m2 en la mejor ubicación Alcanzar la mayor rentabilidad con la menor rentabilidad /m2 inversión. % información Alcance Número incidencias # incidencias calidad % eficiencia Facility Managament Plan de mantenimiento Análisis de sistemas y rendimientos Gestión de activos inmobiliarios Calidad información Gestión de espacios Tiempo Plan de contigencia Planos As Built Recorridos virtuales Pos venta Lograr contar con la mayor información en el menor tiempo posible Lograr tener menor incedencias y mayor calidad entregables/tiemp Lograr contar la mayor calidad en los entregables y o activos en el menor tiempo posible. Nota: Elaboración propia pág. 4755 Figura 2. Cuadro resumen CUADRO RESUMEN DE LAS INTERACCIONES DE LOS COMPONENTES DEL MODELO DE GESTIÓN PROPUESTO PROCESO APLICACIÓN COMPONENTE INICIO En este proceso se busca realizar el análisis de la factibilidad del proyecto, BIM con las condicionante planteadas por la VDC inmobiliaria ,para lo cual se plantea SESIONES ICE realizar modelos tridimensionales para MÉTRICAS el desarrollo de la factibilidad, cabida y FACTORES CONTROLABLES diseños preliminares. Información integrada Organización integrada Procesos integrados En este proceso se busca planificar y programar todos los componentes del BIM proyecto, para que logren un nivel de VDC detalle, orientados a la automatización, SESIONES ICE fabricación, montaje y control para la PLANIFICACIÓN MÉTRICAS ejecución, para ello se plantea el uso de FACTORES CONTROLABLES los modelos BIM para realizar las LEAN CONSTRUCTION simulaciones 4D, y prerparar IPD alternativas y secuencias eficientes para la construcción. Información integrada Organización integrada Procesos integrados INTERACCIÓN USOS Y APLICACIONES Factibilidad Evaluación del Terreno Búsqueda de Inversionistas Diseño alternativas conceptuales Desarrollo de diseño Partida de diseño Desarrollo diseño Arquitectónico Desarrollo diseño especialidades Elaboración Anteproyecto Elaboración proyecto Detección de Interferencias Colaboración concurrente (ICE) Cronograma maestro y simulación 4D Gestión de logística y seguridad EJECUCIÓN BIM En este proceso se busca a generar una VDC construcción virtual, interacción y SESIONES ICE coordinción entre los diferentes actores del proyecto; para asi generar flujos de MÉTRICAS FACTORES CONTROLABLES trabajo más eficientes que permitan realizar las partidas de la construcción LEAN CONSTRUCTION sin retrabajos y en el tiempo estipulado IPD Información integrada Organización integrada Procesos integrados CONTROL En este proceso se busca realizar un BIM seguimiento detallado y controlado de VDC las actividades en la pre construcción y MÉTRICAS construcción y así identificar de forma FACTORES CONTROLABLES oportuna los problemas en potencia, VDC reportarlos y particiapar en la gestión SESIONES ICE de sus soluciones. Información integrada Organización integrada Procesos integrados Gestión y centralización de la informacíón Control de calidad de instalaciones Información integrada Organización integrada Procesos integrados Facility Managament Plan de mantenimiento Análisis de sistemas y rendimientos Gestión de activos inmobiliarios Gestión de espacios Plan de contigencia Planos As Built Recorridos virtuales Pos venta CIERRE BIM El proceso se busca se busca obtener VDC un modelo BIM con la información SESIONES ICE actualizada y planos As buit necesarios MÉTRICAS para la gestión de proyectos en la FACTORES CONTROLABLES operación y mantenimiento. IPD Gestión de la producción Gestión de la productividad Documentación detallada Esquemas y planos de trabajos Sectorizaciones Visualizaciónes 3D Nota: Elaboración propia Se plantea un cuadro resumen de interacciones de los componentes del modelo de gestión propuesto; es asi que durante la etapa del desarrollo del diseño se utilizarán modelos tridimensionales paramétricos para la toma de decisiones rápidas y posterior validación; además de ello se verificará el cumplimiento de las normas establecidas en los proyectos de construcción y análisis técnicos respectivamente. pág. 4756 Figura 3. Centralización de información en modelos BIM Nota: Seystic (2020) El modelo de gestión propuesto optimiza el control de información y de cambios al centralizar la información de cronogramas, así como el metrado, simulación 4D y tareas diarias en un solo modelo tridimensional que puede consultado y revisado en cualquier momento. En la imagen inferior se muestra un ejemplo de la centralización de modelos. Figura 4. Cuadro de métricas componente BIM ANÁLISIS DEL REPORTE DE INTERFENCIAS Y/O CONSULTAS: CATEGORIA DE CONSULTA CANTIDAD PORCENTAJE POR CATEGORIA CATEGOR Í A DE CON SU LTA 70 Interferencia Incomp. Información Propuesta de mejora Falta de información 59 17 8 10 53% 15% 7% 60 Interferencia 50 40 Incomp. Información 30 Propuesta de mejora 20 Falta de información 9% Consulta Información 10 Consulta Información 17 15% Nota: Elaboración propia 0 1 ESTRUCTURAS 67 60.4% ARQUITECTURA 60 54.1% ELÉCTRICAS 40 36.0% TECNOLÓGICO 11 9.9% TOTAL DE CONSULTAS : 100% SANITARIAS En todo proceso es importante obtener métricas para evaluar el avance 34y alcance 30.6% de las herramientas 111 aplicadas en una determinada actividad. El modelo de gestión enfatiza en la necesidad de realizar GRAVEDAD DE LA GRAVEDAD DE LAS INCOMPATIBILIDADES CANTIDAD métricasCONSULTA de cada uno de los pasos%o acciones a tomar en la implementación para contar con una Muy grave 0 0% Grave 4 4% Moderada 57 51% Leve 28 25% 0% 4% perspectiva real y tomar acciones preventivas o correctivas, según amerite. 20% Muy grave Grave Moderada 25% Desistimada 22 TOTAL 111 ERROR 20% 51% Leve Desistimada pág. 4757 Figura 5. Planos coordinados AS Built Nota: Cosapi (2015) Figura 6. Modelos tridimensionales de diseño BIM Nota: BIM Projects Perú (2018) pág. 4758 CONCLUSIONES Primera Se concluye que, en el sector inmobiliario, aún existe una demanda elevada de viviendas y una oferta que aún no compensa ese déficit; siendo agravada por la pandemia del COVID-19. Es por ello, que las políticas de gobierno deben de establecer nuevos mecanismos para lograr incrementar la oferta y suplir el déficit agravado por la pandemia. A su vez estos mecanismos de modelos de gestión como el presentado en la tesis, representan una ventaja al propiciar la colaboración y reducción de tiempo y costo e incrementando la calidad Segunda El modelo de gestión realizado afirma que el modelo de gestión propuesto integra los modelos tradicionales y las nuevas metodologías colaborativas de gestión, con enfoques tecnológicos, adaptados a los cinco procesos tradicionales de la gestión de proyectos como: inicio, planificación , ejecución, control y cierre; reforzando sus fortalezas y supliendo sus falencias; enfocándose en sus áreas de oportunidades para potencializarlas y crear alianzas y sinergias con otras; y así lograr proyectos eficientes. REFERENCIAS BIBLIOGRÁFICAS Almonacid, K., Navarro, J., & Rodas, B. I. (2015). Propuesta de Metodología para la Implementación de la Tecnología BIM en la empresa Constructora e Inmobiliaria "IJ PROYECTA". (Tesis de licenciatura, Universidad Peruana de Ciencias Aplicadas) https://repositorioacademico.upc.edu.pe/bitstream/handle/10757/659383/Angulo_SJ.pdf?seq uence=3&isAllowed=y Autodesk (2023). ¿Qué incluye la AEC Collection de Autodesk? https://www.autodesk.es/collections/architecture-engineering-construction/includedsoftware. BIM Forum Chile- Grupo Técnico de trabajo de gestión de proyectos (2018). Gestión documental para BIM https://bimforum.cl/wp-content/uploads/2018/05/BIM-Forum-Chile-2017-11-AT-03- Gesti%C3%B3n-Documental.pdf Banco Central de Reserva del Perú. (2018). Notas de Estudio del BCRP nª39-7 de junio de 2018. pág. 4759 http://www.bcrp.gob.pe/publicaciones/notas-de-estudios.html Barco, D. (2018). Diario de un BIM Manager. Perú: Costos Educa. https://guia-bim.costosperu.com/ Bassam, A.O. (2017). Challenges and setbacks in the implementation of building information Modelling (BIM): A case study. WITPRESS. (169). 10.2495/BIM170021 Bravo, A., Mendoza, J. y Ramírez, H. (2020). Application of Integrated Project Delivery and Virtual Design and Construction to reduce the impacto f incompatibilities in the design stage in residential building. (Tesis de licenciatura). https://repositorioacademico.upc.edu.pe/bitstream/handle/10757/628154/Bravo_DA.pdf?seq uence=3&isAllowed=y BIM 360 (2023). Gestión de proyectos https://team.bim360.com/pricing/index.html. BIM SUMMIT 2018-Management & Technology. (2018). http://bimsummit.pe/avances-de-laadopcion-bim-en-el-peru/ Chingay, A. (2015). Diseño y Construcción Virtual (VDC) para superar problemas de ingeniería en la Fase de Construcción de Edificaciones de Oficinas. Lima-Perú: Universidad Nacional de Ingeniería. http://cybertesis.uni.edu.pe/handle/uni/4404 EDITECA. (2018). El BIM en Latinoamérica. https://editeca-com.cdn.ampproject.org/c/s/editeca.com/bim-en-latinoamerica/amp/ Espacio LEAN BIM-Construcción colaborativa. (2016). Episodio 9: Los pasos BIM. 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Introducción de Lean Construction. Madrid: Fundación Laboral de la Construcción. http://www.juanfelipepons.com/wp-content/uploads/2017/02/Introduccion-al-LeanConstruction-1.pdf Porras, H. (2014). Filosofía Lean Construction para la Gestión de Proyectos: Una revisión actual. Revistas.unilibre, 22. https://revistas.unilibre.edu.co/index.php/avances/article/view/298 Reinholdt, M., Lædre, O. y Hjelseth, E. (2019). Metrics in VDC Projects. 27th Annual Conference of the International Group for Lean Construction (IGLC). pp 1129-1140. Recuperado de https://doi.org/10.24928/2019/0167 Suwal, S., Laukkanen, M., Javaja, P., Hakkinen, T y Kubicki, S. (2019). BIM and EnergyEfficiency training requirement for the construction industry. IOP Conference Series: Earth and Environmental Science (19), 297. https://doi.org/10.1088/1755-1315/297/1/012037 The American Institute of Architects (2023). The American Institute of Architects- AIA. https://aiasf.org/ TYCH-Ingeniería & Construcción (s). 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https://openalex.org/W1996243786	https://bg.copernicus.org/articles/12/2327/2015/bg-12-2327-2015.pdf	English	null	Predicting the denitrification capacity of sandy aquifers from in situ measurements using push–pull &lt;sup&gt;15&lt;/sup&gt;N tracer tests	Biogeosciences	2,015	cc-by	17,515	Predicting the denitriﬁcation capacity of sandy aquifers from in situ measurements using push–pull 15N tracer tests W. Eschenbach1, R. Well1, and W. Walther,* 1Johann Heinrich von Thünen-Institut, Federal Research Institute for Rural Areas, Forestry and Fisheries, Institute of Climate-Smart Agriculture, Bundesallee 50, 38116 Braunschweig, Germany formerly at: Institute for Groundwater Management, Dresden University of Technology, 01062 Dresden, Germany retired Correspondence to: W. Eschenbach (w.eschenbach@gmx.de) Correspondence to: W. Eschenbach (w.eschenbach@gmx.de) Received: 21 October 2014 – Published in Biogeosciences Discuss.: 2 December 2014 Revised: 5 March 2015 – Accepted: 10 March 2015 – Published: 17 April 2015 Received: 21 October 2014 – Published in Biogeosciences Discuss.: 2 December 2014 Revised: 5 March 2015 – Accepted: 10 March 2015 – Published: 17 April 2015 Abstract. Knowledge about the spatial variability of in situ denitriﬁcation rates (Dr(in situ)) and their relation to the denitriﬁcation capacity in nitrate-contaminated aquifers is crucial to predict the development of groundwater quality. Therefore, 28 push–pull 15N tracer tests for the measurement of in situ denitriﬁcation rates were conducted in two sandy Pleistocene aquifers in northern Germany. set. However, the predictability of Dcum(365) and SRC from Dr(in situ) data clearly increased for aquifer samples from the zone of NO− 3 -bearing groundwater. 3 In the NO− 3 -free aquifer zone, a lag phase of denitriﬁca- tion after NO− 3 injections was observed, which confounded the relationship between reactive compounds and in situ den- itriﬁcation activity. This ﬁnding was attributed to adaptation processes in the microbial community after NO− 3 injections. It was also demonstrated that the microbial community in the NO− 3 -free zone just below the NO− 3 -bearing zone can be adapted to denitriﬁcation by NO− 3 injections into wells for an extended period. In situ denitriﬁcation rates were 30 to 65 times higher after pre-conditioning with NO− 3 . Results from this study suggest that such pre-conditioning is crucial for the measurement of Dr(in situ) in deeper aquifer material from the NO− 3 -free groundwater zone and thus for the prediction of Dcum(365) and SRC from Dr(in situ). The 15N analysis of denitriﬁcation-derived 15N-labelled N2 and N2O dissolved in water samples collected during the push–pull 15N tracer tests was performed using isotope ra- tio mass spectrometry (IRMS) in the lab and additionally for some tracer tests online in the ﬁeld with a quadrupole mem- brane inlet mass spectrometer (MIMS) in order to test the feasibility of on-site real-time 15N analysis. Predicting the denitriﬁcation capacity of sandy aquifers from in situ measurements using push–pull 15N tracer tests Aquifer material from the same locations and depths as the push–pull injection points was incubated, and the initial and cumulative denitri- ﬁcation after 1 year of incubation (Dcum(365)) as well as the stock of reduced compounds (SRC) was compared with in situ measurements of denitriﬁcation. This was done to de- rive transfer functions suitable to predict Dcum(365) and SRC from Dr(in situ). Dr(in situ) ranged from 0 to 51.5 µg N kg−1 d−1. Deni- triﬁcation rates derived from on-site isotope analysis using MIMS satisfactorily coincided with laboratory analysis by conventional IRMS, thus proving the feasibility of in situ analysis. Dr(in situ) was signiﬁcantly higher in the sulﬁdic zone of both aquifers compared to the zone of non-sulﬁdic aquifer material. Overall, regressions between the Dcum(365) and SRC of the tested aquifer material with Dr(in situ) ex- hibited only a modest linear correlation for the full data 1 Introduction Denitriﬁcation, the microbially mediated reduction of nitrate (NO− 3 ) and nitrite (NO− 2 ) to the nitrogen gasses nitric ox- ide (NO), nitrous oxide (N2O) and dinitrogen (N2), is im- portant to water quality and chemistry at landscape, regional and global scales (Groffman et al., 2006). NO− 3 is quanti- tatively the most abundant reactive nitrogen (Nr1) species. Diffuse NO− 3 emissions from the agricultural sector are the W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 2328 dominant source of Nr ﬂuxes to aquifers.1 Denitriﬁcation in aquifers, reviewed, for example, by Korom (1992), His- cock et al. (1991), Burgin and Hamilton (2007), and Rivett et al. (2008), ranges from 0 to 100 % of total NO− 3 input, with high spatial variability (Seitzinger et al., 2006). This leads to the question of how individual aquifers will respond to the anthropogenic NO− 3 pollution in groundwater. This problem depends not only on how rates of denitriﬁcation will respond to Nr loading (Seitzinger et al., 2006) but also on where and how long denitriﬁcation in aquifers can remediate NO− 3 pol- lution (Kölle et al., 1985). Continuous NO− 3 input via seep- age water leads to ongoing exhaustion of the reductive ca- pacity of aquifers. This can be a problem for keeping NO− 3 in drinking water below the limit of 50 mg L−1 (Drinking Water Directive 98/83/EC) and can also be problematic due to pos- sible eutrophication of surface waters (Vitousek et al., 1997). However NO− 3 can also mobilise deposits of uranium (U) in aquifers, which can be mobilised if NO− 3 reaches reduced aquifer zones (Senko et al., 2002; Istok et al., 2004). There- fore, knowledge about the denitriﬁcation capacity of aquifers is needed to predict the possible development of groundwater quality. microbial community is adapted to denitriﬁcation (Eschen- bach and Well, 2013). In situ denitriﬁcation rates can be measured using single- well push–pull tests where a test solution containing solutes of interest is rapidly injected into a well (push phase) and process information is obtained from analysing the mixture of groundwater and test solution collected during the subse- quent pull phase. These tests, perhaps ﬁrst used for in situ measurement of denitriﬁcation rates by Trudell et al. (1986), have proven to be a relatively low-cost technique to ob- tain quantitative information about several aquifer properties. This method has been applied in a variety of studies to derive in situ denitriﬁcation rates indirectly by the measurement of NO− 3 depletion during push–pull tests (Trudell et al., 1986; Istok et al., 1997, 2004; Schroth et al., 2001; McGuire et al., 2002; Harris et al., 2006). W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers In comparison, only a limited num- ber of studies have directly measured denitriﬁcation rates from the gaseous denitriﬁcation products (Sanchez-Perez et al., 2003; Kneeshaw et al., 2007; Well and Myrold, 2002, 1999; Addy et al., 2002, 2005; Well et al., 2003; Kellogg et al., 2005; Konrad, 2007). Aside from the study of Kon- rad (2007), these push–pull tests have only been conducted in the uppermost groundwater. The presented study continues previous research on deni- triﬁcation rates measured in two sandy Pleistocene aquifers in northern Germany (Fuhrberger Feld aquifer (FFA) and the aquifer of Großenkneten (GKA)). Frind et al. (1990) reported that, due to lithotrophic denitriﬁcation, NO− 3 has a half-life of 1 to 2 years in the deeper zone (below 5 to 10 m) of the well- investigated FFA. Weymann et al. (2010) reported very low denitriﬁcation rates with values as low as 4 µg N kg−1 d−1 in the uppermost groundwater, in the organotrophic denitriﬁ- cation zone of the same aquifer. In a recent study, Eschen- bach and Well (2013) measured median denitriﬁcation rates of 15.1 and 9.6 mg N kg−1 yr−1 during 1 year of anaerobic incubations of FFA and GKA aquifer samples, with signiﬁ- cantly higher denitriﬁcation rates in the deeper parts of both aquifers. This study showed that the cumulative denitriﬁca- tion after prolonged incubation of aquifer samples is corre- lated with the stock of reduced compounds (SRC). Similar results had been obtained earlier for other aquifers in north- ern Germany (Konrad, 2007). While we found close correla- tions between initial laboratory denitriﬁcation rates and the SRC in aquifer zones where NO− 3 is present in groundwa- ter, samples from the NO− 3 -free groundwater zone showed a time lag of denitriﬁcation of several weeks during incuba- tions (Eschenbach and Well, 2013), possibly due to the initial absence of denitrifying enzymes. These ﬁndings demonstrate that the SRC can be estimated from denitriﬁcation rates if the Well et al. (2005) showed that in situ denitriﬁcation rates measured with the push–pull 15N tracer method in the satu- rated zone of hydromorphic soils agreed relatively well with denitriﬁcation rates measured in parallel soil samples. 1The term reactive nitrogen is used in this work in accordance with Galloway et al. (2004) and includes all biologically or chem- ically active N compounds like reduced forms (e.g. NH3, NH+ 4 ), oxidized forms (e.g. NOx, HNO3, N2O, NO− 3 ) and organic com- pounds (e.g. urea, amines, proteins). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 2.1 Study sites In situ measurements of denitriﬁcation were conducted in the Fuhrberger Feld aquifer (FFA) and the Großenkneten aquifer (GKA). Both aquifers are located in drinking water catch- ment areas in the north of Germany. The FFA is situated about 30 km NE of the city of Hanover and the GKA about 30 km SW of the city of Bremen. Both aquifers consist of carbonate-free Quaternary sands and the deeper parts of the GKA additionally of carbonate-free marine sands (Pliocene). The thickness of the FFA and GKA is 20 to 40 and 60 to 100 m, respectively. Both aquifers are unconﬁned and con- tain unevenly distributed amounts of microbially available sulﬁdes and organic carbon. Intensive agricultural land use leads to considerable NO− 3 inputs to the groundwater of both aquifers (Böttcher et al., 1989; van Berk et al., 2005; Schuchert, 2007). Groundwater recharge is 250 mm yr−1 in the FFA (Wessolek et al., 1985) and 200 to 300 mm yr−1 in the GKA (Schuchert, 2007). Overall, the performance of previous push–pull studies suggests that this approach may be suitable to deliver in situ denitriﬁcation data that reﬂect the reduction capacity of the aquifer, i.e. it might be used to estimate SRC without the need for collecting aquifer material. Nevertheless, individual aquifer samples should always be analysed to verify these estimates repeatedly. p y To test whether 15N push–pull tests could be evaluated during the course of experiments directly in the ﬁeld, a mem- brane inlet mass spectrometer (MIMS) was used during ﬁve push–pull tests at two monitoring wells for direct ﬁeld mea- surements of 15N-labelled denitriﬁcation products (see Sup- plement). The main advantages of MIMS with respect to the conventional IRMS approach is that MIMS is low-priced compared to IRMS and results can be obtained during exper- iments directly in the ﬁeld. Sampling intervals can thus be adapted to get more precise rates. Moreover, the length of the pull phase can be limited to the duration of clearly increas- ing (N2+ N2O)den concentrations to save hours of labour. Finally, the relatively low cost and simple operation of the MIMS system are favourable to enable extensive application of the 15N push–pull approach to explore denitriﬁcation ca- pacities of aquifers. Evidence of an intense ongoing denitriﬁcation within the FFA is given by NO− 3 and redox gradients (Böttcher et al., 1992) as well as excess-N2 measurements (Weymann et al., 2008). 2 Materials and methods munity needed a certain time to adapt to the electron acceptor NO− 3 before denitriﬁcation could proceed at a rate equivalent to the availability of reduced compounds. So far, the effect of different ambient redox conditions, i.e. the presence or ab- sence of NO− 3 in groundwater, on the outcome of push–pull tests has been insufﬁciently considered. 2.1 Study sites The FFA can be divided into two hydro-geochemical zones: the zone of organotrophic denitriﬁcation near the groundwater surface with organic carbon (Corg) as an elec- tron donor and a deeper zone of predominantly lithotrophic denitriﬁcation with pyrite as the dominant electron donor (Böttcher et al., 1991, 1992). Detailed information about the FFA is given by Strebel et al. (1992), Frind et al. (1990) and von der Heide et al. (2008). The geological structure of the GKA is described in Howar (2005) and Wirth (1990). Ex- tended zones with oxidising and reducing conditions in the groundwater are evident in the GKA (van Berk et al., 2005) but their distribution within the aquifer is more complex than in the FFA and denitriﬁcation is known to occur in the zone of reduced groundwater (van Berk et al., 2005). Own excess-N2 measurements (Well et al., 2012) at monitoring wells prove intense denitriﬁcation within the GKA. But there are no stud- ies on the type of denitriﬁcation in this aquifer. p q This study is the second part of a combined approach (a) to quantify exhaustibility of the denitriﬁcation capacity in aquifers, (b) to investigate controlling factors and derive predictive models during incubation experiments, and (c) to check whether the cumulative denitriﬁcation measured af- ter 1 year of incubation (Dcum(365)) (Eschenbach and Well, 2013) can be derived from in situ denitriﬁcation rates mea- sured with push–pull tracer tests. Here a study on objective (c) is presented. The speciﬁc objectives of this study are (1) to measure in situ denitriﬁcation rates with push–pull 15N tracer tests at groundwater monitoring wells, (2) to develop regression models to predict Dcum(365) as well as the stock of reduced compounds from in situ denitriﬁcation rates, and (3) to test an approach to adapt the microbial community in NO− 3 -free aquifer zones to NO− 3 as a newly available elec- tron acceptor during experiments as a means of conditioning prior to subsequent push–pull 15N tracer tests. Additionally, as a fourth objective, the suitability of MIMS for online ﬁeld analysis during 15N tracer tests was tested (in the Supple- ment). W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers Kon- rad (2007) reported a close correlation between in situ den- itriﬁcation rates and the cumulative denitriﬁcation after at least 1 year of incubation based on a small number of com- parisons (ﬁve), so only a relatively small data set was used to derive transfer functions. Since denitriﬁcation is a microbially mediated reaction, the composition, activity and amount of microbes in aquifers should directly inﬂuence the measured denitriﬁcation rates during single-well push–pull tests. It is known that steep gra- dients in the composition of microbial communities occur in aquifers resulting from the distribution and availability of electron donors and acceptors in aquifers (Kölbelboelke et al., 1988; Griebler and Lueders, 2009; Santoro et al., 2006). Law et al. (2010) reported substantial changes in the micro- bial community composition after the initiation of denitriﬁ- cation and the transition from denitriﬁcation to Fe(III) reduc- tion within incubated aquifer material. Higher microbial ac- tivities after bio-stimulation of indigenous microorganisms by the injection of electron donors into aquifers were re- ported by Istok et al. (2004), Kim et al. (2005) and Kim et al. (2004). Compared with preceding push–pull tests at the same groundwater monitoring wells, the multiple injec- tion of electron donors increased the reduction rates of NO− 3 , pertechnetate (Tc(VII)) and U(VI) measured during subse- quent push–pull tests in a shallow unconﬁned silty–clayey aquifer (Istok et al., 2004). Trudell et al. (1986) found in- creasing denitriﬁcation rates during a 12-day push–pull test in NO− 3 -free groundwater suggesting that the microbial com- www.biogeosciences.net/12/2327/2015/ Biogeosciences, 12, 2327–2346, 2015 2329 W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers Push phase To prepare the tracer solution, 50 L of groundwater was extracted from multilevel wells (FFA and GKA) or 220 L at common groundwater monitoring wells (GKA) for each push–pull test (Fig. 1). The groundwater was pumped to a stainless steel storage container (type BO 220 L, SPEIDEL, Ofterdingen, Germany), which was equipped with a ﬂoating lid to avoid gas exchange with the atmosphere and thus main- tain the dissolved gas composition of the extracted ground- water. After extraction, a stock solution of deionised wa- ter (100 mL) with dissolved 15N-labelled potassium nitrate (KNO3 with 60 atom % 15N) and potassium bromide (KBr) was added to attain a concentration of 10 mg 15N-labelled NO− 3 -N L−1 and 10 mg Br−L−1, respectively. The mixture of the stock solution and the extracted groundwater is here- inafter referred to as tracer solution. The tracer solution was mixed for 1 h with a submersible pump (Gigant, Eijkelkamp, Giesbeek, Netherlands) within the stainless steel storage con- tainer. The extracted groundwater from the NO− 3 -bearing groundwater zone (NO− 3 -bearing zone) contained varying concentrations of NO− 3 (Table 2). Consequently, the NO− 3 in the tracer solution of these push–pull tests was a mixture of natural and 15N-enriched NO− 3 , and NO− 3 concentrations in these tracer solutions were > 10 mg NO− 3 -N L−1 (see discus- sion about inﬂuence of NO− 3 concentrations on denitriﬁca- tion rates in Sect. 4.2 and in Eschenbach and Well, 2013). For sampling multilevel wells, groundwater and tracer so- lution were extracted with a peristaltic pump (Masterﬂex COLE-PARMER, Vernon Hills, USA). A submersible pump (GRUNDFOS MP1, Bjerringbro, Denmark) was used for common groundwater monitoring wells. During sampling, an outﬂow tube with the extracted groundwater or tracer so- lution was placed at the bottom of 26 or 120 mL serum bot- tles (multilevel wells and common groundwater monitoring wells, respectively). After an overﬂow of at least 3 times the volume of these bottles, the tubing was removed and the bot- tles were immediately sealed airtight with grey butyl rubber septa (ALTMANN, Holzkirchen, Germany) and aluminium crimp caps. Four replications were collected per sampling. Groundwater was sampled from the injection depth prior to each push–pull test. During injection, the outﬂow of the stainless steel storage container was connected with Tygon® tubings to the selected depths of the multilevel wells. Push phase For common groundwater monitoring wells the submersible pump was connected with a pump riser pipe and an inﬂatable packer (packer set, UIT Umwelt- und Ingenieurtechnik GmbH, Dresden, Germany). The packer was installed within the groundwater monitor- ing well to prevent mixing of the injected tracer solution with the water column in the groundwater monitoring well W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 2330 Table 1. Overview of the conducted push–pull 15N tracer tests, the used wells and the depth range of the respective ﬁlter screens in both aquifers. Push–pull test with and without pre-conditioning were conducted at multilevel well B4. Table 1. Overview of the conducted push–pull 15N tracer tests, the used wells and the depth range of the respective ﬁlter screens in both aquifers. Push–pull test with and without pre-conditioning were conducted at multilevel well B4. Fuhrberg Großenkneten (multilevel wells) (conventional monitoring and multilevel wells) Monitoring well B1 B2 B4 B6 N10 Gro 326 Gro 327 S1 S2 CMT1 CMT2 ﬁlter screen, metres below ground surface Non-sulﬁdic zone 2.95–3.05 3 8.0–10.0 8.15–8.40 (NO− 3 -bearing zone) 4.15–4.25 6 22.65–22.90 Transition zone 7.95–8.05 5 (NO− 3 -bearing zone) 8.95–9.05 9.95–10.05 8 Sulﬁdic zone 6.95–7.05 13.95–14-05 6.95–7.05∗ 35.0–39.0 66.0–67.0 26.0–27.0 26.65–26.90 (NO− 3 -free zone) 7.95–8.05 8.75–8.85 29.15–29.40 9.85–9.95∗ 31.15–31.40 9.95–10.05∗ 33.35–33.60 ∗Push–pull tests with pre-conditioning. ∗Push–pull tests with pre-conditioning. ter monitoring wells (101 mm ID) with 1 to 4 m long ﬁl- ter screens and (2) multilevel wells (CMT multilevel sys- tem, Soilinst, Georgetown, Canada) consisting of PE pipes with three individual channels (13 mm ID) with 25 cm long ﬁlter screens at the end. Each channel ended at a different depth. To allow for a direct comparison with a previous labo- ratory incubation study (Eschenbach and Well, 2013), wells from the same locations and with ﬁlter screens at the same depth where the aquifer samples had been collected were se- lected in the FFA and GKA. In situ experiments were con- ducted principally as described in previous studies (Addy et al., 2002; Trudell et al., 1986; Well et al., 2003). www.biogeosciences.net/12/2327/2015/ 2.2.1 Well types and sampling procedure To quantify in situ denitriﬁcation rates (Dr(in situ)), a total of 28 single-well push–pull 15N tracer tests, afterwards re- ferred to as push–pull tests, were performed in the FFA and GKA (Table 1) by injecting 15N-labelled NO− 3 tracer solu- tion into groundwater monitoring wells. In the FFA, push– pull tests were conducted at multilevel wells consisting of PE tubings (4 mm ID) (Böttcher et al., 1985). Each of these tubes was connected to a ﬁlter element at the respective depth. In the GKA, two types were used: (1) conventional groundwa- www.biogeosciences.net/12/2327/2015/ Biogeosciences, 12, 2327–2346, 2015 2330 W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers Table 1. Overview of the conducted push–pull 15N tracer tests, the used wells and the depth range of the respective ﬁlter screens in both aquifers. Push–pull test with and without pre-conditioning were conducted at multilevel well B4. Fuhrberg Großenkneten (multilevel wells) (conventional monitoring and multilevel wells) Monitoring well B1 B2 B4 B6 N10 Gro 326 Gro 327 S1 S2 CMT1 CMT2 ﬁlter screen, metres below ground surface Non-sulﬁdic zone 2.95–3.05 3 8.0–10.0 8.15–8.40 (NO− 3 -bearing zone) 4.15–4.25 6 22.65–22.90 Transition zone 7.95–8.05 5 (NO− 3 -bearing zone) 8.95–9.05 9.95–10.05 8 Sulﬁdic zone 6.95–7.05 13.95–14-05 6.95–7.05∗ 35.0–39.0 66.0–67.0 26.0–27.0 26.65–26.90 (NO− 3 -free zone) 7.95–8.05 8.75–8.85* 29.15–29.40 9.85–9.95∗ 31.15–31.40 9.95–10.05∗ 33.35–33.60 ∗Push–pull tests with pre-conditioning. W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 2.2.2 Push–pull tests A single-well push–pull test consists of the injection of a tracer solution into a monitoring well (push phase) and the extraction of the mixture of test solution and groundwater from the same well (pull phase). Biogeosciences, 12, 2327–2346, 2015 www.biogeosciences.net/12/2327/2015/ W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 233 Table 2. Background conditions of the groundwater from the injection depths of the push–pull 15N tracer tests. Location Inj. deptha Aquifer zone O2 NO− 3 N2O SO2 4 − pH Redox Cond.b m mg L−1 mg N L−1 µg N L−1 mg S L−1 mV µS cm−1 FFA B1 6.95–7.05 sulﬁdic 0.67 < 0.25 n.d. 27.64 6.00 −171 473 FFA B1 7.95–8.05 sulﬁdic 0.76 < 0.25 n.d. 24.73 6.04 −175 440 FFA B2 2.95–3.05 non-sulﬁdic 3.66 41.47 1.59 15.07 4.66 273 563 FFA B2 4.15–4.25 non-sulﬁdic 0.96 27.59 68.31 36.94 4.83 209 564 FFA B2 7.95–8.05 transition zone 0.16 12.58 0.03 32.52 4.48 341 553 FFA B2 8.95–9.05 transition zone 0.13 7.09 0.05 38.41 4.65 367 488 FFA B2 9.95–10.05 transition zone 0.06 1.0 n.d. 43.30 4.75 374 458 FFA B2 13.95–14.05 sulﬁdic 0.40 0.63 n.d. 42.51 6.75 117 453 FFA B4 7.95–8.05 sulﬁdic 0.22 < 0.25 1.14 42.30 5.28 −38 432 FFA B4 8.95–9.05 sulﬁdic 0.12 < 0.25 0.70 51.19 5.43 − − FFA B6 2.95–3.05 non-sulﬁdic 9.51 6.10 0.02 13.95 5.70 365 255 FFA B6 5.95–6.05 non-sulﬁdic 1.28 19.55 10.66 22.45 5.18 349 441 FFA N10 4.95–5.05 transition zone 0.12 13.12 184.8 59.87 4.61 341 660 FFA N10 7.95–8.05 transition zone 0.16 0.4 1.03 52.03 5.60 3 463 GKA 326 8.0–10.0 non-sulﬁdic 6.30 3.06 0.12 4.67 4.10 374 105 GKA CMT2 8.15–8.40 non-sulﬁdic 6.10 3.14 0.12 5.06 4.40 387 100 GKA CMT2 22.65–22.90 non-sulﬁdic 5.70 3.98 0.56 12.09 5.10 276 163 GKA CMT2 26.65–26.90 sulﬁdic 0.10 < 0.25 0.01 18.57 5.40 30 221 GKA S2 26.0–27.0 sulﬁdic 0.30 < 0.25 n.d. 17.85 5.30 161 217 GKA CMT1 29.15–29.40 sulﬁdic 0.20 < 0.25 n.d. 18.16 5.50 −24 240 GKA CMT1 31.15–31.40 sulﬁdic 0.14 < 0.25 n.d. 17.91 5.20 134 195 GKA CMT1 33.35–33.60 sulﬁdic 0.20 < 0.25 n.d. 2.2.2 Push–pull tests 18.60 5.10 122 272 GKA 327 35.0–39.0 sulﬁdic 0.10 < 0.25 0.13 10.85 5.30 26 275 GKA S1 66.0–67.0 non-sulﬁdic 0.13 < 0.25 0.02 5.10 5.72 −54 103 FFA: Fuhrberger Feld aquifer; GKA: Großenkneten aquifer; a injection depth (the absolute depth can vary by a few centimetres); b conductivity; c n.d.: not detected. W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 2331 FFA: Fuhrberger Feld aquifer; GKA: Großenkneten aquifer; a injection depth (the absolute depth can vary by a few centimetres); b conductivity; c n.d.: not detected. and 30 to 60 L was extracted per sample from multilevel and groundwater monitoring wells, respectively. For com- mon groundwater monitoring wells the sampling volume dif- fered because of different lengths of ﬁlter screens and re- sulting different dead volumes. During extraction, ground- water temperature, dissolved oxygen, pH and electrical con- ductivity were measured with sensors (pH/Oxi 340i and pH/Cond 340i, WTW Wissenschaftlich-Technische Werk- stätten GmbH, Weilheim, Germany) installed in a ﬂow- through chamber. (Fig. 1). The packer was inﬂated with air to a pressure of 1 bar above the pressure of the overlying water column. The inﬂated packer and the pump riser pipe remained within the groundwater monitoring well during the entire tracer test. The pump riser pipe was connected with a PVC hose (13 mm ID) to the stainless steel container. For both types of moni- toring wells, the tracer solution was injected gravimetrically. Injections took 30–45 min for common wells, 45–80 min for the CMT multilevel system and 150–240 min for the multi- level wells in the FFA. Pull phase Laboratory experiments were performed to compare deni- triﬁcation rates measured during laboratory anaerobic incu- bation (Dr(365)) with in situ denitriﬁcation rates. The in- cubated aquifer material originated from the same location and depths as the ﬁlter screens of the push–pull test injection points. The aquifer material was sampled and incubated as described in detail in Eschenbach and Well (2013). The common groundwater monitoring wells in the GKA were constantly sampled at 12 h intervals. The multilevel wells in the FFA were sampled every 12 h during night and every 3 to 4 h during day to investigate more detailed tem- poral patterns. The multilevel wells were more suitable for this, due to their smaller dead volumes and lower extraction rates. The pull phases of the conducted tracer tests lasted a maximum of 72 h. The ﬁrst sampling was performed im- mediately after injection. Prior to each sampling, an amount of tracer solution sufﬁcient to replace the dead volume of the groundwater monitoring well was extracted. In total, 4 Brieﬂy, aquifer material from both aquifers was collected between 2 and 68 m below ground surface. The aquifer sam- ples were incubated in transfusion bottles, in three to four replications. 15N-labelled KNO3 solution was added and the www.biogeosciences.net/12/2327/2015/ 2.5.1 Isotope analysis of dissolved N2 and N2O Water samples sampled during push–pull tests were adjusted to 25 ◦C and a headspace was generated within the serum bottles by the injection of 15 or 40 mL of ambient air into the 26 and 115 mL serum bottles, respectively, replacing the same volume of sample solution. The replaced solution was directly transferred into 20 mL PE vials and frozen for later NO− 3 and SO2− 4 analysis. After headspace generation the serum bottles were agitated for 3 h on a horizontal shaker at a constant temperature of 25 ◦C to equilibrate the dis- solved gases with the headspace gas. Finally, 13 mL of the headspace gas of each serum bottle was extracted with a plas- tic syringe and then transferred to an evacuated 12 mL sam- pling vial (Exetainer® Labco, High Wycombe, UK), giving a slight positive pressure within the sampling vial. The sam- pled nitrogen gases in the 12 mL vials were then a mixture of N2 and N2O gained from atmosphere and denitriﬁcation, respectively. 2.5 Analytical techniques transfusion bottles were sealed airtight. To ensure anaerobic conditions during incubation, the headspaces of the transfu- sion bottles were evacuated and ﬂushed with pure N2. Af- terwards, the samples were incubated for 1 year in the dark at 10 ◦C, which is approximately the groundwater temper- ature in both aquifers. The transfusion bottles were shaken manually two times a week to mix sediment and batch solu- tion. The headspace and the supernatant batch solution in the transfusion bottles were sampled at days 1, 2, 7, 84, 168 and 365 of incubation. W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 2332 Figure 1. Schematic of push–pull 15N tracer tests at groundwater monitoring and multilevel wells. with a ﬁlter screen at 7 to 8 m depth below ground surface, which is located 30 m west of multilevel well B4, into an 800 L tank (IBC Tank Wassertank Container 800 L, Barrel Trading GmbH and Co. KG, Gaildorf, Germany) using a drill pump (Wolfcraft Bohrmaschinenpumpe 8 mm Schaft, Wolfcraft GmbH, Kempenich, Germany). The drill pump was connected with a PVC hose (13 mm ID) to the ground- water monitoring well and to the 800 L tank. The extracted groundwater was supplemented with KNO3 of natural 15N abundance to a concentration of 10 mg NO− 3 -N L−1. Approx- imately 40 L of this mixture was injected weekly into each of the depths 7, 8, 9 and 10 m below ground surface at multilevel well B4. The injection rate was approximately 1 L min−1. For 7 and 8 m depth the peristaltic pump was used for in- jection, and for 9 and 10 m depth the drill pump was used; both pumps were connected with Tygon® tubings to the se- lected depths of the multilevel well. The ﬁrst injection took place on 22 February 2011 and the last on 22 March 2011. In total, ﬁve pre-conditioning injections were conducted at the four depths. Subsequently, four push–pull tests were per- formed in the previously pre-conditioned injection depths as described above between 29 March and 1 April 2011. Figure 1. Schematic of push–pull 15N tracer tests at groundwater monitoring and multilevel wells. www.biogeosciences.net/12/2327/2015/ www.biogeosciences.net/12/2327/2015/ Biogeosciences, 12, 2327–2346, 2015 W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers derived from the measured 29 / 28 molecular ion mass ra- tio. We analysed replicate samples; one was equilibrated by electrodeless discharge and the other untreated (Well et al., 1998). This allowed for calculation of (N2+ N2O)den as well as the 15N abundance in NO− 3 undergoing denitriﬁca- tion. N2O was measured using a gas chromatograph (Fisons GC 8000, Milan, Italy) equipped with a split injector and an electron capture detector and a HP-Plot Q column (50 m length × 0.32 mm ID; Agilent Technologies, Santa Clara, USA) kept at 38 ◦C. Gas analysis was completed within 2 weeks after the respective push–pull tests. The concentra- tions of denitriﬁcation-derived 15N-labelled N2 and N2O in the gas samples were calculated as described by Well and Myrold (1999) and Well et al. (2003), respectively. The con- centration of N2O in the added atmospheric air was taken into account when calculating denitriﬁcation-derived N2O in the sample. The measured molar concentrations of N2 and N2O in the headspace samples were converted into dis- solved gas concentrations using gas solubilities given by Weiss (1970) and Weiss and Price (1980) and taking into account the temperature, headspace pressure and the liquid- to-headspace volume ratio during equilibration of dissolved gases with the headspace gases in the serum bottles. The concentrations of (N2+ N2O)den measured during the push–pull tests were corrected for dilution caused by disper- sion, diffusion and the tortuosity of the pores. To do this the dilution factor (Fdil(ti)) (Eq. 1) was derived from the con- centration changes of the conservative tracer Br−during the push–pull tests as proposed by Sanches-Perez et al. (2003): Fdil(ti) = [Br−]t0 [Br−]ti , (1) Fdil(ti) = [Br−]t0 [Br−]ti , (1) where Br− t0 and Br− ti are the Br−concentrations of the in- jected tracer solution and the sampled tracer solution at sam- pling time ti, respectively. The encountered dilution factors ranged from 1 to 20 and were below 5 in 18 push–pull tests. Only during four push–pull tests were the dilution factors be- tween 5 and 10, and only during two they were in the range of 10 to 20. The conventional wells (GKA) showed on average higher dilution factors compared with the CMT multilevel system and the multilevel wells in the FFA. Dilution factors were near 1 for most of the push–pull tests in the FFA, i.e. the injected tracer solution interfered little with the surrounding ambient groundwater. 2.6 Calculation of denitriﬁcation rates Measured concentrations of (N2+ N2O)den were converted from (µg N L−1) to (µg N kg−1) under the following assump- tions: (1) the average density of the solid aquifer material is 2.65 g cm−3 and (2) the effective porosity of the aquifer ma- terial was estimated to be 0.3 from literature values for sed- iments of similar grain size distribution (Kollmann, 1986), with a range of uncertainty of 0.2 to 0.4, respectively. 2.5.2 Analysis of NO− 3 , SO2− 4 and Br− NO− 3 concentrations in the water samples were determined photometrically with a continuous ﬂow analyser (Skalar, Erkelenz, Germany). SO2− 4 concentrations were analysed us- ing potentiometric back-titration of excess Ba2+ ions remain- ing in the solution after addition of a deﬁned amount of BaCl2 in excess to SO2− 4 . SO2− 4 precipitated as BaSO4. The original SO2− 4 concentration was then analysed by potentio- metric back-titration of the excess Ba2+ ions remaining in the solution using EDTA as a titrant. Possible interfering metal cations were removed from the samples prior to this analy- sis by cation exchange. Bromide (Br−) was analysed with an inductively coupled plasma–atomic emission spectrome- ter (ICP-AES; Spectro Analytical Instruments, Kleve, Ger- many) after stabilising the aliquot of the analysed water sam- ples with 10 % HNO3. 2.4 Pre-conditioning of wells in the NO− 3 -free zone of the FFA To stimulate denitriﬁcation in the NO− 3 -free zone with sus- pected lack of active denitriﬁers (Eschenbach and Well, 2013), groundwater monitoring wells were amended by re- peated injections of groundwater with added NO− 3 of nat- ural 15N abundance. Injections were designed to maintain elevated NO− 3 levels in the vicinity of the ﬁlter screens during a period of several weeks. This was done to test whether in situ denitriﬁcation rates measured in these wells after pre-conditioning would reﬂect the average denitriﬁca- tion rates measured during 1 year of incubation of corre- sponding aquifer samples (Eschenbach and Well, 2013). The 15N analysis of gas samples was performed via iso- tope ratio mass spectrometry (IRMS) at the Centre for Sta- ble Isotope Research and Analysis in Göttingen, Germany, using a Delta V advantage isotope ratio mass spectrom- eter (Thermo Scientiﬁc, Bremen, Germany) following the method described in Well et al. (2003). Analysis included reduction of N2O to N2 prior to the mass spectrometer en- trance. The sum of N2 and N2O isotopologues was thus detected as N2 in the mass spectrometer. In the following, the sum of denitriﬁcation-derived N2 and N2O is referred to as (N2+ N2O)den. The 15N abundance of (N2+ N2O) was Pre-conditioning was performed at four depths in the NO− 3 -free groundwater zone at multilevel well B4 in the FFA, from which two had been previously tested without pre- conditioning. Therefore 800 L of NO− 3 -free reduced ground- water was extracted from a groundwater monitoring well, www.biogeosciences.net/12/2327/2015/ Biogeosciences, 12, 2327–2346, 2015 2333 W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers The corrected concentrations of (N2+ N2O)den are then obtained by multiplying the uncorrected concentrations of (N2+ N2O)den at time ti with Fdil(ti). Denitriﬁcation rates were calculated from the tangent of dilution-corrected time courses of (N2+ N2O)den concentrations at time intervals with the steepest increase during the respective push–pull test (Sanchez-Perez et al., 2003; Istok et al., 2004). This method was used because we suppose that the section of the steepest increase in measured denitriﬁcation products during a push– pull test is the best approximation of the maximal denitriﬁ- cation rate possible in the aquifer at the very location of the respective push–pull test. The rationale behind this is that we suppose the maximal possible denitriﬁcation rate mea- surable at a certain point in an aquifer is dependent on the amount of reduced compounds in the aquifer material capa- ble of supporting denitriﬁcation. Also, the measured deni- triﬁcation rate during a push–pull test is dependent on the state of the microbial community in the aquifer material at the location of the conducted push–pull test. For example, if in situ denitriﬁcation rates are measured in the zone of NO− 3 -free groundwater, microbes might need time to express the appropriate enzymes to start to denitrify after injection of the NO− 3 -containing tracer solution. Since it is unknown how long this adaption time might be, the highest measurable denitriﬁcation rate during a push–pull test should give an ap- proximation for the maximal possible denitriﬁcation rate at the very point of the push–pull test (see also Sects. 4.1.2 and 4.2). In addition to the standard IRMS analysis of (N2+ N2O)den, 15N-labelled denitriﬁcation products were measured with a MIMS during ﬁve push–pull tracer tests directly in the ﬁeld (see the Supplement). 2.5.2 Analysis of NO− 3 , SO2− 4 and Br− 2.7 Detection limit and precision of (N2+ N2O)den measurements The detection limit of 15N analysis was calculated as the min- imum amount of 15N-labelled (N2+ N2O)den mixed with the (2) (2) rsa −rst ≥3 × sdrst where rsa and rst are the 29N2 / 28N2 ratios in sample and standard, respectively, and sdrst is the standard deviation of repeated rst measurements. The rst values were analysed with IRMS by measuring repeated air samples. Under the ex- perimental conditions, the detection limit for the amount of (N2+ N2O)den was 5 and 1 µg N L−1 for samples in 26 and 115 mL serum bottles, respectively, depending on the dif- ferent ratio of liquid sample to headspace in the respective serum bottles. 2.8 Statistical analysis and modelling Statistical analysis and regression modelling was conducted with WinSTAT for MS Excel version 2000.1 (R. Fitch Soft- ware, Bad Krozingen, Germany). Experimental data (x) were converted into Box–Cox-transformed data (f B–C(x)) ac- cording to Eq. (3) using different lambda coefﬁcients (λ) to achieve a normal-like distribution of experimental data within the different data sets. The aquifer samples were collected from drilled material obtained during construction of groundwater monitoring and multilevel wells in the FFA and GKA. The analysed aquifer samples originated from depth intervals of approximately 1 m above to 1 m below ﬁlter screens or ﬁlter elements of respective groundwater monitoring or multilevel wells used for push–pull tests (Table 1). f B−C(x) = (xλ −1) λ (3) (3) 3 Results Box–Cox transformations were performed with the statis- tic software STATISTICA 8 (StatSoft, Tulsa, USA). Simple linear regression analysis was conducted to evaluate quan- titative relations between in situ denitriﬁcation rates (Dr(in situ)) and various sediment parameters of corresponding aquifer material measured in the laboratory (Eschenbach and Well, 2013). Normal distribution of the measured parame- ters within the different data sets and the residuals of linear regressions were tested with the Kolmogorov–Smirnov test; normal distribution was assumed at the P > 0.05 level, with the null hypothesis that the tested parameter was normally distributed. The uniform distribution of residuals of regres- sions was checked with scatter plots of residuals vs. inde- pendent variables of the respective regression analysis. This was done to ensure homoscedasticity during regression anal- ysis, i.e. to ensure that the least-squares method yielded best linear estimators for the modelled parameter. To use the re- gression functions given in the result section with own data, the experimental values have to be transformed according to 2.9 Model sediment properties using regression functions with Dr(in situ) In situ denitriﬁcation rates (Dr(in situ)) measured during push–pull tests were used to model parameters of the investi- gated aquifer samples measured in the laboratory. These pa- rameters were (1) the cumulative denitriﬁcation after 1 year of incubation (Dcum(365)); (2) the SRC; and (3) several sed- iment parameters like water-soluble organic carbon (Chws), the fraction of KMnO4 labile organic carbon (Cl), total sulfur (total S) and total organic carbon (Corg). Dcum(365) is the cu- mulative amount of denitriﬁcation products per kilogram dry weight of incubated aquifer material at the end of 1 year of anaerobic incubation (mg N kg−1). The SRC is the amount of sulﬁdes and Corg converted into N equivalents (mg N kg−1) according to their potential ability to reduce NO− 3 to N2 (Es- chenbach and Well, 2013). These sediment parameters and denitriﬁcation rates were analysed during a laboratory incu- bation study with aquifer samples from the FFA and GKA (Eschenbach and Well, 2013). The mean coefﬁcient of variation (CV) of concentration measurements of (N2+ N2O)den (µg N L−1) in three repli- cates per sampling event during all push–pull tests was 0.18. The conversion of concentration data from (µg N L−1) to (µg N kg−1) increased the mean CV signiﬁcantly to 0.49. (The mean CV after conversion to (µg N kg−1) was calcu- lated from the three concentrations resulting from the range of effective porosity values (in the Supplement).) www.biogeosciences.net/12/2327/2015/ Biogeosciences, 12, 2327–2346, 2015 2334 W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers given background of headspace N2 of natural 15N abundance necessary to increase the measured 29N2 / 28N2 ratio to fulﬁl the following equation: Eq. (3) with the lambda coefﬁcients given in Table S2 in the Supplement. Eq. (3) with the lambda coefﬁcients given in Table S2 in the Supplement. Differences between partial data sets were considered sig- niﬁcant at the P < 0.05 level (Kruskal–Wallis test (kw) with the null hypothesis that both partial data sets belong to the same population). rsa −rst ≥3 × sdrst (2) Biogeosciences, 12, 2327–2346, 2015 3.2 In situ denitriﬁcation rates and time courses of denitriﬁcation products Dr(in situ) ranged from 0.0 to 51.5 µg N kg−1 d−1. Mean Dr(in situ) in the FFA (9.1 µg N kg−1 d−1) was almost 4 to 5 times higher than in the GKA, but differences between aquifers were not signiﬁcant (Figs. 2 and 3, Tables 3 and 4). The non-sulﬁdic zone of both aquifers exhibited the lowest mean Dr(in situ) (1.04 µg N kg−1 d−1) of all partial data sets (Table 4) and statistical signiﬁcant differences (kw: P < 0.05) occurred with the full and all partial data sets except Dr(in situ) measured in the GKA and in the NO− 3 -bearing zone of both aquifers. The other partial data sets exhibited no signif- icant differences between one another. Mean Dr(in situ) of the transition zone (9.32 µg N kg−1 d−1) was slightly higher than in the sulﬁdic zone of both aquifers. Dr(in situ) ranged from 0.0 to 51.5 µg N kg−1 d−1. Mean Dr(in situ) in the FFA (9.1 µg N kg−1 d−1) was almost 4 to 5 times higher than in the GKA, but differences between aquifers were not signiﬁcant (Figs. 2 and 3, Tables 3 and 4). The non-sulﬁdic zone of both aquifers exhibited the lowest mean Dr(in situ) (1.04 µg N kg−1 d−1) of all partial data sets (Table 4) and statistical signiﬁcant differences (kw: P < 0.05) occurred with the full and all partial data sets except Dr(in situ) measured in the GKA and in the NO− 3 -bearing zone of both aquifers. The other partial data sets exhibited no signif- icant differences between one another. Mean Dr(in situ) of the transition zone (9.32 µg N kg−1 d−1) was slightly higher than in the sulﬁdic zone of both aquifers. Except for the multilevel well B6 at 6 m depth, all push–pull injection points with O2 concentrations above 1 mg O2 L−1 in the groundwater exhibited Dr(in situ) be- low 0.75 µg N kg−1 d−1 (Tables 2 and 3) and aquifer material from this locations were assigned to non-sulﬁdic aquifer ma- terial during laboratory incubations (Eschenbach and Well, 2013). Dr(in situ) after pre-conditioning (well B4, FFA) was com- parable to or higher than Dr(365) with Dr(in situ)-to-Dr(365) ratios of 0.73 to 2.76 (Fig. 3 and Table 4). Dr(in situ) was 30 to 65 times higher compared to values obtained without pre- conditioning at the same wells (Fig. 5 and Table 3). 3.3.1 Comparison of Dr(in situ) and Dcum(365) Dr(in situ) was compared with mean denitriﬁcation rates dur- ing 365 days of laboratory incubation (Dr(365)) (Eschen- bach and Well, 2013) with aquifer material collected from the locations of the monitoring wells (see Sect. 2.3). Dr(365) was obtained by dividing cumulative (N2+ N2O)den produc- tion (Dcum(365)) by incubation time (365 d). Dr(in situ) was generally lower than Dr(365) (Fig. 3 and Table S1 Supple- ment). The means of the Dr(in situ)-to-Dr(365) ratio were calculated for the different partial data sets giving a range of 0.05 to 0.47, with the lowest and highest ratios for the data sets of GKA and transition zone push–pull tests, respectively (Table 4). In the transition zone, Dr(in situ)-to-Dr(365) ra- tios were signiﬁcantly higher compared to the other data sets (kw:p < 0.05). Statistically, Dr(in situ) of FFA aquifer ma- terial was signiﬁcantly more closely related to Dr(365) than Dr(in situ) measured in the GKA. The mean Dr(in situ)-to- Dr(365) ratio from the NO− 3 -bearing zone of both aquifers (0.23) was signiﬁcantly larger than in the NO− 3 -free zone of both aquifers (0.1) (Table 4). W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 2335 exponential increase in (N2+ N2O)den. All other push–pull tests in the NO− 3 -bearing zone exhibited almost linear trends. After pre-conditioning at the same depths of multilevel well B4 in the NO− 3 -free zone, the time course of denitriﬁcation products was drastically different compared to the initial tests with a much steeper and initially almost linear trend (Fig. 4). groundwater to be considered nitrate-bearing in this study. Sulﬁdic and non-sulﬁdic aquifer material was distinguished using the sulfate formation capacity (SFC, (mg S kg−1 yr−1)) of incubated aquifer material from the vicinity of the respec- tive ﬁlter screen of the used monitoring wells (Eschenbach and Well, 2013). Aquifer samples with a SFC > 1 mg SO2− 4 - S kg−1 yr−1 during incubation were assigned sulﬁdic, and push–pull tests conducted at wells with ﬁlter screens in this zone were accordingly assigned to the sulﬁdic zone. The transition zone was deﬁned as the zone within the aquifer where aquifer material still contains sulﬁdes and ground- water still contains NO− 3 . It follows that the NO− 3 -bearing groundwater zone comprises the zone of non-sulﬁdic aquifer material and the transition zone. 3.3 Relationship between Dr(in situ), Dcum(365) and aquifer parameters 3.3 Relationship between Dr(in situ), Dcum(365) and aquifer parameters 3.1 Grouping of push–pull test measuring points Push–pull tests were grouped into data subsets according to the redox state of groundwater and chemical properties of the aquifer material from the vicinity of the ﬁlter screens of groundwater monitoring wells used for the respective push– pull tests (aquifer material was collected during well con- struction) (see also Eschenbach and Well (2013) Sect. 3.1). These data subsets consist of data from wells with ﬁlter screens in the NO− 3 -bearing and NO− 3 -free groundwater zone (NO− 3 -bearing and NO− 3 -free zone, respectively) and wells in the zone of non-sulﬁdic, sulﬁdic and transition zone aquifer material (Tables 1 and 2). 0.4 mg NO− 3 -N L−1 was the lowest measured NO− 3 con- centration above the limit of detection of 0.2 mg NO− 3 -N L−1 in the various monitoring wells (Table 2). Therefore, 0.4 mg NO− 3 -N L−1 was the lowest NO− 3 concentration of www.biogeosciences.net/12/2327/2015/ Biogeosciences, 12, 2327–2346, 2015 3.3.2 Regression models to predict Dcum(365), SRC and denitriﬁcation-relevant aquifer parameters from Dr(in situ) Dr(in situ) measured after pre-conditioning of push– pull injection points at multiple well B4 (FFA) (67.83 to 152.70 µg N kg−1 d−1) were 30 to 65 times higher than Dr(in situ) measured 1 year before without pre-conditioning (2.76 and 2.28 µg N kg−1 d−1) (Table 3). Simple linear regression analysis was applied to obtain re- gression models for the prediction of Dcum(365) from Dr(in situ) for the full and partial data sets. The correlation coef- ﬁcient (R) and the average ratio of calculated Dcum(365) to measured Dcum(365) are used to evaluate the goodness of ﬁt of the regression models. Among the total 28 push–pull tests, 24 were conducted without pre-conditioning, from which 12 were located in the NO− 3 -bearing and 12 in the NO− 3 -free zone of both aquifers. Among the 12 push–pull tests in the NO− 3 -free zone all of the ﬁve FFA locations showed an exponential increase in (N2+ N2O)den during push–pull tests, whereas in the GKA this was only the case in two to three of the seven GKA loca- tions. In contrast to this, only 2 out of 12 push–pull tests in the NO− 3 -bearing zone of both aquifers exhibited exponential increases and these push–pull tests were located in the tran- sition zone of multilevel well B2. The two push–pull tests at multilevel well B4 (NO− 3 -free zone of the FFA) showed an The goodness of ﬁt of regression models to predict Dcum(365) by Dr(in situ) varied for the various data sub- sets from no ﬁt in the sulﬁdic zone to a good approxima- tion of Dcum(365) by Dr(in situ) in the NO− 3 -bearing zone (R = 0.04 and R = 0.84, respectively, Table 5). For the full data set, the quality of the ﬁt was modest (R = 0.62) result- ing in a wide range of deviations between calculated and measured Dcum(365) from −49.1 to 18.1 mg N kg−1 in the www.biogeosciences.net/12/2327/2015/ www.biogeosciences.net/12/2327/2015/ www.biogeosciences.net/12/2327/2015/ Biogeosciences, 12, 2327–2346, 2015 W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 2337 Table 3. In situ denitriﬁcation rates Dr(in situ) and minimum and maximum values of Dr(in situ). Minimum and maximum values correspond to the range of possible effective porosities (0.2 to 0.4). Dr(in situ) were calculated from a regression line through the (N2+ N2O)den concentrations at time intervals with the steepest increase in (N2+ N2O)den during the respective push–pull test. Tracer tests after pre- conditioning are marked with . Location Injection deptha Aquifer zone Dr(in situ) Dr(in situ) Dr(in situ) Rb max min m µg N kg−1 d−1 FFA B1 6.95–7.05 sulﬁdicd 17.59 27.361 10.261 0.94 FFA B1 7.95–8.05 sulﬁdicd 1.512 2.352 0.882 0.92 FFA B2 2.95–3.05 non-sulﬁdicc 0.120 0.186 0.070 0.14 FFA B2 4.15–4.25 non-sulﬁdicc 0.065 0.102 0.038 0.01 FFA B2 7.95–8.05 transition zonec 0.429 0.667 0.250 0.95 FFA B2 8.95–9.05 transition zonec 1.415 2.201 0.825 0.90 FFA B2 9.95–10.05 transition zonec 8.650 13.456 5.046 0.99 FFA B2 13.95–14.05 sulﬁdicd 51.47 80.078 30.029 0.82 FFA B4 7.95–8.05 sulﬁdicd 2.755 4.286 1.607 0.98 FFA B4 8.95–9.05 sulﬁdicd 2.278 3.544 1.329 0.86 FFA B6 2.95–3.05 non-sulﬁdicc 0.057 0.089 0.033 0.02 FFA B6 5.95–6.05 non-sulﬁdicc 4.998 7.774 2.915 0.96 FFA N10 4.95–5.05 transition zonec 12.89 20.052 7.520 0.95 FFA N10 7.95–8.05 transition zonec 23.19 36.074 13.528 0.99 FFA B4 6.95–7.05 sulﬁdicd 152.6 237.527 89.073 0.94 FFA B4* 7.95–8.05 sulﬁdicd 67.83 105.514 39.568 0.99 FFA B4* 8.95–9.05 sulﬁdicd 145.5 226.481 84.930 0.98 FFA B4* 9.95–10.05 sulﬁdicd 150.7 234.530 87.949 1.00 GKA 326 8.0–10.0 non-sulﬁdicb 0.747 1.162 0.436 0.96 GKA CMT2 8.15–8.40 non-sulﬁdicb 0.051 0.079 0.030 0.02 GKA CMT2 22.65–22.90 non-sulﬁdicb 0.009 0.013 0.005 0.00 GKA CMT2 26.65–26.90 sulﬁdicd 1.233 1.918 0.719 0.70 GKA S2 26.0–27.0 sulﬁdicd 0.860 1.338 0.502 0.99 GKA CMT1 29.15–29.40 sulﬁdicd 4.427 6.886 2.582 0.78 GKA CMT1 31.15–31.40 sulﬁdicd 0.504 0.784 0.294 0.63 GKA CMT1 33.35–33.60 sulﬁdicd 2.002 3.114 1.168 0.77 GKA 327 35.0–39.0 sulﬁdicd 6.192 9.632 3.612 0.99 GKA S1 66.0–67.0 non-sulﬁdicd 2.271 3.533 1.325 1.00 FFA: Fuhrberger Feld aquifer; GKA: Großenkneten aquifer; a(the absolute depth can vary by a few cm); b correlation coefﬁcient of the regression line; c NO− 3 -bearing zone; d NO− 3 -free zone. FFA: Fuhrberger Feld aquifer; GKA: Großenkneten aquifer; a(the absolute depth can vary by a few cm); b correlation coefﬁcient of the regression line; c NO− 3 -bearing zone; d NO− 3 -free zone. W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 2336 Figure 2. Time courses of denitriﬁcation-derived (N2+ N2O)den and dissolved O2 during 15N push–pull tests in the FFA (a) and (c) and GKA (b) and (d). FFA: Fuhrberger Feld aquifer; GKA: Großenkneten aquifer; ns: non-sulﬁdic; s: sulﬁdic; tZ: transition zone aquifer material. Figure 2. Time courses of denitriﬁcation-derived (N2+ N2O)den and dissolved O2 during 15N push–pull tests in the FFA (a) and (c) and GKA (b) and (d). FFA: Fuhrberger Feld aquifer; GKA: Großenkneten aquifer; ns: non-sulﬁdic; s: sulﬁdic; tZ: transition zone aquifer material. Figure 3. Relation between in situ denitriﬁcation rates determined by 15N push–pull tracer tests and average denitriﬁcation rates during 1 year of incubation (Eschenbach and Well, 2013). FFA: Fuhrberger Feld aquifer; GKA: Großenkneten aquifer; ns: non-sulﬁdic; s: sulﬁdic; tZ: transition zone aquifer material. Figure 3. Relation between in situ denitriﬁcation rates determined by 15N push–pull tracer tests and average denitriﬁcation rates during 1 year of incubation (Eschenbach and Well, 2013). FFA: Fuhrberger Feld aquifer; GKA: Großenkneten aquifer; ns: non-sulﬁdic; s: sulﬁdic; tZ: transition zone aquifer material. different data subsets. Linear relationships between Dr(in situ) and Dcum(365) were better for GKA in comparison to FFA aquifer material. Aquifer material which was not yet in contact with NO− 3 -bearing groundwater (NO− 3 -free zone and most of sulﬁdic zone material) exhibited Dr(in situ) val- ues which were clearly less correlated with Dcum(365) than aquifer material which was already in contact with NO− 3 - bearing groundwater (non-sulﬁdic zone, transition zone and NO− 3 -bearing zone) (Table 5). 3 The goodness of the ﬁt of regression models to calcu- late the SRC from Dr(in situ) was on average slightly worse than the one of regression models to predict Dcum(365) from Dr(in situ). As for the prediction of Dcum(365), the best goodness of ﬁt of regression models was obtained from the Biogeosciences, 12, 2327–2346, 2015 www.biogeosciences.net/12/2327/2015/ W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers tween Dr(in situ) and SRC exhibited a modest goodness of ﬁt (R = 0.54 and R = 0.53, respectively) (Table 5). GKA data sets, the transition zone and the NO− 3 -bearing zone, with coefﬁcients of determination of R = 0.75, 0.77 and 0.50 (Table 5). Like Dcum(365), the prediction for SRC was also best for zones of both aquifers where the aquifer ma- terial had already been in contact with NO− 3 -bearing ground- water in situ prior to the push–pull tests. In contrast with other partial data sets, the data subset of Dr(in situ) mea- sured in sulﬁdic aquifer material exhibited a clearly better goodness of ﬁt between Dr(in situ) and SRC than between Dcum(365) and Dr(in situ), R = 0.41 and R = 0.04, respec- tively. Regression analysis between several denitriﬁcation- relevant parameters of aquifer material (Eschenbach and Well, 2013) and Dr(in situ) revealed that, for some partial data sets, the linear regressions between some of these pa- rameters and Dr(in situ) were even better than the regres- sion between Dr(in situ) and Dcum(365) (Table S3 Supple- ment in comparison to Table 5). For GKA aquifer material, Dcum(365) was in closest linear correlation with Dr(in situ). In contrast with this, for FFA aquifer material Dr(in situ) was more closely related to SO42− extr and Chws than to Dcum(365) or SRC. For data subsets grouped according to the sulfate formation capacity of the incubated aquifer material, several parameters had better or at least equal linear correlation to As already mentioned above, pre-conditioning of multi- level well B4 strongly increased the measured Dr(in situ). Here, regressions between Dr(in situ) and Dcum(365) and be- W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 2338 Table 4. Means, standard deviation and ranges of Dr(in situ) of the data sets. Statistical signiﬁcant differences (kw: P < 0.05) between Dr(in situ) values measured in the various data subsets occurred only between Dr(in situ) measured in the non-sulﬁdic zone and some other partial data sets Dr(in situ)a Dr(in situ) / Dr(365)b (µg kg−1 N d−1) non- Data set Nc means range sulﬁdicd Ne means range Whole data set 24 6.07 ± 11.36 0.00–51.48 s1 34 0.15 ± 0.20 0.00–0.60 FFA 14 9.10 ± 14.20 0.06–51.48 s1 16 0.26 ± 0.24 0.01–0.60 GKA 10 1.83 ± 2.02 0.00–6.19 ns 18 0.06 ± 0.06 0.00–0.20 Non-sulﬁdic zone 8 1.04 ± 1.78 0.00–5.00 − 11 0.05 ± 0.08 0.00–0.23 Sulﬁdic zone 14 8.59 ± 13.67 0.43–51.48 s2 23 0.20 ± 0.22 0.01–0.60 Transition zone 5 9.32 ± 9.32 0.43–23.19 s1 8 0.47 ± 0.14 0.25–0.60 NO− 3 -bearing zone 12 4.38 ± 7.24 0.00–23.19 ns 17 0.23 ± 0.24 0.00–0.60 NO− 3 -free zone 16 7.76 ± 14.53 0.50–51.48 s1 17 0.10 ± 0.10 0.01–0.37 B4 pre-conditioned 4 128.1 ± 43.4 67.8–152.7 − 4 1.87 ± 0.84 0.72–2.76 B4 un-conditioned 2 2.52 ± 0.34 2.28–2.76 − 2 0.04 ± 0.02 0.02–0.05 a All Dr(in situ) measurements; b only Dr(in situ) measurements with corresponding incubated aquifer samples; c number of Dr(in situ) measurements; d statistical differences between non-sulﬁdic and other data sets (s: signiﬁcant differences; ns: non-signiﬁcant differences; 1 differences signiﬁcant at the 0.05 probability level; 2 differences signiﬁcant at the 0.01 probability level; 3 differences signiﬁcant at the 0.001 probability level); e number of comparisons between Dr(in situ) and corresponding incubated aquifer samples. a All Dr(in situ) measurements; b only Dr(in situ) measurements with corresponding incubated aquifer samples; c number of Dr(in situ) measurements; d statistical differences between non-sulﬁdic and other data sets (s: signiﬁcant differences; ns: non-signiﬁcant differences; 1 differences signiﬁcant at the 0.05 probability level; 2 differences signiﬁcant at the 0.01 probability level; 3 differences signiﬁcant at the 0.001 probability level); e number of comparisons between Dr(in situ) and corresponding incubated aquifer samples. Figure 4. Time courses of (N2+ N2O)den during push–pull tests without pre-conditioning (a) (grey diamonds) and with pre-conditioning (b) (black diamonds) at multilevel well B4 in the FFA. The push–pull tests without pre-conditioning at B4 were conducted in April 2010. 4.1 Quantifying Dr(in situ) with push–pull tests 4.1 Quantifying Dr(in situ) with push–pull tests W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers One year later, in April 2011, the aquifer material of the respective depths was conditioned over 5 weeks with NO− 3 amended groundwater of natural 15N abundance prior to the 15N push–pull tests. Figure 4. Time courses of (N2+ N2O)den during push–pull tests without pre-conditioning (a) (grey diamonds) and with pre-conditioning (b) (black diamonds) at multilevel well B4 in the FFA. The push–pull tests without pre-conditioning at B4 were conducted in April 2010. One year later, in April 2011, the aquifer material of the respective depths was conditioned over 5 weeks with NO− 3 amended groundwater of natural 15N abundance prior to the 15N push–pull tests. Dr(in situ) than Dcum(365). These parameters were Corg and total S in the non-sulﬁdic zone, SO42− extr and total S in the sul- ﬁdic zone, Corg and total S in the transition zone, Corg and SO42− extr in the NO− 3 -bearing zone, and SO42− extr and Cl in the NO− 3 -free zone. www.biogeosciences.net/12/2327/2015/ www.biogeosciences.net/12/2327/2015/ Biogeosciences, 12, 2327–2346, 2015 4.1.1 Ranges of Dr(in situ) and comparison with previous studies To compare previous Dr(in situ) data with our measure- ments, all denitriﬁcation rates were converted to the dimen- sion µg N kg−1 d−1 assuming an effective pore space of 0.3 and an average density of dry aquifer solids of 2.65 g cm−3. Dr(in situ) values measured in the FFA and GKA (Table 3) Biogeosciences, 12, 2327–2346, 2015 www.biogeosciences.net/12/2327/2015/ W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 2339 W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 233 Table 5. Simple regressions between Dr(in situ) and Dcum(365) and SRC from anaerobic incubations with corresponding aquifer materia f B−C(X) = A + B × f B−C(Dr(in situ)). 4.1.1 Ranges of Dr(in situ) and comparison with previous studies For these data pairs the following equation applies: X = A + B × Dr(in situ). a Independent sediment parameter; b number of samples; c correlation coefﬁcient. Experimental data of pre-conditioned push–pull tracer tests were not Box–Cox-transformed before regression analysis because of the small n data pairs the following equation applies: X = A + B × Dr(in situ). a Independent sediment parameter; b number of samples; c correlation coe Figure 5. Dr(in situ) after 5 weeks of pre-conditioning of aquifer material (black diamonds) in comparison to Dr(in situ) without pre- conditioning. The small graph shows the difference between Dr(in situ) after pre-conditioning and unconditioned Dr(in situ) at multi- level well B4 in the FFA. are comparable with Dr(in situ) (2.3–27.1 µg N kg−1 d−1) measured by Konrad (2007) in two Pleistocene sandy aquifers in northern Germany (aquifers of Thülsfelde and Sulingen, about 40 km west and 30 km south of the city of Bremen, respectively). Also, Dr(in situ) reported by Addy et al. (2002, 2005) shows a similar range of denitriﬁcation rates, with 2.1–121.2 and 0.5–87.9 µg N kg−1 d−1, respec- tively. Those values were measured in two riparian sites and a site with marsh sediments in Rhode Island, USA. Somewhat larger spans of Dr(in situ) were reported by Well et al. (2003) for water-saturated mineral sub-soils from various locations in northern Germany and by Konrad (2007) for the sandy to silty aquifer of Wehnsen (about 30 km southeast of the FFA) with Dr(in situ) from 0 to 300 and 45 to 339 µg N kg−1 d−1, respectively. These larger spans also cover the full range of Dr(in situ) values measured at multilevel well B4 in the FFA after pre-conditioning (Table 3). Sanches-Perez (2003) mea- sured Dr(in situ) from 22.1 to 7646.4 µg N kg−1 d−1 with the acetylene inhibition method in two shallow sandy aquifers in France and Spain. Overall, there is a wide range of reported Dr(in situ) in aquifers. Figure 5. Dr(in situ) after 5 weeks of pre-conditioning of aquifer material (black diamonds) in comparison to Dr(in situ) without pre- conditioning. The small graph shows the difference between Dr(in situ) after pre-conditioning and unconditioned Dr(in situ) at multi- level well B4 in the FFA. Denitriﬁcation rates can also be derived from the anal- ysis of groundwater samples from monitoring-well tran- sects along hypothesised groundwater ﬂow paths. 4.1.1 Ranges of Dr(in situ) and comparison with previous studies There- fore, Tesoriero and Puckett (2011) selected 12 study sites with monitoring-well transects within the US. The study areas represented a wide range of sedimentary environ- 4.1.1 Ranges of Dr(in situ) and comparison with previous studies Calculated/measured Deviation (mg N kg−1 yr−1) Data set Xa Nb A B Rc mean range mean range Whole data set Dcum(365) 34 2.878 0.603 0.62 2.29 ± 4.19 0.16–22.96 −3.07 ± 14.67 −47.2–12.8 Whole data set SRC 34 6.123 0.152 0.40 1.51 ± 1.31 0.12–5.19 −671.2 ± 2091 −7734–1379 FFA Dcum(365) 16 2.640 0.578 0.52 2.83 ± 4.90 0.13–19.18 −3.08 ± 14.71 −49.1–7.0 FFA SRC 16 3.772 0.006 0.07 1.22 ± 0.82 0.11–2.92 −377.8 ± 1375 −5317–413.7 GKA Dcum(365) 18 3.046 0.818 0.82 1.34 ± 0.92 0.26–3.85 −2.25 ± 12.28 −30.8–5.5 GKA SRC 18 8.024 0.613 0.75 1.43 ± 1.23 0.178–4.47 −617.0 ± 2179 −5780–2390 Non-sulﬁdic Dcum(365) 11 1.050 0.156 0.40 2.25 ± 3.20 0.26–10.65 −0.10 ± 2.41 −5.2–1.8 Non-sulﬁdic SRC 11 8407 752.8 0.43 1.50 ± 0.84 0.46–3.19 31.54 ± 240.7 −553–272.6 Sulﬁdic Dcum(365) 23 4.185 −0.033 0.04 1.33 ± 0.90 0.30–4.19 −3.32 ± 15.13 −39.4–13.1 Sulﬁdic SRC 23 21.40 −1.372 0.41 0.30 ± 0.18 0.03–0.61 −1823 ± 2313 −8564–144 Transition zone Dcum(365) 8 1.109 0.581 0.53 1.03 ± 0.26 0.74–1.43 −0.36 ± 2.84 −4.5–3.3 Transition zone SRC 8 5.349 −0.602 0.77 1.05 ± 0.41 0.58–1.92 −50.11 ± 340.6 −518.7–561 NO− 3 -bearing Dcum(365) 17 2.132 0.454 0.84 2.21 ± 3.76 0.13–15.17 −0.67 ± 2.52 −6.3–2.7 NO− 3 -bearing SRC 17 193.3 16.32 0.55 1.36 ± 0.75 0.41–2.76 −19.35 ± 365.2 −929–462.6 NO− 3 -free Dcum(365) 17 7.774 2.036 0.36 1.47 ± 0.88 0.31–3.00 −1.69 ± 16.23 −38.7–18.1 NO− 3 -free SRC 17 77.61 8.421 0.21 1.78 ± 1.46 0.27–4.47 −485.4 ± 2494 −6077–2095 Pre-conditioned1 Dcum(365) 4 14.402 0.099 0.54 1.06 ± 0.35 0.62–1.47 0.12 ± 9.49.79 −12.95–9.41 Pre-conditioned1 SRC 4 319.5 4.895 0.53 1.12 ± 0.52 0.51–1.77 5.5 ± 462 −638.0–464 1 Experimental data of pre-conditioned push–pull tracer tests were not Box–Cox-transformed before regression analysis because of the small number of data pairs. For these data pairs the following equation applies: X = A + B × Dr(in situ). a Independent sediment parameter; b number of samples; c correlation coefﬁcient. Table 5. Simple regressions between Dr(in situ) and Dcum(365) and SRC from anaerobic incubations with corresponding aquifer material. f B−C(X) = A + B × f B−C(Dr(in situ)). ween Dr(in situ) and Dcum(365) and SRC from anaerobic incubations with corresponding aquifer material. r(in situ)). 1 Experimental data of pre-conditioned push–pull tracer tests were not Box–Cox-transformed before regression analysis because of the small number of data pairs. 4.1.2 Temporal and spatial variability of in situ denitriﬁcation rates In addition to possible systematic differences between differ- ent methods with respect to the derived denitriﬁcation rates, it has to be taken into account that Dr(in situ) can show a considerable temporal variability during push–pull tests it- self. This was evident during the 12-day-long pull phase of a push–pull test conducted by Trudell et al. (1986) in the O2 and NO− 3 -free groundwater zone of a shallow sandy aquifer in southwestern Ontario Canada, where Dr(in situ) increased from 30.3 to 504.6 µg N kg−1 d−1 (Trudell et al., 1986). Green et al. (2010) showed that groundwater mixing due to advection and mechanical dispersion can strongly inﬂuence the derived apparent denitriﬁcation rates along ﬂow paths in such a way that these transport processes tend “to cre- ate the appearance of lower reaction rates and fractionation parameters when measured at larger scales and longer ﬂow paths” (Green et al., 2010, p. 12). Green et al. (2010) showed that mixing effects increase with the mean travel distances of groundwater and they conclude that “effects of transport and scale should be considered when comparing reaction rates in different aquifer systems, or when comparing reaction rates in different parts of the same system”. µg g ( , ) In this study most of the push–pull tests in the NO− 3 -free zone showed an exponential increase in (N2+ N2O)den with time, i.e. increasing denitriﬁcation rates, which is compara- ble to the results of Trudell et al. (1986). Periods of an ex- ponential increase in dilution-corrected denitriﬁcation prod- ucts during tracer tests were also previously reported (Es- chenbach and Well, 2011; Konrad, 2007). In the study of Konrad (2007), 5 out of 13 push–pull tests showed an ex- ponential increase in dilution-corrected denitriﬁcation prod- ucts. Four of these ﬁve push–pull tests were located in the NO− 3 -free groundwater zone. Conversely, push–pull tests in the NO− 3 -free zone (consisting of the data subsets of non- sulﬁdic aquifer material and the transition zone) showed ap- proximately constant denitriﬁcation rates during the push– pull tests. The non-sulﬁdic aquifer material exhibited very low denitriﬁcation rates during the push–pull tests, presum- ably because the aquifer material was depleted in reduced compounds capable of supporting denitriﬁcation (Table S1 in the Supplement and Eschenbach and Well (2013) Sect. 4.2) and dissolved O2 in groundwater inhibited NO− 3 reduc- tion. W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers The O2 concentrations in the ambient groundwater at these push–pull locations were mostly clearly above 1 mg L−1, which is near the reported apparent threshold for the onset of denitriﬁcation in aquifers (Green et al., 2008, 2010; McMahon et al., 2004; Tesoriero and Puckett, 2011) (see Sect. 4.3). Mean Dr(in situ) values of data subsets of push–pull test at locations with low O2 con- centrations (transition zone and NO− 3 -free zone) (Table 2) were 9 and 8 µg N kg−1 d−1, respectively, and thus a factor of 10 higher than the high rates reported by Tesoriero and Puckett (2011). which is very short compared to ﬂow paths of hundreds of metres or several kilometres in the case of monitoring-well transects. (Additionally, the mixing of the injected tracer so- lution with ambient groundwater was taken into account by the addition of Br−as conservative tracer to the tracer solu- tion (see Sect. 2.6) to minimise the inﬂuence of mixing ef- fects.) The observed differences in denitriﬁcation rates mea- sured in this study with denitriﬁcation rates derived from monitoring-well transects (Tesoriero and Puckett, 2011) might thus be attributed to effects of transport along long ﬂow paths. We think that these effects should also be consid- ered when denitriﬁcation rates are compared that have been derived with different methods. www.biogeosciences.net/12/2327/2015/ Biogeosciences, 12, 2327–2346, 2015 W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 2340 ments and climatic conditions. Tesoriero and Puckett (2011) generalised the determined denitriﬁcation rates broadly into three categories: low rates (< 0.02 µg N kg−1 d−1), medium rates (0.02–0.06 µg N kg−1 d−1) and high rates (> 0.6 µg N kg−1 d−1). Low rates were found in areas with el- evated O2 concentrations in the groundwater, medium rates in the presence of low O2 concentrations and high denitri- ﬁcation rates when changes in the lithology resulted in a sharp increase in the supply of electron donors (Tesoriero and Puckett, 2011, p. 13). Overall, the denitriﬁcation rates obtained from theses monitoring-well transects are below the mean Dr(in situ) of the various data subsets in this study (Table 4). For example, the mean Dr(in situ) of non- sulﬁdic aquifer material was 1µg N kg−1 d−1 (Table 4) and thus even higher than the high denitriﬁcation rates reported by Tesoriero and Puckett (2011). The O2 concentrations in the ambient groundwater at these push–pull locations were mostly clearly above 1 mg L−1, which is near the reported apparent threshold for the onset of denitriﬁcation in aquifers (Green et al., 2008, 2010; McMahon et al., 2004; Tesoriero and Puckett, 2011) (see Sect. 4.3). Mean Dr(in situ) values of data subsets of push–pull test at locations with low O2 con- centrations (transition zone and NO− 3 -free zone) (Table 2) were 9 and 8 µg N kg−1 d−1, respectively, and thus a factor of 10 higher than the high rates reported by Tesoriero and Puckett (2011). ments and climatic conditions. Tesoriero and Puckett (2011) generalised the determined denitriﬁcation rates broadly into three categories: low rates (< 0.02 µg N kg−1 d−1), medium rates (0.02–0.06 µg N kg−1 d−1) and high rates (> 0.6 µg N kg−1 d−1). Low rates were found in areas with el- evated O2 concentrations in the groundwater, medium rates in the presence of low O2 concentrations and high denitri- ﬁcation rates when changes in the lithology resulted in a sharp increase in the supply of electron donors (Tesoriero and Puckett, 2011, p. 13). Overall, the denitriﬁcation rates obtained from theses monitoring-well transects are below the mean Dr(in situ) of the various data subsets in this study (Table 4). For example, the mean Dr(in situ) of non- sulﬁdic aquifer material was 1µg N kg−1 d−1 (Table 4) and thus even higher than the high denitriﬁcation rates reported by Tesoriero and Puckett (2011). 4.1.2 Temporal and spatial variability of in situ denitriﬁcation rates All in all the measured denitriﬁcation rates during 1 year of incubation (Dr(365)) were on average higher in compar- ison to denitriﬁcation rates derived with normal push pull tests (Dr(in situ)). This may have resulted from several fac- tors, including the stimulation of denitriﬁcation in the lab due to disturbance of aquifer material, establishment of strictly anaerobic conditions, and the adaptation of the microbial community over time. The ratio between Dr(in situ) and Dr(365) was highly variable within the data set. Interestingly, it was lowest in the non-sulﬁdic and NO− 3 -free zones of both aquifers (Table 4). In the case of non-sulﬁdic aquifer mate- rial, dissolved O2 (Table 2) might have inhibited NO− 3 re- duction. Dr(365) of non-sulﬁdic aquifer material measured during anaerobic incubation in the laboratory (Eschenbach and Well, 2013) can therefore be seen as a potential activ- ity which is only partly effective under in situ conditions due to a low reduction rate of dissolved O2 in groundwa- ter. This is also reﬂected by the low Dr(in situ)-to-Dr(365) ratio in the non-sulﬁdic wells (Table 4). The mean Dr(in situ)-to-Dr(365) ratio in the NO− 3 -bearing zone was twice as high compared to the NO− 3 -free zone (Table 4 and Fig. 3). This probably reﬂects the need for microbial adaptation to NO− 3 in the NO− 3 -free zone discussed in the previous sec- tion. Mean Dr(in situ) and the ratio of Dr(in situ) to Dr(365) of 0.47 were highest in the transition zone, showing that, in the transition zone, Dr(in situ) and Dr(365) were in closer agreement compared with other zones. During the push–pull tests in the transition zone, the ambient concentration of dis- solved O2 was always below 0.13 mg L−1 and NO− 3 was al- ways detectable in the ambient groundwater at the ﬁve in- jection points in the transition zone (Table 2). Denitriﬁca- tion was therefore presumably not inhibited by dissolved O2 and the microbial population had already adapted to NO− 3 as an available electron acceptor. Hence, denitrifying conditions during push–pull tests and during laboratory incubation were similar, resulting in closer agreement in denitriﬁcation rates. 4.1.2 Temporal and spatial variability of in situ denitriﬁcation rates Dissolved O2 concentrations in the ambient ground- water and therefore also in the injected test solutions were > 1 mg O2 L−1 at six out of eight injection points in the non- sulﬁdic zone of both aquifers (Table 2), which is near or above the apparent threshold for the onset of denitriﬁcation in aquifers (see Sect. 4.3 below), whereas O2 concentrations in the transition zone were far below this threshold. In rela- tion to the amount of reduced compounds of transition zone In contrast, Korom et al. (2005) reported a clearly higher zero-order denitriﬁcation rate of 35.6 µg N kg−1 d−1 mea- sured in an aquifer mesocosm; this rate is comparable with the highest Dr(in situ) measured in this study (Table 2). Ko- rom et al. (2012) argued that, in contrast with monitoring- well transects, such transport-dependent mixing processes would not inﬂuence denitriﬁcation rates measured by aquifer mesocosms, since advection and mechanical dispersion are negligible. The inﬂuence of advection and mechanical dis- persion on the measured apparent denitriﬁcation rates from push–pull tests should be higher compared to in situ meso- cosms. However, during push–pull tests, mixing processes by advection and mechanical dispersion should be signiﬁcantly lower in comparison to monitoring-well transects, since the ﬂow path of the injected tracer solution in the aquifer is in a decimetre or, at most, metre range during a push–pull test, Biogeosciences, 12, 2327–2346, 2015 Biogeosciences, 12, 2327–2346, 2015 www.biogeosciences.net/12/2327/2015/ W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 2341 laboratory denitriﬁcation rates compared to in situ denitriﬁ- cation rates measured with normal push-pull tests (Fig. 5). aquifer material (Table S1 in the Supplement and Fig. 2 in Eschenbach and Well, 2013), which was almost as low as the one of non-sulﬁdic aquifer material, the denitriﬁcation rates measured in situ were comparatively high (Table 3). Despite the clearly lower SRC content in situ denitriﬁcation rates in the transition zone were on average higher than in the NO− 3 -free zone (Table 3). We suspect that the clearly dif- ferent activity of denitriﬁcation in relation to the SRC in the transition zone compared to the NO− 3 -free zone is because the microbial community in the NO− 3 -free zone is not ready to denitrify since it needs time to adapt to NO− 3 as a possible electron acceptor. 4.1.2 Temporal and spatial variability of in situ denitriﬁcation rates Therefore, it is concluded that the exponential increase in denitriﬁcation products observed during push–pull tests in our study and previous studies can probably be attributed to growth and stimulation of denitriﬁers by the injection of NO− 3 into aquifer zones that have previously not been in con- tact with NO− 3 . Trudell et al. (1986) found an increase in den- itrifying bacteria species during the 12-day-long tracer test which was accompanied by a 17-fold increase in measured denitriﬁcation rates. Several other investigations have shown increasing microbial activity after bio-stimulation of aquifer sediments by the injection of electron donors into monitor- ing wells (Istok et al., 2004; Kim et al., 2004, 2005). Istok et al. (2004) reported that the viable biomass on solid samplers installed in monitoring wells more than doubled compared with samplers installed in monitoring wells without electron donor addition. To establish an active denitrifying microbial community in the strict anaerobic zone of an aquifer, we injected NO− 3 as the newly available electron acceptor in the NO− 3 -free zone at multilevel well B4 in the FFA. To our knowledge, this study is the ﬁrst to have used pre-conditioning of aquifer material prior to a push–pull 15N tracer test by the injection of only NO− 3 . Pre-conditioning at multilevel well B4 (see Sect. 2.4) resulted in a 30- to 65-fold increase in measured in situ den- itriﬁcation rates compared with push–pull tests without pre- conditioning at the same depths of multilevel well B4 (Ta- ble 3 and Fig. 5). It can be concluded that pre-conditioning in the NO− 3 -free zone of the FFA led to growth of the commu- nity of active denitriﬁers in the aquifer material in the vicinity of the respective injection points. The increase in Dr(in situ) due to pre-conditioning might be a combined effect from the increase in active denitriﬁers and a higher denitriﬁcation rate per microbial cell due to synthesis of enzymes for denitriﬁ- cation. Pre-conditioning does not only lead to higher denitri- ﬁcation rates; the time course of (N2+ N2O)den also did not show a period of a distinct exponential increase compared with prior measurements without pre-conditioning (Fig. 4). This might show that denitriﬁers in the tested aquifer material after pre-conditioning were ready to denitrify and that there was a stable denitrifying community (see also Sect. 4.2). Pre- conditioning also improved the comparability of in situ and www.biogeosciences.net/12/2327/2015/ 4.3 Predicting Dcum(365) and SRC of aquifer sediments from Dr(in situ) The main objective of this study is to predict the cumulative denitriﬁcation measured during 1 year of laboratory incuba- tion of aquifer samples (Dcum(365)) and the SRC from in situ denitriﬁcation rates (Dr(in situ)). In comparison to costly drilling of aquifer material and laboratory measurement of Dcum(365) and SRC, Dr(in situ) can be measured with rela- tively low-cost push–pull tests at existing groundwater moni- toring wells, which would thus allow spatial mapping of den- itriﬁcation activity within aquifers. There are only scarce data comparing the SRC or longer- term denitriﬁcation rates (e.g. Dr(365)) with Dr(in situ)). Well et al. (2003) showed for denitriﬁcation in the saturated zone of hydromorphic soils that laboratory-derived denitriﬁ- cation rates after 24 h of anaerobic incubation were in good agreement with in situ denitriﬁcation rates, but the study was limited to near-surface groundwater. Konrad (2007) tested this approach in deeper aquifer zones with a small data set of pairs of Dr(in situ) vs. Dcum(four push–pull 15N tracer tests and incubations of corresponding aquifer material) and found that both quantities were related (Spearman rank cor- relation coefﬁcients of R ≥0.8). Figure 6. Schematic time courses of denitriﬁcation during push– pull tests in the NO− 3 -free groundwater zone. (Dr: measured in situ denitriﬁcation rates; saRC: surface area of reduced compounds present in the investigated aquifer.) area of reduced compounds (saRC) present in the aquifer ma- terial. If the denitrifying community is adapted to NO− 3 and had colonised the saRC, denitriﬁcation rates should be rel- atively constant. Hence a zero-order reaction model should ﬁt the measured data during the relatively short duration of a push–pull test (Fig. 6, linear response phase). It is sus- pected that these conditions apply to the NO− 3 -bearing zone but not to the NO− 3 -free zone. After pre-conditioning at mul- tilevel well B4, (N2+ N2O)den was initially high and there was no subsequent exponential increase, while the opposite was the case during previous tests at the same well with- out pre-conditioning (Fig. 4). This probably reﬂects the more constant activity of denitriﬁers during the push–pull tests af- ter pre-conditioning (Fig. 6, linear response phase). 4.3 Predicting Dcum(365) and SRC of aquifer sediments from Dr(in situ) Similar adaptation effects have been reported previously, where bio- stimulation by injecting electron donors like ethanol, glu- cose, propane or fumarate resulted in constant activity, thus allowing the use of zero-order reaction models to derive re- duction rates during push–pull tests (Istok et al., 2004; Kim et al., 2004, 2005). This supports our interpretation that pre- conditioning leads to a kind of equilibrium between the den- itrifying community, the injected NO− 3 and the saRC present in the aquifer material, ultimately resulting in relatively con- stant reaction rates while NO− 3 is not limiting (Fig. 6, linear response phase). In our experiments, the latter condition was fulﬁlled, because NO− 3 concentrations during the pull phase were always clearly above 1.0 mg NO− 3 -N L−1, which is as- sumed to be the threshold of NO− 3 concentrations limiting denitriﬁcation rates reported by Wall et al. (2005). In this study, transfer functions were developed to pre- dict Dcum(365) from Dr(in situ) measurements with a larger data set in different redox zones typically present in aquifers. Moreover, pre-conditioning was evaluated through addition of NO− 3 to aquifer material and the subsequent measurement of in situ denitriﬁcation rates. Only a modest goodness of ﬁt (R = 0.62) was found using linear regression between Dr(in situ) and Dcum(365) for the full data set (Table 5). Without Box–Cox transformations of input data the correlation coefﬁcient was even lower (R = 0.1). This shows that it was necessary to transform the input data to approach normal distribution and homoscedasticity for regression analysis. Otherwise the ordinary least-squares method did not ﬁnd the best or efﬁcient linear estimators for regression coefﬁcients. Like in the previous laboratory study (Eschenbach and Well, 2013), grouping of Dr(in situ) measuring points by locality or according to hydro-geochemical zones increased the predictive power of Dr(in situ) with respect to the mea- sured Dcum(365) and SRC of aquifer material for some par- tial data sets. Altogether, Dr(in situ) was the best predictor for Dcum(365) and SRC of the partial data set of GKA aquifer material, with correlation coefﬁcients of 0.82 and 0.75, re- spectively. For the FFA the predictive power of Dr(in situ) for Dcum(365) and SRC was signiﬁcantly lower compared to the GKA (Table 5). W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 2342 Figure 6. Schematic time courses of denitriﬁcation during push– pull tests in the NO− 3 -free groundwater zone. (Dr: measured in situ denitriﬁcation rates; saRC: surface area of reduced compounds present in the investigated aquifer.) zone of NO− 3 -free groundwater is needed to allow for the SRC to be estimated from in situ denitriﬁcation rates. zone of NO− 3 -free groundwater is needed to allow for the SRC to be estimated from in situ denitriﬁcation rates. 4.3 Predicting Dcum(365) and SRC of aquifer sediments from Dr(in situ) 4.2 Interpretation of observed time courses of produced (N2+ N2O)den Figure 6 sums up our interpretation of the results from push– pull tests in the NO− 3 -free zone. Immediately after the injec- tion of the 15N tracer in the NO− 3 -free zone of both aquifers there seems to follow a time interval with little to no produc- tion of 15N-labelled (N2+ N2O)den (i.e. lag phase) (compare with Figs. 2 and 4). During this time, denitriﬁers might still have to synthesise enzymes for denitriﬁcation and are not yet ready to denitrify. After the lag phase follows a phase of exponential increase in (N2+ N2O)den during which the amount of active denitri- ﬁers and or their activity might adapt to the newly available electron acceptor NO− 3 . The growth of denitriﬁers might de- pend on the microbially available SRC, i.e. on the surface www.biogeosciences.net/12/2327/2015/ www.biogeosciences.net/12/2327/2015/ Biogeosciences, 12, 2327–2346, 2015 4.3 Predicting Dcum(365) and SRC of aquifer sediments from Dr(in situ) This ﬁnding mirrors results of laboratory incubations with FFA and GKA material reported by Eschen- bach and Well (2013) (Table 4 of the cited study), in which From the dynamics of microbial adaptation outlined above it follows that pre-conditioning prior to push–pull tests in the Biogeosciences, 12, 2327–2346, 2015 www.biogeosciences.net/12/2327/2015/ W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 2343 initial denitriﬁcation rates (Dr(7)) of GKA material were a better predictor of Dcum(365) than in the case of FFA ma- terial. In contrast with the GKA aquifer samples, the SRC of the FFA samples was not predictable by Dr(in situ). One reason might be a different microbial availability of organic carbon (Corg), which is one major constituent of SRC in both aquifers (Eschenbach and Well, 2013). The ratio of KMnO4 labile organic carbon (Cl) to Corg was almost twice as high in the GKA material compared to the FFA material (Eschen- bach and Well, 2013), suggesting that the proportion of Corg available for microbes is higher in the GKA aquifer material and that a signiﬁcant proportion of Corg is unavailable for denitriﬁcation in the FFA. ited content of reduced compounds. This might explain the poor ﬁt between calculated and measured values (Table 5) in the non-sulﬁdic zone. In the NO− 3 -free zone the groundwater was almost O2-free and, in comparison to the other zones, the aquifer material had a larger SRC (Table S1 in the Sup- plement). Nonetheless, the correlation coefﬁcients between Dr(in situ) and Dcum(365) and Dr(in situ) and the SRC were very low and the developed regression functions underesti- mated Dcum(365) and SRC of deeper aquifer samples with high values of Dcum(365) and SRC to a large extent (Table 5). We suppose the reason for this is the lack of adaptation of the microbial community to NO− 3 , as already discussed above. 3 Pre-conditioning at multilevel well B4 led to a clearly bet- ter ﬁt of Dr(in situ) and Dr(365) (Table 4). This indicates that pre-conditioning should increase the predictability of Dcum(365) and probably also SRC from Dr(in situ) measure- ments in the NO− 3 -free zone. Grouping of aquifer material according to hydro- geochemical zones or sediment parameters resulted in better regressions between Dr(in situ) and Dcum(365) and SRC for partial data sets where NO− 3 is still present in the ground- water, i.e. 5 Conclusions The possibility to predict the capacity of aquifer zones to remove NO− 3 inputs over extended time periods based on in situ measurement of denitriﬁcation rates was evaluated in two Pleistocene aquifers in northern Germany. This was done by comparison of Dr(in situ) with denitriﬁcation param- eters determined in aquifer material samples, i.e. the stock of reduced compounds (SRC) and the cumulative denitriﬁca- tion measured during 1 year of incubation in the laboratory (Dcum(365)). Prediction of Dcum(365) and SRC from Dr(in situ) for data sets containing data from both aquifers was only satisfactory in the aquifer zones where NO− 3 was present. This type of in situ test might thus be suitable for mapping Dcum(365) and SRC in NO− 3 -bearing zones of Pleistocene sandy aquifers using existing monitoring wells. It is thus a promising and low-cost method to estimate Dcum(365) of aquifer material from aquifer zones where NO− 3 is still present in the ground- water. Our results also indicate that the push–pull technique (without pre-conditioning) is not suited for deriving the SRC or Dcum(365) of aquifer samples from in situ denitriﬁcation rates under conditions where the groundwater is nitrate-free. The goodness of ﬁt in the modelling of Dcum(365) and SRC using linear regression functions was highly vari- able among partial data sets. The mean ratios of calculated Dcum(365) to measured Dcum(365) and calculated SRC and measured SRC were best for the transition zone with ratios near 1. We suppose the reasons for this might be (1) that residual reduced compounds that could support denitriﬁca- tion were still present in the aquifer material, (2) the O2 con- centrations in the ambient groundwater (Table 2) were far below the reported apparent threshold of < 40–60 µmol L−1 (≈1.5–2.3 mg O2 L−1) for the onset of denitriﬁcation in aquifers (Green et al., 2008, 2010; McMahon et al., 2004; Tesoriero and Puckett, 2011) (see also Sect. 4.1 in Eschen- bach and Well, 2013), and (3) NO− 3 was present in the ambi- ent groundwater of the transition zone. Therefore we expect that the microbial community was already adapted to NO− 3 , i.e. ready to denitrify, and denitriﬁcation was not inhibited by dissolved O2. 4.3 Predicting Dcum(365) and SRC of aquifer sediments from Dr(in situ) in the transition and NO− 3 -bearing zone (Table 5). Konrad (2007) reported similar relationship between Dr(in situ) and Dcum(365) under comparable conditions. Relatively weak ﬁts were obtained for data sets with push–pull measur- ing points located completely or mostly in the zone of NO− 3 - free groundwater (NO− 3 -free zone and sulﬁdic aquifer mate- rial, respectively) and in the non-sulﬁdic zone (Table 5). For the NO− 3 -free zone this is attributed to a missing adaptation of the microbial community to NO− 3 as an electron accep- tor as discussed above. In the study of Trudell et al. (1986) it took at least 8 days until measured denitriﬁcation rates stopped increasing during the push–pull test. In our study, such long pull periods were not possible because of com- paratively higher groundwater velocities in both aquifers. At some injection points in the FFA, the tracer plume had al- ready moved away with groundwater within 35 h of the in- jection. www.biogeosciences.net/12/2327/2015/ 5 Conclusions Conversely, in the non-sulﬁdic zone, higher O2 concentrations might have inhibited denitriﬁcation and this might have been more limiting for Dr(in situ) than the lim- Moreover, future routine applications of this approach could be facilitated by online ﬁeld analysis using membrane inlet mass spectrometry (MIMS), which we demonstrated to be feasible and precise. Still, the correction for dilution of the injected tracer solution with ambient groundwater is nec- essary when using MIMS in the ﬁeld (see Sect. 2.6 and the Supplement). In the NO− 3 -free aquifer zone, increasing denitriﬁcation rates were observed during the conducted push–pull tests, which were interpreted as the result of adaptation processes of the denitrifying communities following NO− 3 injections. Also Dr(in situ) without pre-conditioning was generally lower than average denitriﬁcation rates after 1 year of incu- www.biogeosciences.net/12/2327/2015/ W. Eschenbach et al.: Predicting the denitriﬁcation capacity of sandy aquifers 2344 bation (Dr(365)) in the laboratory. This was especially the case for Dr(in situ) measurements in the NO− 3 -free ground- water zone. In this study it was demonstrated that the micro- bial community in the NO− 3 -free zone just below the NO− 3 - bearing zone can be adapted to denitriﬁcation by amending wells with NO− 3 injections for an extended period. In situ denitriﬁcation rates measured after this pre-conditioning re- ﬂected the Dcum(365) and SRC more satisfactorily. From these ﬁndings it is assumed that microbial adaptation after NO− 3 injection confounded the relationship between reactive compounds present in the tested aquifer material and Dr(in situ) measured during push–pull tests, which resulted in poor prediction of Dcum(365) and SRC from Dr(in situ). There- fore we assume that pre-conditioning is a prerequisite for the measurement of in situ denitriﬁcation rates using push–pull tracer tests in the NO− 3 -free groundwater zone. Further re- search is needed to check whether this microbial adaptation would also work in deeper layers far below the NO− 3 -bearing zone. bation (Dr(365)) in the laboratory. This was especially the case for Dr(in situ) measurements in the NO− 3 -free ground- water zone. In this study it was demonstrated that the micro- bial community in the NO− 3 -free zone just below the NO− 3 - bearing zone can be adapted to denitriﬁcation by amending wells with NO− 3 injections for an extended period. In situ denitriﬁcation rates measured after this pre-conditioning re- ﬂected the Dcum(365) and SRC more satisfactorily. From these ﬁndings it is assumed that microbial adaptation after NO− 3 injection confounded the relationship between reactive compounds present in the tested aquifer material and Dr(in situ) measured during push–pull tests, which resulted in poor prediction of Dcum(365) and SRC from Dr(in situ). There- fore we assume that pre-conditioning is a prerequisite for the measurement of in situ denitriﬁcation rates using push–pull tracer tests in the NO− 3 -free groundwater zone. Further re- search is needed to check whether this microbial adaptation would also work in deeper layers far below the NO− 3 -bearing zone. Böttcher, J., Strebel, O., and Duijnisveld, W. H. M.: Kinetik und Modellierung gekoppelter Stoffumsetzungen im Grundwasser eines Lockergesteins-Aquifers., Geol. Jahrb. Reihe C, 51, 3–40, 1989. Böttcher, J., Strebel, O., and Duijnisveld, W. H. 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https://openalex.org/W2800379732	https://revistas.ucu.edu.uy/index.php/paginasdeeducacion/article/download/1551/1535	English	null	What’s new about ‘fake news’? Critical digital literacies in an era of fake news, post-truth and clickbait	Páginas de educación/Páginas de educación	2,018	cc-by	8,655	INTRODUCTION In the Republic of Macedonia on the banks of the river Varda, lies the city of Veles, population 43,716. The 19th century wooden houses that line the steep streets are reminders of a time when the city was at its peak, forming part of the trade route that connected the Balkan Peninsula and Aegean sea via the river. While globalisation might be responsible for the deindustrialisation of the city, it is the unexpected consequences of globalisation that have made Veles famous in more recent months. Veles is a city that now hosts over 100 US politics websites; the city has started trading in a new industry - fake news. And it is the digitally savvy teenagers of the city who are reaping the rewards. While most of the news websites that are based in Veles are pro-Trump, the teenagers responsible for them are reported as caring little about US politics or the outcome of the election. Their goal is to create controversial content, to be shared 'virally' across social media platforms, in order to make money through the advertising that can be embedded alongside these news articles. While the average wage in the city is approximately 4200 Euro a year, some teenagers are earning up to 20 times that through their Google AdSense accounts. It is perhaps not surprising that the Mayor of Veles, Slavco Chediev, describes the monetisation of fake news sites as a success story for the city. The teenagers behind the websites are paying their taxes, which, as one teenager quipped, would keep two of his teachers employed for a year. While bias in the news is not new, the opportunities brought about by the democratization, monetisation and circulation of 'news' via digital platforms has brought this issue to a critical point, highlighted by Trump’s surprising election victory. The goal for the teenagers of Veles however is simply to attract clicks in whichever way they can, whether through clickbait, polls or sensational news stories. As one teenager explained: “Polls work best because you don’t need to write much and people always click through” (Byrne, 2016). While the sites appear legitimate with domain names designed to imitate genuine news sites, like ABCnews.com.co and Bloomberg.ma, the news articles are cobbled together from other false news articles and online content. WHAT’S NEW ABOUT ‘FAKE NEWS’? CRITICAL DIGITAL LITERACIES IN AN ERA OF FAKE NEWS, POST-TRUTH AND CLICKBAIT ¿Qué hay de nuevo en las noticias falsas? Alfabetizaciones digitales críticas en la era de las noticias falsas, la posverdad y el cebo de clics DOI: https://doi.org/10.22235/pe.v11i1.1551 LUCI PANGRAZIO Deakin University. Australia. luci.pangrazio@deakin.edu.au Recibido: 20-10-2017 Revisado: 21-11-2017 Aceptado: 02-01-2018 Recibido: 20-10-2017 Revisado: 21-11-2017 Aceptado: 02-01-2018 Recibido: 20-10-2017 Revisado: 21-11-2017 Aceptado: 02-01-2018 Abstract: The 2016 Facebook fake news scandal has highlighted the difficulty in determining the credibility and reliability of news. As a result, there have been calls for individuals to adopt a more informed and critical stance toward the sources of their news. This paper considers what might be involved in cultivating critical digital literacies in an era of post-truth, fake news and clickbait. Using the platform as the framework for study, the paper examines how the architecture, algorithms and network effects of the platform have changed the way news is created and disseminated, and how audiences are positioned to engage with it. This theoretical critique provides insight into the technical, political and social issues surrounding how individuals engage with online news. Keywords: critical digital literacies; fake news; digital platforms; social media. Resumen: El escándalo de noticias falsas de Facebook de 2016 puso de relieve la dificultad para determinar la credibilidad y la confiabilidad de las noticias. Como resultado, se instó a individuos a que adoptaran una posición más informada y crítica frente de la fuente de sus noticias. El presente trabajo considera qué podría estar relacionado con el cultivo de alfabetizaciones digitales críticas en la era de la posverdad, las noticias falsas y el cebo de clics. A través de la utilización de la plataforma como marco para el estudio, el trabajo examina cómo la arquitectura, los algoritmos y los efectos de red de la plataforma han cambiado la manera en que las noticias se crean y se diseminan, y cómo las audiencias se posicionan frente a ellas. La presente crítica teórica arroja luz sobre los aspectos técnicos, políticos y sociales alrededor de la manera en que los individuos se involucran con las noticias en línea. Palabras clave: alfabetismos digitales esenciales; noticias falsas; plataformas digitales; redes sociales. Palabras clave: alfabetismos digitales esenciales; noticias falsas; plataformas digitales; redes sociales. 6-22 What’s New about ‘Fake News’? Critical Digital Literacies in an Era of Fake News… Pangrazio Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 WHAT IS FAKE NEWS? Fake news emerged as an issue after the 2016 US election, in which the most widely circulated stories in the last three months of the campaign came from false websites and hyperpartisan blogs. These stories were shared with much greater frequency than any of the top news articles from major news media, generating over 8.7 million shares, comments and likes (Silverman, 2016). While much has been made of this being a critical moment in media manipulation, it could be argued that this was more likely the point at which we became critically aware of these issues. Indeed, various forms of media bias have long existed (see for example Darnton, 2017). In addition, fake news has emerged against a backdrop of ongoing societal changes, such as an increasing distrust of public institutions and news media (Nicolaou & Giles, 2017) as well as a decline in professional news journalists (Clark & Marchi, 2017). This essay however is focused on the role played by digital platforms in this current moment of media manipulation. With the emergence of the ‘participatory web’ (Jenkins, 2006) user generated content has become an increasingly important part of digital culture (Grossman, 2006; Mitchem, 2008). This has brought significant changes to the news media industry. Specifically, the ways in which news is reported and shared across populations are expanded through connective media platforms, which has had a positive influence on engaging young people with news and current affairs (Greenhow & Reifman, 2009). At the same time, it is the monetisation and rapid circulation of ‘news’ through digital platforms that have led to such widespread and effective forms of media manipulation. Digital platforms might democratise the creation and circulation of news, however, in doing so questions around what news is, how it gets made, shared and read in online contexts are also raised. One of the challenges to this issue is defining what is meant by the term fake news. This term is not only misleading, but also generalizes the different ways in which news can be manipulated. Wardle (2017) has come up with seven types of mis- and disinformation in an attempt to distinguish the different types of ‘problematic content’ (n.p.) that exist within the current news media ecosystem. This includes categories such as false connection; false context; manipulated content; satire or parody; misleading content; imposter content and fabricated content. INTRODUCTION However, once shared by 'friends' on social media platforms these fake news stories acquire a legitimacy that exploits the affective relations between users and their predetermined political bias. In light of this, rather than being social nuisances, the teenagers of Veles' ability to manipulate the technical affordances of digital platforms and the affective reasoning of social media users, make them models par excellence for what it means to be digitally literate in an era of platform politics. This essay is about the most recent instantiation of news bias - namely fake news - and the critical questions it raises for digital literacies education. Using digital platforms as a framework for analysis, the purpose of this essay is to focus explicitly on what is new about this moment of fake news. As such, I consider the social, technical and political 7 7 Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 What’s New about ‘Fake News’? Critical Digital Literacies in an Era of Fake News… 6-22 Pangrazio milieu that has led to what has been called the 'post-truth' era – an era in which emotion and personal belief are more influential in shaping opinions than objective facts. While this issue has implications for all individuals, I am particularly interested in what this moment signifies for digital literacies education of young people. As such the paper concludes by considering the critical digital literacies necessary for everyday lived experiences of social media use and the implications for digital literacies educators. Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 WHAT IS FAKE NEWS? The type of 'problematic content' used depends on the creator and their motivation. While heuristics like this are helpful, the more recent instantiation of fake news not only raises questions about the content, but also the way 8 Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 What’s New about ‘Fake News’? Critical Digital Literacies in an Era of Fake News… Pangrazio 6-22 in which digital texts are disseminated through digital platforms. Through social media platforms, digital texts become a conduit for relational work between users. These relations affect the interpretive processes of individuals, positioning them to engage with the news article or headline in particular ways. So far there have been a range of responses to the supposed fake news crisis. News outlets and universities have been quick to respond to the most recent moment of fake news by launching online ‘fact checking’ apps and digital tools that check the ‘truth’ and validity of particular facts and ideas presented in articles. In some instances, this approach might be helpful, however, it only addresses a small part of a much broader and more complicated challenge. The fact-checking app might have a detrimental effect as not only is critique ‘outsourced’ to a digital tool, but the fact that these are arbitrated by traditional ‘authorities’ undermines individual agency and the role of critical digital literacies in everyday life. Facebook and Google on the other hand have introduced different tools to their platforms to help users identify and report fake news. Facebook has avoided outright censorship of news and information and instead circulated a series of tips to help users spot fake news, as well as adding the option to report content that users believe to be fake (Pogue, 2017). Ironically, Google have introduced human editors to evaluate the content of their search results in an attempt to train algorithms to detect low quality content (Leong, 2017). While Google appear to be doing more to correct the situation, both companies still develop algorithms that are designed to deliver information that they believe users want to read, regardless of its truthfulness. I see the issue of fake news as having particular significance to researchers and educators working in the field of digital literacies. WHAT IS FAKE NEWS? My aim in this essay is not to replicate the writing and research already taking place on fake news in other disciplines, such as media and communication (Mihailidis & Viotty 2017; Balmas, 2012), information technology (Dale, 2017), journalism (Marchi, 2012; Khaldarova & Pantii, 2016) and even science (Spinney, 2017). Instead, this essay will focus on what the most recent moment of news media bias means for digital literacies educators. Using the platform as a framework for study, this essay will explore the features that lay the foundation for the current moment in fake news, with a particular focus on the digital literacies required to navigate these challenges. Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 1. Platforms produce and rely upon network effects The theory of network effects or network externalities comes from economics, and claims that the value of a platform depends on the number of users it has (Katz & Shapiro, 1986; Rohn, 2013). In the context of social media platforms, the more users a platform has the more valuable it becomes as a communication service to its members. Facebook, for example, has become the default social networking service in many parts of the world because the sheer number of users means that it is the most logical place for individuals to connect with friends online. However, institutions, companies and other community groups, are also drawn to using the platform because it connects them to the public in useful ways. As participation increases, so too does the amount of data generated, which improves the reliability of the platform algorithms to suggest, recommend and match information between all parties. As Belleflamme and Peitz (2016, p.5) explain this ‘enhances the quality of the platform service and, thereby, the utility of all users’. For this reason there is a natural tendency toward platform monopolization. However, Facebook’s monopolization of communicative media has had the residual effect of changing the way people find and read news. Most notably it has limited the number of news sources individuals consult to remain informed about what is going on in the world. According to the Pew Research Centre, 62% of American adults now get their news through social media. Of this 62%, 64% report that they only get their news from Facebook (Gottfried & Shearer, 2016). In Australia, 52.2% of adults receive their news through social media, however, only 18.5% of these people use only social networking sites or blogs (Park, 2016). While these data only represent two countries they indicate a broader trend toward engagement with news on social media platforms, most notably Facebook. On Facebook, users are more likely to stumble upon news shared by friends, rather than actively seek it out (Oeldorf-Hirsch & Sundar 2015). While this might increase young people’s engagement with news and information, it also introduces different news reading practices and encourages different processes of interpretation in individuals. A PLATFORM APPROACH TO FAKE NEWS Research into the dominance of the platform structure (Srnicek, 2017; Bratton, 2015) and the platformization of the web (Helmond, 2015) explains how platforms provide both the technical and economic infrastructure and the discursive framing for social and communicative practices. In line with this more critical approach, this paper uses the platform as an analytical framework to offer a more technical and reflexive account of the role that platforms play in the fake news phenomena. While I argue that the digital literacies required to critique fake news require an understanding of the computational, Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 9 What’s New about ‘Fake News’? Critical Digital Literacies in an Era of Fake News… 6-22 Pangrazio cultural and social layers of digital platforms, which facilitate the widespread and rapid dissemination of such digital texts. To identify exactly what these literacies might be, I analyse seven features of platforms that have contributed to the current moment of news bias. Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 2. Platforms work as a framing device There are two ways in which platforms frame information for users – through the architecture of the platform and the social networks that mediate the content that is shared. The 'architecture' of a platform can be taken to mean the 'system's overall structure and function', including the interface specifications, as well as the algorithms 10 Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 What’s New about ‘Fake News’? Critical Digital Literacies in an Era of Fake News… 6-22 Pangrazio and processes that ‘govern relationships among components and allow them to interoperate' (Baldwin & Woodard, 2008, p.7). While platforms might appear decentralized, in reality it is only content creation that is decentralized. The platform position is one of an intermediary, managing or governing the terms of the relationships between parties through the manner and volume of interactions. This means that the platform is essentially a ‘drawing and framing machine’ (Bratton, 2015, p.85), accentuating particular aspects and modes of communication and relegating others. On Facebook, for example, links to the outside web are deprioritized in the Newsfeed, as operators aim to keep users on the platform for as long as possible (Tufekci 2016). Official news organisations therefore have a difficult time connecting individuals to their content on Facebook, as the architecture of the platform mediates the distribution of information. In mid-2016 Facebook changed its algorithms to preference posts from friends and family and de-emphasise those from mainstream news media. As a result of Facebook’s decision, traffic to these news media sites fell by numbers as significant as 25% (Ingram, 2016), making it less likely for users to read news from traditional news media sources. When a news article, or digital resource is shared on a social media platform, it is done so via some kind of social relationship, be it a strong or weak tie. Unlike other media, such as the newspaper or television news, the platform and the relationships it sustains, also act as a framing device for digital content. It matters that we are familiar with the individual who shared the article, as this can abate critical faculties and position the reader to engage with the text in particular ways. On social media the underlying relationship or impression one has of the person sharing becomes particularly significant in how that information is interpreted. 2. Platforms work as a framing device As Apperley and Parikka (2015, p.5) explain, platforms ‘are not just technologies but techniques that sustain interactions as well as offer an epistemological framework'. When disseminated via a social media platform, a news article becomes more than just information; it becomes a conduit for affective relations between individuals. Specifically, social networks tend to be made up of like-minded people, meaning the phenomenon of confirmation bias, in which we seek out or more readily believe information that confirms what we know or value (Braucher, 2016), is enhanced. to suit their needs While overall governance of the platform belongs with the platform operators, the user can manipulate the structure to suit their needs. Indeed, the utility and adaptability of digital platforms have helped secure their dominance on the internet. As Srnicek (2017) explains, platforms come with tools that enable users and developers to build their own services, products and marketplaces. The Macedonian teenagers, for example, were able to create Facebook identities that enabled them to reach American audiences and Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 11 What’s New about ‘Fake News’? Critical Digital Literacies in an Era of Fake News… 6-22 Pangrazio purportedly influence US politics. While these individuals were not recognized as political ‘citizens’ of the US, as Bratton (2015, p.87) points out, they are ‘nevertheless included in communication by platforms that are agnostic to the legal status of its users’ (p.87). Despite this, the credibility of the users disseminating these news articles was increased if their profiles were based in the US. Not surprisingly US based Facebook profiles can now be bought on the black market (Subramanian, 2017). Despite enabling and promoting the participation of other parties, the platform operators hold little responsibility for what takes place on the service. Indeed, no news organization has ever had as much power to influence public opinion as Facebook. Despite this, current CEO Mark Zuckerberg remains adamant that it is a ‘tech company’ and not a ‘media company’ because such a label would mean greater responsibility to regulate the users and content on the platform (Roberts, 2016). Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 5. Platform architecture facilitates frictionless sharing of digital content In 2011 Facebook introduced an external like button - a plugin that can be included on any website. With the advent of this social button all pages on the web became potentially ‘likeable’. As Gerlitz and Helmond (2013) explain, this means that Facebook and the external web are increasingly interconnected, as the practices in one space affect another, ‘rendering both more open and relational’ (p.1358). Indeed, the architecture of the Facebook platform – including the interface design and the proliferation of social buttons across the internet – mean news and information are easily shared both with an array of social and commercial actors. This ‘alternative fabric’ (Gerlitz & Helmond, 2013, p.1361) of social media platforms is dependent on data flows generated through users sharing, recommending, commenting and liking posts and pages across various social media platforms. Despite the control afforded to platform operators through the Facebook architecture users have, in the main, embraced its design, values and practices. In 2012, there was an estimated 3.2 billion likes and comments on Facebook everyday (McGee, 2012), creating a culture of participation based around sharing and liking. As Sumner et al. (2017) explain one of the main benefits of the like button is its ambiguity, meaning its interpretation is highly dependent on the context and audience. While the like is often used to share content, it is the relational work that it performs which is most significant. As various studies report (Sumner et al, 2017; Eranki & Lonkila, 2015), the like has become an integral part of facilitating relationships and self-representation. However, its pervasiveness has led to expectations around use. Indeed, sharing has become the ‘fundamental and constitutive activity’ of social media (John, 2012, p.167). Research by Egebark and Ekstrom (2011) suggests that the Facebook platform is an environment that constitutes conformity because it is highly visual and it is based around expression of beliefs and attitudes symbolised by the like button. 4. Platforms ensure every user's experience of the platform is different One of the most significant features of the internet is the large amount of news and information that users have access to. While this has obvious benefits, the constant stream of information can be difficult for users to navigate. This ‘infoglut’, as Andrejevic (2013) terms it, has led to the creation of an array of digital tools, data mining strategies and algorithms that filter information in order to establish a more personalised, streamlined experience of the web for users (Mobasher, Cooley & Srivastava, 2000). Many digital platforms aim for increased levels of personalisation. Google, for example, provides personalised results for search queries based on browsing histories and social connections (Google, 2009). On social media platforms such as Facebook, information is not only filtered through user curated social networks, but also interface design and the News Feed algorithm. While humans have always tended towards homophily (i.e. selectively interacting with like-minded people) the architecture of digital platforms and their focus on personalising user experience, only enhances this tendency. Research by Del Vicario et al. (2016) showed that the Facebook platform helps users find, follow and focus on certain people while excluding others, encouraging the emergence of polarized communities. Their findings on two hyperpartisan community pages suggest that whether a news item is accepted as true is strongly affected by the social norms of the group or how much 'it coheres with the community's shared belief system and values' (Del Vicario et al., 2016, p.9). However, the News Feed algorithm also makes assumptions about the future content that users will be interested in based on which content they have engaged with in the past and which users they have most connections with (Bucher, 2012). In a similar way, Google search will start to include and prioritise particular content that matches the users social networks and browsing history. As such the goal of many platforms to ‘personalize’, ‘customize’ and ‘tailor’ user experience, means individuals become aligned with social groups that can lead to a kind of 'group think' approach to news and 12 sta Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 What’s New about ‘Fake News’? Critical Digital Literacies in an Era of Fake News… Pangrazio 6-22 information. This also means factually incorrect information, or fake news, can be rapidly spread through social groups and networks which share the same or similar beliefs and values. Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 6. Platforms depend on data extraction and monetisation While many of these platforms are ostensibly ‘free’ to users, the business model relies on data extraction and monetization. The platforms that have most significance to fake news are Facebook and Google – platforms that Srnicek (2017) categorises as 'advertising platforms' or platforms that 'extract information on users, undertake a labour of analysis, and then use the products of that process to sell ad space' (p.49). The Google platform, for example, enables any website to be connected to a Google Ad sense account, regardless of the content that website disseminates. The teenagers of Veles were able to 13 Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 What’s New about ‘Fake News’? Critical Digital Literacies in an Era of Fake News… Pangrazio 6-22 tap into the data assemblage and yield a profit through their Google AdSense accounts, which monetise the clicks on the ads that are embedded alongside the false and misleading articles. The content itself matters little. In some cases manipulating the headline is enough for an article to be shared widely on social media and attract attention and clicks to the external website. In the lead up to the US election the articles that were circulated the most often had an outrageous or exaggerated headline, however, the actual content of the article was sometimes contradictory or even true (Silverman, 2016). Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 7. User input on platforms results in an increase in value On social media, individuals are encouraged to like, share and comment on digital texts. In the process these texts accrue credibility as well as value for those who circulate them, such as increased social connections and personal morale. The practice of sharing texts not only disseminates information across social networks, but also helps to distil and project a sense of self to the world. Adami (2012) argues that the culture of sharing often requires greater effort on the part of the viewer to retrieve and interpret the implied and intertextual meaning bound up in shared texts. As such, interpretation leads to a sense of reward in that the viewer becomes an insider or ‘part of an elite’ (Adami, 2012, p.132). While those who create fake news articles might be motivated by the need to make money, the user who shares the article through their social network is mostly seeking to maintain or perhaps expand their socialities, or associations with other individuals in society. Sharing the article becomes part of their ‘identity work’, which demonstrates their understanding of the implied and intertextual meanings of the article, as well as their belonging to a particular political group. These digital texts gather credibility as they are shared across social networks acquiring likes, shares and comments from users. As these links and shares have value for the author and viewer, the motivation to prevent the spread of misinformation is diminished. Through the platform, people’s sociality becomes imbricated with the creation and sharing of digital texts in ways that can be difficult to identify and unpack. In considering the complex features of digital platforms the sophisticated nature of the Macedonian teenager’s digital literacies is evident. Not only did they create news articles that would gather the attention of audiences across the United States and the world, but they were able to exploit the network effects of digital platforms to disseminate the news widely, and subsequently generate a personal income. While their success relied upon identity theft and willfully creating factually inaccurate news articles, it also required a complex understanding of the potentialities of digital texts and their affective ‘work’ across digital platforms. Many of the fake news websites coming out of Macedonia have now been shut down, however, the broader concern of how digital platforms can be manipulated to spread misinformation still remains an issue. Given that social media are Revista Páginas de Educación. Vol. 7. User input on platforms results in an increase in value 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 14 6-22 What’s New about ‘Fake News’? Critical Digital Literacies in an Era of Fake News… Pangrazio playing an increasingly important role in how people encounter news and information, there are important implications for digital literacies and digital literacies education. IMPLICATIONS FOR DIGITAL LITERACIES AND DIGITAL LITERACIES EDUCATION Digital platforms have not only introduced new reading practices, but they have also changed the interpretive processes individuals typically bring to reading news and information articles. Many of these changes have taken place in a subtle way as readers have adjusted to the news context without considering the specific challenges it raises. In light of this, there is clearly a need to identify the digital literacies required to address the challenges brought about by the most recent moment of fake news. The features of digital platforms identified in the previous section highlight some of the literacies required to understand the technical, political and social layers implicit in the creation and dissemination of fake news. Indeed, to critique fake news in the context of social media, one needs an intertextual, affective and networked reading of the content in question. But first, digital literacies educators need to acknowledge that it is not just the content on digital platforms that is significant, but the platform itself needs to be approached as an object of study. Schools and educational institutions have been afraid to focus on digital platforms, perhaps because they are often associated with recreational or social uses. However, as social media platforms and their infrastructure are increasingly a part of news and information practices it is essential they are critically evaluated. Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 1. Identify the changing nature of news reading practices In analysing the role of platforms in the fake news phenomena there are at least two significant changes to reading practices that are important for digital literacies educators to be aware of. First, in relation to sourcing news and information individuals are less likely to seek news directly from news sources and instead come across information through their social networks. According to Matsa and Mitchell (2014), 78% of users see news when they are using Facebook for other reasons. While only 34% of users subscribe to a news media source on social media. Encountering news articles via the social sphere rather than the sphere of news and information means the article is not read in context, giving people less opportunity to compare the structure, style and voice to other news articles. Related to this is that discovering news and information is no longer an individual pursuit but instead a social endeavour (Nikolov et al., 2015). This not only changes how people find out about news and information, but also their fundamental disposition towards engaging with these articles. Typically, users do not feel the need to be critical in this space, as a social media platform like Facebook is a site for sharing news and information with friends. However, the most recent instantiation of fake news has shown that the more relaxed and open disposition of users can be exploited, not only by the 15 Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 What’s New about ‘Fake News’? Critical Digital Literacies in an Era of Fake News… 6-22 Pangrazio platform operators, but other parties as well. Making educators aware of these changes will encourage them to find ways to support young people to develop critical news reading practices as part of their everyday lived experience of social media use. Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 2. Knowledge of platform architecture Also required is an understanding of the platform architecture, including its structure and function, as well as the algorithms and processes that govern relationships allowing the various components to interoperate. Young people need to be discouraged from seeing platforms as neutral conduits of news and information and instead analyse the structure and function of the architecture. While this might seem an obvious point, many people fail to intuit the fact that the architecture of the platform is designed to encourage participation from users that will benefit the platform – the production of an individual with agency is not necessarily an ambition. Introducing a more critical disposition toward the presentation of information at the interface is an important first step toward developing critical digital literacies. Analysing Facebook’s Newsfeed algorithm (Bucher, 2012) and Google’s PageRank algorithm (Rieder, 2012) would help young people think about what is not prioritized or even shown on the interface, which is a powerful way to critique the motivations of platform operators. Being aware of these specific design issues is useful to understanding the way in which fake news articles are presented and circulated across the platform. For example, with regard to fake news it might also be helpful to think about the function of the like button and the role that it has played in opening connections between Facebook and the wider web. Specifically, the fact that the like button now mediates connections and interactions for users is a key point to consider. Rather than accepting the ambiguity implicit to the like button, being more conscious and aware of its function on the platform would also be a useful step toward more conscious and thoughtful sharing. Pangrazio Pangrazio 6-22 What’s New about ‘Fake News’? Critical Digital Literacies in an Era of Fake News… Visualisations of the underlying network can be a helpful way to understand how platforms connect people, institutions and information (NetSciEd, 2017). Helping young people to visualize how news articles enter and spread out across networks, as well as the various points of incentivisation, would develop a more critical approach to the role played by social media platforms in the fake news phenomena. In doing so, they may discover that the network is not as equally or well distributed as first thought. Indeed, Galloway (2011) reminds us mapping information in this way cannot visualise or represent the ‘social totality’ of the information age. Bearing this in mind, when visualizing networks and the role of digital platforms within these, a consideration of which aspects of the network are difficult to capture and why is important. Identifying and exploring the ‘blindspots’ within networks would help individuals to critically evaluate structures and functions that are typically beyond perspicuity. 3. Understand digital platforms as part of broader social and technical It is also important to build an understanding of how digital platforms fit into a broader network of technological and semantic systems. Elsewhere this has been called ‘network literacy’ (NetSciEd, 2017), which can be thought of as ‘basic knowledge about how networks can be used as a tool for discovery and decision making’, including an understanding of the ‘potential benefits and pitfalls’ of networks (p.2). Importantly this definition acknowledges that networks can be both beneficial and problematic, and that even the same feature of a platform can be seen in different ways depending on the situation. For example, in the case of a natural disaster, network effects are obviously advantageous because alerts and warnings can be quickly shared across populations. However, in the case of misinformation the same feature of networks is problematic. Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 16 5. Explore how affect circulates and condenses across digital platforms Finally, individuals should be encouraged to think about how the sharing of news and information relates to their socialities through affect. Affect can be thought of as the more subliminal forces that drive behaviours, but which defy categorization into typical emotions, such as happiness and sadness. It explains ‘how individual, collective, discursive, and networked bodies, both human and machine, affect and are modified by one another’ (Paasonen, Hillis & Petit 2015, p.3). In this way, technology ‘mediates desires’ and creates a series of ‘travelling affects’ (Kofoed & Ringrose, 2012, p.16), or emotional responses that circulate through people and digital media. In light of this, consideration needs to be given to how the information presented on platforms develops an affective response in individuals, shaping their digital identities and online relationships. Economic historian Philip Mirowski (2014), for example, argues that the interface is ‘continuously destabilizing identity’, distilling an individual’s identity ‘to a jumble of unexplained tastes and alliances’, in such a way that requires ‘constant care and management’ (p.113). This creates a kind of perpetual lack, which can only be quelled through visiting and maintaining the digital identities presented on the platform. Cultivating an awareness of the way affect condenses through metrics and notifications might help to create a critical distance from the platform that is necessary to evaluate fake news articles. 4. Identify and critique the ideologies implicit to digital platforms Critical digital literacies encourage young people to analyse the ideologies implicit to a text. However, it is not just the ideology of the text that requires scrutiny, but the digital platforms that disseminate this content. These require sophisticated digital literacies due to the opacity of the architecture behind digital platforms. Indeed, being opaque means ‘control, ownership and ideological uses of these new [information] flows’ remain ‘volatile and dynamic’ (Luke 2013, p. 137), which no doubt has benefits to platform operators. One way to develop literacies of digital platforms is through an understanding of their political economy. While this might sound difficult, particularly in relation to cultivating the digital literacies of young people, it could be something as simple as unpacking the role that metrics play on the platform and understanding the role between metrics and commodification. As Beer (2016, p.24) explains understanding the role of metrics helps to reveal the ideology upon which these systems are built: Metrics then play a central role in the formations of neoliberalism and its limits. Systems of measurement are the means by which the shift can be made towards calculation and away from judgment and critique. But, it is important that we see these metrics as cultural and political objects as well as being infrastructural by-products. Digital literacies for fake news also need to consider the way data are extracted and monetized, as well as the way in which online advertising attracts revenue for website operators. While many young people claim to be unfazed by online advertising, the fact that revenue is generated through advertising clicks has material implications for website operators that users should know about. Identifying these opportunities for revenue generation means individuals can understand why particular practices and behaviours are encouraged. Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 17 What’s New about ‘Fake News’? Critical Digital Literacies in an Era of Fake News… Pangrazio 6-22 Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 CRITICAL DIGITAL LITERACIES FOR NEWS AND INFORMATION ON SOCIAL MEDIA PLATFORMS – TOWARDS A RESEARCH AGENDA Given the features of digital platforms and their role in disseminating news and information that have been outlined in this paper there is clearly a need for future research in this area. However, with recent initiatives directed toward ‘fact checking’ tools and platform based governance in the form of check lists and flags, there is the possibility that research and education developing social and political understandings of digital platforms in individuals will be de-prioritised. This article has outlined the specific digital literacies required to help individuals to be responsive to the changing nature of news and information as it appears on digital platforms. This is not to replace the literacies needed to critically engage with the content that is presented in these articles, but to draw attention to the infrastructure that enables the creation and dissemination of misinformation. From this perspective the following questions point to some areas that require further in-depth research and investigation: - How do digital platforms reconfigure news and information practices? - How do digital platforms reconfigure news and information practices? kinds of critical digital literacies are necessary to understand infrastructure - What kinds of critical digital literacies are necessary to understand infrastructure of digital platforms and how might these be practiced? 18 18 Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 What’s New about ‘Fake News’? Critical Digital Literacies in an Era of Fake News… 6-22 Pangrazio Pangrazio - What sort of critical understandings do young people have of digital platforms and their role in disseminating news and information? In what ways are these applied in daily digital practices? - What sorts of practices and techniques have successfully developed critical approaches to news and information in the past? Can these lessons be translated to the challenges initiated by digital platforms? - What are the short- and long-term consequences for society and democracy as news and information are increasingly disseminated through social networks? - What are the short- and long-term consequences for society and democracy as news and information are increasingly disseminated through social networks? This paper has analysed the features of digital platforms that are pertinent to the fake news phenomena. In doing so, I have raised epistemological and ontological concerns that are difficult to address. CRITICAL DIGITAL LITERACIES FOR NEWS AND INFORMATION ON SOCIAL MEDIA PLATFORMS – TOWARDS A RESEARCH AGENDA However, in identifying these features and the literacies required to critically evaluate the way in which content is created, disseminated and circulated it is hoped that an evidence-based framework for critical studies of digital platforms will soon follow. Revista Páginas de Educación. Vol. 11, Núm. 1 (2018) ISSN: 1688-5287; e-ISSN: 1688-7468 REFERENCES Adami, E. (2012). The rhetoric of the implicit and the politics of representation in the age of copy-and-paste. Learning, Media and Technology, 37(2), 131-144. Andrejevic, M. (2013). Infoglut: How Too Much Information is Changing the Way We Think and Know. New York, NY: Routledge. Andrejevic, M. (2013). Infoglut: How Too Much Information is Changing the Way We Think and Know. New York, NY: Routledge. Apperley, T., & Parikka, J. (2015). Platform studies' epistemic threshold. Games and Culture, 1(21), 1-14. 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https://openalex.org/W4386695799	https://russjcardiol.elpub.ru/jour/article/download/5467/4045	Russian	null	Pulse oximetry methods: opportunities and limitations	Rossijskij kardiologičeskij žurnal	2,023	cc-by	7,050	Pulse oximetry methods: opportunities and limitations Garanin A. A., Dyachkov V. A., Rubanenko A. O., Reprintseva O. A., Duplyakov D. V. Ключевые слова: пульсоксиметрия, трансмиссионная пульсоксиметрия, рефракционная пульсоксиметрия, сатурация. Для цитирования: Гаранин А. А., Дьячков В. А., Рубаненко А. О., Репринцева О. А., Дупляков Д. В. Методы пульсоксиметрии: возможности и ограничения. Российский кардиологический журнал. 2023;28(3S):5467. doi:10.15829/1560- 4071-2023-5467. EDN LWXJYA Отношения и деятельность: нет. ФГБОУ ВО Самарский государственный медицинский университет Минздрава России, Самара, Россия. Relationships and Activities: none. The aim of this review was to evaluate the current literature on various methods of pulse oximetry, their advantages and disadvantages. Modern pulse oximetry methods generally correlate well with invasive saturation monitoring, which makes it possible to be widely used in clinical practice. At the same time, in order to impro ve the accuracy of saturation measurements, existing limitations of various pulse oximetry methods should be taken into account. The emergence and introduc tion into clinical practice of reflectance pulse oximetry can largely compensate for the limitations of traditional transmission pulse oximetry regarding peripheral hypoperfusion, low response time, and features of patient's nails. In the event of special situations when pulse oximetry may not be accurate enough (carbon monoxide poisoning, methemoglobinemia, severe anemia, severe tricuspid insufficiency, etc.), a thorough clinical assessment of the patient is recommended, as well as invasive saturation monitoring. Методы пульсоксиметрии: возможности и ограничения Гаранин А. А., Дьячков В. А., Рубаненко А. О., Репринцева О. А., Дупляков Д. В. Гаранин А. А.* — к.м.н., директор НПЦ дистанционной медицины, ORCID: 0000-0001-6665-1533, Дьячков В. А. — к.м.н., доцент кафедры пропедевти- ческой терапии, ORCID: 0000-0001-8891-6088, Рубаненко А. О. — к.м.н., до цент кафедры пропедевтической терапии, ORCID: 0000-0002-3996-4689, Репринцева О. А. — врач по медицинской профилактике, ORCID: 0009-0004- 8167-5655, Дупляков Д. В. — д.м.н., профессор, зав. кафедрой пропедевти- ческой терапии, ORCID: 0000-0003-2773-1682. Целью описательного обзора явилась оценка современных литературных дан- ных, посвященных различным методам пульсоксиметрии, их преимуществам и недостаткам. Современные методы определения сатурации с помощью пуль- соксиметрии в целом хорошо соотносятся с инвазивными методами оценки данного показателя, что позволяет широко использовать ее в реальной клини- ческой практике. В то же время для повышения точности измерения сатурации необходимо учитывать имеющиеся ограничения различных методик пульсо ксиметрии. Появление и внедрение в клиническую практику методики реф- ракционной пульсоксиметрии позволяет в значительной мере компенсировать ограничения традиционной трансмиссионной пульсоксиметрии, касающиеся периферической гипоперфузии, невысокого времени отклика, ограничений, связанных с особенностью ногтей пациента. В случае возникновения особых ситуаций, при которых измерение сатурации кислорода с помощью пульсо ксиметрии может оказаться недостаточно точным (отравление угарным газом, метгемоглобинемия, выраженная анемия, выраженная трикуспидальная недо- статочность и т.д.), рекомендуется тщательная клиническая оценка пациента, а также контроль данных пульсоксиметрии с помощью инвазивных методик. Автор, ответственный за переписку (Corresponding author): sameagle@yandex.ru ДИ — доверительный интервал. Рукопись получена 18.05.2023 Рецензия получена 15.06.2023 Принята к публикации 02.08.2023 Российский кардиологический журнал 2023;28(3S):5467 doi:10.15829/1560-4071-2023-5467 https://russjcardiol.elpub.ru Российский кардиологический журнал 2023;28(3S):5467 doi:10.15829/1560-4071-2023-5467 https://russjcardiol.elpub.ru Российский кардиологический журнал 2023;28(3S):5467 doi:10.15829/1560-4071-2023-5467 https://russjcardiol.elpub.ru ОБЗОРЫ ЛИТЕРАТУРЫ ISSN 1560-4071 (print) ISSN 2618-7620 (online) ISSN 2782-2257 (online) Методы пульсоксиметрии: возможности и ограничения Методология поиска Поиск литературных источников осуществлял- ся в литературных базах ClinicalTrials, PubMED, eLIBRARY на русском и английском языках за пери- од 2000-2023гг. Поисковые запросы включали в себя слова: "pulse oximetry" ("пульсоксиметрия"), "oxygen saturation" ("сатурация кислорода"), "limitations" ("ограничения"). Всего было проанализировано 750 источников, поиск включал систематические обзоры, опубликованные и неопубликованные ран- домизированные контролируемые исследования и репрезентативные контролируемые наблюдательные исследования, в которых сообщалось о скорректиро- ванных оценках эффекта. В окончательный анализ публикаций не включались постерные доклады, дис- сертации, симпозиумы, клинические случаи и серии клинических случаев, письма читателям, исследова- ния на животных, рекомендации. Как было указано выше, пульсоксиметрия может использоваться не только у взрослых, но также и у детей, в частности, у новорожденных. Таким обра- зом, одним из перспективных направлений ее при- менения является раннее выявление критических врожденных пороков сердца. В проведенном систе- матическом обзоре в 2018г, включившем 19 исследо- ваний и 436758 новорожденных детей, было показа- но, что чувствительность пульсоксиметрии в выявле- нии критических пороков сердца у новорожденных составила 76,3% (95% доверительный интервал (ДИ): 69,5-82,0), специфичность — 99,9% (95% ДИ: 99,7- 99,9), по сравнению с эхокардиографией [11]. При этом частота ложноположительных результатов со- ставила всего 0,14 (95% ДИ: 0,07-0,22) [11]. По данным Jullien S, et al. (2021), в результате анализа имеющих- ся рекомендаций и мнений экспертных сообществ, проведенные исследования свидетельствуют о том, что пульсоксиметрия обладает достаточной точ- ностью в выявлении критических врожденных поро- ков сердца в дополнении к антенатальному ультразву- ковому исследованию и физикальному обследованию [12]. Таким образом, проведение пульсоксиметрии может дать ценную информацию у пациентов с раз- личными врожденными пороками сердца [13-15]. Области применения метода пульсоксиметрии Пульсоксиметрия может успешно использоваться у детей, взрослых, а также людей пожилого и старче- ского возраста, причем нормальные значения сату- рации, измеренные с помощью пульсоксиметрии, у людей разного возраста не отличаются. Данный ме- тод широко используется у пациентов с хронической обструктивной болезнью легких [8], бронхиальной астмой [9], внебольничной пневмонией [10] и други- ми заболеваниями. К настоящему времени известно несколько методов пульсоксиметрии, различные ва- рианты конструкции датчиков и самих приборов [6]. Samara State Medical University, Samara, Russia. В связи с этим для реализации мето- дики пульсоксиметрии применяют датчик, который включает в себя два элемента: в первом располага- ются два излучателя, испускающих свет в красном и инфракрасном диапазонах, а во втором, расположен- ном с противоположной стороны, находится фото- детектор, регистрирующий интенсивность светового потока, попадающего на него [6]. Определяя разницу между количеством поглощаемого света в систолу и диастолу, существует возможность определить пока- затели артериальной пульсации. Исходя из изложен- ного выше, проводя пульсоксиметрию, можно вычис- лить сатурацию, которая определяется соотношением количества оксигемоглобина к общему количеству гемоглобина и выражается в процентах. В норме ее диапазон составляет 95-100%. Невзирая на то, что са- турация взаимосвязана с его парциальным давлением в крови, зависимость эта не является линейной. Существующие портативные модели для неин- вазивной пульсоксиметрии не лишены недостатков, некоторые из которых являются критическими и бу- дут рассмотрены ниже. Авторы полагают, что актуа лизация обозначенной темы может быть полезной для ученых и клиницистов, чьи усилия направлены на реализацию научно-практических проектов по разработке и внедрению в практику новых приборов для определения уровня сатурации. Практический интерес к данной теме может быть обусловлен необ- ходимостью импортозамещения и стимулированием технологического суверенитета нашей страны, в т.ч. в части разработки отечественных изделий медицин- ского назначения. Цель настоящего описательного обзора — актуа- лизация сведений, посвященных различным методам пульсоксиметрии, их преимуществам и недостаткам, а также возможности применения у пациентов с хро- ническими неинфекционными заболеваниями и, прежде всего, с болезнями системы кровообращения. Samara State Medical University, Samara, Russia. Garanin A. A. ORCID: 0000-0001-6665-1533, Dyachkov V. A. ORCID: 0000-0001- 8891-6088, Rubanenko A. O. ORCID: 0000-0002-3996-4689, Reprintseva O. A. ORCID: 0009-0004-8167-5655, Duplyakov D. V. ORCID: 0000-0003-2773-1682. Garanin A. A.* ORCID: 0000-0001-6665-1533, Dyachkov V. A. ORCID: 0000-0001- 8891-6088, Rubanenko A. O. ORCID: 0000-0002-3996-4689, Reprintseva O. A. ORCID: 0009-0004-8167-5655, Duplyakov D. V. ORCID: 0000-0003-2773-1682. *Corresponding author: sameagle@yandex.ru Received: 18.05.2023 Revision Received: 15.06.2023 Accepted: 02.08.2023 Received: 18.05.2023 Revision Received: 15.06.2023 Accepted: 02.08.2023 For citation: Garanin A. A., Dyachkov V. A., Rubanenko A. O., Reprintseva O. A., Duplyakov D. V. Pulse oximetry methods: opportunities and limitations. Russian Journal of Cardiology. 2023;28(3S):5467. doi:10.15829/1560-4071-2023-5467. EDN LWXJYA Keywords: pulse oximetry, transmission pulse oximetry, reflectance pulse oximetry, saturation. симетры, представляющие собой приборы для инва- зивного измерения показателей переноса кислорода гемоглобином в образце крови [3]. Инвазивные мето- ды оценки вышеуказанных показателей используют- ся, как правило, в отделениях реанимации и интен- сивной терапии и малоприменимы в амбулаторных условиях. В связи с этим в клиническую практику в настоящее время широко внедрен неинвазивный В настоящее время "золотым" стандартом опреде- ления газообменной функции легких является оцен- ка газового состава крови с измерением парциально- го давления кислорода и углекислого газа, выполня- ющаяся инвазивно (исследование артериальной или венозной крови) с использованием газоанализаторов [1, 2]. В то же время в некоторых исследованиях в ка- честве референсного метода использовались СО ок- 59 Российский кардиологический журнал 2023; 28 (3S) метод оценки насыщения крови кислородом, полу- чивший название пульсоксиметрия [4-6]. метод оценки насыщения крови кислородом, полу- чивший название пульсоксиметрия [4-6]. в то время как дезоксигемоглобин больше поглощает красный свет. В связи с этим для реализации мето- дики пульсоксиметрии применяют датчик, который включает в себя два элемента: в первом располага- ются два излучателя, испускающих свет в красном и инфракрасном диапазонах, а во втором, расположен- ном с противоположной стороны, находится фото- детектор, регистрирующий интенсивность светового потока, попадающего на него [6]. Определяя разницу между количеством поглощаемого света в систолу и диастолу, существует возможность определить пока- затели артериальной пульсации. Исходя из изложен- ного выше, проводя пульсоксиметрию, можно вычис- лить сатурацию, которая определяется соотношением количества оксигемоглобина к общему количеству гемоглобина и выражается в процентах. В норме ее диапазон составляет 95-100%. Невзирая на то, что са- турация взаимосвязана с его парциальным давлением в крови, зависимость эта не является линейной. в то время как дезоксигемоглобин больше поглощает красный свет. Таблица 1 Таблица 1 Области применения пульсоксиметрии Возрастная группа/нозология Возможности Примечание Новорожденные Раннее выявление критических врожденных пороков сердца Высокая специфичность; Целесообразно использовать в дополнении к антенатальному ультразвуковому исследованию и физикальному обследованию ХОБЛ Выявление признаков гипоксемии Метод рекомендован у всех пациентов с ХОБЛ, в т.ч. для определения показаний к проведению дополнительной оксигенотерапии БА Использование в качестве одного из критериев тяжести обострения Метод рекомендован у всех пациентов с БА при лечении обострения Внебольничная пневмония Выявление пациентов с гипоксемией, нуждающихся в респираторной поддержке Метод рекомендован у всех пациентов с внебольничной пневмонией ОРДС Использование в определении степени тяжести синдрома Метод рекомендован у пациентов с ОРДС Хроническая сердечная недостаточность Определение показаний к проведению оксигенотерапии Метод рекомендован у пациентов с острой декомпенсацией хронической сердечной недостаточности Сокращения: БА – бронхиальная астма, ОРДС – острый респираторный дистресс-синдром, ХОБЛ – хроническая обструктивная болезнь легких. Таблица 1 Области применения пульсоксиметрии Возрастная группа/нозология Возможности Примечание Новорожденные Раннее выявление критических врожденных пороков сердца Высокая специфичность; Целесообразно использовать в дополнении к антенатальному ультразвуковому исследованию и физикальному обследованию ХОБЛ Выявление признаков гипоксемии Метод рекомендован у всех пациентов с ХОБЛ, в т.ч. для определения показаний к проведению дополнительной оксигенотерапии БА Использование в качестве одного из критериев тяжести обострения Метод рекомендован у всех пациентов с БА при лечении обострения Внебольничная пневмония Выявление пациентов с гипоксемией, нуждающихся в респираторной поддержке Метод рекомендован у всех пациентов с внебольничной пневмонией ОРДС Использование в определении степени тяжести синдрома Метод рекомендован у пациентов с ОРДС Хроническая сердечная недостаточность Определение показаний к проведению оксигенотерапии Метод рекомендован у пациентов с острой декомпенсацией хронической сердечной недостаточности Сокращения: БА – бронхиальная астма, ОРДС – острый респираторный дистресс-синдром, ХОБЛ – хроническая обструктивная болезнь легких. Области применения пульсоксиметрии Различные особенности проведения пульсокси- метрии в первую очередь исследовались на пациен- тах, находящихся в отделениях реанимации и интен- сивной терапии. снижается [18]. По данным Perkins GD, et al. (2003), при исследовании 41 пациента, находившихся в отде- лении интенсивной терапии, и анализе 1085 парных измерений (неинвазивным и инвазивным способом) была продемонстрирована умеренная корреляция между данными пульсоксиметрии и инвазивным определением сатурации с помощью СО-оксиметра (r=0,6; p<0,01) [3]. Авторы показали, что методика пульсоксиметрии имела тенденцию к завышению истинных значений сатурации, определенных ин- вазивным способом [3]. Сравнение вышеуказанных методов измерения сатурации проводилось также в проспективном обсервационном исследовании Van de Louw A, et al. (2001), в которое было включено 102 пациента, находившихся в отделении интенсив- ной терапии [19]. Сущность метода пульсоксиметрии Пульсоксиметрия — неинвазивный метод оценки степени насыщения крови кислородом, т.е. процент- ного содержания оксигемоглобина в артериальной крови (сатурации). Принцип метода основан на спо- собности связанного и несвязанного с кислородом гемоглобина абсорбировать световые волны различ- ного диапазона [6]. Первые сведения о возможности использования пульсоксиметрии датируются 1874г, один из первых подобных приборов был создан в 1936г, а первый пульсоксиметр — в 1975г [7]. Как известно, оксигемоглобин способен в большей мере абсорбировать свет в инфракрасном диапазоне, 60 60 ПЕРЕДОВАЯ СТАТЬЯ ОБЗОРЫ ЛИТЕРАТУРЫ Таблица 2 Таблица 2 Особенности методов пульсоксиметрии Особенности метода Трансмиссионная пульсоксиметрия Рефракционная пульсоксиметрия Характеристика метода Регистрация светового потока, который проникает сквозь ткани Регистрация светового потока, который отражается от ткани Основные места наложения датчиков Палец, крыло носа, мочка уха Лоб Возможность выбора места исследования Отсутствует Имеется Необходимость располагать излучающий и отражающий датчики симметрично друг напротив друга Имеется Отсутствует Использование при наличии лака, клея на ногтях, деформации ногтевых пластин Ограничено (для пальцевых датчиков) Возможно (для ушных датчиков) Возможно Фиксация датчика Чаще всего не затруднена Может быть затруднена Особенности методов пульсоксиметрии Особенности методов пульсоксиметрии ксиметрии является необходимость правильного расположения датчика для корректного определения значений сатурации, обе части датчика должны рас- полагаться симметрично по отношению друг к другу. Рефракционная (отраженная) пульсоксиметрия основана на регистрации световых волн, которые не поглощаются оксигенированным гемоглобином и отражаются от ткани. Данный метод удобен возмож- ностью применения на разнообразных участках тела, где расположить датчики симметрично друг напро- тив друга не представляется технически возможным, или расстояние между датчиками будет превышать возможность для регистрации светового потока, на- пример, на животе, лице, предплечье, плече. К пре- имуществам рефракционной пульсоксиметрии от- носится возможность выбора места исследования, отсутствие необходимости располагать излучающий и отражающий датчики друг напротив друга, а также возможность использования при деформации ногте- вых пластин, наличии лака для ногтей, в отличие от пальцевых трансмиссионных датчиков. Однако при использовании рефракционной пульсоксиметрии определенным ограничением могут стать трудности фиксации датчиков при некоторых вариантах их рас- положения, например, на коже лба. Основные особенности методов пульсоксиметрии может привести к различиям в показаниях вышеука- занных методов, которые могут иметь клиническое значение и обусловливать различную тактику веде- ния пациента. Также необходимо принимать во вни- мание тот факт, что вероятность ошибки измерения при проведении пульсоксиметрии низкая при исход- ных значениях сатурации >90%, однако она возрас- тает при снижении ее уровня <90% и значительно возрастает при значениях ниже 70%, ввиду отсут- ствия контрольных значений. Такие низкие значе- ния сатурации довольно часто могут наблюдаться у пациентов, находящихся в отделении интенсивной терапии. С другой стороны, в вышеуказанных от- делениях мониторинг сатурации часто проводится в т.ч. с использованием инвазивных методик. При использовании методики пульсоксиметрии в амбу- латорной практике при наблюдении за пациентами вероятность наличия исходных значений сатурации <90% небольшая, следовательно, ценность этой ме- тодики возрастает. ксиметрии является необходимость правильного расположения датчика для корректного определения значений сатурации, обе части датчика должны рас- полагаться симметрично по отношению друг к другу. полагаться симметрично по отношению друг к другу. Таблица 1 В целом авторы получили низкую вероятность ошибки в 0,02%, при этом стандартное отклонение различий измерений составило 2,1%, 95% ДИ находился в диапазоне от -4,22% до 4,18% [19]. Так, в обзоре Wick KD, et al. (2022) авторы приш- ли к заключению, что применение пульсоксиметрии для постановки диагноза, а также для лечения паци- ентов с острым респираторным дистресс-синдромом может способствовать более раннему его распозна- ванию во всем мире [16]. Rackley CR в своей работе (2020) сделал вывод, что пульсоксиметрия является точным средством контроля у пациентов, подвергаю щихся механической вентиляции легких, принимая во внимание тот факт, что у них может наблюдаться быстрое ухудшение состояния ввиду развития дыха- тельной недостаточности [17]. Сведения о некоторых областях применения пуль- соксиметрии представлены в таблице 1. В целом необходимо отметить, что применение пульсоксиметрии у пациентов с различными заболе- ваниями обусловлено необходимостью раннего вы- явления гипоксемии, что может помочь в диагности- ке, а также определением показаний к проведению оксигенотерапии, что может, в свою очередь, опти- мизировать тактику лечения. В ряде исследований проводилось сравнение дан- ных сатурации, полученных с помощью пульсокси- метрии с инвазивным способом измерения сатура- ции с помощью газоанализатора. Так, в исследовании Rauniyar N, et al. (2020) при изучении 101 пациента, находившихся в блоке интенсивной терапии, было получено, что процент совпадений между вышеука- занными двумя методами составил 83,2%, при этом пульсоксиметрия имела 84,6% чувствительность и 83% специфичность в измерении сатурации [20]. Авторы пришли к выводу, что пульсоксиметрия имеет высокую точность измерения сатурации при исход- ном ее уровне >90% и в этом случае может исполь- зоваться вместо анализа газового состава крови [20]. Сравнение пульсоксиметрии с инвазивными мето- дами В литературе имеются данные, что у пациентов в критическом состоянии средние различия между неинвазивным определением сатурации с помощью пульсоксиметра и инвазивным референсным мето- дом (с помощью CO-оксиметра) были менее чем 2% при исходном значении сатурации 90% и более, при этом стандартное отклонение различий измерений двух вышеуказанных методов было менее, чем 3% [18]. В то же время при сатурации у пациента <90%, величина ошибки измерения этого показателя с по- мощью пульсоксиметра увеличивается, а точность — В целом следует отметить, что по данным боль- шинства исследований, пульсоксиметры имеют не- большую ошибку измерения сатурации, по сравне- нию с инвазивными методами, однако достаточно высокие значения доверительных интервалов, что В целом следует отметить, что по данным боль- шинства исследований, пульсоксиметры имеют не- большую ошибку измерения сатурации, по сравне- нию с инвазивными методами, однако достаточно высокие значения доверительных интервалов, что 61 Российский кардиологический журнал 2023; 28 (3S) Таблица 2 Рефракционная (отраженная) пульсоксиметрия основана на регистрации световых волн, которые не поглощаются оксигенированным гемоглобином и отражаются от ткани. Данный метод удобен возмож- ностью применения на разнообразных участках тела, где расположить датчики симметрично друг напро- тив друга не представляется технически возможным, или расстояние между датчиками будет превышать возможность для регистрации светового потока, на- пример, на животе, лице, предплечье, плече. К пре- имуществам рефракционной пульсоксиметрии от- носится возможность выбора места исследования, отсутствие необходимости располагать излучающий и отражающий датчики друг напротив друга, а также возможность использования при деформации ногте- вых пластин, наличии лака для ногтей, в отличие от пальцевых трансмиссионных датчиков. Однако при использовании рефракционной пульсоксиметрии определенным ограничением могут стать трудности фиксации датчиков при некоторых вариантах их рас- положения, например, на коже лба. Методики пульсоксиметрии Таблица 3 Сравнительная характеристика многоразовых и одноразовых датчиков для проведения пульсоксиметрии Особенность Многоразовый датчик Одноразовый датчик Место наложения датчика Палец, ухо, крыло носа Палец, лоб Вариант исполнения Датчик-прищепка или резиновый Адгезивный датчик с пластырем Возможность повреждения Высокая (особенно для датчика-прищепки) Низкая Соотношение цена/качество Более высокое, особенно в амбулаторных условиях (в случае необходимости однократного измерения сатурации кислорода большому числу пациентов) Более низкое (невозможность измерений сатурации кислорода у разных пациентов одним и тем же датчиком) Скорость наложения датчика Более быстрая Более медленная Возможность регистрации сатурации с разных участков тела в случае получения низкоамплитудных волн Имеется Отсутствует Риск передачи инфекции Более высокий Низкий Надежность крепления датчика Менее надежно Более надежно (особенно при движении пациента) Возможность использования при наличии вазоконстрикции Ограничена Имеется Сравнительная характеристика многоразовых и одноразовых датчиков для проведения пульсоксиметрии всего используются адгезивные датчики или датчики меньшего размера. В ряде случаев датчик-прищеп- ка для взрослых может использоваться на большом пальце у ребенка [10]. Сравнение между собой одно- разовых и многоразовых датчиков представлено в таблице 3 [21]. Методики пульсоксиметрии На современном этапе в медицине нашли при- менение два способа пульсоксиметрии: трансмис сионная и рефракционная. Проведение трансмиссион- ной пульсоксиметрии основано на способности про- никновения светового потока через ткани человека, в связи с этим для определения значений сатурации излучатель и воспринимающий датчик должны рас- полагаться строго на противоположных сторонах, между которыми должна находиться исследуемая об- ласть. Для комфортного проведения данного иссле- дования необходимо накладывать датчики на такие небольшие участки тела, как палец, мочка уха, кры- ло носа. Однако при проведении трансмиссионной пульсоксиметрии могут наблюдаться ряд ограниче- ний при использовании пальцевого датчика. К ним относятся накладные ногти, лак для ногтей, изме- нения ногтевого ложа при некоторых заболеваниях (симптом "барабанных палочек", симптом "часовых стекол"). В такой ситуации более предпочтительным является применение ушного датчика. Также одним из ограничений трансмиссионного метода пульсо Основные особенности методов пульсоксиметрии представлены в таблице 2. В настоящее время существует достаточно боль- шое количество различных датчиков для проведения пульсоксиметрии, которые могут иметь различные конструктивные особенности в зависимости от места их размещения, например, на пальце, на ухе, на коже лба (обычно используется датчик-прищепка и рези- новый датчик), используемой длины волны излуча- телей, возрастной категории пациентов (взрослый, новорожденный, младенец), санитарной обработке (одно- и многоразовые). Многоразовые датчики обычно исполнены в ви- де зажимов (прищепок), которые крепятся на палец, мочку уха, крыло носа, одноразовые, как правило, представлены адгезивными датчиками, которые при- клеиваются на палец или лоб пациента. У детей чаще 62 62 ПЕРЕДОВАЯ СТАТЬЯ ОБЗОРЫ ЛИТЕРАТУРЫ — неправильное позиционирование датчика (на- рушается симметрия, путь световых волн из двух све- тодиодов неодинаковый); (2007), подтвердивших, что использование рефракционного пульсоксиметра с креплением на лбу сопровождается меньшей вероятностью ошибок измерения и большей точностью, по сравнению с традиционным пальцевым пульсоксиметром [25, 26]. Также определенный интерес представляет исполь- зование внутренних датчиков для рефракционной пульсоксиметрии, например, пищеводных датчиков, которые вводятся с помощью зондов [27, 28]. В двух вышеуказанных исследованиях результаты, получен- ные с помощью пищеводного датчика, хорошо соот- носились с результатами СО-оксиметра, а также име- ли меньшую погрешность измерений, по сравнению с пальцевым трансмиссионным пульсоксиметром. Авторы пришли к выводу, что такие датчики могут успешно применяться как альтернатива датчикам с традиционными местами крепления, особенно у па- циентов с плохой периферической перфузией, в т.ч. у пациентов с распространенными ожогами, или же при проведении открытых сердечно-сосудистых опе- раций и операций на грудной клетке. Также в лите- ратуре имеются сведения об использовании прямо- го измерения, а также мониторирования сатурации с поверхности внутренних органов, таких как пи- щевод, желудок, кишечник, печень, для оценки их перфузии при проведении открытых хирургический операций [29], что также может использоваться как альтернатива традиционной пульсоксиметрии. Таким образом, при сравнении трансмиссионных датчиков пульсоксиметрии с креплением на палец и ухо, а также рефракционного датчика с креплением на лоб, использование последнего представляется более предпочтительным у большинства пациентов, особенно при ухудшении периферической циркуля- ции, а также при необходимости мониторирования сатурации у пациентов, у которых предполагается быстрое ее изменение, например, во время хирур- гических операции под общим наркозом. В каче- стве примера можно назвать операцию коронарного шунтирования, во время которой может наблюдать- ся быстрое изменение сатурации кислорода, а также наблюдается периферическая гипоперфузия. Более предпочтительное использование датчика с крепле- нием на лбу при наличии у пациента гипоперфузии обуславливается тем, что при таком расположе- нии датчик регистрирует сатурацию с надглазнич- ной артерии, которая имеет обильный кровоток и меньше подвержена вазоконстрикции, в отличии от периферических артерий. При сравнении между собой пальцевого и ушного датчиков, последний представляется более предпочтительным выбором вследствие более быстрой реакции на изменение сатурации кислорода и меньшей зависимости от периферической перфузии. Кроме того, ушной датчик и датчик с фиксацией на лбу лишены недо- статка пальцевого датчика — затруднение измере- ния сатурации при использовании пациентами лака для ногтей, накладных ногтей, а также деформации ногтевых пластин при ряде заболеваний/состояний (например, при симптоме барабанных палочек и ча- совых стекол). При проведении открытых хирургических опе- раций можно использовать прямое измерение са- турации различных органов, что может дать хирур- гу дополнительную информацию об их функцио- нировании. Анемия. Далее рассмотрим другое ограничение использования метода пульсоксиметрии — наличие у пациента анемии. — неправильное позиционирование датчика (на- рушается симметрия, путь световых волн из двух све- тодиодов неодинаковый); (2018) при изучении 15 здо- ровых добровольцев было показано, что пальцевой датчик был чувствительным к развитию перифериче- ской гипоперфузии, которая моделировалась путем — яркий свет, направленный на прибор (в пуль- соксиметрах используется фотодетектор); — движения и дрожь пациента (затруднение де- текции сигнала); — периферическая гипоперфузия при шоке, ги- потермии, гиповолемии (уменьшение или исчезно- вение пульсовой волны); — нарушения ритма сердца (затруднение воспри- ятия пульсового сигнала); — отравление угарным газом, например, при по- жаре (монооксид углерода вытесняет кислород и об- разует карбоксигемоглобин — ярко-красное соеди- нение); — анемия (поглощение света зависит от концен- трации гемоглобина); — выраженная трикуспидальная регургитация (определение венозной сатурации); 63 Российский кардиологический журнал 2023; 28 (3S) го измерения, а также мониторирования сатурации с поверхности внутренних органов, таких как пи- щевод, желудок, кишечник, печень, для оценки их перфузии при проведении открытых хирургический операций [29], что также может использоваться как альтернатива традиционной пульсоксиметрии. локальной гипотермии в 10 С [23]. В этом исследо- вании локальная гипотермия приводила к падению сатурации, измеренной с помощью пальцевого пуль- соксиметра, менее 90% у 5 добровольцев, особенно в последние 4 мин воздействия (из 10) по сравнению с 1 добровольцем при использовании внутриушного пульсоксиметра [23]. При использовании рефракци- онной пульсоксиметрии возможно использовать дру- гие локализации для позиционирования датчиков, например, кожу лба. В исследовании Choi SJ, et al. (2010) при сравнении между собой трансмиссион- ного пальцевого датчика, а также рефракционного датчика с размещением на лбу у двух пациентов, у которых применялся общий наркоз, было показано, что после эпизода апноэ время наступления деса- турации кислорода до 95% составляло 82,0 с (IQR: 67,0-98,5) vs 94,0 с (IQR: 84,0-106,5) (p<0,001), а де- сатурации до 90% — 94,0 с (IQR: 75,5-109,5) vs 100,0 с (IQR: 84,5-114,5) (p<0,001) для рефракционного и трансмиссионного пульсоксиметра, соответственно [24]. Рефракционный пульсоксиметр в данном ис- следовании также быстрее реагировал и на ресатура- цию, которая возникала при дыхании 100% кислоро- дом через маску, по сравнению с трансмиссионным пульсоксиметром (23,2±5,6 vs 28,9±7,6 с; p<0,001). По результатам проведенного исследования авторы указывают на то, что в ситуациях, когда предпола- гаются быстрые изменения сатурации, например, при проведении операций в условиях общего нарко- за, предпочтительным будет выбор рефракционного пульсоксиметра в связи с его более быстрым откли- ком на де- и ресатурацию кислорода [24]. В литера- туре также имеются данные исследований, прове- денных Fernandes N, et al. (2007), а также Schallom L, et al. — неправильное позиционирование датчика (на- рушается симметрия, путь световых волн из двух све- тодиодов неодинаковый); — неправильное позиционирование датчика (на- рушается симметрия, путь световых волн из двух све- тодиодов неодинаковый); — наличие лака на ногтях, накладные ногти, де- формация ногтевых пластин (затруднение прохожде- ния светового потока); Кроме того, существуют различные варианты ис- полнения и самих пульсоксиметров, они могут быть напалечными, ручными и стационарными, а также обычными и цифровыми, и т.д. — внешнее электромагнитное излучение (могут возникать помехи вследствие влияния электрома гнитных полей, например, от физиотерапевтической аппаратуры и т.д.). Ограничения и недостатки пульсоксиметрии Таким образом, на результаты измерений сатура- ции с помощью пульсоксиметрии могут влиять раз- личные факторы, которые, в некоторых случаях, сле- дует учитывать в клинической практике. Как известно, пульсоксиметрия — это непрямой неинвазивный метод оценки легочной вентиляции, следовательно, она не дает представление об уровне парциального давления углекислого газа (PaCO2). Таким образом, при проведении пульсоксиметрии невозможно оценить степень выраженности гипер- капнии, что может оказать влияние на результаты из- мерений. Также на показания пульсоксиметра могут повлиять следующие основные факторы [6, 10, 21]: Позиционирование датчика пульсоксиметрии. В ли- тературе имеются данные, что ушной датчик пуль- соксиметра быстрее реагирует на изменение сату- рации, по сравнению с пальцевым датчиком [22]. Авторы вышеуказанного исследования при обследо- вании 7 мужчин в возрасте от 18 до 35 лет показали, что средняя задержка между появлением наимень- ших значений сатурации при использовании ушных и пальцевых датчиков пульсоксиметра составила 15±3,5 с, при этом, когда ушные датчики показывали наименьшее ее значение (78±3,5%), пальцевые дат- чики в этот момент демонстрировали гораздо более высокие цифры (94,6±3,5%) [22]. В этом исследова- нии снижение сатурации у участников происходи- ло на фоне задержки дыхания на 60 сек. Поскольку апноэ провоцирует развитие вазоконстрикции, ис- пользование ушных датчиков для проведения пуль- соксиметрии в данном случае является предпочти- тельным ввиду более высокой точности измерения. В работе Budidha K, et al. — неправильное позиционирование датчика (на- рушается симметрия, путь световых волн из двух све- тодиодов неодинаковый); Как известно, поглощение света при проведении пульсоксиметрии зависит от кон- центрации гемоглобина, таким образом, низкие его значения могут приводить к искажениям результатов данного метода. При анемии, как известно, требуется более высокий уровень кислорода для обеспечения его транспорта, таким образом, у данных пациентов показатель сатурации может быть неоправданно за- вышенным. В исследовании Perkins GD, et al. (2003) авторы продемонстрировали, что наличие анемии 64 64 ПЕРЕДОВАЯ СТАТЬЯ ОБЗОРЫ ЛИТЕРАТУРЫ хоть и увеличивало вероятность ошибки измерения сатурации в большую сторону, однако данная вероят- ность увеличивалась незначительно [3]. В исследова- нии Osborn ZT, et al. (2019) было показано, что мето- дику пульсоксиметрии можно использовать в качестве скрининга на наличие анемии у пациентов, причем чувствительность и специфичность данной методики оказались 81,6% и 75,4%, соответственно [30]. хоть и увеличивало вероятность ошибки измерения сатурации в большую сторону, однако данная вероят- ность увеличивалась незначительно [3]. В исследова- нии Osborn ZT, et al. (2019) было показано, что мето- дику пульсоксиметрии можно использовать в качестве скрининга на наличие анемии у пациентов, причем чувствительность и специфичность данной методики оказались 81,6% и 75,4%, соответственно [30]. -0,3 до -3; p=0,03), что, скорее всего, не будет иметь самостоятельного значения в клинической практи- ке [32]. В то же время некоторые авторы указыва- ют на возможную переоценку значений сатурации у пациентов с серповидно-клеточной анемией при образовании большого количества карбоксигемогло- бина вследствие гемолиза эритроцитов, однако эти данные оказались противоречивыми. Таким образом, исходя из данных проведенных исследований, наличие анемии, вероятнее всего, не будет приводить к значительному увеличению веро- ятности ошибки определения сатурации с помощью пульсоксиметрии. В то же время при проведении пульсоксиметрии пациентам с выраженной анеми- ей, а также признаками гипоксии, т.е. исходным ожидаемым низким значением сатурации, оцени- вать эти данные необходимо аккуратно, принимая во внимание увеличение вероятности ошибок из- мерения пульсоксиметров при исходных значениях сатурации <90%. Путем решения вышеуказанной проблемы с неко- торым возможным увеличением количества ошибок в определении сатурации у пациентов с качественны- ми изменениями гемоглобина может быть использо- вание инвазивного измерения сатурации с помощью современных СО-оксиметров, которые используют множество различных длин волн света, тем самым позволяя измерять концентрацию оксигемоглобина, дезоксигемоглобина, карбоксигемоглобина и метге- моглобина [31]. В то же время следует отметить, что некоторые современные пульсоксиметры способны измерять метгемоглобин и карбоксигемоглобин. Так, в исследовании Barker SJ, et al. (2006) использование пульсоксиметра Masimo Rad-57 позволяло измерять уровень карбоксигемоглобина с погрешностью в ±2% с разбросом от 0% до 15%, а также уровень мет- гемоглобина с погрешностью в 0,5% и разбросом от 0% до 12% [33]. Качественные изменения гемоглобина. Таблица 4 Таблица 4 Характеристика основных факторов, которые могут влиять на измерение сатурации кислорода при проведении пульсоксиметрии Фактор, приводящий к неправильному измерению сатурации кислорода пульсоксиметром Причина Примеры Влияние на измерение сатурации Возможности преодоления действия фактора Уменьшение или исчезновение пульсовой волны Периферическая гипоперфузия Вазоконстрикция Гиповолемия Гипотермия Шок Низкие значения или невозможность измерения Использование ушного датчика или датчика с креплением на лоб Наличие в крови других форм гемоглобина, имеющих аномальный спектр поглощения Качественные изменения гемоглобина Высокая концентрация карбоксигемоглобина, метгемоглобина, сульфгемоглобина, гемоглобина-S Ложно-нормальные или ложно-высокие значения при отравлениях угарным газом. Возможны ложно-низкие или ложно-высокие значения при метгемоглобинемии, сульфгемоглобинемии. Возможны ложно-низкие значения сатурации при серповидно-клеточной анемии, особенно при кризах Использование инвазивных способов измерения сатурации, в т.ч. современных СО-оксиметров Высокий уровень кислорода для обеспечения его транспорта Количественные изменения гемоглобина и эритроцитов Анемия Ложно-высокие значения Использование инвазивных способов измерения сатурации Затруднение прохождения светового потока Лак для ногтей, деформация ногтей Черный, коричневый лак для ногтей, накладные ногти, деформация ногтей при симптоме барабанных палочек и часовых стекол Ложно-низкие значения при использовании пальцевых датчиков (особенно устаревших датчиков) Использование ушного датчика или датчика с креплением на лоб Регистрация венозных пульсаций пульсоксиметром Низкая сатурация венозной крови Трикуспидальная регургитация, артериовенозные шунты Сепсис Ложно-низкие или ложно- высокие значения Использование инвазивных способов измерения сатурации Попадание дополнительного света на фотодетектор или увеличение количества света, проходящего сквозь ткани Яркий свет, направленный на прибор Измерения при ярком свете Ложно-высокие или ложно- низкие значения Ограничение попадания света на пульсоксиметр/ пациента Механические колебания датчика Движение и дрожь пациента Физическая нагрузка Паркинсонизм Ложно-низкие значения Измерение сатурации в состоянии покоя, использование датчика с креплением на лоб Затруднение восприятия светового сигнала Нарушения ритма сердца Фибрилляция, трепетание предсердий, частая экстрасистолия и т.д. Ложно-низкие значения Использование инвазивных способов измерения сатурации Несимметричное расположение частей датчика (датчиков) друг относительно друга Неправильное расположение трансмиссионного датчика Ошибки при расположении датчика, механическая неисправность датчика Ложно-низкие значения Использование рефракционных датчиков, изменения положения трансмиссионного датчика рактеристика основных факторов, которые могут влиять на измерение сатурации кислорода при проведении пульсоксиметрии щихся способов определения сатурации, их преиму- ществ и недостатков. щихся способов определения сатурации, их преиму- ществ и недостатков. лей пульсоксиметров дополнительными функция- ми, например, определением частоты дыхания для всесторонней оценки дыхательной недостаточности и определения ее возможных причин. В плане пер- спективы разработки темы стоит отметить необходи- мость выполнения поисковых прикладных исследо- ваний по определению новых и оптимальных мето- дик пульсоксиметрии с учетом накопленного опыта по использованию в клинической практике имею- — неправильное позиционирование датчика (на- рушается симметрия, путь световых волн из двух све- тодиодов неодинаковый); К ограни- чениям метода пульсоксиметрии можно отнести не только количественные, но и качественные изме- нения гемоглобина, например, наличие у пациента высокой концентрации в крови карбоксигемогло- бина, а также метгемоглобина. Традиционные пуль- соксиметрические датчики используют две длины волны — 660 и 940 нм для того, чтобы определить соотношение между окси- и дезоксигемоглобином на основании их различных спектров поглощения [31]. Таким образом, точность измерения сатурации с помощью пульсоксиметрии может быть нарушена в присутствии других форм гемоглобина, имеющих аномальный спектр поглощения. Например, погло- щение карбоксигемоглобина аналогично оксигемо- глобину при длине волны 660 нм. Следовательно, при высоких концентрациях в крови карбоксигемо- глобина, наблюдающиеся, например, у людей, по- страдавших при пожаре, показатели сатурации при проведении пульсоксиметрии будут неоправданно завышены [31]. Также наличие у пациента в крови высокой концентрации метгемоглобина при метге- моглобинемии будет приводить к его поглощению при длинах волн 660 и 940 нм, сопровождаясь завы- шенными значениями сатурации в данном случае. Теоретически, к ошибкам в измерении сатурации может приводить использование пульсоксиметрии у пациентов с серповидно-клеточной анемией. При этом заболевании у пациентов образуется аномаль- ный гемоглобин — S, неизмерение которого при проведении пульсоксиметрии может приводить к ложному снижению сатурации, что было подтверж- дено в исследовании Fitzgerald RK, et al. (2001) [32]. С другой стороны, хотя недооценка истинного значе- ния сатурации в данном исследовании имела место, однако ошибка составила всего -1,6% (95% ДИ: от Трикуспидальная регургитация. Еще одним огра- ничением метода пульсоксиметрии может быть ее использование у пациентов с выраженной трикуспи- дальной регургитацией. Как известно, в данном слу- чае у пациентов возникает застой крови в большом круге кровообращения с появлением венозной пуль- сации (положительный венный пульс). В этом случае наполнение артерий и вен будут происходить в одно и то же время и, следовательно, датчик пульсокси- метра в данном случае будет регистрировать в т.ч. венозную пульсацию и, соответственно, сатурацию. Как известно, венозная сатурация меньше арте риальной, следовательно, у пациентов с выраженной трикуспидальной регургитацией можно ожидать не- оправданное низкое значение показателя сатурации [34]. Для преодоления данного ограничения у таких пациентов рекомендуется использовать инвазивные методы оценки сатурации. Характеристика основных факторов, которые мо- гут влиять на измерение сатурации при проведении пульсоксиметрии, представлена в таблице 4. Принимая во внимание ограничения применения "классической" пальцевой пульсоксиметрии, необхо- дим поиск новых возможных вариантов для опре- деления сатурации и разработка соответствующих девайсов для их реализации. Особенно актуальной эта проблема представляется в свете необходимости импортозамещения и достижения технологического суверенитета, в т.ч. в сфере медицинских изделий. — неправильное позиционирование датчика (на- рушается симметрия, путь световых волн из двух све- тодиодов неодинаковый); Важным является дополнение существующих моде- 65 Российский кардиологический журнал 2023; 28 (3S) Литература/References Possibilities of pulse oximetry application in clinical practice (literature review). 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Respir Care. 2020;65(6):832-46. doi:10.4187/respcare.07812. 17. Rackley CR. Monitoring During Mechanical Ventilation. Respir Care. 2020;65(6):832-46. doi:10.4187/respcare.07812. 5. Sigal ZM, Bryndin VV, Meshchanov SYu, et al. Литература/References Gajnitdinova VV, Avdeev SN, Pozdnyakova AA, et al. Bronchial asthma and COVID-19 in elderly patients: features of the course, survival, predictors of mortality. Pulmonology. 2022;32(2):151-61. (In Russ.) Гайнитдинова В. В., Авдеев С. Н., Позднякова А. А. и др. Бронхиальная астма и COVID-19 у пожилых пациентов: особенности тече- ния, выживаемость, предикторы летальности. Пульмонология. 2022;32(2):151-61. doi:10.18093/0869-0189-2022-32-2-151-161. 25. Fernandez M, Burns K, Calhoun B, et al. Evaluation of a new pulse oximeter sensor. Am J Crit Care. 2007;16:146-52. 26. Schallom L, Sona C, McSweeney M, et al. Comparison of forehead and digit oximetry in surgical/trauma patients at risk for decreased peripheral perfusion. Heart Lung. 2007;36:188-94. doi:10.1016/j.hrtlng.2006.07.007. 27. Pal SK, Kyriacou PA, Kumaran S, et al. Evaluation of oesophageal reflectance pulse oximetry in major burns patients. Burns. 2005;31(3):337-41. doi:10.1016/j.burns.2004.10.025. 10. Bajsultanova RE, Rachina SA, Kotidis IM, et al. The practice of treating community- acquired pneumonia in adult patients with type 2 diabetes mellitus in a multidisciplinary hospital. Pulmonology. 2022;32(4):568-75. (In Russ.) Байсултанова Р. Э., Рачина С. А., Котидис И. М. и др. Практика лечения внебольничной пневмонии у взрослых пациен- тов с сахарным диабетом 2-го типа в многопрофильном стационаре. Пульмонология. 2022;32(4):568-75. doi:10.18093/0869-0189-2022-32-4-568-575. 28. Kyriacou PA. Direct pulse oximetry within the esophagus, on the surface of abdominal viscera, and on free flaps. Anesth Analg. 2013;117(4):824-33. doi:10.1213/ ANE.0b013e3182a1bef6. 29. Kyriacou PA, Hickey M, Phillips JP. Pulse oximetry of body cavities and organs. Annu Int Conf IEEE Eng Med Biol Soc. 2013;2013:2664-7. doi:10.1109/EMBC.2013.6610088. 11. Plana MN, Zamora J, Suresh G, et al. Pulse oximetry screening for critical congenital heart defects. Cochrane Database Syst Rev. 2018;3(3):CD011912. doi:10.1002/14651858. CD011912.pub2. 30. Osborn ZT, Villalba N, Derickson PR, et al. Accuracy of Point-of-Care Testing for Anemia in the Emergency Department. Respir Care. 2019;64(11):1343-50. doi:10.4187/ respcare.06364. 12. Jullien S. Newborn pulse oximetry screening for critical congenital heart defects. BMC Pediatr. 2021;21(Suppl 1):305. doi:10.1186/s12887-021-02520-7. 31. Verhovsek M, Henderson MP, Cox G, et al. Unexpectedly low pulse oximetry measurements associated with variant hemoglobins: a systematic review. Am J Hematol. 2010;85(11):882- 5. doi:10.1002/ajh.21810. 13. Sadykova DI, Sabirova DR, Kustova NV, et al. Pulse oximetric screening for early detection of critical conditions in newborns. Modern science: actual problems of theory and practice. Series: Natural and Technical Sciences. 2017;10:90-3. (In Russ.) Садыкова Д. И., Сабирова Д. Р., Кустова Н. В. и др. Пульсоксиметрический скрининг для раннего выявления критических состояний у новорожденных. Современная наука: актуальные проблемы теории и практики. Серия: Естественные и технические науки. 2017;10:90-3. 32. Заключение Современные методы определения сатурации с помощью пульсоксиметрии в целом хорошо соот- носятся с инвазивными методами оценки данного показателя, что позволяет ее широко использовать в реальной клинической практике. В то же время для 66 66 ПЕРЕДОВАЯ СТАТЬЯ ОБЗОРЫ ЛИТЕРАТУРЫ повышения точности измерения сатурации необхо- димо учитывать имеющиеся ограничения различных методик пульсоксиметрии. Появление и внедрение в клиническую практику методики рефракционной пульсоксиметрии позволяет в значительной мере компенсировать ограничения традиционной транс- миссионной пульсоксиметрии, касающиеся пери- ферической гипоперфузии, невысокого времени отклика, ограничений, связанных с особенностью ногтей пациента. В случае возникновения особых ситуаций, при которых измерение сатурации с по- повышения точности измерения сатурации необхо- димо учитывать имеющиеся ограничения различных методик пульсоксиметрии. Появление и внедрение в клиническую практику методики рефракционной пульсоксиметрии позволяет в значительной мере компенсировать ограничения традиционной транс- миссионной пульсоксиметрии, касающиеся пери- ферической гипоперфузии, невысокого времени отклика, ограничений, связанных с особенностью ногтей пациента. В случае возникновения особых ситуаций, при которых измерение сатурации с по- мощью пульсоксиметрии может оказаться недоста- точно точным (отравление угарным газом, метге- моглобинемия, выраженная анемия, выраженная трикуспидальная недостаточность и т.д.), рекомен- дуется тщательная клиническая оценка пациента, а также контроль данных пульсоксиметрии с по- мощью инвазивных методик. мощью пульсоксиметрии может оказаться недоста- точно точным (отравление угарным газом, метге- моглобинемия, выраженная анемия, выраженная трикуспидальная недостаточность и т.д.), рекомен- дуется тщательная клиническая оценка пациента, а также контроль данных пульсоксиметрии с по- мощью инвазивных методик. Литература/References Fitzgerald RK, Johnson A. Pulse oximetry in sickle cell anemia. Crit Care Med. 2001;29(9):1803-6. doi:10.1097/00003246-200109000-00025. 33. Barker SJ, Curry J, Redford D, et al. Measurement of carboxyhemoglobin and methemoglobin by pulse oximetry: a human volunteer study. Anesthesiology. 2006;105(5):892-7. doi:10.1097/00000542-200611000-00008. Erratum in: Anesthesiology. 2007;107(5):863. 34. Hart GK, Warrillow S. Misleading pulse-oximetry in a patient with tricuspid valve incompetence. Anaesth Intensive Care. 2006;34(2):282-3. 14. Karpova AL, Spivak EM, Pyhanceva AN, et al. Pulse oximetry as a method of early neonatal screening for the presence of critical heart defects in children. Neonatology: news, 67
https://openalex.org/W4379769183	https://jogh.org/wp-content/uploads/2023/06/jogh-13-04044.pdf	English	null	Understanding effective approaches to addressing the common challenges faced by global health networks: Mobilising multi-stakeholder networks to address the upstream determinants of maternal health in five low- and middle-income countries	Journal of global health	2,023	cc-by	11,422	Electronic supplementary material: The online version of this article contains supplementary material. Cite as: Gausman J, Langer A, Jolivet RR. Understanding effective approaches to addressing the common challenges faced by global health networks: Mobilising multi-stakeholder net works to address the upstream determinants of maternal health in five low- and middle-in come countries. J Glob Health 2023;13:04044. © 2023 The Author(s) Electronic supplementary material: The online version of this article contains supplementary material. Cite as: Gausman J, Langer A, Jolivet RR. Understanding effective approaches to addressing the common challenges faced by global health networks: Mobilising multi-stakeholder net works to address the upstream determinants of maternal health in five low- and middle-in come countries. J Glob Health 2023;13:04044. © 2023 The Author(s) Electronic supplementary material: © 2023 The Author(s) © 2023 The Author(s) Cite as: Gausman J, Langer A, Jolivet RR. Understanding effective approaches to addressing the common challenges faced by global health networks: Mobilising multi-stakeholder net works to address the upstream determinants of maternal health in five low- and middle-in come countries. J Glob Health 2023;13:04044. Understanding effective approaches to addressing the common challenges faced by global health networks: Mobilising multi- stakeholder networks to address the upstream determinants of maternal health in five low- and middle-income countries Jewel Gausman , Ana Langer , R Rima Jolivet Jewel Gausman , Ana Langer , R Rima Jolivet Department of Global Health & Population, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA Background Past case studies on global initiatives to address maternal health and survival have focused on global health networks, identifying four essential tasks that define their ability to successfully enact change. We applied the con ceptual framework of global health networks at the country level to organisations sharing concerns on how to address national maternal health and the upstream determinants of maternal survival in five countries and explored how they ad dressed these four essential tasks. Department of Global Health & Population, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA Methods We conducted focus group discussions and key informant interviews with 20 members of national maternal health multi-stakeholder networks in Ban gladesh, India, Mexico, Nigeria, and Pakistan. We drew on the principles and essential components of appreciative inquiry, an assets-based action research methodology that emerged from positivist theories of organisational development to understand how the networks addressed the four tasks. We used a deductive content analysis approach, developing initial themes based on pre-designed codes corresponding to the four tasks faced by global health networks and later identi fying emergent themes in the four areas of the framework. Results We identified themes related to each of the four tasks. Correspondence to: Jewel Gausman, ScD, MHS Department of Global Health and Population, Harvard TH Chan School of Public Health 677 Huntington Ave, Boston, MA 02115 USA jgausman@mail.harvard.edu Electronic supplementary material: Electronic supplementary material: The online version of this article contains supplementary material. Cite as: Gausman J, Langer A, Jolivet RR. Understanding effective approaches to addressing the common challenges faced by global health networks: Mobilising multi-stakeholder net works to address the upstream determinants of maternal health in five low- and middle-in come countries. J Glob Health 2023;13:04044. © 2023 The Author(s) Participants em phasised the need for structure and focus in defining the problem, strengths as sociated with network diversity, and the network’s ability to pivot and redefine the problem to align with other sweeping priorities, such as COVID-19 pandem ic. Themes related to inspiring action centred on aligning the issue with ongoing local and global initiatives, cultivating a sense of group ownership, and defining success incrementally. Themes related to forging alliances emphasised needing to engage high-level leadership, being opportunistic about timing, reducing barri ers to participation by external players, and identifying rewards for participants. Themes related to establishing a governance structure centred on needing strong structure and organisation, cultivating individual commitment, sustaining advo cacy efforts, and obtaining funding. Conclusions Our results demonstrate that challenges commonly faced by global health networks are also relevant to networks operating on a national scale and may offer them strategies for future national networks to consider adopting to address these challenges. Sustainable Development Goal (SDG) 3 focuses on ensuring healthy lives for all [1]. A crucial step to achieving it is ending preventable maternal mortality is critical, which is why target 3.1 is reducing the global maternal mortality ratio to less than 70 per 100 000 live births by the year 2030. 2023 • Vol. 13 • 04044 1 Gausman et al. In 2015, the World Health Organization (WHO) released a report entitled “Strategies toward ending preventable maternal mortality” (EPMM Strategies), which outlined global targets and strategies for re ducing maternal mortality in the 2015-2030 SDGs era [2], with a special focus on human rights and system performance to eliminate disparities in access, quality, and outcomes of maternal care both within and between countries. The strategies highlight 11 EPMM key themes grounded in fundamental human rights principles of equity, non-discrimination, transparency, participation, and accountability. They represent the full, broad spectrum of determinants of maternal health and survival, including so cial/structural, political, economic, and health system-level determinants. A comprehensive monitoring framework tied to each of these themes was developed to track national and global progress in improv ing maternal health [3,4]. PAPERS In September 2017, the Women & Health Initiative of the Harvard T. H. Electronic supplementary material: The online version of this article contains supplementary material. Cite as: Gausman J, Langer A, Jolivet RR. Understanding effective approaches to addressing the common challenges faced by global health networks: Mobilising multi-stakeholder net works to address the upstream determinants of maternal health in five low- and middle-in come countries. J Glob Health 2023;13:04044. © 2023 The Author(s) Chan School of Public Health launched the Improving Maternal Health Measurement Capacity and Use (IMHM) project aimed at help ing countries and global development partners plan, track and accelerate progress towards EPMM by fur ther strengthening the framework’s indicators and their use through a variety of activities. Through the project, seven consultations (known as National Dialogues) were conducted to better understand national priority areas for improvement related to maternal health and to support the adoption and use of EPMM indicators in national-level monitoring frameworks to drive improvement in those self-identified priority areas. Each National Dialogue included approximately 40-50 stakeholders representing expertise for and commitment to each of the 11 EPMM key themes, including representatives from the Ministry of Health, UN and donor agencies, development partners, civil society advocates, and others from outside the health sector. The National Dialogues took place in seven countries: Bangladesh (February 2019), Cote d’Ivoire (November 2018), India (April 2019), Kenya (July 2018), Mexico (July 2019), Nigeria (March 2020), and Pakistan (October 2019). The National Dialogue organisers hoped to use the platform as a catalyst to create a lasting multi-stakehold er network that would continue to drive the achievement of the established priorities and commitments beyond the one-day event. The Dialogues were organised by planning committees consisting of global and country-level project staff, representatives from each country’s Ministry of Health, representatives from UN Agencies and local government entities from relevant sectors outside health, as well as other various ac tive country stakeholders who were selected to reflect the range of issued highlighted in the EPMM 11 key themes; all participants were national actors dedicated to advancing maternal and child health priorities. The planning committees were central to bringing stakeholders from participating countries together to en gage them in the National Dialogues, strengthening commitments and supporting the shared agenda. While the agenda in each National Dialogue was country-specific, the desire to increase the attention paid to the upstream determinants of maternal health was part of a larger, global initiative. Global initiatives to address maternal health and survival have been the subject of past case studies fo cused on understanding global health networks [5,6]. Global health networks are defined as “cross-na tional webs of individuals and organisations linked by a shared concern to address a particular health problem global in scope” [7]. Methodological approach Our methodology is grounded in the principles and essential components of appreciative inquiry (AI), an assets-based action research methodology that emerged in the 1980s out of positivist theories of organisa tional development [10]. Traditional deficit-based methodologies of organisational development focus on “the root causes of failure” [11], viewing organisations as broken entities that are defined by their weaknesses and problems, leading critics to argue that such approaches may become self-fulfilling and hamper future progress [12]. In contrast, AI encourages a deeper understanding of the “root causes of success” [11] to con struct a future reality that embraces positive elements of change. It is based on the idea that understand ing organisational challenges by focusing on achievements, peak experiences, and best practices is a more straightforward approach to identifying ways that organisations can improve than traditional approaches that are characterised by a preoccupation with problems and weaknesses [13]. By engaging stakeholders in transformative dialogue, AI values local knowledge and indigenous solutions first to create a belief that or ganisations have the power to change and then to empower actors to create that change [14]. While outlining a wide variety of approaches, most current literature includes four essential components of AI, which underlie what is known as the “AI 4-D process.” The four “Ds” are discovery (appreciating and valuing the best of what is), dream (envisioning what might be), design (dialoguing what should be), and destiny (innovating what will be) [15]. The implementation of these four components is flexible and has been widely adapted to meet the specific needs of various settings and participants [16]. For each of the four “Ds”, AI emphasises the use of narrative discussion and storytelling through which participants can develop a rich understanding of past experiences and visions for the future [17,18]. AI has been used extensively as an approach to programme evaluation across disciplines, including in appli cations related to health service delivery and system performance involving diverse stakeholder groups [16]. One such example is the Nepal Safer Motherhood Project, designed to change attitudes and improve account ability of health workers; the project used AI to harness group cooperation and to encourage new ideas and learning [14,19]. Elsewhere, AI was used to engage a broad group of actors during a multi-stakeholder con ference focused on eliminating racial disparities in birth outcomes and to set a future agenda for action [20]. Methodological approach We used the principles of AI to understand how the IMHM National Dialogue planning committees ad dressed the four essential tasks that must be realised by successful global health networks. By using a posi tivist rather than a deficit-based approach, we aimed to capture achievements, positive experiences, and best practices with the goal of using these data to fuel continued progress. Rather than framing the four essential tasks of global health networks as challenges or limitations that inevitably impede progress, we used AI to engage the planning committees working to foster and sustain global health networks in their settings in a strength-finding reflection on their assets and positive experiences to date and what future progress may look like. We expected this process to generate important lessons to strengthen the current health networks and inform future programming in countries where the National Dialogues occurred and in those that may wish to undertake similar work in the future. Electronic supplementary material: The online version of this article contains supplementary material. Cite as: Gausman J, Langer A, Jolivet RR. Understanding effective approaches to addressing the common challenges faced by global health networks: Mobilising multi-stakeholder net works to address the upstream determinants of maternal health in five low- and middle-in come countries. J Glob Health 2023;13:04044. © 2023 The Author(s) They simultaneously serve policy, knowledge creation, and advocacy func tions to bring about improvement in their domain of focus [8]. Past research has explored how glob al health networks seeking to address issues related to safe motherhood have encountered difficulty in gaining traction, partially because of issues relating to the severity of the problem on a population level, a lack of widely accepted approaches to measurement and monitoring, and limited consensus on inter vention prioritisation [5]. Four essential tasks have been identified that define the ability of global health networks to successfully enact change: generating consensus on defining the problem and how it should be addressed, positioning the issue in a way that inspires action, forging alliances with players within and outside of the health sector, and establishing a persistent organisational structure to facilitate col lective action on the issue [9]. We aimed to apply the conceptual framework of global health networks by mapping their four essential tasks to national webs of individuals and organisations sharing concerns on how to address national ma ternal health and survival so as to understand its relevance to national-level efforts. By focusing on how the National Dialogue planning committees view the effort to foster and sustain effective health networks in each of their contexts, we compare experiences and lessons learned across countries and explore whether and how they have been able to achieve the four essential tasks facing global health networks, to rally stake holders over time in support of a common objective: the integration of EPMM themes and indicators into national level strategic plans, policies, programming, and/or monitoring frameworks. 2023 • Vol. 13 • 04044 www.jogh.org • doi: 10.7189/jogh.13.04044 www.jogh.org • doi: 10.7189/jogh.13.04044 2 Effective approaches to challenges to global health networks www.jogh.org • doi: 10.7189/jogh.13.04044 Data collection We used mini focus group discussions (FGDs) consisting of three participants per setting [21] and key in formant interviews (KIIs) to maximise stakeholder participation. All FGDs were conducted in English, ex cept for those in Mexico, which were conducted in either Spanish or English, depending on the fluency of the participants. We collected data between March and June 2021. Due to the limitations of the COVID-19 pandemic, all FGDs and KIIs were held remotely using Zoom. The facilitator followed a semi-structured discussion guide based on the four “Ds” to enable participants to reflect on their experiences. The facilita tor had been involved at the global level in supporting the organisation and implementation of the National Dialogues and was known by the participants. Participants were informed that we were interested in con ducting a strength-finding exercise from a positivist perspective, rather than focusing on deficits. Each FGD was organised in a way that aligned with the AI 4-D process and was cantered on the four essen tial tasks of global health networks. For example, during the discovery phase, participants were asked about their experience defining the problem of maternal mortality in their country and how they thought the Na tional Dialogues were effective in addressing it. As part of the dream phase, participants were asked to reflect on how the National Dialogues inspired action towards priority issues related to maternal mortality in their country and what ideas that have fostered continuing progress towards accomplishing country-level goals. 2023 • Vol. 13 • 04044 www.jogh.org • doi: 10.7189/jogh.13.04044 3 Gausman et al. The discussions during the design phase focused on how the National Dialogues forged connections within and outside the health sector, what experiences were most successful, and how existing networks could be expanded to create a stronger alliance. Last, the destiny phase focused on any past and ongoing efforts to establish a formal institutional body or informal network to drive progress in achieving the commitments made in the National Dialogues, while generating ideas for how to strengthen collective action and account ability. KIIs took approximately one hour to complete and FGDs took approximately one and a half hours. d Study participants Due to the COVID-19 pandemic, we only included five of the sev en countries in which the National Dialogues were held (B l d h I di M i Ni i d P ki t ) All of participants in each country in focus group dis informant interviews PAPERS Table 1. Number of participants in each country in focus group dis cussions and key informant interviews Country Number of participants Mode of data collection Bangladesh 4 1 FGD, 1 KII India 7 2 FGDs, 1 KII Mexico 2 2 KIIs Nigeria 3 1 FGD Pakistan 4 1 FGD, 1 KII FGD – focus group discussion, KII – key informant interview Country Number of participants Mode of data collection Bangladesh 4 1 FGD, 1 KII India 7 2 FGDs, 1 KII Mexico 2 2 KIIs Nigeria 3 1 FGD Pakistan 4 1 FGD, 1 KII FGD – focus group discussion, KII – key informant interview Ethical approval We obtained ethical approval from the Institutional Review Board at the Harvard TH Chan School of Pub lic Health. All participants provided verbal informed consent to participate. Data management and analysis We audio/video recorded each FGD and asked the participants to use video when internet connectivity al lowed. The content of each FGD was transcribed and subsequently verified by another member of the re search team for quality assurance. We used a deductive content analysis approach in the data analysis, developing initial themes based on pre-defined codes, designing them to correspond to the four tasks faced by global health networks. Pre-de signed codes were as follows: “Essential Task 1: Generating consensus on defining the problem and how it should be addressed”, “Essential Task 2: Positioning the issue in a way that inspires action”, “Essential Task 3: Forging alliances with players within and outside of the health sector”, and “Essential Task 4: Establish ing some sort of governance or institutional body to facilitate collective action on the issue”. Following the initial coding using pre-designed codes, the data were coded a second time for each of the four challenges identified a priori to identify emergent themes in the four framework areas [22]. We con ducted data analysis using Dedoose [23]. Our research and reporting the Standards for Reporting Qualitative Research (SRQR) [24]. Ethical approval Study participants Study participants were members of the planning committees in countries where the IMHM National Dia logues took place. Planning committees ranged in composition, but typically included members of the gov ernment, academia, non-governmental organisations, United Nations agency counterparts, and maternal health advocates. We purposefully selected the participants to include a range of perspectives. Due to the COVID-19 pandemic, we only included five of the sev en countries in which the National Dialogues were held (Bangladesh, India, Mexico, Nigeria, and Pakistan). All members of the planning committees were invited to par ticipate in either a KII or FGD, depending on their avail ability. We aimed to include at least two participants from each country, but included anyone from the planning committees who expressed interest. Participants were in vited by email, and we followed up once in the event of non-response. Table 1 provides the number of partici pants from each of the five countries. mber of participants in each country in focus group dis d key informant interviews Number of participants Mode of data collection 4 1 FGD, 1 KII 7 2 FGDs, 1 KII 2 2 KIIs 3 1 FGD 4 1 FGD, 1 KII group discussion, KII – key informant interview Study participants were members of the planning committees in countries where the IMHM National Dia logues took place. Planning committees ranged in composition, but typically included members of the gov ernment, academia, non-governmental organisations, United Nations agency counterparts, and maternal health advocates. We purposefully selected the participants to include a range of perspectives. Due to the COVID-19 pandemic, we only included five of the sev en countries in which the National Dialogues were held (Bangladesh, India, Mexico, Nigeria, and Pakistan). All members of the planning committees were invited to par ticipate in either a KII or FGD, depending on their avail ability. We aimed to include at least two participants from of participants in each country in focus group dis informant interviews mber of participants Mode of data collection 4 1 FGD, 1 KII 7 2 FGDs, 1 KII Study participants were members of the planning committees in countries where the IMHM National Dia logues took place. Planning committees ranged in composition, but typically included members of the gov ernment, academia, non-governmental organisations, United Nations agency counterparts, and maternal health advocates. We purposefully selected the participants to include a range of perspectives. www.jogh.org • doi: 10.7189/jogh.13.04044 Essential task 1: Generating consensus on defining the problem and how it should be addressed Essential task 1: Generating consensus on defining the problem and how it should be addressed Theme 1.1: Need for focus and structure Theme 1.1: Need for focus and structure Participants described that starting with a clear focus and applying a structure for deliberations helped them better understand the problem and prioritise next steps for action. Several participants thought that using an evidence-based framework (i.e. the EPMM key themes and indicators) gave participants a com mon understanding on which to begin building consensus on defining the problem of maternal mortality in their own country. 2023 • Vol. 13 • 04044 4 www.jogh.org • doi: 10.7189/jogh.13.04044 Effective approaches to challenges to global health networks In terms of the [EPMM] framework, these are the key things that need to be discussed, and certainly they’ve already been researched, so I think that that was a helpful framework for us to think through [the issue]. – Key informant, Bangladesh In terms of the [EPMM] framework, these are the key things that need to be discussed, and certainly they’ve already been researched, so I think that that was a helpful framework for us to think through [the issue]. – Key informant, Bangladesh Others emphasised that having a shared background framework was especially important given that there was such a wide range of stakeholders present. …that overall understanding was very helpful which put everybody in the same platform, because there were people from different backgrounds. – Key informant, India …that overall understanding was very helpful which put everybody in the same platform, because there were people from different backgrounds. – Key informant, India …that overall understanding was very helpful which put everybody in the same platform, because there were people from different backgrounds. – Key informant, India Participants thought that focus and structure were equally important in developing an action plan or a “road map.” Participants reflected on how building consensus within the group was eased by having: Participants thought that focus and structure were equally important in developing an action plan or a “road map.” Participants reflected on how building consensus within the group was eased by having: …a really focused agenda with some clear short and midterm goal posts and accomplishments. Essential task 1: Generating consensus on defining the problem and how it should be addressed – Key informant, Mexico In Nigeria, participants reflected on how having a clear plan for action supported their advocacy efforts af ter the meeting to get high-level support: In Nigeria, participants reflected on how having a clear plan for action supported their advocacy efforts af ter the meeting to get high-level support: After this meeting, we went back to the [Federal Ministry of Health]... and presented our key feedback from this meeting...he was supportive... – Key informant, Nigeria After this meeting, we went back to the [Federal Ministry of Health]... and presented our key feedback from this meeting...he was supportive... – Key informant, Nigeria Structuring the action plan in a way that it reflected different stakeholders’ strengths was thought to be par ticularly effective in coordinating efforts within the network. Structuring the action plan in a way that it reflected different stakeholders’ strengths was thought to be par ticularly effective in coordinating efforts within the network. [W]e were very able to clearly kind of map that and try to support and help each other so that we can complement each other’s activities, and support the government in achieving their overall goals globally, to which they have committed. – Key informant, Pakistan Further, it was thought that drawing on each other’s strengths to build on repeated efforts by multiple stakeholders was a benefit of having a cohesive, long-term action plan, recognising that part of the goal is to have conversations, especially with government, that can evolve over time as the networks’ activi ties progress. Especially the ministry looks at the evidence and make decisions on the basis of what what’s a priority, then it is important...to sit with them..[to] plan some of these analytical pieces and ask the right questions in that format and then come up with the solutions that could fit...If we are actually looking for how the network or multiple stakeholders can actually come together to mobilize the conversations, which may influence the thinking and the government, then that’s a kind of another route that one can take... and that will be a little longer, and the conversations can still happen. – Key informant, India Theme 1.2: Diversity within the network helped to reshape the problem Theme 1.2: Diversity within the network helped to reshape the problem Similarly, in Bangladesh, the network’s ability to identify new opportunities to come together in the face of COVID-19 reinforced the consensus generated from the meeting: Similarly, in Bangladesh, the network’s ability to identify new opportunities to come together in the face of COVID-19 reinforced the consensus generated from the meeting: COVID is really a very challenging situation for Bangladesh, but it is also true that our history says that when our country has faced some challenge, we actually stood up boldly, so COVID, is actually giving that opportunity to strengthen our digital system... All issues that need to be connected with [priorities identified at the National Dialogue]. And more you can connect... all of us will be in common under standing and [develop] a common way to move. – Key informant, Bangladesh Finally, in Nigeria, participants described that the COVID-19 pandemic made the priorities defined in the National Dialogue more urgent, and by pivoting to focus on the immediate concerns brought about by COVID-19, they were able to position their own agenda in a way that fostered action. A participant de scribed that the members of the network hosted a listening session to better understand the issues faced by health workers and citizens, which ultimately were very closely aligned with the priorities that emerged from the National Dialogue: So that dialogue, the feedback from that [listening session] formed a presentation that we took back to the Ministry of Health, telling them okay, this is what we have heard from the citizens and the health workforce. And it was accepted, the Department of Family Health, accepted that and we are hoping that they are going to act on that submission. – Key informant, Nigeria Essential task 2: Positioning the issue in a way that inspires action Theme 2.1: Aligning with other local and global priorities and strategies Finding ways to align the issues identified within national priorities and strategies was important for the networks to inspire action within themselves to make progress on their action plans. By first coming to gether under a shared problem statement with a joint action plan, the multi-stakeholder networks were able to make greater progress towards meeting their commitments jointly than they would have in acting alone, as it allowed them to leverage a larger programmatic platform while fostering joint ownership. Theme 1.2: Diversity within the network helped to reshape the problem Broad representation from multiple stakeholders with different backgrounds and expertise was thought to enrich how the group defined the problem. Participants with different backgrounds highlighted new dimensions that may not have otherwise been discussed or addressed by the group. Diversity within the group helped to expand the space to engage more players in a meaningful way, as described by a participant in Mexico. In Nigeria, a participant emphasised that having a clear understanding of the issue structured on a framework and built upon a diverse group of stakeholder perspectives made this attempt at cross-sec toral collaboration more successful than past ones due to its creation of a cohesive group with a deep un derstanding of the problem. Planning the EPMM dialogue, emphasized to us the importance of multi-sectoral approach to address ing maternal health, especially in Nigeria. So [in the past], we’ve had cases where different sectors are doing different things, and there is no coherent result. During the planning, we saw the importance of bringing in all the sectors, the Ministries of Data Research and Planning, the Ministries of Health, the Ministries of Education, those in finance, and the Bureau of Statistics themselves, and they actively par ticipated in that meeting. Now, what that has done for us is that since the meeting we’ve had a closer working relationship with the Department of Family Health, in the Ministry of Health...and most of in dicators that came up in our advocacy plan in that meeting, have often resonated, and the discussions [have been] moving forward. – Key informant, Nigeria A diverse network was described as key to fully understanding the problem from multiple angles in a way that supported complex action. 2023 • Vol. 13 • 04044 www.jogh.org • doi: 10.7189/jogh.13.04044 5 Gausman et al. [I]f you want to carry a woman from one place to another, you need to have good roads, right? And so these are the things which are very, very key so you know a lot of these stakeholders need to be there to understand [the issue]... because there are areas where your network does not work. – Key infor mant, India Similarly, involving diverse stakeholders from across sectors was viewed as necessary to bring new ideas into the group to tackle problems. Theme 1.2: Diversity within the network helped to reshape the problem For example, a participant in Nigeria explained, once the issue is estab lished as a priority across the diverse network, achievements can be magnified: So it just shows the wonders of having this kind of dialogues and partnering with different stakeholders, especially people that have this as a priority in their programming. And because they can actually take ideas – people actually come in to look for ideas ...and decide to push these in their different programs. So, for me that was one key learning actually having to work with the stakeholders to see how this is playing out. – Key informant, Nigeria PAPERS Last, having participants in the network from different levels, not only different sectors, also helped enrich the group’s understanding of the issue as it was being defined. I remember in that workshop, the participants’ level was different, I mean there were participants from leadership-level, then managers-level, then mid-level, then also practitioners, so in that sense, there was a [deep] understanding of the issue. – Key informant, Bangladesh I remember in that workshop, the participants’ level was different, I mean there were participants from leadership-level, then managers-level, then mid-level, then also practitioners, so in that sense, there was a [deep] understanding of the issue. – Key informant, Bangladesh a [deep] understanding of the issue. – Key informant, Banglades Theme 1.3: The network’s ability to pivot and stay relevant in the face of new challenges Theme 1.3: The network’s ability to pivot and stay relevant in the face of new challenges Participants from all countries emphasised that the network’s ability to pivot to new issues was fundamen tal. The COVID-19 pandemic was a pervasive example of how the multi-stakeholder networks saw the pri orities identified at the National Dialogues reflected in a new and overwhelming challenge. A participant from India reflected that: Participants from all countries emphasised that the network’s ability to pivot to new issues was fundamen tal. The COVID-19 pandemic was a pervasive example of how the multi-stakeholder networks saw the pri orities identified at the National Dialogues reflected in a new and overwhelming challenge. A participant from India reflected that: All those stakeholders reinitiated dialogue on some of the recommendations from the past to see what new challenges have posed, and how those challenges link to the recommendation from the National Dialogue. www.jogh.org • doi: 10.7189/jogh.13.04044 Theme 2.2: Building on external opportunities to reinvigorate action Participants discussed that taking advantage of opportunities or platforms from outside the network was one way in which they have successfully continued to keep the network focused on realising the actions set out in the meeting. For example, participants said that they leveraged meetings that engage certain members of the network to reinvigorate the discussion on the commitments agreed upon in the National Dialogues. For example, a participant from Nigeria described efforts to engage stakeholders at the regular meeting of a working group focused on a separate initiative: [That working group] brings most of the stakeholders into one meeting. The meeting holds quarterly so we are also able to reenter it... So there’s room for discussions and you know, I remember the last meet ing, we’re able to make reference that “this dialogue happened and this is what was said,”...So the good thing is that we have this network where these stakeholders are actually able to come together and I think somehow the [onus] is on also us to make sure we are able to retrace the learnings from the dia logue. – Key informant, Nigeria Theme 1.2: Diversity within the network helped to reshape the problem So, [the Sustainable Development Goals] SDGs are really kind of landmark thing in Bangladesh...Ma ternal health is not only a Ministry of Family Planning health issue; this is beyond that. It has to cover education, awareness, social safety net, where a poor mother can receive voucher, a maternal health voucher. So, our understanding that this is not a health focused ministry, it goes beyond. So, SDG is ac tually helping us in that. – Key informant, Bangladesh So, [the Sustainable Development Goals] SDGs are really kind of landmark thing in Bangladesh...Ma ternal health is not only a Ministry of Family Planning health issue; this is beyond that. It has to cover education, awareness, social safety net, where a poor mother can receive voucher, a maternal health voucher. So, our understanding that this is not a health focused ministry, it goes beyond. So, SDG is ac tually helping us in that. – Key informant, Bangladesh Theme 1.2: Diversity within the network helped to reshape the problem Further, it caused the issue of maternal health measurement, a topical area that participants described as typically appealing to only a small group of technical stakeholders, to be institutionalised into regular discussions 2023 • Vol. 13 • 04044 www.jogh.org • doi: 10.7189/jogh.13.04044 www.jogh.org • doi: 10.7189/jogh.13.04044 6 Effective approaches to challenges to global health networks when complementary activities were being planned. A participant from Bangladesh described it as a dom ino effect, with one success leading to further ones across the network: when complementary activities were being planned. A participant from Bangladesh described it as a dom ino effect, with one success leading to further ones across the network: All the stakeholders, plus NGOs who are involved…could all be there, then once the indicators are se lected, they could make it as part of their indicators of their projects. So, the government sees that yes, these projects are getting these indicators right, we can improve it within our system also. Because with out some evidence, government won’t suddenly introduce something. – Key informant, Bangladesh Finding a way to align the network’s goals with government priorities came up repeatedly as a way to in spire action. A participant from Nigeria explained that building on a shared agenda had become more im portant in recent years: “Initially, we would say ‘Hey, government, this is what we plan to do!’ We’re doing this!’ but now the question is, what strategic plan does this contribute to and if you can’t find where this contributes, it’s actually hard for you to just come in you know, with your agenda and all of that to drive that.” – Key informant, Nigeria “Initially, we would say ‘Hey, government, this is what we plan to do!’ We’re doing this!’ but now the question is, what strategic plan does this contribute to and if you can’t find where this contributes, it’s actually hard for you to just come in you know, with your agenda and all of that to drive that.” – Key informant, Nigeria Aligning the relevant issues at national level within the context of global priorities was also seen as a strat egy to drive progress towards achieving the network’s agreed upon action plan. Several participants ref erenced the SDGs and how the cross-sectoral focus on them has helped garner attention to the network’s goals across a diverse network. Theme 2.2: Being at the forefront of a wave Network members were more likely to feel motivated when they felt that the issues they were focused on belonged to a process or initiative larger than the network itself. Some participants described this as forging new connections between the network and individuals who were pushing out new information or research on the topics prioritised in the National Dialogue. A participant in Mexico linked this to: …the excitement of being able to work where you feel that you’re at the forefront of a wave. – Key in formant, Mexico Participants in Bangladesh thought that timing the National Dialogue with the increased high-level political attention being given to maternal health was important. In India, one participant gave an example of how the National Dialogue aligned with large scale efforts to establish a cadre of midwives, which enabled the network to engage with an expanded group of technical experts who were pushing that issue while con tinuing to reinforce the consensus that was built during the meeting. Theme 2.3: Defining success in bits and pieces Feeling successful was also thought to be important to keep the network motivated and focused; howev er, several participants acknowledged the challenges involved in defining success in a multi-sectoral group 2023 • Vol. 13 • 04044 7 7 www.jogh.org • doi: 10.7189/jogh.13.04044 Gausman et al. given that successes are not immediately visible and are the result of many different actors. Further, the network’s successes were not typically on a large scale, but rather occurred in a piecemeal fashion. Some participants described that the network would be reinvigorated when small successes were achieved, such as seeing certain priorities or indicators being integrated into government programmes; however, there was recognition that specific attribution was not possible. Maintaining motivation was integral to maintaining action. To achieve this, some participants suggested finding a way to provide feedback to the network when achievements occurred. given that successes are not immediately visible and are the result of many different actors. Further, the network’s successes were not typically on a large scale, but rather occurred in a piecemeal fashion. Some participants described that the network would be reinvigorated when small successes were achieved, such as seeing certain priorities or indicators being integrated into government programmes; however, there was recognition that specific attribution was not possible. Maintaining motivation was integral to maintaining action. Theme 2.2: Being at the forefront of a wave To achieve this, some participants suggested finding a way to provide feedback to the network when achievements occurred. [W]hat I feel is that the process should be a continuous process where whatever the steps have been taken, or whatever the achievements have been gained, they should be displayed or disseminated at certain forum because people... they are involved in the initial process, and then what comes out of that or what prog ress is there, they are not informed, timely, and this causes lack of interest. – Key informant, Pakistan PAPERS Essential task 3: Forging alliances with players within and outside of the health sector Essential task 3: Forging alliances with players within and outside of the heal sector Theme 3.1: Simplifying language to make network appealing to broader audience One strategy echoed by participants across countires was the need to simplify the language used to facili tate understanding across sectors so as to increase broad participation in the network. They agreed that the language used within the health sector and (more narrowly) within groups of stakeholders focused only on the issue of maternal health tends to be overly technical, which ultimately excludes participants or causes those from other sectors to disengage. Because the technical language we use is not the same language they use... so if you want to really talk about these themes and the indicators – cross cutting issues – we have to use very simple language. – Key informant, Bangladesh Theme 3.2: Establishing linkages at the right time with high-level decision makers Engaging high-level leaders, especially individuals with political power or access to financial resources, was thought to be essential to the network’s success. I think a critical success factor was a clear link to decision-makers and people who had access to budget so that it felt like a policy advocacy group where discussions could translate into actionable change that had an impact on the field level. – Key informant, Mexico I think a critical success factor was a clear link to decision-makers and people who had access to budget so that it felt like a policy advocacy group where discussions could translate into actionable change that had an impact on the field level. – Key informant, Mexico However, simply engaging high-level decision makers was not enough; participants described that both strategy and timing were important factors to consider when bringing them into the network. Theme 3.4: Need for catalytic/transactional exchange – Key informant, Pakistan I think that there are a number of forums or networks already available, and all you need to do is to basically, get in touch with them and invite the key person responsible...and then, once they are part of the discussion...you basically form the network... – Key informant, Pakistan the discussion...you basically form the network... – Key informant, Pakistan As an example of a more defined platform, a participant in Mexico described a network structure built on a national programme: [The network] links many institutions that are also law enforcing institutions, so it’s a large multi-sec toral effort. And we’re legally bound by a national program and by the...law and so we’re all working towards that common agenda because we’re legally bound to it and there is actually no funding to co ordinate these efforts, but there is a structure for it, so even though there’s no dedicated funding, there is a structure that allows for the system to work together. – Key informant, Mexico [The network] links many institutions that are also law enforcing institutions, so it’s a large multi-sec toral effort. And we’re legally bound by a national program and by the...law and so we’re all working towards that common agenda because we’re legally bound to it and there is actually no funding to co ordinate these efforts, but there is a structure for it, so even though there’s no dedicated funding, there is a structure that allows for the system to work together. – Key informant, Mexico Theme 2.2: Being at the forefront of a wave Similarly, participants described the need to continually engage in outreach to new institutions and organ isations to keep the network engaged in the field and relevant. Such a strategy was thought to be especially useful in expanding the network beyond even groups that were targeted by outreach. Bring[ing] people [in] from different sectors...help us build new partnerships, which we have contin ued to engage since the [National] Dialogue. It has also made some organizations that don’t even real ly know what we do, really understand...it also amplified what we do and who we are to a lot of oth er organizations. I remember somebody from the Ministry who we haven’t engaged with before came and said, ‘Oh, I really like your organization, you guys seem really organized,’ and he had invited us for subsequent meetings after that... It has helped us build new partnerships; it has also kept us in that space. – Key informant, Nigeria PAPERS Theme 2.2: Being at the forefront of a wave However, simply engaging high-level decision makers was not enough; participants described that both strategy and timing were important factors to consider when bringing them into the network. Regarding strategy, participants referenced the need to be prepared with evidence when approaching poli ticians and other high-level politicians in order to successfully engage them in the network. You know, evidence takes time and they these [politicians] are in a hurry. They come with a plan that you know, ‘we will make changes in a particular area,’ like that, and they try to find out and once they do, they do not get that [research takes time], and then they think that, ‘Okay, there is [no evidence]...’But the problem is that you know, we as technical people can say that, but for politicians, if they have five years and they can’t wait for the evidence to come up... – Key informant, India Timing was also key when engaging high-level decision makers. Timing stakeholder engagement when sim ilar issues were already on the agenda was thought to be an important part of the strategy, and helpful in focusing attention on the issues. The timing of it was a clear direction politically of a consolidated national prioritization for maternal health, which brought a lot of stakeholders together... – Key informant, India The timing of it was a clear direction politically of a consolidated national prioritization for maternal health which brought a lot of stakeholders together Key informant India Theme 3.3: Bringing in new members over time to re-energise the group Participants emphasised that the network should not be stagnant, but should have a strategy to bring in new members from across different sectors over time to re-energise the group’s thinking and inject new ex citement. One person leaves that institution, or a young person graduates and leaves that University, [if] the Uni versity is on board, they’re going to give us another person, and we can involve them, we can institution alize this process. – Key informant, Pakistan 2023 • Vol. 13 • 04044 www.jogh.org • doi: 10.7189/jogh.13.04044 8 Effective approaches to challenges to global health networks Similarly, participants described the need to continually engage in outreach to new institutions and organ isations to keep the network engaged in the field and relevant. Such a strategy was thought to be especially useful in expanding the network beyond even groups that were targeted by outreach. www.jogh.org • doi: 10.7189/jogh.13.04044 Theme 3.4: Need for catalytic/transactional exchange A final way participants described for bringing in new members to the network was to ensure opportu nities for members to get something tangible back for their participation. While generally membership in the network came from being passionate about the issue, it was also recognised that it had a transactional component. One of the important areas is the question that people might ask, especially in the private sector, NGOs, and the media, is ‘What is in it for them?’ This is a sort of a question that we normally face, that why should they spend time with public sector people if there’s nothing for them? – Key informant, Ban gladesh Another participant in Mexico described that clear rewards for participation in networks was an importan incentive for members to join and stay engaged: Another participant in Mexico described that clear rewards for participation in networks was an importan incentive for members to join and stay engaged: And then I saw that engaging in that group was like a ticket to something else, maybe it was ticket to go to “Women Deliver.” Maybe it was a ticket to get X, you know, but it was kind of like this idea of a catalytic exchange or a way or whatever, but there was a transactional thing there. – Key informant, Mexico Essential task 4: Establishing some sort of governance or institutional body to facilitate collective action on the issue Essential task 4: Establishing some sort of governance or institutional body to facilitate collective action on the issue Theme 4.1: Building a platform on an existing initiative Theme 4.1: Building a platform on an existing initiative Creating a clear group structure was thought to promote accountability within the network. Some partic ipants described an ideal group structure to be more like a platform, and their descriptions of that struc ture varied from being relatively loosely defined to being more established. A common strategy to obtain a cohesive structure was to build it upon either an existing initiative or network, as described by a partic ipant in Pakistan: I think that there are a number of forums or networks already available, and all you need to do is to basically, get in touch with them and invite the key person responsible...and then, once they are part of the discussion...you basically form the network... Theme 4.4: Funding was a missing piece in establishing a governance structure Nearly all participants reflected on the need for funding to support a network’s ongoing governance struc ture. Many participants said that the networks often relied on favours to move their agenda forward in the absence of funding, which was not sustainable. As described by a participant in Mexico: It takes a lot of [effort to] coordinate these networks, even smaller networks, takes dedicated time and takes funding, and I think that’s a critical ingredient. – Key informant, Mexico It takes a lot of [effort to] coordinate these networks, even smaller networks, takes dedicated time and takes funding, and I think that’s a critical ingredient. – Key informant, Mexico When funding is available, then it works. And, most of the time, funding is not there...There is a lead ership crisis ticking. – Key informant, Bangladesh Theme 4.2: Creating a sense of individual commitment to the group Participants believed that ensuring that members felt committed to the network’s success was essential to facilitating collective action. Sharing successes was one way to do this; however, participants thought that finding ways to enable network members to feel ownership over the successes achieved was im portant. Establishing a mechanism for network members to share their individual contributions and discuss their specific role in the group’s achievements would help foster that sense of individual owner ship that participants thought was necessary, while simultaneously helping develop a shared commit ment to the network. 2023 • Vol. 13 • 04044 www.jogh.org • doi: 10.7189/jogh.13.04044 9 Gausman et al. I was quite comfortable in my space and I used to give space to other people. That’s some very major thing, let me tell you, because technical people are quite insecure, let me be honest. And if you don’t give that space, people don’t want to come to you. And when you allow and you give platform to these guys, who are doing a lot of good work... and actually take them and expose them to the limelight which, is due to them... a lot of good work is being done. – Key informant, India Establishing an institutional body was also a way in which participants thought the network could better cultivate a sense of commitment to the group, as it would make the agenda more achievable. Some partici pants thought such an approach may be more easily realised by establishing a core team within the network so that roles and responsibilities are easily defined. PAPERS A sense of ownership or individual commitment to the group, a clear sense of roles and responsibilities, so a small enough group of people that there’s a sense of role… that you’re not being a participant, but there’s this sense of agency. – Key informant, Mexico A sense of ownership or individual commitment to the group, a clear sense of roles and responsibilities, so a small enough group of people that there’s a sense of role… that you’re not being a participant, but g g p f p p f y g p p , there’s this sense of agency. – Key informant, Mexico Theme 4.3: Generating a driving force Theme 4.3: Generating a driving force Participants agreed that a leader or some force capable of pushing the group towards accomplishing its shared agenda was needed for the networks to be effective. Several different types of leadership structures were proposed, ranging from individual leaders to secretariats, but all believed that having some sort of for mal direction was necessary to keep the network functioning and accountable. There was also a lot of em phasis on linking the network to established government initiatives to serve this function, in the absence of funding for a standalone leader. In Bangladesh, a participant described how such a leadership role could be played indirectly by an external government body: Commitments from the government, who is the regulatory body... not in terms of, ‘We’re going to do this’ [but] in terms of saying, ‘Yes, we’ll follow up. We’ll have more meetings to see how it can be inte grated, how [it] can be more focused. – Key informant, Bangladesh However, a participant in Mexico described differences between how local networks function vs interna tional networks on establishing an accountable leadership structure: I’ve seen the groups that work internationally, where there is a secretariat or there’s someone, that is moving the agenda forward, and [there is] usually someone [who has] been paid for that time. Then... locally, in terms of other networks that are national...[they] are usually linked to legal issues, so...there is something that legally binds you to responding in a network forum [to] some sort of common agen da. – Key informant, Mexico Theme 4.4: Funding was a missing piece in establishing a governance structure Theme 4.4: Funding was a missing piece in establishing a governance structure DISCUSSION Global health networks are critiqued for being often led by actors from the global north and having limited representation in their leadership by members from the South. Our results demonstrate, however, that the challenges commonly faced by global health networks are also relevant to networks operating on a national scale, which are all cultivated and led by local actors. The internal framing of an issue within the network and its external positioning through how said issue is publicly portrayed are important factors related to network effectiveness [9]. It has been suggested that the maternal health community has been unable to frame the issue effectively across multi-stakeholder networks in order to gain traction; such challenges have been identified in relation to measuring progress, articulating precise strategies to address the problem, and understanding the issue in relation to the health and health 2023 • Vol. 13 • 04044 www.jogh.org • doi: 10.7189/jogh.13.04044 www.jogh.org • doi: 10.7189/jogh.13.04044 10 Effective approaches to challenges to global health networks systems concerns [5]. We found that, by centring the National Dialogues on the EPMM key themes and in dicators and then leveraging the meeting to engage stakeholders in establishing consensus on a clear action plan, the networks in this study were able to overcome some of the challenges related to internal framing that have been faced by the global community. Having a common framework enabled the network to have an initial platform on which to build consensus in understanding the problem locally and agreeing on con text-specific priorities for action, which was thought to be especially useful given the wide range of network actors present at each National Dialogue. Our results also provide insight on how national networks to address maternal mortality have succeeded in positioning the issue externally to non-experts to gain support and motivate stakeholders to act. Despite ef forts of global networks to position the issue of maternal mortality as a literal matter of life and death, they have often had limited success in gaining traction outside of the health sector [5]. Two important dimen sions of external positioning emerged from our results. First, others within global governance frameworks have argued that policy coherence and inter-sectoral cooperation can come from the alignment of national priorities with global responsibilities [25]. By positioning the network’s goals within the SDGs, networks were able to effectively generate cross-sectoral interest. DISCUSSION Second, past research has found that high-stakes is sues, such as those associated with high mortality, morbidity, and social disruption, are likely to generate more robust networks [6]. Aligning the upstream determinants of maternal mortality within the context of the COVID-19 pandemic enabled the stakeholders to associate their issue with an issue that was of crit ical, global importance. In our study, participants described the catalysing effect that the COVID-19 pan demic had on strengthening their networks and gaining traction in achieving their agenda, which appears to be a policy window – a moment at which conditions aligned to present a strong opportunity to engage with leaders on an issue [5]. The fact that successful networks were able to pivot and re-frame the issue of maternal health to highlight its relevance within the pandemic response appears to have helped them to strengthen their standing. Historically, global health networks seeking to address maternal mortality struggled to gain traction due to stove-piped initiatives that were perceived to be led by technical actors from the global north [5,6], as well as challenges involved in engaging a diverse network of stakeholders. Similar difficulties emerged in our re search, but at a local level. Network members recognised that, to be effective in driving progress, their net works needed to reflect diverse expertise and interests, while also making outside actors feel welcome. Our research highlighted the strategies that the multi-stakeholder networks had to put in place to overcome the barriers they faced in diversifying their networks, such as by reducing the technicality of the language used. Last, in terms of leadership, establishing the network’s legitimacy by linking its goals with ongoing gov ernment initiatives of platforms helped the network cultivate power to act and make progress towards its agenda. By comprising both state and non-state actors, it seemed that the networks in our study were able to more effectively harness the state power. While the subject of much debate in the literature on global health networks [26], our results point to an underlying need for buy-in from the government to strengthen the national networks described here. Despite the networks’ ability to obtain government buy-in, ensuring the network itself had formalised lead ership, either through individuals or a secretariat, was also seen as critical to its success and survival. DISCUSSION While passionate individuals were needed to give the network a vision, there also needed to be a persistent driving force and abiding structure to ensure that network members were organised in a way that leveraged their strength to act efficiently and effectively. This study has several strengths and limitations. It included diverse stakeholders from a wide range of coun tries facing different, context-dependant challenges related to maternal mortality. However, it was conduct ed during the COVID-19 pandemic, which made participation for some stakeholders difficult and likely re duced the number of participants. While we believe that our sample size is sufficiently large in each country to represent a wide range of voices – especially given the convergence of themes that were found across the study countries – our research could have benefitted from additional perspectives. www.jogh.org • doi: 10.7189/jogh.13.04044 CONCLUSIONS JG, RRJ, and AL contributed intellectually to the conceptualisation and drafting of the manuscript, and reviewed and approved the final version. Authorship contributions: JG and RRJ conceived and designed the consultation and drafted the manuscript. JG devel oped the methodology and performed the analysis. JG, RRJ, and AL contributed intellectually to the conceptualisation and drafting of the manuscript, and reviewed and approved the final version. Authorship contributions: JG and RRJ conceived and designed the consultation and drafted the manuscript. JG devel oped the methodology and performed the analysis. JG, RRJ, and AL contributed intellectually to the conceptualisation and drafting of the manuscript, and reviewed and approved the final version. PAPERS Disclosure of interest: The authors completed the ICMJE Disclosure of Interest Form (available upon request from the corresponding author) and disclose no relevant interests. Disclosure of interest: The authors completed the ICMJE Disclosure of Interest Form (available upon request from the corresponding author) and disclose no relevant interests. 1 Sustainable Development Goals Knowledge Platform. Sustainable Development Goal 3. Available: https://sustainablede velopment.un.org/sdg3. Accessed: 9 May 2023.. 2 World Health Organization. Strategies towards ending preventable maternal mortality (EPMM). Geneva: W Organization; 2015. 3 Jolivet RR, Moran AC, O’Connor M, Chou D, Bhardwaj N, Newby H, et al. Ending preventable maternal mortality: phase II of a multi-step process to develop a monitoring framework, 2016–2030. BMC Pregnancy Childbirth. 2018;18:258. Med line:29940890 doi:10.1186/s12884-018-1763-8 4 Moran AC, Jolivet RR, Chou D, Dalglish SL, Hill K, Ramsey K, et al. A common monitoring framework for ending pre ventable maternal mortality, 2015–2030: phase I of a multi-step process. BMC Pregnancy Childbirth. 2016;16:250. Med line:27565428 doi:10.1186/s12884-016-1035-4 5 Shiffman J, Smith S. Generation of political priority for global health initiatives: a framework and case study of maternal mortality. Lancet. 2007;370:1370-9. Medline:17933652 doi:10.1016/S0140-6736(07)61579-7 6 Smith SL, Rodriguez MA. Agenda setting for maternal survival: the power of global health networks and norms. Health Policy Plan. 2016;31:i48-59. Medline:26273062 doi:10.1093/heapol/czu114 n J, Quissell K, Schmitz HP, Pelletier DL, Smith SL, Berlan D, et al. A framework on the emergence and effectiv 7 Shiffman J, Quissell K, Schmitz HP, Pelletier DL, Smith SL, Berlan D, et al. A framework on the emergence ness of global health networks. Health Policy Plan. 2016;31:i3-16. Medline:26318679 doi:10.1093/heapol/cz 8 Shiffman J, Quissell K, Schmitz HP, Pelletier DL, Smith SL, Berlan D, et al. A framework on the emergence and effective ness of global health networks. Health Policy Plan. 2016;31:i3-16. CONCLUSIONS Medline:26318679 doi:10.1093/heapol/czu046 8 Shiffman J, Quissell K, Schmitz HP, Pelletier DL, Smith SL, Berlan D, et al. A framework on the emergence and effective ness of global health networks Health Policy Plan 2016;31:i3 16 Medline:26318679 doi:10 1093/heapol/czu046 9 Shiffman J. Four challenges that global health networks face. Int J Health Policy Manag. 2017;6:183. Medline:28812801 doi:10.15171/ijhpm.2017.14 10 Whitney D, Cooperrider D. Appreciative inquiry: A positive revolution in change. Oakland: Berrett-Koehler Publishers; 2011. 11 Cooperrider DL, Sorensen PF, Yaeger TF, Whitney D. Appreciative inquiry: An emerging direction for organization devel opment. Champaign: Stipes Publishing Co; 2001. 12 Cooperrider DL, Whitney D. A positive revolution in change: Appreciative inquiry. In: Golembiewski RT, editor. Hand book of Organizational Behavior, Revised and Expanded. New York: Routledge; 2000. 13 Ludema JD, Cooperrider DL, Barrett FJ. Appreciative inquiry: The power of the unconditional positive book of action research. 2006:155-65. 14 Barker CE, Bird CE, Pradhan A, Shakya G. Support to the Safe Motherhood Programme in Nepal: an integrated approach. Reprod Health Matters. 2007;15:81-90. Medline:17938073 doi:10.1016/S0968-8080(07)30331-5 15 Cooperrider DL, Srivastva S. A Contemporary Commentary on Appreciative Inquiry in Organizational Life. In: Woodman R, Pasmore W, editors. Research in organizational change and development, vol. 1. Bingley: Emerald Group Publishing Limited; 2013. p. 129-169. 16 Trajkovski S, Schmied V, Vickers M, Jackson D. Implementing the 4D cycle of appreciative inquiry in health care: a meth odological review. J Adv Nurs. 2013;69:1224-34. Medline:23317408 doi:10.1111/jan.12086 17 Grant S, Humphries M. Critical evaluation of appreciative inquiry: Bridging an apparent paradox. Action Res. 2006;4:401- 18. doi:10.1177/1476750306070103 18 Ludema JD. Appreciative storytelling: A narrative approach to organization development and change. In: Fry RE, Whit ney D, Seiling JG, Barrett F. Appreciative Inquiry and Organizational Transformation: Reports from the Field. Westport: Praeger; 2002. 18 Ludema JD. Appreciative storytelling: A narrative approach to organization development and change. In: Fry RE, Whit ney D, Seiling JG, Barrett F. Appreciative Inquiry and Organizational Transformation: Reports from the Field. Westport: Praeger; 2002. g 19 Thomas D, Kc LM, Messerschmidt D, Devkota B. Increasing access to essential obstetric care: a review of progress and process. Kathmandu: Department for International Development. 2004. 19 Thomas D, Kc LM, Messerschmidt D, Devkota B. Increasing access to essential obstetric care: a review of progress and process. Kathmandu: Department for International Development. 2004. 20 Jackson FM, Saran AR, Ricks S, Essien J, Klein K, Roberts D, et al. CONCLUSIONS Our study illustrates the applicability of the conceptual framework of global health networks at national lev els, highlighting several effective approaches for national multi-stakeholder networks to address common challenges faced by global health networks. The themes we identified may be offer helpful strategies for fu ture national networks in addressing these challenges. 2023 • Vol. 13 • 04044 11 www.jogh.org • doi: 10.7189/jogh.13.04044 Gausman et al. Acknowledgements: We would like to thank Dea Oviedo for her support with data management of the transcripts and logistical support. We are also thankful to our organisational partners for the National Dialogues: White Ribbon Alli ance (WRA) and Management Sciences for Health (MSH). We are grateful to Kim Whipkey (WRA) and Martha Mur dock (MSH) for their help identifying stakeholders. Ethics statement: Ethical approval was obtained from the Institutional Review Board at the Harvard TH Chan School of Public Health. All participants provided verbal informed consent to participate. Funding: This work was supported by the Bill and Melinda Gates Foundation with grant number OPP1169546. Authorship contributions: JG and RRJ conceived and designed the consultation and drafted the manuscript. JG devel oped the methodology and performed the analysis. JG, RRJ, and AL contributed intellectually to the conceptualisation and drafting of the manuscript, and reviewed and approved the final version. Disclosure of interest: The authors completed the ICMJE Disclosure of Interest Form (available upon request from the corresponding author) and disclose no relevant interests. Acknowledgements: We would like to thank Dea Oviedo for her support with data management of the transcripts and logistical support. We are also thankful to our organisational partners for the National Dialogues: White Ribbon Alli ance (WRA) and Management Sciences for Health (MSH). We are grateful to Kim Whipkey (WRA) and Martha Mur dock (MSH) for their help identifying stakeholders. Ethics statement: Ethical approval was obtained from the Institutional Review Board at the Harvard TH Chan School of Public Health. All participants provided verbal informed consent to participate. Ethics statement: Ethical approval was obtained from the Institutional Review Board at the Harvard TH Chan School of Public Health. All participants provided verbal informed consent to participate. unding: This work was supported by the Bill and Melinda Gates Foundation with grant number OPP1169546. Authorship contributions: JG and RRJ conceived and designed the consultation and drafted the manuscript. JG devel oped the methodology and performed the analysis. 22 Hsieh H-F, Shannon SE. Three approaches to qualitative content analysis. Qual Health Res. 2005;15:1277-88. Med line:16204405 doi:10.1177/1049732305276687 24 O’Brien BC, Harris IB, Beckman TJ, Reed DA, Cook DA. Standards for Reporting Qualitative Research: A Synthesis of Rec ommendations. Acad Med. 2014;89:1245-51. Medline:24979285 doi:10.1097/ACM.0000000000000388 Effective approaches to challenges to global health networks 22 Hsieh H-F, Shannon SE. Three approaches to qualitative content analysis. Qual Health Res. 2005;15:1277-88. Med line:16204405 doi:10.1177/1049732305276687 23 SocioCultural Research Consultants. Dedoose. 2022. Available: https://www.dedoose.com/. Accessed: 9 May 2023. 24 O’Brien BC, Harris IB, Beckman TJ, Reed DA, Cook DA. Standards for Reporting Qualitative Research: A Synthesis of Rec ommendations. Acad Med. 2014;89:1245-51. Medline:24979285 doi:10.1097/ACM.0000000000000388 25 Kickbusch I, Szabo MMC. A new governance space for health. Glob Health Action. 2014;7:23507. Medline:24560259 doi:10.3402/gha.v7.23507 26 Shiffman J. Agency, structure and the power of global health networks. Int J Health Policy Manag. 2018;7:879. Med line:30316239 doi:10.15171/ijhpm.2018.71 g 26 Shiffman J. Agency, structure and the power of global health networks. Int J Health Policy Manag. 2018;7:879. Med line:30316239 doi:10.15171/ijhpm.2018.71 23 SocioCultural Research Consultants. Dedoose. 2022. Available: https://www.dedoose.com/. Accessed: 9 May 2023. Effective approaches to challenges to global health networks www.jogh.org • doi: 10.7189/jogh.13.04044 25 Kickbusch I, Szabo MMC. A new governance space for health. Glob Health Action. 2014;7:23507. Med doi:10.3402/gha.v7.23507 p y 24 O’Brien BC, Harris IB, Beckman TJ, Reed DA, Cook DA. Standards for Reporting Qualitative Research: A Synthesis of Rec ommendations. Acad Med. 2014;89:1245-51. Medline:24979285 doi:10.1097/ACM.0000000000000388 25 Kickbusch I, Szabo MMC. A new governance space for health. Glob Health Action. 2014;7:23507. Medline:24560259 doi:10.3402/gha.v7.23507 CONCLUSIONS Save 100 Babies©: engaging communities for just and equitable birth outcomes through Photovoice and Appreciative inquiry. Matern Child Health J. 2014;18:1786-94. Med line:24474593 doi:10.1007/s10995-014-1436-9 20 Jackson FM, Saran AR, Ricks S, Essien J, Klein K, Roberts D, et al. Save 100 Babies©: engaging communities for just and equitable birth outcomes through Photovoice and Appreciative inquiry. Matern Child Health J. 2014;18:1786-94. Med line:24474593 doi:10.1007/s10995-014-1436-9 21 Krueger RA. Focus groups: A practical guide for applied research. Thousand Oaks: SAGE Publications; 2014. 21 Krueger RA. Focus groups: A practical guide for applied research. Thousand Oaks: SAGE Publications; 2014. 2023 • Vol. 13 • 04044 12 www.jogh.org • doi: 10.7189/jogh.13.04044 Effective approaches to challenges to global health networks REFERENCES PAPERS 2023 • Vol. 13 • 04044 www.jogh.org • doi: 10.7189/jogh.13.04044 13
https://openalex.org/W4292454246	https://www.frontiersin.org/articles/10.3389/fped.2022.809523/pdf	English	null	An in-depth discussion of cholesteatoma, middle ear Inflammation, and langerhans cell histiocytosis of the temporal bone, based on diagnostic results	Frontiers in pediatrics	2,022	cc-by	5,055	TYPE Original Research PUBLISHED 09 August 2022 DOI 10.3389/fped.2022.809523 TYPE Original Research PUBLISHED 09 August 2022 DOI 10.3389/fped.2022.809523 COPYRIGHT © 2022 Duan, Pan, Chen, Qiao and Xu. 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. Introduction inﬂuence the choice of surgical methods and techniques is the available preoperative information concerning underlying diseases. Enhancing the imaging modality for identifying temporal bone tumors and otitis media can, thus, have a signiﬁcant impact on clinical medicine, and eﬀorts should be made to achieve this. Middle ear inﬂammation (MEI) is a common condition among children. The incidence of otitis media is 8.1% (1), and imaging is not typically required in cases of MEI. However, for patients with MEI who present with hearing loss, and where the tympanic membrane cannot be visualized, imaging may be required (2). The speciﬁc imaging features will vary depending on the form. Radiomics is a method that is applied in the ﬁeld of medicine that uses data representation algorithms to extract a large number of features from medical images. These features are known as “radiographic” data and can be used to observe several disease characteristics that are invisible to the naked eye. A radiology approach considers that the signal intensity patterns in medical images are closely related to the genetic, molecular, and biological characteristics of the tissue. These methods include T-scans and MRI scans. The incidence of congenital cholesteatoma of the middle ear, once thought to be relatively rare, is on the rise and presently accounts for 2–5% of all the cholesteatomas (3). Langerhans cell histiocytosis (LCH) is a relatively rare condition that primarily aﬀects children. Soft tissue sarcoma and rhabdomyosarcoma are the most common among infants and young children. The head and neck are relatively common parts of the body involved in this condition; the percentage of cases with temporal bone involvement is approximately 7%. The focus of the current study was to develop a deep learning framework for the automatic CT scan diagnosis of temporal bone diseases in children. To date, the radiographic features that are used to assess the utility of temporal bone disease have been rarely studied in the context of imaging. In this context, the current study aimed to provide a better analysis of these image scans to provide a more accurate diagnosis. The performance of artiﬁcial intelligence (AI) network model was tested on a separate dataset. The study also compared clinical specialists, including otologists, otolaryngologists, and radiologists. Currently, high-resolution CT (HRCT) is typically used for the diagnosis and preoperative examination of suspected temporal bone tumors in clinical patients (4). An in-depth discussion of cholesteatoma, middle ear Inﬂammation, and langerhans cell histiocytosis of the temporal bone, based on diagnostic results OPEN ACCESS EDITED BY Stephen I. Pelton, Boston University, United States REVIEWED BY Hui Jiang, Fudan University, China Yougan Saman, University of Leicester, United Kingdom CORRESPONDENCE Zheng-Min Xu zhengmin98987@111.com Zhong-Wei Qiao zhongweiqiaomd@163.com SPECIALTY SECTION This article was submitted to Pediatric Otolaryngology, a section of the journal Frontiers in Pediatrics RECEIVED 11 January 2022 ACCEPTED 14 July 2022 PUBLISHED 09 August 2022 Bo Duan1, Li-Li Pan2, Wen-Xia Chen1, Zhong-Wei Qiao2 and Zheng-Min Xu1* Bo Duan1, Li-Li Pan2, Wen-Xia Chen1, Zhong-Wei Qiao2* and Zheng-Min Xu1* Bo Duan1, Li-Li Pan2, Wen-Xia Chen1, Zhong-Wei Qiao2* and Zheng-Min Xu1* 1Department of Otolaryngology-Head and Neck Surgery, Children’s Hospital of Fudan University, Shanghai, China, 2Department of Radiology, Children’s Hospital of Fudan University, Shanghai, China Objective: This study aimed to conduct an in-depth investigation of the learning framework used for deriving diagnostic results of temporal bone diseases, including cholesteatoma and Langerhans cell histiocytosis (LCH). In addition, middle ear inﬂammation (MEI) was diagnosed by CT scanning of the temporal bone in pediatric patients. CITATION Duan B, Pan L-L, Chen W-X, Qiao Z-W and Xu Z-M (2022) An in-depth discussion of cholesteatoma, middle ear Inﬂammation, and langerhans cell histiocytosis of the temporal bone, based on diagnostic results. Front. Pediatr. 10:809523. doi: 10.3389/fped.2022.809523 Design: A total of 119 patients were included in this retrospective study; among them, 40 patients had MEI, 38 patients had histology-proven cholesteatoma, and 41 patients had histology-proven LCH of the temporal bone. Each of the 119 patients was matched with one-third of the disease labels. The study included otologists and radiologists, and the reference criteria were histopathology results (70% of cases for training and 30% of cases for validation). A multilayer perceptron artiﬁcial neural network (VGG16_BN) was employed and classiﬁed, based on radiometrics. This framework structure was compared and analyzed by clinical experts according to CT images and performance. Results: The deep learning framework results vs. a physician’s diagnosis, respectively, in multiclassiﬁcation tasks, were as follows. Receiver operating characteristic (ROC) (cholesteatoma): (0.98 vs. 0.91), LCH (0.99 vs. 0.98), and MEI (0.99 vs. 0.85). Accuracy (cholesteatoma): (0.99 vs. 0.89), LCH (0.99 vs. 0.97), and MEI (0.99 vs. 0.89). Sensitivity (cholesteatoma): (0.96 vs. 0.97), LCH (0.99 vs. 0.98), and MEI (1 vs. 0.69). Speciﬁcity (cholesteatoma): (1 vs. 0.89), LCH (0.99 vs. 0.97), and MEI (0.99 vs. 0.89). Frontiers in Pediatrics frontiersin.org 01 Duan et al. An in-depth discussion of cholesteatoma, middle ear Inﬂammation, and langerhans cell histiocytosis of the temporal bone, based on diagnostic results 10.3389/fped.2022.809523 10.3389/fped.2022.809523 10.3389/fped.2022.809523 Conclusion: This article presents a research and learning framework for the diagnosis of cholesteatoma, MEI, and temporal bone LCH in children, based on CT scans. The research framework performed better than the clinical experts. PyTorch, cholesteatoma, langerhans cell histiocytosis, computed tomography (CT) scan, deep learning Methods and materials Techniques that may be helpful for diagnosing tumors in the temporal bone include diﬀusion-weighted imaging or contrast- enhanced MRI; however, these methods may also aﬀect the diagnostic results due to the high level of protein ﬂuid present in inﬂammatory tissue and cases of otitis media (6). Introduction Chronic otitis media and temporal bone tumors are more prone to erosion around the bone structure, which may require surgical treatment. Cholesteatoma is generally manifested in the location of the tympanic sinus (5). These two entities may appear in diﬀerent positions of the tympanic chamber, which will also limit HRCT and diﬀerentiation between them. Computed tomography imaging All the registered patients required one or more CT scans of the temporal bone, and a total of 119 scans were available for analysis. These temporal bone scans were obtained using a 128-channel multidetector Somatom Deﬁnition Edge with z-SharpTM Technology CT scanners. The procedure for conducting each scan was as follows. A full scan was required for each patient from the lower edge of the external auditory canal to the upper edge of the petrous bone. The collimation was set at 128 mm × 0.625 mm, the ﬁeld of view was 220 mm × 220 mm, the spacing was 0.8 mm, the matrix size was 512 × 512, the voltage was 120 kV, 240 mass, and the axial section was 0.625 mm. The scans were performed on sections where lesions were present, and the number of axial CT sections per scan ranged from 30 to 50. The total number of scans performed was 2,588. All the images were downloaded from the doctor’s workstation and saved in the prescribed 224 × 224-pixel jpg format for subsequent analysis (Figure 1). Study participants The Institutional Review Committee of Children’s Hospital of Fudan University approved the current study. In procedural terms, a search of the clinical database identiﬁed 161 children who had undergone middle ear surgery between May 2013 and October 2020. Medical records were rigorously checked to exclude patients who had received CT scans and were subsequently diagnosed with acute otitis media or inner ear disease, and who lacked temporal bone. A ﬁnal group of 119 patients was included in the collected retrospective data. Early stage/small tumors may not always be easily identiﬁable. In the medical ﬁeld, a ﬁnal accurate diagnosis must currently include an analysis of surgical tissue histopathology. Accurate preoperative diﬀerentiation between the two above- noted diseases is of signiﬁcant clinical importance because most cases of temporal bone tumors require surgical treatment; chronic otitis media, on the other hand, can typically be treated conservatively (7). In addition, one of the key factors that can Frontiers in Pediatrics frontiersin.org 02 10.3389/fped.2022.809523 Duan et al. FIGURE 2 VGG16_BN structure diagram. FIGURE 1 LCH of the temporal bone. The bone has not been destroyed. LCH of the temporal bone. The bone has not been destroyed. Professional performance evaluation Four clinical specialists were selected to evaluate the diagnostic performance of their test datasets. Two of the specialists were registered otologists with 15 years of combined experience, and the remaining two specialists were radiologists trained in ear diseases with 16 years of combined experience. Each of the four experts could only select one of the three categories, based on the CT images, to diagnose a case. Clinical labeling The distribution of clinical labels for each ear was ﬁrst arranged according to each patient’s diagnostic record. All the participants’ medical records were examined independently by two experienced otolaryngologists and radiologists, who, concurrently, resolved any diﬀerences that arose to reach a consensus. The facts that were selected in the training and testing sessions for the classiﬁcation network were reﬂected in these clinical labels. On closer examination of these labels, the three most common types of cases were found to be “cholesteatoma,” “MEI,” and “LCH.” These three categories were based on web-based training and required in-depth learning to achieve adequate performance. The present article will summarize the distribution of clinical labels at a later stage. The labels for ear surgery were allocated according to pathology. increased, additional lesion information was at risk of becoming lost in the image (8). Consequently, the current authors adopted a neural network with fewer layers. A convolutional neural network known as VGG16 (9) is an architecture with 16 layers of weights and 3 × 3 ﬁlters. After the convolutional layer, there are two interconnected layers, followed by the soft maximum output. The VGG16_BN network adds a batch normalization function to the VGG16 network. The matrix is then added to the network and outputted as three categories (see Figure 2). It has about 138 million network parameters. In temporal bone CT images, the lesion area accounted for a relatively small proportion of the overall image. During the training of a deep learning network, as the number of layers Frontiers in Pediatrics frontiersin.org 03 Duan et al. 10.3389/fped.2022.809523 FIGURE 3 CT of the temporal bone with cholesteatoma: Original image (left) and heat map (right). FIGURE 4 CT of the temporal bone with LCH: Original image (left) and heat map (right). FIGURE 3 CT of the temporal bone with cholesteatoma: Original image (left) and heat map (right). FIGURE 3 CT of the temporal bone with cholesteatoma: Original image (left) and heat map (right). FIGURE 4 CT of the temporal bone with LCH: Original image (left) and heat map (right). FIGURE 4 CT of the temporal bone with LCH: Original image (left) and heat map (right). Data enhancement technology of the classiﬁcation task evaluate the performance of the model after the training was complete. Clinical experts could also use these datasets to assess performance. To ensure the eﬃciency of deep learning, all the images required a mirror image, minor rotation, cropping, scaling, and side-length moving to increase the training data for the classiﬁcation task. The implementation model of the data volume enhancement was carried out in real-time by the classiﬁcation network. Finally, the Bicubic interpolation algorithm was used to scale all the images to 512 × 512- pixel size. Results The model that was adopted in the current study is used in discrete networks. For multivariate classiﬁcation tasks, the test results were satisfactory when the dataset was tested for the classiﬁcation of cholesteatoma, LCH, and MEI. Based on the total weight parameters included in the pretraining, the heat map was created according to gradient-weighted class activation mapping. The heat map indicated the diﬀerences related to where the computer focused the imaging scan, in which the orange area was signiﬁcant (Figures 3–5). The area under the curve for this network was 0.99 (see Figure 6). The network derived a better conclusion than the four experts who were included in the clinical trial. Deep learning framework detection of cholesteatoma (class 0), LCH (class 1), and MEI (class 2). Deep learning framework detection of cholesteatoma (class 0), LCH (class 1), and MEI (class 2). TABLE 1 Multi-classiﬁcation confusion matrix for deep learning. TABLE 1 Multi-classiﬁcation confusion matrix for deep learning. Predict cholesteatoma LCH MEI Cholesteatoma 185 2 4 LCH 0 190 1 MEI 0 0 191 TABLE 2 Confusion matrix comparison of AI vs. clinical experts. Precision Recall F1 score Accuracy Precision Recall F1 score Accuracy Precision Recall F1 score Accuracy The overall accuracy of the network was high (98.7 vs. 88.5%) for the three task categories, and the network achieved a higher F1 score (98.7 vs. 88.2%) compared to the clinical experts. These results are shown in Table 2. tasks (and to a higher quality than physicians) has been very satisfactory. For example, machine learning is currently used in CT scanning to detect abnormalities in the head, classify masses in the liver, and predict amyotrophic lateral sclerosis. In the ﬁelds of neurology and otology, many leading ﬁgures have applied basic machine learning techniques for predicting the hearing results of patients experiencing sudden hearing impairment (9), or to make predictions regarding the hearing and language perception of children with cochlear implants (10). Analyzing and compiling statistics Based on the basic VGG16_BN classiﬁcation network model, the Softmax function acted as its ﬁnal classiﬁcation layer. The probability distribution of cholesteatoma otitis media was trained using the PyTorch application in Python. A random selection of 85% of the dataset (n = 2,070) was used during the validation process, and ﬁve diﬀerent parameters were included. The duration value of the training course was set to 100 (from the initial 0.01-bit speed). The remaining 15% of the data (n = 388) were stored and could be used to To accurately evaluate the diagnostic ability of the AI model, the performances of the deep learning algorithm and clinical experts during data testing were summarized using the confusion matrix method, i.e., binarizing all the labels of the AI model and drawing the receiver operating characteristic (ROC) curve. The prediction probability was applied to measure the area under the ROC curve of the deep learning algorithm, and Frontiers in Pediatrics frontiersin.org 04 Duan et al. 10.3389/fped.2022.809523 FIGURE 5 CT of the temporal bone with MEI: Original image (left) and heat map (right). FIGURE 5 CT of the temporal bone with MEI: Original image (left) and heat map (right). FIGURE 6 Deep learning framework detection of cholesteatoma (class 0), LCH (class 1), and MEI (class 2). a 95% CI was estimated using the Tak Long Estate Algorithm. Python and Excel were used to conduct all the statistical analyses (alpha level, 0.05). a 95% CI was estimated using the Tak Long Estate Algorithm. Python and Excel were used to conduct all the statistical analyses (alpha level, 0.05). Classiﬁcation network and clinical experts’ performance The training accuracy was 99%, and the validation accuracy was 98.7% at its most balanced time. The confusion matrix is shown in Table 1. TABLE 2 Confusion matrix comparison of AI vs. clinical experts. TABLE 2 Confusion matrix comparison of AI vs. clinical experts. Discussion With the rapid development of deep learning technology and the use of big data to develop the training of these networks, healthcare AI technology has undergone rapid development. Radiomics analysis of diﬀerent types of imaging data has yielded valuable results that can be used to isolate diﬀerent tumor entities throughout the human body. In some areas that rely on medical imaging, particularly radiology and pathology, the use of machine learning methods to perform a variety of medical The current article is the ﬁrst study to apply a deep learning network to temporal bone tumors in children, Frontiers in Pediatrics frontiersin.org 05 10.3389/fped.2022.809523 Duan et al. correctly identiﬁed more of the normal ears, they proved their greater particularity. Accordingly, to a large extent, they can help eliminate case results that have been misclassiﬁed and analyzed by AI to achieve the goal with minimal labor costs. based on CT images. The performance of the adopted model was excellent, and its sensitivity and speciﬁcity were superior to those of medical and clinical experts. These advantages, as well as the absolute consistency and high eﬃciency of AI in decision-making, indicated that the clinical application of AI in the diagnosis of temporal bone tumors, based on CT scans, represents broad development prospects. One of the most important obstacles to the broad application of deep learning in medicine is the inability to understand the basic principles of algorithmic conclusions. In a recent research experiment (11), the researchers constructed a framework based on algorithmic components that could generate a tissue image in a digital optical coherence tomography scan using a segmented network while concurrently creating a classiﬁcation network; this network was then used for establishing a diagnosis and for providing treatment recommendations. This workﬂow divided the decision-making process into two distinct steps, i.e., explaining the initial scan, and establishing a judgment, based on the ﬁndings of the ﬁrst step. In the current study, the authors did not design an automatic separation framework. Tumors may not always be conﬁned to the mastoid process and, as such, may involve other parts of the temporal bone. The clinician should, thus, inspect and extract particular regions of interest. In the ﬁeld of deep learning, overﬁtting is a classic problem, particularly when the number of samples is limited or the training steps are extensive. Discussion In response to the problem of overﬁtting, the following methods can be used to avoid problems and improve the versatility of a model. Using data enhancement technology, the application of this technology can greatly increase the number of images that can be used for network training. A network model can use this method to achieve 98, 99, and 99% accuracy in the three classiﬁcation tasks (training, verifying, and testing datasets, respectively). The accurate diagnosis of otitis media is a challenging task. The key factor for a correct diagnosis in this regard is using guidelines and practices that are currently implemented by the country in which the research is conducted. This includes accurately conﬁrming the length of time of infection and determining each TM that expands or contracts. The present study showed that an automated system, developed using deep learning methods, could be useful for identifying otitis media. Various CT imaging features were quantitatively evaluated by applying image processing methods, including segmentation and image feature extraction. The deep learning classiﬁers used image features to distinguish patients with temporal bone tumors from those with otitis media. This study was designed to examine the value of an automatic method, based on deep learning, for determining patient candidates for tympanotomy. Acquired cholesteatoma has a history dating back three centuries, but the nature of this disease remains unconﬁrmed. If cholesteatoma is not detected and treated in time, it can become very dangerous by rapidly expanding and invading the internal structure of the temporal bone, causing various intra- and extracranial complications. As shown in Figure 1, according to a CT scan, a diagnosis of otitis media may be assumed. However, the patient in question actually suﬀered from LCH, the diagnosis of which is histology- based. Employing deep learning methods may expand the number of candidates for tympanotomy. The ﬁndings of this study concurred with recent research on a deep learning approach (12), in which machine-learning models were developed to classify patients with chronic otitis media and cholesteatoma. The classiﬁer and an optimal area that was less than 0.99, sensitivity was 0.97, and speciﬁcity was 0.80. The accuracy rate of the AI model was approximately 90%, but the average accuracy rate of clinical experts was relatively low (approximately 73.8%). It is diﬃcult to identify a temporal mass using the naked eye without the presence of bone damage. Conclusion The reviewer HJ declared a shared parent aﬃliation with the authors to the handling editor at the time of review. The purpose of this study was to establish a deep learning research framework for the diagnosis of LCH of the temporal bone, cholesteatoma, and MEI, based on CT scanning of the temporal bone in children. An in-depth research framework, based on CT scanning of the temporal bone in children, was proposed for the in-depth study of temporal bone fractures, cholesteatoma, and conﬁrming a temporal lobe tumor diagnosis. The results of the study also showed that the clinical application of AI in CT imaging presented good development prospects. In the future, temporal bone research, as well as 3D CT imaging technology, will be used to expand this method to the larger- scale treatment of temporal bone diseases. Ethics statement The studies involving human participants were reviewed and approved by the Children’s Hospital of Fudan University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Discussion With this model, an early diagnosis can be made before the temporal bone is entirely destroyed. Following further analysis and inspection of misclassiﬁed cases, the network logic can be observed. Clinical experts and AI models may attempt to classify both the typical and non- challenging cases. However, in some situations, neither may be able to correctly classify these cases. When this happens, CT scanning will be required to highlight atypical and extremely subtle patterns. Although the strategy that was applied in the AI classiﬁcation tasks was not clear, when classifying cases of cholesteatoma, LCH, and MEI, the higher recall rate that was obtained reﬂected that AI was in the CT image. When reading abnormal patterns, there was a high level of sensitivity. This ability is of great value in terms of providing a ﬁrst step in the screening and diagnosis process because it minimizes the risk of overlooked cases. However, when clinical experts In the research process, the AI model obtained very good results. The accuracy of the model classiﬁcation (99%) in the presence of otitis media and temporal bone tumors was 0.99, which was equivalent to/better than the results reported in studies based on ear mirroring, which ranged from 80 to 85.6% (13, 14). The deep learning model presented in the current article has several possible advantages. The system used a computerized method to quantitatively evaluate various CT parameters. Multiple image features may enhance the ability of the model to predict pathological tissue. Pretrained deep learning models may signiﬁcantly reduce the time required to determine whether symptom onset had been within the therapeutic time Frontiers in Pediatrics frontiersin.org 06 Duan et al. 10.3389/fped.2022.809523 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. Data availability statement window. These characteristics may enable an otolaryngologist to determine whether patients require tympanotomy or another type of intervention. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The following limitations are present in the current study and must be noted. First, only moderate datasets were available for inclusion, and these temporal tumor samples were too small to support creating a complete classiﬁer, or there was a slight imbalance. Therefore, based on the currently known properties of deep learning techniques, the AI model presented herein can be used in two-dimensional CT images. Two-dimensional CT images are less detailed than their three-dimensional (3D) conunterparts because the latter includes the temporal bone and the middle ear region. Accordingly, for the validated AI model, using 3D images will improve the model’s diagnostic accuracy. The above algorithm is also consistent with the CT scan explained by the clinician. 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. Author contributions All the study participants were sourced from the same clinical center. Further veriﬁcation resulting from multicenter research settings and diﬀerent patient populations is warranted. Furthermore, the scientiﬁc data employed in the current article had been optimized and, accordingly, showed only the current population. As such, the results of this study should be interpreted with care. BD and Z-MX: conception and design of the research. BD, L-LP, W-XC, and Z-WQ: acquisition of data. BD, L-LP, and W-XC: analysis and interpretation of the data. BD, Z-WQ, and Z-MX: statistical analysis. BD: writing of the manuscript. Z-MX: critical revision of the manuscript for intellectual content. All authors read and approved the ﬁnal draft. Compared with the conclusions derived by the deep learning model, the outcomes derived by clinicians may vary too much for the former to be considered the gold standard. Additional validation using a large database is, thus, necessary. 2. Kalcioglu MT, Sallavaci S, Hrncic N, Beishenova M, Davcheva Cakar M, Vlaski L, et al. Prevalence of and factors aﬀecting otitis media with eﬀusion in children in 1. Gates GA, Klein JO, Lim DJ, Mogi G, Ogra PL, Pararella MM, et al. Recent advances in otitis media. 1. Deﬁnitions, terminology, and classiﬁcation of otitis media. Ann Otol Rhinol Laryngol Suppl. 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Prevalence of otitis media and risk-factors for sensorineural hearing loss among infants attending Child Welfare Clinics in the Solomon Islands. Int J Pediatr Otorhinolaryngol. (2018) 111:21–5. doi: 10.1016/j.ijporl.2018.05.021 14. Kaspar A, Newton O, Kei J, Driscoll C, Swanepoel W, Goulios H. Prevalence of otitis media and risk-factors for sensorineural hearing loss among infants attending Child Welfare Clinics in the Solomon Islands. Int J Pediatr Otorhinolaryngol. (2018) 111:21–5. doi: 10.1016/j.ijporl.2018.05.021 9. Simonyan K, Zisserman A. Very deep convolutional networks for large-scale image recognition. ArXiv[Preprint]. (2015) Available online at: https://doi.org/10. 48550/arXiv.1409.1556 (accessed April 10, 2015). 08 Frontiers in Pediatrics Frontiers in Pediatrics 08 frontiersin.org
W2413300501.txt	https://journals.plos.org/ploscompbiol/article/file?id=10.1371/journal.pcbi.1004924&type=printable	en		EGFR Signal-Network Reconstruction Demonstrates Metabolic Crosstalk in EMT	PLOS computational biology/PLoS computational biology	2,016	cc-by	12,217	RESEARCH ARTICLE EGFR Signal-Network Reconstruction Demonstrates Metabolic Crosstalk in EMT Kumari Sonal Choudhary1,2, Neha Rohatgi1,2, Skarphedinn Halldorsson1,2, Eirikur Briem2,3,4, Thorarinn Gudjonsson2,3,4, Steinn Gudmundsson1, Ottar Rolfsson1,2* 1 Center for Systems Biology, University of Iceland, Reykjavik, Iceland, 2 Biomedical Center, University of Iceland, Reykjavik, Iceland, 3 Stem Cell Research Unit, Department of Anatomy, School of Health Sciences, University of Iceland, Reykjavík, Iceland, 4 Department of Laboratory Hematology, Landspitali-University Hospital, Reykjavik, Iceland a11111 * ottarr@hi.is Abstract OPEN ACCESS Citation: Choudhary KS, Rohatgi N, Halldorsson S, Briem E, Gudjonsson T, Gudmundsson S, et al. (2016) EGFR Signal-Network Reconstruction Demonstrates Metabolic Crosstalk in EMT. PLoS Comput Biol 12(6): e1004924. doi:10.1371/journal. pcbi.1004924 Editor: Jeffrey J. Saucerman, University of Virginia, UNITED STATES Received: February 12, 2016 Accepted: April 17, 2016 Published: June 2, 2016 Copyright: © 2016 Choudhary 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. Data Availability Statement: All data are fully available. The EMT model used in this study for analyzing the effect of AKT signaling on metabolism can be accessed from Biomodels database: http:// www.ebi.ac.uk/biomodels/, Model ID: MODEL1602080000. All the other data has been provided in main and supporting files. Funding: This project is supported by Icelandic Research Fund (RANNIS) project grants (Grant no. 130591-051 and 152358-051). KSC was supported by Icelandic Research Fund (RANNIS) postdoctoral grant (Grant no. 152369-051). The funders had no Epithelial to mesenchymal transition (EMT) is an important event during development and cancer metastasis. There is limited understanding of the metabolic alterations that give rise to and take place during EMT. Dysregulation of signalling pathways that impact metabolism, including epidermal growth factor receptor (EGFR), are however a hallmark of EMT and metastasis. In this study, we report the investigation into EGFR signalling and metabolic crosstalk of EMT through constraint-based modelling and analysis of the breast epithelial EMT cell model D492 and its mesenchymal counterpart D492M. We built an EGFR signalling network for EMT based on stoichiometric coefficients and constrained the network with gene expression data to build epithelial (EGFR_E) and mesenchymal (EGFR_M) networks. Metabolic alterations arising from differential expression of EGFR genes was derived from a literature review of AKT regulated metabolic genes. Signaling flux differences between EGFR_E and EGFR_M models subsequently allowed metabolism in D492 and D492M cells to be assessed. Higher flux within AKT pathway in the D492 cells compared to D492M suggested higher glycolytic activity in D492 that we confirmed experimentally through measurements of glucose uptake and lactate secretion rates. The signaling genes from the AKT, RAS/MAPK and CaM pathways were predicted to revert D492M to D492 phenotype. Follow-up analysis of EGFR signaling metabolic crosstalk in three additional breast epithelial cell lines highlighted variability in in vitro cell models of EMT. This study shows that the metabolic phenotype may be predicted by in silico analyses of gene expression data of EGFR signaling genes, but this phenomenon is cell-specific and does not follow a simple trend. Author Summary The epidermal growth factor receptor (EGFR) signaling cascade is one of the key signaling pathways that are involved in the induction of Epithelial Mesenchymal Transition (EMT) and tumor metastasis. These signaling cascades often affect metabolic fate in tumor cells PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 1 / 26 Modelling EGFR Signaling in Human Breast Epithelium role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. and control their progression. Here we demonstrate a method to build a mathematical model of the EGFR signaling cascade and use it to study signaling in EMT and how signaling affects metabolism. The model was used to obtain a list of potential signaling and metabolic targets of EMT. These targets may aid in the understanding of the molecular mechanisms that underlie EMT and metastasis. Our results further highlight the heterogeneity of cell models used to study EMT and support the idea of cell specific anti-cancer interventions. Introduction Epithelial to mesenchymal transition (EMT) is a developmental process where polarized epithelial cells transition to an invasive mesenchymal-like phenotype through molecular reprogramming that leads to degradation of the extra-cellular matrix (ECM) and the loss of cell polarity. Following recruitment to specific sites at distant locations within the developing embryo, the mesenchymal cells may revert back to the epithelial phenotype by a process known as mesenchymal to epithelial transition (MET), thereby seeding new epithelial tissues [1]. Although EMT is fundamental for several developmental processes and wound healing, dysregulation of EMT may cause cancer cells to initiate metastasis and form secondary tumors at distant sites [1–3]. EMT is induced by a number of distinct molecular processes [1]. These include the binding of several growth factors, including the platelet derived growth factor (PDGF), insulin-like growth factor (IGF), neuregulin and epidermal growth factor (EGF) to their cognate cell-surface receptors, leading to receptor activation [4]. This activates downstream signaling pathways that regulate the control of specific transcription factors, cell-surface proteins and microRNAs [5]. EMT is also involved in reorganization and expression of cytoskeletal proteins and production of ECM-degrading enzymes [1]. This series of events leads to increased expression of mesenchymal markers like N-cadherin and vimentin and decreased expression of epithelial markers such as E-cadherin [6]. Binding of EGF to its cognate epidermal growth factor receptor (EGFR) family has been shown to stimulate EMT in breast cancer cells [7,8], leading to altered expression of E-cadherin and vimentin [8,9]. Activated EGFR signaling suppresses Ecadherin expression either by promoting its endocytosis [10] or by enhancing the expression of transcription factors (TFs) like Snail and Twist [11,12]. As a result, the cells may transition from epithelial to mesenchymal phenotype with spindle like morphology [8]. EGFR regulates mammary gland development and in certain aggressive breast cancer cells has been shown to regulate invasion and migration [8]. The most common signaling cascades activated downstream of EGFR are PI3K/Akt, Ras/Raf/Mek and DAG/IP3 and CaM signaling, that affect cell cycle progression, inhibition of apoptosis, angiogenesis, tumor cell motility, and metastases (Fig 1) [13,14]. EMT is likely to impact metabolism, but the effects are not as widely studied as cancer metabolism [15,16] Cancer cells exhibit a shift of ATP generation from oxidative phosphorylation to aerobic glycolysis known as the Warburg effect [17]. This leads to a higher rate of glycolysis in cancer cells. Cancer cells also tend to show enhanced glutamine metabolism which has been shown to contribute cancer cell migration [18]. Signaling pathways have often been associated with metabolic consequences, but can themselves be influenced by metabolism. Interestingly, up-regulated glycolysis has been linked with higher AKT signaling in cancer cells [19,20]. However, the mechanistic manner in which metabolism is affected during EMT is unknown. PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 2 / 26 Modelling EGFR Signaling in Human Breast Epithelium Fig 1. An overview of downstream signaling pathways induced by EGFR signaling. Three main pathways: AKT, RAS/MAPK and CaM and DAG/IP3 are induced downstream of active EGFR signaling. doi:10.1371/journal.pcbi.1004924.g001 Computational approaches such as Constraint-based modeling and analysis (COBRA) techniques are very useful in analysis of the complex biological networks like signaling networks [21–23]. Prior efforts of modeling of signaling cascades include modeling of the EGFR pathway [21,22,24–26], TLR signaling [27–29], JAK-STAT signaling [23], MAPK pathway [30] and interleukin 1 signaling [31]. COBRA techniques mainly focus on the use of physio-chemical and biological constraints and are sparsely dependent on kinetic data that has limited availability. Protocols for the generation of biochemical networks and computational algorithms/methods for querying these networks are now well established [32,33]. They involve conversion of biological data (e.g. genomic, metabolic, and regulatory) to a mathematical reaction format. This allows better definition of regulatory changes associated with specific events such as EMT and exploration of metabolic alterations associated with the process. For example, how altered EGFR signaling is propagated to a metabolic phenotype can be investigated using COBRA methodology. In this study, we built a computational signaling network of EGFR to query how the expression of signaling genes can affect metabolic alterations during EMT in human breast epithelial cells. An EGFR signalling network was reconstructed (EGFR_SN) and was constrained with the transcriptomics data of the breast epithelial cell line D492 and its mesenchymal counterpart D492M [34] to form EGFR_E and EGFR_M networks. D492 is an E6/E7 viral oncogene immortalized human breast epithelial basal cell line with stem cell like properties that differentiates into both myoepithelial and luminal cells and has the ability to undergo branching morphogenesis when grown in a 3D reconstituted basement membrane matrix [35]. The 3D coculture of D492 cells with human endothelial cells led to establishment of mesenchymal cells with spindle-like morphology called D492M. The D492M cells have high expression of N-cadherin and vimentin and low expression of E-cadherin that is typical for cells that have undergone EMT [34]. PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 3 / 26 Modelling EGFR Signaling in Human Breast Epithelium The EMT specific signaling network of D492 and D492M enabled us to investigate differential gene expression of EGFR signalling genes. This was subsequently extended to other breast epithelial cell lines and their mesenchymal counterparts. Through extensive literature review, the EGFR_SN network was linked to metabolic genes that were likely to be affected. The differential flux values in EGFR_E and EGFR_M allowed the assessment of how the altered signaling affects metabolic gene expression and metabolism in D492 and D492M cells. Increased flux within the AKT and RAS/MAPK signaling pathways was predicted in D492 as compared to D492M. The in silico predicted increase in flux of the AKT pathway induced higher glycolytic activity in D492 cells. This suggested that there may be an EMT-related decrease in glycolysis in D492M as compared to D492 cells, which was confirmed in vitro by glucose uptake and lactate secretion measurements. Comparative analysis of EGFR signaling networks in three other breast epithelial cell lines showed that regulation of signaling pathways are cell specific and follow no simple trend. Results and Discussion EGFR signaling network reconstruction In order to capture how altered EGFR signaling is propagated through metabolic pathways in the breast epithelium, we built a constraint based EGFR network, EGFR_SN. To generate EGFR_SN, the EGFR pathway map (Reactome ID: R-HSA-177929) was downloaded from the Reactome database [36]. Several modifications such as incorporation of gene-protein reaction (GPRs) rules/association, additions of modifiers, inhibitors and activators and removal of dead ends were made to the Reactome pathway to make it feasible for analysis with the COBRA methodology (methods section). Due to the incorporated GPRs, experimental data (e.g, gene expression, proteomic, fluxomic data) can now be mapped onto EGFR_SN, thereby relating genomic information to the reactions in the network. The resulting reconstructed network EGFR_SN accounts for 182 reactions, 216 genes, 152 reacting species, 11 inhibitors and 2 activators. The 182 reactions were divided between 83 internal reactions and 99 exchange reactions. Exchange reactions were added in order to remove dead-ends in the network and acquire feasibility. The number of exchange reactions added here is large compared to what is typically present in metabolic networks, where exchange reactions are added mainly to allow for accumulation of metabolites and secretion of wastes. Internal reactions represent connections between internal signaling components, while the exchange reactions represent connections of the system boundary with the environment. Fig 2 shows a sub-network of EGFR_SN representing AKT signaling. D492 and D492M specific EGFR signaling networks Microarray gene expression profiles of the human breast epithelial cell line D492 and the mesenchymal like D492M were used to constrain the EGFR_SN network to build an EMT specific signaling model. D492 can be used as a model for studying EMT for which biological data is available: mRNA, micro-RNA, cell phenotypic data, growth curves etc. [34,37]. D492 has previously been used in determining the role of microRNAs in EMT [37], studies related to branching morphogenesis [38] and more recently it was used to investigate the role of EGFR as a tumor suppressor [39]. To derive epithelial and mesenchymal specific signaling networks, gene expression data from D492 and D492M was mapped onto the EGFR_SN network. The pipeline is described in Fig 3. Differentially regulated (up-regulated and down-regulated) genes in the two cell lines led to two different signaling networks: EGFR_E and EGFR_M for D492 and D492M respectively, as described in the methods section. PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 4 / 26 Modelling EGFR Signaling in Human Breast Epithelium Fig 2. Sub-network of EGFR_SN network. A part of the EGFR signaling network representing AKT signaling. Nodes indicate the reacting component and edges denote the reactions. Red nodes indicate inhibitors which were manually added to the model in the form of GPRs (information obtained from the Reactome database), white nodes indicate signaling components present in the original SBML file downloaded from the Reactome database and green nodes indicate exchange reactions which were added to the model to remove dead ends. Node ‘B’ and ‘P’ indicates binding, and phosphorylation, respectively. Edges: ! transition, ⟞ inhibition, ⫯ catalysis. doi:10.1371/journal.pcbi.1004924.g002 Network simulation of D492 and D492M Flux analysis discriminates between D492 and D492M EGFR signaling phenotypes. EGFR_E and EGFR_M networks were investigated through flux analysis. Flux distribution in EGFR_E and EGFR_M networks were estimated using random sampling [21,40] (Fig 4). Fig 4A shows the probabilty density for the flux in selected reactions, where red and blue lines denote EGFR_M and EGFR_E, respectively. Fig 4A, panel A1 shows that the most probable flux for this reaction in EGFR_E is higher than in EGFR_M, indicating this reaction is more active in EGFR_E. The panels A2-A6 belonging to AKT and RAS/MAPK pathways show that PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 5 / 26 Modelling EGFR Signaling in Human Breast Epithelium Fig 3. The pipeline employed to generate context specific signaling networks EGFR_E and EGFR_M of an immortalized breast epithelial cell line (D492) and mesenchymal cell line (D492M). SBML pathway map from Reactome was converted to mathematical format using the COBRA toolbox. This network was further modified to remove dead ends and include GPRs for modifiers, activators and inhibitors, resulting in the formation of the EGFR_SN network. The EGFR_SN network was constrained with the transcriptomic data of D492 and D492M to form EGFR_E and EGFR_M networks. Flux differences between EGFR_E and EGFR_M were used to further predict metabolic phenotype and target reactions critical for the reversal of mesenchymal to epithelial phenotype. doi:10.1371/journal.pcbi.1004924.g003 the most probable flux in EGFR_M is lower than the most probable flux in EGFR_E. The graphs in panels A7-A9, belong to the CaM pathway and are in contrast to panels A2-A4, indicating higher flux in EGFR_M. Fig 4B shows the relative mean flux values in each reaction within the AKT, RAS/MAPK and DAG/IP3 and CaM pathways. The EGFR_E network had higher flux through the AKT and RAS/ MAPK pathways. In contrast, the transition from epithelial (EGFR_E) to mesenchymal (EGFR_M) phenotype promotes higher flux through the calcium CaM signaling pathway in the EGFR_M network. Moreover, flux was also increased in the di-acyl glycerol (DAG) and inositol trisphosphate (IP3) pathway in the EGFR_M network, which constitutes an important part of the CaM pathway. Predicting reversal of mesenchymal to epithelial phenotype We next studied how the flux in the EGFR_M network could be modified so that it became similar to the EGFR_E flux, in order to identify how the EMT process could be reversed. This PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 6 / 26 Modelling EGFR Signaling in Human Breast Epithelium Fig 4. Flux differences between epithelial network (EGFR_E) and mesenchymal network (EGFR_M). A) Probability density estimates for the flux values in selected reactions as obtained by random sampling. The blue curve represents the flux distribution of EGFR_E and the red curve that of EGFR_M. Vertical axis denote probability and flux values are represented on the horizontal axis. AU: arbitrary units B) Relative mean flux for each reaction in EGFR_E and EGFR_M through the AKT, RAS and DAG/IP3 and CaM pathways. Higher flux within reactions in AKT and RAS/MAPK pathways are observed in the EGFR_E network compared to EGFR_M while CaM and DAG/IP3 have higher flux in EGFR_M. Negative values denote higher flux in EGFR_E and positive values denotes higher flux in EGFR_M. Numerical values of these fluxes are given in supplementary file (S1 File). doi:10.1371/journal.pcbi.1004924.g004 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 7 / 26 Modelling EGFR Signaling in Human Breast Epithelium was achieved by using an optimization algorithm similar to the MOMA algorithm which is frequently used to study perturbations in metabolic networks [41]. The algorithm searches for a flux distribution in EGFR_M, which most closely resembles the average flux values obtained previously for the EGFR_E model with random sampling, with minimum relaxation in the reaction bounds in EGFR_M while maintaining steady state conditions, see the methods section for more details. The algorithm highlighted five reactions whose bounds needed to be relaxed such that the flux distribution of EGFR_M is close to that of EGFR_E. These included three internal reactions: 1) phosphorylation of the MAP2K dimer by RAF, 2) phosphorylation of MAPKs by MAP2Ks (RAF/MAPK pathway), 3) PIP2 conversion to PIP3 by PIK3 (AKT pathway), and two exchange reactions, belonging to the RAF/MAPK pathway. Through GPRs we identified that there are 22 genes associated with the reactions predicted above (S1 Table). However, among these 22 genes, MAPK1, NRAS, HRAS and EGFR genes were overexpressed in D492 as compared to D492M and the inhibitor PTEN was overexpressed in D492M. Based on these predictions and by analysing the flux differences between EGFR_E and EGFR_M, we hypothesize that increased AKT and RAS/MAPK signaling in D492 epithelial cells promotes their transition to the D492M mesenchymal like phenotype. However, after attainment of a mesenchymal state, AKT and RAS/MAPK signaling is reduced and alternative pathways such as CaM signaling gets activated which may be involved in the maintenance of the mesenchymal state. We also hypothesize that up-regulation of MAPK1, NRAS, HRAS and EGFR and down-regulation of PTEN inhibitor in EGFR_M may lead to its transformation into EGFR_E. Interestingly, most of the in silico predicted targets from our study have previously been implicated to play a role in EMT. The activation of MAPK1 protein has been shown to induce EMT in MCF10 breast epithelial cells by phosphorylation and consequent stabilization of Twist1 [42]. In another study, the silencing of MAPK1 led to increased expression of E-cadherin and a decrease in vimentin and Snail expression in human cervical cancer cells [43], suggesting its role in the transition from epithelial to mesenchymal phenotype. HRAS and Slug together have been shown to induce the expression of vimentin and enhance cell migration in pre-malignant MCF10A breast epithelial cells [43]. Further, activation of EGFR has been shown to induce the expression of Twist by activating STAT3, suggesting a prominent role of EGFR in EMT [11]. In a recent study where mesenchymal cells were generated from D492 by overexpressing HER2 (ErBb2) [39], subsequent overexpression of EGFR promoted mesenchymal to epithelial transition. In light of this finding, and based on our in silico predictions, we made EGFR overexpressing D492M cell line (D492MEGFR) (method section). D492MEGFR cells showed higher phosphorylation of AKT and ERK1/2 (Fig 5), although reversal of mesenchymal to epithelial phenotype was not observed (S1 Fig). Thus, we hypothesize that for complete reversal of mesenchymal to epithelial phenotype, the MAPK1, NRAS and HRAS genes may need to be overexpressed in addition to EGFR, along with PTEN inhibition in D492M. Likewise, maintaining expression of the MAPK1, NRAS, HRAS and EGFR in epithelial cells while suppressing the expression of PTEN is expected to inhibit EMT. At present, a number of EGFR inhibitors have been approved by the FDA for cancer treatment. These include Cetuximab and Panitumumab that are monoclonal antibodies against EGFR, and Erlotinib, Gefitinib and Lapatinib which are specific tyrosine kinase inhibitors against EGFR. All these compounds are prescribed against advanced, late-stage or metastatic cancers [44,45]. The findings we present here suggest that inhibition of EGFR may contribute to or increase the risk of EMT in cancers of epithelial origin. Indeed, the overexpression of HER2 in D492 cells has recently been shown to suppress EGFR expression and induce EMT [39]. Our predictions also indicate that PTEN inhibition can help maintain the epithelial phenotype, thereby preventing EMT and metastasis. However, recent findings suggest that loss of PTEN function may promote tumor PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 8 / 26 Modelling EGFR Signaling in Human Breast Epithelium Fig 5. EGFR overexpression in D492M. (A) Real-Time Quantitative Reverse Transcription PCR of EGFR in D492, D492MEGFR and D492MEmpty, normalized to GAPDH. EGFR transcription level is significantly higher in D492MEGFR compared to D492MEmpty but does not reach the D492 EGFR transcription level. (B) Protein expression of EGFR, Phospho-p44/42 MAPK (ERK1/2) and Phospho-AKT determined by Western blotting. Overexpression of EGFR in D492M leads to increased protein expression of EGFR in D492MEGFR compared to D492MEmpty, but EGFR protein expression does not reach the D492 level. Overexpression of EGFR in D492M leads to increased MAPK (Erk1/2) and Akt phosphorylation which is higher than in both D492MEmpty and D492. doi:10.1371/journal.pcbi.1004924.g005 progression in a mouse model [46] and EMT in human colon cancer cells [47], highlighting the fact that further research on the role of PTEN in EMT is needed. Taken together, these above results demonstrate the EGFR_SN network can be used to study in detail the differences in EGFR signaling between epithelial and mesenchymal cells during EMT and identify gene targets which could possibly be used to hinder or even revert EMT. Crosstalk of the signaling and metabolism in D492 and D492M We next studied how changes in AKT signaling could influence metabolic gene expression in EMT. The AKT pathway was chosen since it has previously been shown to affect various metabolic pathways, e.g. glycolysis and fatty acid metabolism in cancer cells [19,48]. To reflect how PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 9 / 26 Modelling EGFR Signaling in Human Breast Epithelium Table 1. Predicted expression of metabolic genes regulated by AKT in D492 and D492M cells. No. References Metabolic Genes Proposed Expression Microarray Expression 1 [50] GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) #M #M 2 [51] GLUT1 (facilitated glucose transporter) #M #E 3 [48,52] GYS1 (Glycogen [starch] synthase, muscle) #M #M 4 [53,54] HK1 (Hexokinase-1) #M #M 5 [53,54] HK2 (Hexokinase-2) #M #M 6 [52] G6PC (Glucose-6-phosphatase) #E NA 7 [52] PCK1 (Phosphoenolpyruvate carboxykinase 1) #E NA 8 [48] ACLY (ATP-citrate synthase) #M #M 9 [48] ME1 (Malic enzyme) #M #M 10 [19,55] PFKFB2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2) #M NA 11 [48] HMGCR (3-hydroxy-3-methylglutaryl coenzyme A reductase) #M #M 12 [48] HMGCS1 (Hydroxymethylglutaryl-CoA synthase, cytoplasmic) #M #M 13 [56] ACC (acetyl-CoA carboxylase alpha) #M NA 14 [56] SREBF1 (Sterol regulatory element-binding protein 1) #M #E 15 [48,56] SREBF2 (Sterol regulatory element-binding protein 2) #M NA 16 [56] FASN (Fatty acid synthase) #M #M 17 [57] ATIC (Bifunctional purine biosynthesis protein PURH) #M #M 18 [57] HPRT1 (Hypoxanthine-guanine phosphoribosyltransferase) #M #M 19 [57] TALDO1 (Transaldolase) #M #M 20 [58] TKT (Transketolase) #M #M Increased ﬂux through the AKT pathway in EGFR_E network as compared to EGFR_M suggested up-regulated expression of genes involved in glycolysis, fatty acid and purine/pyrimidine metabolism in D492 cells and down-regulated expression of genes involved in gluconeogenesis pathway. The “References” column lists the studies from which the inﬂuence of AKT signaling on the expression of the corresponding metabolic genes was derived. No: 1–7 belong to Carbohydrate metabolism, 8–16 to fatty acid metabolism and 17–20 to purine/pyrimidine metabolism. NA: gene expression data is not present in the microarray data set. doi:10.1371/journal.pcbi.1004924.t001 alterations in the AKT signaling pathway may affect metabolism, a literature based survey was conducted to identify the connections between this pathway and its target metabolic genes. This suggested that active AKT signaling induces the expression of metabolic genes involved in glycolysis, fatty acid metabolism and purine and pyrimidine metabolism, while it suppresses the expression of metabolic genes involved in gluconeogenesis (Table 1). The ratio of average flux in each signaling reaction of the AKT pathways in the EGFR_M and EGFR_E network was used to identify differences in relative metabolic gene expression in the D492 and D492M cells. The higher flux observed in the AKT pathway in the EGFR_E network suggested increased expression of genes belonging to glycolysis, fatty acid and purine/ pyrimidine metabolism in D492 in comparison to D492M (Table 1). Higher expression of glycolytic genes suggested higher glycolytic activity in the D492 cells. To test these in silico predictions, we compared the predicted expression of the metabolic genes with their corresponding relative expression values in the microarray data set [34] and compared relative expression of metabolic genes in D492 and D492M. Up- and down-regulated metabolic genes were identified based on differential expression and significance measurements, as analyzed by SAM [49], included in the microarray dataset. A cut-off of 0.05 on the significance measure was used. The relative gene expression of 13 out of 15 metabolic genes (86.6%) affected by AKT signaling were in agreement with the predicted in silico expression (Table 1). Since the experimental expression values of the metabolic genes were not used to PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 10 / 26 Modelling EGFR Signaling in Human Breast Epithelium generate the EGFR_E and EGFR_M networks, this data corresponds to an independent validation set for the in silico predictions. However, the gene expression of 2 out of 15 (13.3%) of the metabolic genes (GLUT1 and SREBF1) from the microarray data and in silico predictions were not in agreement. This suggests an alternate level of regulation that metabolic genes may encounter during EMT, in addition to the direct regulation by molecular signaling pathways. For accuracy measure we did not take into account metabolic genes which did not have any detectable expression values (denoted NA). Ultimately, we observed that the expression of most of the metabolic genes directly affected by AKT signaling during EMT was correctly predicted in our D492 model system. Glycolytic flux is increased in D492 as compared to D492M and consistent with model predictions Gene expression measurements of metabolic genes are not necessarily a quantitative predictor of their metabolic activity. We therefore tested our in silico prediction that glycolytic activity is higher in D492 than in D492M by measuring the proliferation rates and glucose consumption and lactate secretion rate to estimate glycolytic activity in vitro. Cells with a higher rate of proliferation may have higher nutrient and energy requirements and consequently greater metabolic demand [59]. Cell proliferation assays showed that the D492 cells had a higher growth rate than D492M (Fig 6A). Spent medium of D492 and D492M cells was analyzed to measure glucose and lactate levels. Higher glucose levels and lower lactate levels in cultured supernatants of the D492M cells indicated a lower rate of glucose consumption and lactate secretion rates in D492M cells (Fig 6B). This indicates a lower glycolytic rate in D492M cells compared to D492 and suggests a shift in the glycolytic capacity of the cells in response to EMT and is in agreement with the in silico predictions that indicated higher gene expression of glycolytic enzymes in D492 cells. We conclude that this EMT related decrease in aerobic glycolysis appears to be driven by an overall decrease in the expression of glycolytic enzymes due to down-regulated AKT signaling in D492M cells. To the best of our knowledge, this is the first report that demonstrates that in silico network predictions can be used to study the influence of a molecular signaling pathways, such as AKT, on the metabolic outcome during EMT in breast epithelial cells. Our findings are in agreement with a previous study on human non-small cell lung carcinoma (NSCLC) cells [60] which demonstrated a decrease in aerobic glycolysis during EMT but are in contrast to results obtained for MCF7 breast epithelial cells that have undergone EMT [61]. EMT metabolic network constrained with AKT signaling regulated metabolic genes Results from the previous section showed that the metabolic phenotype could be accurately predicted by in silico analyses of the changes in the expression of AKT signaling genes. Further, we investigated how these changes in AKT signaling, that impact the expression of metabolic genes (Table 1), are propagated through other metabolic pathways during EMT. Based on RECON2 [62], we have also built an EMT metabolic network (MODEL1602080000). This metabolic network was built by constraining RECON2 with microarray gene expression data [34] of metabolic genes during EMT in D492 and D492M. RECON2 is a global human metabolic reconstruction that has been used previously to investigate regulation of metabolism in diseases like obesity and diabetes [40,62]. In this study, we constrained our EMT metabolic network with the metabolic genes that were predicted to be regulated by changes in AKT signaling (Table 1). This led to the formation of epithelial metabolic (Met_E) and mesenchymal PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 11 / 26 Modelling EGFR Signaling in Human Breast Epithelium Fig 6. Proliferation rate and glycolytic activity are higher in D492 than in D492M cells. A) Cell proliferation assay demonstrated a higher growth rate of D492 cells compared to D492M cells. B) Spent medium analysis of glucose (dashed lines) and lactate (solid lines) shows higher glucose uptake and lactate secretion in D492 cells (blue) than in D492M cells (red). C) Calculated glucose uptake and lactate secretion rates indicate higher glycolytic flux rates per cell per hour in D492 cells than D492M cells. Data represents results from 3 independent experiments. Error bars represent standard deviation in a single experiment done in triplicate. mM: milli molar, fmol: femto molar. doi:10.1371/journal.pcbi.1004924.g006 PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 12 / 26 Modelling EGFR Signaling in Human Breast Epithelium metabolic (Met_M) networks, specific for AKT signaling regulated metabolism during EMT (described in methods section). Metabolic differences between the Met_E and Met_M models were identified based on differences in their relative flux span (methods section). Reactions carrying higher flux in Met_E compared to Met_M included reactions that are involved in N-glycan metabolism, Glycolysis, Fatty acid synthesis, Fatty acid oxidation, nucleotide interconversion and pentose phosphate pathway. Reactions carrying higher flux in Met_M involved Glutathione, glycerophospholipid, and inositol phosphate metabolism. Alteration of metabolic pathways, including N-glycan, glutathione metabolism, glycolysis, fatty acid and purine metabolism that we observed from our constructed Met_E and Met_M metabolic networks, have been shown to play an important role in the regulation of EMT [19,48,57,60,63,64]. However, the details of the mechanism are still unknown. A list of the metabolic reactions similarly predicted to be affected by the AKT pathway in the Met_E and Met_M networks are provided in the supplementary file (S2 File). In conclusion, this method was able to predict metabolic pathways that may be affected downstream upon activation of AKT signaling in breast epithelial cells during EMT. This method extends the approach of associating metabolic phenotype with regulation of signaling pathways. Further, it also suggests the possibility of determining the metabolic regulation in cases that are limited by the availability of metabolomic data or the gene expression data of metabolic genes. Although these predictions are context specific in relation to AKT signaling, the integration of other signaling pathways affected during EMT may give a more coherent picture of altered metabolism. Comparative analysis of the EGFR signaling network between D492 and three other human breast epithelial cell lines We next studied whether a general trend of higher flux in the reactions of the AKT and RAS pathways (downstream of EGFR) was observed in other human breast epithelial cell lines, when compared to their mesenchymal counterparts. We mapped the microarray transcriptomic datasets for the three human breast epithelial cell lines (HMLE, MCF-7 and MCF-10A) onto the EGFR_SN network similar to the method used for the D492 cells (methods section). Comparisons between cell lines were done in terms of the ratio between flux in the mesenchymal network vs flux in the corresponding epithelial network (Fig 7). Numerical values of the fluxes within AKT, RAS and CaM pathways in HMLE, MCF-7 and MCF-10A are given in supplementary file (S3 File). Higher flux through the reactions in the AKT pathway was observed in both the D492 and HMLE epithelial cells, suggesting that the HMLE cells may have similar metabolic phenotype as D492 (Fig 7). In contrast, the mesenchymal counterparts of MCF7 and MCF10A cells had higher flux in the AKT pathway (Fig 7), suggesting that cells that have undergone EMT may have increased glycolytic activity. This is in agreement with Kondaveeti et al. who have reported an increase in glycolytic activity post-EMT in MCF-7 cells as a result of increased expression of glucose transporters and lactate dehydrogenase [61]. The flux in the RAS/MAPK pathway was higher in the D492 and the MCF7 epithelial cells than in their mesenchymal counterparts. Analysis of DAG/IP3 and CaM pathway showed that D492M, MCF7 mesenchymal cells and those of HMLE which have undergone EMT due to induction of Twist, had higher flux as compared to their epithelial counterpart (Fig 7). Thus, no general pattern was observed in the flux distributions between the epithelial and mesenchymal networks for the different breast epithelial cell lines. Induction of EMT by different factors (viral induction of SNAIL, SLUG, TWIST, miR374a or TGFβ1 treatment) also seemed to differentially regulate signaling pathways as was evident in the MCF10A and HMLE cells. For example, the induction of EMT in HMLE cells by overexpression of Slug and Twist resulted in different flux patterns PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 13 / 26 Modelling EGFR Signaling in Human Breast Epithelium Fig 7. Comparison of flux distribution in the reactions within AKT, RAS and CaM pathway in different breast epithelial cell lines. The heat map has been generated using log2 relative mean flux of the reactions within the AKT, RAS and DAG/IP3 and CaM pathways in epithelial and mesenchymal cells of D492, MCF, MCF10A and HMLE cells. Exchange reactions are not included in the heat map. The negative and positive values as depicted on the color scale denote higher flux in reactions of epithelial and mesenchymal cells, respectively. D492M, MCF7_mirna, MCF7_snail, MCF10_snail, MCF10A_tgfb, HMLE_slug, HMLE_twist and HMLE_snail are EMT derived mesenchymal cells. doi:10.1371/journal.pcbi.1004924.g007 in the RAS/MAPK and DAG/IP3 and CaM pathways (Fig 7). Similar effect was seen by Deshiere et al., where they have shown that TGFβ1 treatment and CK2b silencing activate divergent signaling pathways, that ultimately lead to EMT in MCF-10A cells [65]. Finally, we compared the predicted metabolic phenotypes to the metabolic gene expression data for the MCF7, MCF10A and HMLE cell lines similar to the method used for the D492 cells, however the gene expression data of metabolic genes were not statistically significant and hence was not included in our study (S1–S4 Tables). In summary, we observed that different cell lines may affect different signaling regulation during EMT. Moreover, variation in the methods of EMT induction may dictate differential regulation of the signaling and metabolic cross talk. Conclusions Herein we have demonstrated a method to build a stoichiometric model of the EGFR signaling (EGFR_SN) network employing COBRA methods that aids in understanding the differential activation of downstream EGFR signaling pathways during EMT. Epithelial and mesenchymal specific EGFR signaling networks were obtained by integrating microarray transcriptomics data of signaling genes from the D492 breast epithelial and mesenchymal cells with EGFR_SN. The epithelial and mesenchymal networks were used to predict the expression of metabolic genes. The predicted expression values were in agreement with transcriptomics data of PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 14 / 26 Modelling EGFR Signaling in Human Breast Epithelium metabolic genes as well as biochemical data that demonstrated higher glycolytic activity in D492 epithelial cells. Furthermore, signaling genes leading to reversion to the epithelial phenotype (MET) via up- regulation in the mesenchymal cells were predicted. Additional in vitro testing would be required to confirm these in silico predictions. Thus, in this study we showed that the metabolic phenotype can be predicted in silico using gene expression profiles of EGFR signaling components. EGFR_SN is not limited in scope to the investigation of EMT. The signalling network could be used to highlight signaling-metabolic crosstalk in different cell types for which metabolic reconstructions exist [66] or for different conditions [66] where EGFR signalling is known to be influential [67]. Furthermore, the network described herein could be expanded to allow for more comprehensive coverage of signalling pathways of relevance to EMT. Signaling networks for platelet derived growth factor (PDGF), insulin like growth factor (IGF), and vascular endothelial growth factor (VEGF) for example, could be constructed and co-integrated. The co-integration of these signaling networks regulated by different growth factors would give more comprehensive knowledge of cross talk between signaling and metabolic pathways during EMT. The pipeline developed for D492 was used on other cell models representative of breast epithelial cell lines. We assumed that different breast epithelial cells models would have similar signaling patterns and hence could have a general interpretation of regulation of signaling pathways during EMT. In contrast to our hypothesis, the regulation of signaling pathways showed no general pattern and appeared to be a cell-specific phenomenon. Flux values in the AKT pathway from our network, suggest that there may be an EMT related decrease in aerobic glycolysis in both the HMLE and D492 cell lines, while the opposite was observed in the MCF7 and MCF-10A breast epithelial cells. There are several possible explanations for this disparity. First, the highly complex nature of the regulation of EMT, which may be differentially regulated by the cellular micro environment or EMT inducing factors as seen in our in silico predictions. Second, our study of EMT signal transduction is primarily based on transcriptional signatures which may or may not necessarily correlate with the translational output (proteinlevels) [68,69]. This last issue might be addressed by co-integrating transcriptomics and proteomics/phospho-proteomics data in order to obtain more accurate models. Such a strategy was recently reported where it was used to reconstruct a metabolic network for predicting of metabolic signatures in diabetes patients [70]. The disparity between cell models suggests considerable heterogeneity of the cell models used for EMT research in general. Finally, these efforts highlight a lack of comprehensive datasets available that accurately describe EMT and ultimately hinder mechanistic understanding of the genotype phenotype relationship underlying EMT. The direct link between regulation of signaling pathways and the consequent metabolic phenotype may be of clinical interest, as metabolically based therapeutics to combat cancer EMT could be masked by inaccurate metabolic understanding. Methods Reconstruction of signaling network The EGFR pathway network was downloaded from the Reactome database which is curated and peer reviewed [71]. The EGFR pathway was then converted from SBML to COBRA format for further analyses. This conversion is based on the stoichiometric coefficients of the reacting species provided in the Reactome pathway and also requires setting constraints on each reaction in the form of lower and upper bounds, which determine the minimum and maximum allowable reaction rates (fluxes), respectively [33]. Flux in a signaling network is defined as the rates of phosphorylation, de-phosphorylation, dimerization, or binding of proteins. PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 15 / 26 Modelling EGFR Signaling in Human Breast Epithelium The COBRA model was represented by an m by r stoichiometric matrix S, where m denotes the number of network components (metabolites, proteins, and complexes) and r the number of network reactions. Reactions within the network were mass-balanced. The system was assumed to be at steady state, which means that the fluxes v = (v1,. . .,vm) satisfy the equations Sv = 0. The upper and lower bounds of all the internal reactions were set to 1000 and zero, respectively. A lower bound of zero was used since all the reactions are irreversible. The initial COBRA model contained many dead ends and was infeasible due to network gaps [72]. Dead-ends represents those reacting species which are either only produced or only consumed in the network, leading to blocked reactions, i.e. reactions unable to carry flux.To remove the dead ends and obtain a feasible model, exchange reactions were introduced allowing uptake and secretion of components across the system boundary. By adding exchange reaction for all reacting species, a feasible model was obtained. In this model, the network topology becomes irrelevant since all demands on the network can be met by the exchange reactions. To avoid this situation, an optimization algorithm, ‘relax_rxns’ (S1 Dataset) was developed that enabled a feasible steady state network, while minimizing uptake/secretion (exchange) of the dead-end molecules. The methodology is similar to the one used by Vardi et al. [21]. First, the lower bounds of all the internal reactions were set to 1 to force removal of blocked reactions (reactions with zero flux) and consequently removal of the dead-end species. Exchange reactions were then added for every reacting species in the network, initially with all uptake and secretion rates set to zero. The optimization algorithm returns a minimal set of exchange reactions that need to be present in order to remove all dead ends. These reactions were included in the final model, but the other exchange reactions were removed. The optimization problem was formulated as follows: P ð1Þ minimize j2Rr yj Sv ¼ 0 lj nj vj uj þ pj li vi ui pj Myj ; nj Myj pj 0; nj 0 yj 2 f0; 1g ð2Þ j 2 Rr i 2 Rn j 2 Rr j 2 Rr j 2 Rr ð3Þ ð4Þ ð5Þ ð6Þ ð7Þ The decision variables are v, the flux values in individual reactions, pj and nj which represent the amount of relaxation of upper and lower bounds for reaction j, respectively and binary variables yj which indicate whether reaction j is relaxed or not. The objective is to minimize the number of reactions that are relaxed (1). The steady state mass balance constraints are represented by (2), Rr is the set of reactions that are to be relaxed (3) with the corresponding upper and lower bounds set to zero. The set Rn represents all the remaining reactions (4) with the corresponding upper bounds set to 1000 and lower bounds set to 1. The value of the constant M in (5) was set to 1000. The optimization model was implemented in Matlab (Mathworks, Natick, MA, USA) using the CVX modeling language [73,74] and solved using the Gurobi solver [75]. The version of CVX used in this study supports binary variables as those in constraint (7). PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 16 / 26 Modelling EGFR Signaling in Human Breast Epithelium Modelling modifiers, activators and inhibitors The signaling network from section 4.1 was extended to include modifiers, activators and inhibitors. Modifiers are phosphorylated protein entities that further phosphorylate downstream targets. In the original Reactome pathway, the modifiers were not included as reacting species in reactions, such that they were not connected to downstream targets. For this reason modifiers were included as the reacting species in their target reactions. We included the modifiers in the same way as described by Dasika et al. [22]. Briefly, modifier mod1 acts as a modifier for the transition of A to B. In order to avoid unambiguous stoichiometry, we added mod1p, as a product of mod1 during the reaction as shown below. Production of mod1 ! mod1 ðexchange reactionÞ A þ mod1 ! B þ mod1p ðmod1 becomes mod1pÞ mod1p ! consumption of mod1p ðexchange reactionÞ Activators and inhibitors are responsible for positive and negative regulation of the reaction, respectively. Activators and inhibitors were included in the model via gene-protein reaction rules as described in the next section. Addition of gene information The original Reactome signaling network described the transmission of signal, in terms of activation or inhibition of the subsequent downstream entities. This did not include any enzymatic reactions or the gene-protein rules required to map gene expression data [33]. The signaling network was therefore modified to include GPRs by associating each network reaction with genes encoding modifiers, activators and inhibitors. Reactions not having any information of modifiers, activators or inhibitors were not designated with GPRs. For the GPR generation, we employed UniProt IDs of the protein entities within the Reactome pathway to identify genes, which were then associated with reactions using Boolean logic. Multiprotein complexes were represented with an ‘AND’ operator, while isoforms were represented by an ‘OR’ operator to create a corresponding Boolean rule. Wherever an inhibitor was involved, the corresponding genes were prefixed by a ‘NOT’ operator in the GPR. Exchange reactions were also assigned GPRs. If a reaction is catalysed by a modifier and inhibited by an inhibitor, the presence of modifier will activate the reaction, while an inhibitor will inhibit the reaction. GPRs for all the reactions are provided in the S1 File in the sheet named “GPRs”. The blank rows denote that they were not assigned any GPRs. Fig 8 below, illustrates examples of GPR generation. The resulting network is referred to as EGFR_SN. A spreadsheet containing all the network reactions, reacting species, modifiers, inhibitor, activators, and GPRs in different sheets is provided in the supplementary file (S1 File). All the models used in this study are provided in the supplementary dataset (S1 Dataset). Microarray data integration Gene expression data for the D492 and D492M cell lines was obtained from Sigurdsson et al [34]. The microarray expression data for the HMLE, MCF-7 and MCF-10A cell lines was obtained from NCBI GEO [76], GEO IDs: GSE52593 [77], GSE43495 [78], GSE58252 [79], GSE39358 [80], and GSE28569 [65]. Illumina/Affimetrix IDs within the microarray data were mapped with the Uniprot IDs of the genes in the EGFR_SN network using the Python API of bioDBnet, biological DataBase network. Mapped gene expression of the EGFR signaling genes PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 17 / 26 Modelling EGFR Signaling in Human Breast Epithelium Fig 8. The rules for adding GPRs. Reaction1 is catalyzed by a modifier which is a multiprotein complex of gene1 and gene2. The combination of both the genes are required for the reaction to take place. Reaction2 is inhibited in presence of inhibitor which is an isoform of gene3 and gene4. Presence of either gene3 or gene4 will inhibit Reaction2. A ‘NOT’ operator is assigned to the GPR of Reaction2 to indicate inhibition. A GPR is not assigned to Reaction 3 in this example. doi:10.1371/journal.pcbi.1004924.g008 for each cell line is provided in the supplementary material S4 File with sheets named after each cell line. Negative values indicate higher gene expression in epithelial cells and positive values indicate higher gene expression in mesenchymal cells. Integration of expression data with the EGFR_SN network and subsequent analysis was performed in Matlab. The cut-off value on the relative fold change between epithelial and mesenchymal cell lines, to determine up- and down-regulated genes, depended on the relative expression of housekeeping genes and the statistical significance (p-value 0.05) of the fold change. Accordingly, a cut-off of 2 was considered for D492 cells, 0.5 for MCF7 and MCF10A cells and 0.3 for HMLE cells. Since GPRs link genes with reactions, up- and down-regulated genes identified consequently up-regulated and down-regulated reactions. The change in the expression value of each gene in the EGFR_SN signaling network was used to define the upper and lower flux bounds of its associated reactions, such that up-regulated reactions were allowed to have higher flux values and down-regulated reactions were allowed to have lower flux values. Since, the upper bounds on individual fluxes are essentially infinite, up-regulation in an epithelial model was simulated by downregulating the corresponding reaction in its mesenchymal counterpart, and vice versa. Flux bounds of the up-regulated reactions in D492 were constrained by an arbitrary factor one-hundredth of the initial bounds in D492M and vice versa. Similarly, flux bounds of the activated and inhibited reactions were constrained. Activation in the epithelial model was simulated by downregulating the corresponding reaction in its mesenchymal counterpart and vice versa, while inhibition was simulated by downregulating the corresponding reaction in the same model. This led to the formation of EGFR_E and EGFR_M networks for D492 and D492M respectively. Random sampling was used to obtain flux distributions in the networks [81] using the COBRA toolbox [32], technical details of which can be found in Supplementary methods (S1 Methods). PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 18 / 26 Modelling EGFR Signaling in Human Breast Epithelium Flux differences in epithelial and mesenchymal networks and crosstalk to metabolism Flux differences in individual reactions in the mesenchymal and epithelial networks were quantified in terms of fold changes, vM(i) / vE(i) where vM(i) and vE(i) represent the average flux in reaction i for the mesenchymal and epithelial networks, respectively. Reactions which had vM(i) / vE(i) greater than 1 carried higher flux in the mesenchymal network. A ratio below 1 indicates higher flux in the epithelial network. A literature based survey provided evidence of whether a metabolic gene is positively or negatively regulated by AKT signaling (S5 Table). The prediction of the metabolic gene expression derives from whether EGFR_E or EGFR_M have higher flux in AKT signaling. Minimization of the distance between the Mesenchymal and Epithelial flux distributions The optimization algorithm of section 4.1 was modified by replacing the objective function (1) by P ð1aÞ minimize akv vE k þ ð1aÞ j2Rr yj where v and yj represents the decision variables as before, vE are ﬁxed values representing the mean ﬂux distribution of EGFR_E, obtained from random sampling and \|\|. \|\| represents the Euclidean norm. The upper and lower bounds for each ﬂux correspond to the values from the EGFR_M network. The algorithm returns a set of reactions in EGFR_M whose bounds can be relaxed in order to obtain a ﬂux distribution that resembles that of EGFR_E. The effects of active AKT signaling on the EMT metabolic network We constrained RECON 2 [62] using the microarray data from Sigurdsson et al. [34] to generate an EMT metabolic network (submitted to Biomodels: MODEL1602080000). This reconstruction consists of all the metabolic reactions encoded in both the D492 and D492M cell lines. This EMT metabolic network has information on GPRs connecting each reaction with the genes of the enzymes catalyzing the reaction. Since GPRs associate genes with corresponding reactions, the metabolic genes predicted to be up-regulated in epithelial cells due to AKT signaling (Table 1) led to the identification of up-regulated reactions in D492 and similarly were determined in D492M. This information was then used to define the upper and lower flux bounds of the affected reactions in the EMT metabolic network to form Met_E (metabolic epithelial) and Met_M (metabolic mesenchymal) networks (Fig 9). Up- and down-regulation of the Met_E and Met_M models was simulated as described in section 4.4. Specifically, upregulation in an epithelial metabolic model was simulated by downregulating the corresponding reaction in its mesenchymal counterpart, and vice versa. Flux bounds of the up-regulated metabolic reactions due to AKT signaling in D492 were constrained by an arbitrary factor, one-hundredth of the initial bounds in D492M and vice versa. The flux values through each reaction in both the models were determined through random sampling method and the relative flux span, sM(i)/ sE(i) was used to quantify the flux differences between the networks. Here, sM(i) and sE(i) represent the average flux in reaction i for the mesenchymal and epithelial cells, respectively. Cell proliferation assay and metabolite measurements For proliferation assays and spent medium analysis, 1.5 × 104 D492 or D492M cells were cultured in 48-well plates (Costar) in 200μL H14 medium as previously described [35]. Spent PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 19 / 26 Modelling EGFR Signaling in Human Breast Epithelium Fig 9. The pipeline used to determine the effects of active AKT signaling on the EMT metabolic network. The metabolic genes predicted to be altered in D492 and D492M dependent on the AKT signaling network, were used to constrain the EMT metabolic network generated from Recon2, in order to identify downstream metabolic pathways that are affected by AKT activation. doi:10.1371/journal.pcbi.1004924.g009 medium was collected and cells were fixed in ice-cold methanol at 0 h, 24 h, 48 h and 72 h. To estimate proliferation, cells were stained with crystal violet, washed thoroughly with water, dissolved in 30% acetic acid and read in a spectrophotometer at 570 nm. The observed growth rates were 0.0276 h−1 for D492 and 0.0161 h−1 for D492M. The glucose and lactate concentrations in the spent medium were measured in an ABL90 blood gas analyser (Radiometer, Brønshøj, Denmark). Glucose uptake and lactate secretion per cell were calculated for each cell line as described in [82], based on the ABL90 measurements and growth rates. Viral transduction Vectors used for viral production were acquired from Addgene, pBABE-EGFR and empty backbone (#11011, #1764, respectively) and were used as provided. Phoenix HEK293 cells were used for retroviral (EGFR) virus production, using Arrestin transfection (Life Technologies). D492M cells were transduced overnight with viral supernatant containing 8 μg/ml Polybrene (Sigma-Aldrich). EGFR and empty backbone cells were selected using 2 μg/ml puromycin (Life Technologies). Real-Time Quantitative Reverse Transcription PCR Total RNA was isolated using TRI-Reagent solution (Ambion) and reverse transcribed using SuperScript IV (Invitrogen). The resulting cDNA was used for Real-Time Quantitative Reverse Transcription PCR, in Maxima Probe/ROX qPCR Master Mix (Thermo Scientific) with primer pairs and probes for EGFR (Hs00540086_m1, Life Technologies), ZEB1 (Hs00232783_m1, Life Technologies) and GAPDH (Hs99999905_m1, Life Technologies). Experiments were done in triplicates on 7500 Real Time PCR System (Applied Biosystems). EGFR mRNA levels were normalized to GAPDH and relative mRNA differences were calculated using the 2ΔCt method. PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 20 / 26 Modelling EGFR Signaling in Human Breast Epithelium Western blotting Proteins were isolated using RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Life Technologies). For Western blot analysis 5 μg of protein lysates were loaded per lane on NuPage 10% Bis-Tris gels (Life Technologies) in 2-(N-morpholino)ethanesulfonic acid (MES) running buffer (Life Technologies). Samples were denatured using 10% mercaptoethanol at 95°C for 10 minutes before loading. Samples were transferred to Immobilon FL PVDF membranes (Millipore) and blocked in Li-cor blocking buffer for 1 hour. Primary antibodies were incubated overnight at 4°C and secondary IRDye antibodies were incubated at room temperature for 1 hour (Licor). The following primary antibodies were used for Western blotting: Actin antibody (Abcam, ab3280), EGF Receptor (Cell Signaling, CS#4267), PhosphoAkt (Ser473) (Cell Signaling, CS#4060), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling, CS#4370), N-Cadherin (BD Biosciences, 610921), E-Cadherin (BD Biosciences, 610182) and Cytokeratin 14 (Abcam, ab15461). Near-infrared fluorescence visualization was measured using Odyssey CLx scanner (Li-Cor, Cambridge, UK). Supporting Information S1 Dataset. Codes and all the models in COBRA format used in this study. (ZIP) S1 File. A spreadsheet describing EGFR_SN attributes, along with flux distribution in EGFR_E and EGFR_M for D492 cells. (XLS) S2 File. Flux distribution in Met_E and Met_M. (XLS) S3 File. Flux distribution within EGFR signaling cascade in MCF7, MCF10A and HMLE cell lines. (XLSX) S4 File. Expression data mapped to EGFR signaling genes for each cell line. (XLSX) S1 Methods. Supplementary methods. (DOCX) S2 Methods. Modelpipeline: Detailed procedure for generation and simulation of EGFR signaling network. (DOCX) S1 Fig. Epithelial and mesenchymal marker expression. (A) Western blotting for epithelial markers E-Cadherin and CK14 and mesenchymal marker N-Cadherin. Overexpression of EGFR in D492M does not revert the mesenchymal phenotype towards an epithelial phenotype. D492MEGFR retains N-Cadherin expression and does not gain E-Cadherin or CK14 expression. (B) Real-Time Quantitative Reverse Transcription PCR of the EMT transcription factor ZEB1 in D492, D492MEGFR and D492MEmpty normalized to GAPDH. ZEB1 transcription was not detected in D492 and the transcription level of ZEB1 was unchanged in D492MEGFR compared to D492MEmpty. D492MEGFR retains mesenchymal ZEB1 expression. (TIFF) S1 Table. Gene associated with the reactions important for the reversal of EGFR_M to EGFR_E. (DOCX) PLOS Computational Biology \| DOI:10.1371/journal.pcbi.1004924 June 2, 2016 21 / 26 Modelling EGFR Signaling in Human Breast Epithelium S2 Table. Predicted expression of metabolic genes regulated by AKT in HMLE cells. ER: Microarray Expression data in TWIST, SLUG and SNAIL induced HMLE cells respectively. PE: Proposed Expression. Predictions in agreement with microarray data are highlighted in green and that otherwise are highlighted in orange. (DOCX) S3 Table. Predicted expression of metabolic genes regulated by AKT in MCF10A cells. ER: Microarray Expression data in TWIST, SLUG and SNAIL induced HMLE cells respectively. PE: Proposed Expression. Predictions in agreement with microarray data are highlighted in green and that otherwise are highlighted in orange. (DOCX) S4 Table. Predicted expression of metabolic genes regulated by AKT in MCF7 cells. ER: Microarray Expression data in TWIST, SLUG and SNAIL induced HMLE cells respectively. PE: Proposed Expression. Predictions in agreement with microarray data are highlighted in green and that otherwise are highlighted in orange. (DOCX) S5 Table. Regulation of metabolic gene expression by AKT signaling. “Reference” column lists the studies from which the influence of AKT signaling on the expression of the corresponding metabolic genes was derived. +1 and -1 denotes positive and negative regulation, respectively. (DOCX) S1 Appendix. Full form of abbreviations. (DOCX) Acknowledgments The authors would like to acknowledge Miha Skalic and Guðrún Valdimarsdóttir for valuable discussions. We would also like to thank the members of Stem cell research unit, Biomedical Centre for valuable discussion on this manuscript. Author Contributions Conceived and designed the experiments: KSC OR. Performed the experiments: KSC SH EB. Analyzed the data: KSC NR SH EB TG SG OR. Contributed reagents/materials/analysis tools: TG. Wrote the paper: KSC SH SG OR. References 1. Kalluri R, Weinberg RA. 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https://openalex.org/W140755256	https://newcontree.org.za/index.php/nc/article/download/476/572	English	null	The imperial imprint: British colonial towns	Contree	1,998	cc-by	6,757	109 109 NEW CONTREE Linda Brockett (Department of Town and Regional Planning, University of the Witwatersrand) Brockett ment of Town and Regional Planning, University of the Witwatersrand) THE IMPERIAL IMPRINT: BRITISH COLONIAL TOWNS nda Brockett epartment of Town and Regional Planning, University of the Witwatersrand) Opsomming B i li Opsomming Baie voormalige Britse kolonies sacs Australie, Suid-Afrika en die Verenigde State van Amerika, toon merkwaardige ooreenkomste in die uitle van hul dorpe. Die dorpe word gekenmerk deur 'n rooster -uitleg en soortgelyke uitlegte van eiendomsreg, grondverdeling en eienaarskap. Hierdie referaat se doel is om die basiese elemente en die verskeie aanpassings van die Britse model van dorpsontwikkeling in die kolonies te definieer. Die studie sa! fokus op die Britse koloniale vestiging aan die Ooskus van Amerika en Suid- Afrika. Deur die eeue is die rooster-uitleg sinoniem met verhuisende mense as gevolg van die eenvoud van die uitleg, en gemak van uitbreiding om toekomstige groei tegemoet te kom. Terwyl baie voormalige kolonies ook beinvloed is deur die Britse Tuinstad (Garden City) en Nuwe Stad (New Town) bewegings, gal hierdie referaat slegs fokus op die vroee koloniale tydperk en die aanvanklike vestigingspatrone. Die titel van hierdie referaat verwys nie alleen na die uitleg van die stad nie, maar erken ook dat die uitleg daarvan 'n fisiese uitdrukking is van die politieke, ekonomiese en sosiale instellings. Die imperiale stempel is deurdring met die idea Ie van privaatbesit, kapitale formagie, investering, rykdom en individualisme, en die stad is derhalwe meer as net 'n patroon van fisiese ontwikkeling. 1. Introduction The former British Colonies of America and South Africa show remarkable similarities in their town layout, being characterised by grid layouts and similar concepts of land tenure, land division and ownership. The grid (or orthogonal) layout found in these former colonies has become synonymous with migratory people, due to the ease of survey and the layout's ability to expand to meet future growth. This paper seeks to define the basic elements of the British model of town development in the colonies by focussing on the settlements on the east coast of America and South Africa. While many former colonies were influenced by the British Garden City and New Town Movements, this paper will focus exclusively on the early colonial period and the initial settlement patterns. During this period, it is argued, the British military had a fundamental and far reaching impact upon town planning in the colonies. 2. Historical background 2. Historical background Colonisation profoundly influenced urban form around the world and most cities in less developed countries today still reflect the conflict between European urban form and the indigenous settlement patterns. The creation of European enclaves in the newly colonised territories, as with Roman settlements in conquered lands many centuries before, were designed to develop an urban network, a cultural hegemony, a social make-up and a physical imprint that would be recognisable throughout the colony. As Violich has pointed out, cities were focal points of the decision making process; therefore, controlling them in a social sense was the first step to economic and social continuity for those in power.1 2. Historical background Colonisation profoundly influenced urban form around the world and most cities in less developed countries today still reflect the conflict between European urban form and the indigenous settlement patterns. The creation of European enclaves in the newly colonised territories, as with Roman settlements in conquered lands many centuries before, were designed to develop an urban network, a cultural hegemony, a social make-up and a physical imprint that would be recognisable throughout the colony. As Violich has pointed out, cities were focal points of the decision making process; therefore, controlling them in a social sense was the first step to economic and social continuity for those in power.1 These considerations were uppermost in the minds of the Spanish colonialists: The Spanish Kings (and indeed all the colonial powers), aware of the potential wealth and empire building possibilities in the New World, attempted to orchestrate the slow but steady expansion of a settlements in a way that each act of conquest would result in the effective extension of the highly centralised power structure of the Spanish Crown.2 These considerations were uppermost in the minds of the Spanish colonialists: The Spanish Kings (and indeed all the colonial powers), aware of the potential wealth and empire building possibilities in the New World, attempted to orchestrate the slow but steady expansion of a settlements in a way that each act of conquest would result in the effective extension of the highly centralised power structure of the Spanish Crown.2 The primary aims of colonisation offer a clue as to the settlement patterns in most colonies. 1 Violich after Mundigo and Crouch, "The city planning ordinances of the laws of the Indies revisited: Their philosophy and implications.. Town Planning Review, part 1, 48, 3 (1977), p. 259. 2 Mundigo and Crouch, "City planning ordinances", 1, p. 259. 3 A. J. Christopher, Colonial Africa (Canberra, 1984), p. 1. 1. Introduction Th f B iti h 110 NEW CONTREE The city layout is a physical expression of the political, economic and social institutions of a particular historical context, thus the Imperial Imprint imbued the ideals of private ownership, capital formulation, investment, wealth and individualism. British colonial towns differed fundamentally from the existing settlements in the colonies, suggesting that cultural detenninants had a greater impact upon town planning than environmental factors. This paper seeks to analyse the town layout principles during the British colonial period, it tries to define the underlying principles of British colonisation and illustrate the impact these had on the physical form of settlements and indeed on the pattern of human settlement. 2. Historical background Christopher states that: "In simplistic terms the initial Portuguese thrust into Africa in the fifteenth century provides an answer which is valid for later periods."J The Portuguese entered Africa with five basic goals. The first was scientific; the need to explore the continent with the objective of finding a sea route to India. The second was commercial; seeking to open trade within the continent and beyond The third goal was militaty; to assess the strength of the Moslem enemy and the fourth was to I ink up with any Christian powers to be found. Finally, missionary activity was to be directed to the saving of souls." 111 CONTREE [own emphasis] These reasons for colonisation are echoed in the Spanish Laws of the Indies, which specify: [own emphasis] These reasons for colonisation are echoed in the Spanish Laws of the Indies, which specify: p y ...those who are in charge of governing the Indies, whether spiritually or temporally, should inform themselves diligently if within their districts, including lands and provinces bordering them, there is something to be discovered and pacified, of the wealth and quality, of the peoples and t.4 na Ions... p y ...those who are in charge of governing the Indies, whether spiritually or temporally, should inform themselves diligently if within their districts, including lands and provinces bordering them, there is something to be discovered and pacified, of the wealth and quality, of the peoples and t.4 na Ions... Later in the same laws it is stated: "And they (the regions) should be populated by Indians and Natives to whom we can preach the gospels since this is the principal objective for which we mandate that these discoveries and settlements be made.os It was, however the aspect of commercial gain which came to dominate the enterprise and guide most other European powers wishing to colonise.6 Given the commercial nature of many of these colonies, as well as the need to gain access to the newly discovered areas, the primary settlements were mostly ports with stronger links to the colonising country than to the hinterland. 4 Mundigo and Crouch, .City planning ordinances., 1, p. 250. 5 Mundigo and Crouch, .City planning ordnances., 1, p. 250. .Christopher, Colonial Africa, p. 2. 7 Mundigo and Crouch, .City planing ordinances., 1, p. 398. .R.A. DodQshon, The European past: Social evolution of spatial order (London, 1987). .R.A. DodQshon, The European past: Social evolution of spatial order (London, 1987). 2. Historical background The Spanish Laws of the Indies again offer insight on this point: "...according to a centralised system of royal planning which encouraged concentration of wealth and of resources, (each) city was administratively linked directly to the government in Spain, and trade among the New World cities was not encouraged.7 This pattern of development was clearly evident during the British rule in the Cape; communications between towns was tenuous and most trade centred around the harbours at Cape Town and Durban. Early British architecture in South Africa also illustrates a strong dependence on imported materials. .P. J. Bohannan, Social anthropology (USA, 1963). 10 Bohannan, Social anthropology, p. 211. " Dodgshon, European past, p. 5. 12 E. A Walker, Colonies (Cambridge 1944). 3 The pattern of colonial development 3.1 Theoretical base of the study y Dodgshon states that historical geography can gain more from the substantive than from a formalist approach.8 By this he means that we should work from the premise that modern concepts of human spatial organisation are inapplicable outside the specific socio-historical context in which they have been formulated, and that we should seek to clarify how the unfolding human landscape records the successive imprint of different systems of spatial order. These systems of spatial order are defined through the variables and interactions which determined the organisation of society in space. Change from one 5 Mundigo and Crouch, .City planning ordnances., 1, p. 250. R.A. DodQshon, The European past: Social evolution of spatial order (London, 1987). 112 NEW CONTREE system to another is taken to mean a qualitative change in the nature of the system, a change which could involve different levels or spheres of determination, control mechanisms or goals and, in consequence, effects. It is therefore, necessary to understand the political, economic and social aspects of a settlement in order to fully understand the pattern of development. Stated differently, the spatial structure of a settlement is the physical manifestation of the social structure, economy, religion, technology and function of that settlement.9 Bohannan's theory argues that man manipulates and changes the environment instead of merely exploiting it.1O His aim is not mere survival or even comfortable survival, since man has introduced the illusion that he controls his environment. Within the last ten to fifteen thousand years, human culture has vastly reduced the part that raw nature plays in creating the immediate environment of human beings. The geographical environment, in other words, is replaced with the cultural and social environment; more complex cultures limit the degree to which man is dependent on the natural aspects of the environment. From Bohannan's theory one can assume that urban landscapes, although sometimes adapted to better fit the environment, are essentially determined by the culture of the people who created them. Dodgshon adds the concept of understanding the culture of people at a given point in history, as modern interpretations of culture often lead to incorrect interpretations and notions of human spatial organisation.11 3.2 Social underpinnings Colonisation was often seen as an outlet for surplus populations. The British used colonisation as a tried and trusted method of relieving overpopulation at home, as the Greeks had done long before. One of the great arguments endorsing colonisation in late Elizabethan and Early Stuart times was the need to relieve social pressure in England. It was no coincidence that the Elizabethan Poor Law and the foundation of Ulster and Virginia belonged to the same decade. The next spate of emigration from Britain occurred shortly after the Napoleonic Wars. The idea of finding outlets for surplus populations, however, really took off in the scramble for colonies that occurred during the late nineteenth century.12 The colonies also played a vital role in the provision of openings, official and otherwise, for ambitious citizens of the metropolis. Colonies have long been regarded as safety-valves for the energies of the younger sons and daughters, especially of the upper NEW CONTREE 113 and middle classes. With the direct inheritance system in operation in large parts of Europe, only the eldest son of nobility was ensured a title and the wealth of the family estate; colonies offered the other children a possibility of acquiring a decent living and the rank to which they were accustomed. These people formed the core of the officers and administrators in the colonies. In the Laws of the Indies it is specified that all the original settlers in a colonial town would obtain a title, in this case it afforded people with no rank an opportunity to acquire a title of distinction.13 The establishment of colonial towns was also important in terms of the relations between the colonialists and the local population. Colonial towns aimed at portraying a distinctly European imprint which would set them apart from the local settlements. There is little question that one of the primary aims of colonisation was to impart European culture on the newly colonised world. In the case of Spanish colonisation one of the primary objectives was to convert the natives to Catholicism, it was thus, necessary to establish towns in areas already inhabited by locals. This consideration had a direct influence on the layout of the town; ...the ideal town scape that the Spaniard was being ordered to build in the New World added the necessary physical dimension and reinforcement to the more subtle activities of Indian conversion to Catholicism. 1S E. J Owens, The city in the Greek and Roman world (London and New York, 1992), p.124 '3 Mundigo and Crouch," City planning ordinances", 1. '3 Mundigo and Crouch," City planning ordinances", 1. 14 Mundigo and Crouch, "City planing ordinances", 1. 1S E. J Owens, The city in the Greek and Roman world (London and New York, 1992), p.124. 14 Mundigo and Crouch, "City planing ordinances", 1. '6 Mundigo and Crouch, 'City planning ordinances", 1, p. 399. 17 D. K. Fieldhouse, The colonial empires (London, 1966), p. 4. 18 Bohannan, Social anthropology, p. 222. 3.2 Social underpinnings There was no building of more influence and designed to impress the natives more deeply than the main cathedral, which was to be built according to precise specifications which would result in it acquiring more authority. 14 British colonial settlements were somewhat different, being established at a later time, they came after the Renaissance and the break down of centralised religious control. British towns were home to a variety of denominations of Christianity and hence seldom accorded anyone church with a dominant central location. British towns by contrast tend to have administrative centres. The establishment of colonial towns was important from an administrative point of view as Owen's notes of Roman times: "colonies were established both for military security and, in areas without traditions of urban life, to introduce and familiarise the provincials with law-abiding government." 15 Another interesting phenomenon of colonial towns was the dual nature of settlements. Most colonial settlements were kept separate from the settlements of the local inhabitants and integration of the different cultural groups did not take place. Mundigo and Crouch '3 Mundigo and Crouch," City planning ordinances", 1. NEW CONTREE 114 note that in Lima in 1553 a separate settlement for Indians, with smaller buildings and narrower streets, was provided.16 3.3 Economic influences The discoveries and expans 3.3 Economic influences The discoveries and expansion of Europe had practical consequences in terms of trade and conquest. Every colony or trading centre was a new economic stimulus. America for example provided an immense market for European manufactures and agricultural products American bullion increased the supply of money circulating in Europe and intensified existing economic and social developments.17 With wealth goes power and prestige for both the colony and the colonising power. The enhanced economic status of the colony had a profound effect upon colonial settlements, for ties with the colonial metropolis became more pervasive, and hence the European influence remained a powerful determinant in the physical design of settlements. Settlements were commercial and administrative centres; their location was thus often dictated by economic and military advantage. Towns tended to be located at natural harbours, fording points, on trade routes or in regions with abundant natural resources. British colonisation went hand in hand with mineral exploration and extraction as well as the search for raw materials such as timber and food for the European market. The capitalist principles of the colonising power largely dictated the layout of the towns. As Bohannan explains: "...even when land is not scarce for means of production, there tends to be another matter that makes people value it and demarcate it. That is its value as a site."18 In Western society land fulfils two purposes: 1 L d i di i f i t 1. Land is a necessary dimension of society because human beings nee 2 Land is valued for what it can produce and therefore it enters the ma 2. Land is valued for what it can produce and therefore, it enters the market as a factor of production. It is usual for the two aspects of land occupation -production and site to go together. It is also usual for the dominant one to be the site aspect. One exploits that land on which one finds oneself as a member of a localised social group. Only in a contract society, where land enters the market, do we have the opposite situation, in which local groups come into existence 2. Land is valued for what it can produce and therefore, it enters the market as a fact of production. It is usual for the two aspects of land occupation -production and site to go together. 3.3 Economic influences The discoveries and expans It is also usual for the dominant one to be the site aspect. One exploits that land on which one finds oneself as a member of a localised social group. Only in a contract society, where land enters the market, do we have the opposite situation, in which local groups come into existence 2. Land is valued for what it can produce and therefore, it enters the market as a facto of production. It is usual for the two aspects of land occupation -production and site to go together. It is also usual for the dominant one to be the site aspect. One exploits that land on which one finds oneself as a member of a localised social group. Only in a contract society, where land enters the market, do we have the opposite situation, in which local groups come into existence 115 NEW CONTREE because land has been parcelled out in certain ways in order to maximise production, and then sold.19 Bohannan goes on to explain that every social group possesses some view or opinion about the physical milieu in which it find itself- what he calls 'folk-geography'. He reasons that for the past few centuries Westerners have viewed the world as a sphere covered with a grid. The grid can be manipulated in size and scale from the size of the earth to the size of a classroom globe. The grid can even be reduced to a two dimensional map.20 Thus, culturally, land -whatever else it may be is a measurable entity divisible into 'thing like' parcels by means of the mathematical and technical processes of surveying and cartography. This complex notion of land, with its accompanying technology, is an absolute essential to the western system of land tenure. Legally recognised rights can be held in such a parcel of land and these rights can be sold or exchanged for money at a market price.21 Even in the Colonies where land was not scarce it was still divided and allocated in terms of the western notion of land ,. Bohannan, Social anthropology, p. 223 20 Bohannan, Social anthropology, pp. 223-224. 21 Bohannan, Social anthropology, p. 224. 22 R Strausz-Hupe and H.W. Hazard, The idea of colonialism (London, 1958). Even in the Colonies where land was not scarce it was still divided and allocated in terms of the western notion of land Even as late as the colonial settlement at the Cape (1652), Holland and England used a curvilinear method of survey, which was related to the natural and cultural (artificial) features found existing on the ground. When countries were newly colonised, cultural and natural features were few and far between, thus, the beaconed rectilinear boundary system of ancient Egypt was adopted. The grid layout was the easiest layout available which could be replicated in all the colonies, which suited the notion of the division and ownership of land and which was within the technical surveying abilities of the era. 23 Walker, Colonies, p. 28. 3.4 Political influences The meaning and implications of the word "colonialism" and of the closely connected terms "empire" and "imperialism" have undergone a profound transformation in recent times, Until the end of the nineteenth century the words "empire" and "imperialism" were generally used in laudatory and not pejorative terms,22 Walker lists prestige as a major political motive for establishing colonies, "", if Spain and Portugal had colonies, it behoved CONTREE 116 other powers to have colonies also."23 Political influences can also be traced to the desire to expand the host countries citizenry, expand their markets and to protect markets and trade routes All of these aspects had a bearing on the physical layout of colonial towns, they were planned to guard major harbours, defend sea routes and offer access to the raw materials and resources of the colonies. Most British colonial towns were predicated upon a European concept of the powers of municipal government derived from the development of the free town and the charter city during the Middle Ages. Such communities were municipal corporations of considerable authority typically capable of owning and disposing of all vacant land in the city, of holding monopolies on certain aspects of trade and of approving or disapproving physical changes to the city. They were generally authorised to playa leading role in guiding and directing the physical form of the community as well as its social and economic policies!4 27 Whitworth Porter, History of the corps of the Royal Engineers, 1 (London, 1889). 24 Greckens, p. 22. 23 Walker, Colonies, p. 28. 24 Greckens, p. 22. 25 Christopher, Colonial Africa. 26 Christopher, Colonial Africa, p. 3. 27 Whitworth Porter, History of the corps of the Royal Engineers, 1 (London, 1889). 3.5 Military influences y Despite their divergent national origins, the various groups involved in the colonisation process each acted with some degree of international cohesion. The politicians, most of whom never visited the colonies, drew the boundaries. The military carried out the conquests and frequently chose the ports from which to control the population The military also often formed the first colonial governments as coercion was an essential part of the maintenance of colonial administration.25 Indeed the demarcation between soldier and administrator in the colonies was rarely defined. The administrators determined the pattern of urban settlement by selecting the administrative posts and deciding upon the nature of the administrative regime.26 Many British colonial towns were laid out by the administrators; most of whom came from the military, thus, in essence it can be argued that the Royal Engineers were intimately involved in the design and layout of colonial towns. Whithworth Porter7 in his study of the Royal Engineers states that Modern Engineering emerged in the military in the form of the sappers, "a body of experts who made it their profession to supply the scientific 117 CONTREE needs of an army.,,28 Civil Engineering emerged as all non-military engineering and occurred primarily due to the large number of Royal Engineers who were co-opted into the public sector. If one considers the description of what a Principal Engineer was supposed to know, it is evident that the Royal Engineers obtained training in a what today would fall into the disciplines of Architects, Engineers, Land Surveyors and Planners: He ought to be well skilled in all parts of the mathematics, more particularly in Stereometry, Altimetry, and in Geodesia, to take the distances, heights, depths, surveys of land, measure of solid bodies, and to cut any part of ground to a portion given, to be well skilled in all manner of foundations, in the scantlings of all timber and stone and of their several natures, and to be perfect in Architecture, civil and military ",29 It is the contention of this paper that the British military has had a fundamental and far reaching impact upon the planning of their colonies. It is the contention of this paper that the British military has had a fundamental and far reaching impact upon the planning of their colonies. The role of the military in the planning of colonial settlements was well documented in Ancient Roman times. 28 Porter, Cotps of Royal Engineers, 1, p, 8 29 Whitworth Porter, Idea of Colonialism, p, 49. 30 Porter. Idea of Colonialism. P. 123. 3.5 Military influences At its height the Roman Empire was vast and contained a variety of peoples of different cultures, beliefs and traditions; Rome was faced with major problems both in governing this immense area and in maintaining peace and security within it. In order to achieve these aims the central government relied heavily upon the city. Cities were the primary level of administration in the empire. The Romans believed the city to be the institution most capable of maintaining peace and promoting civilisation in the Roman sense. The result was that Romanised towns, usually laid out in the typical grid fashion, and including typical Roman urban buildings sprang up. The military surveyor and the engineer played a major role in the construction of these new cities.30 The Roman influence on the sixteenth century colonial efforts can be traced directly to the Spanish laws of the Indies. The Spanish government relied heavily on the writing of Vitruvius. The manuscript of his Ten Books on Architecture had been rediscovered in the previous century and published by Alberti When Vitruvius wrote his prescriptions, the Roman Empire had been founding colonial towns for at least three centuries, and their NEW CONTREE 118 theories had been based on the Greek experience going back at least to the seventh century BG. 31 Mundigo and Crouch claim that the Ordinances issued by Philip II for the laying out of new towns were based directly on these works.32 They further state that the Spanish ordinances have remained largely unknown to the English speaking world; the only translations dating 1921 and 1922. It is however, highly possible that the laws of the Indies were known to the British military long before this, even if they were not freely available, the direct impact which Roman colonial planning had on Britain can not be dismissed. The Spanish Laws of the Indies specify a Grid layout with regular plots. Although there is no obvious British equivalent of the Laws of the Indies it is the contention of this paper that a very similar philosophy was used by the British. The Spanish Laws of the Indies are described by Mundigo and Crouch as a "practical 'how to' manual" designed to guide untrained city builders in laying out a new settlement. 31 Mundigo and Crouch, "The city planning ordinances of the laws of the Indies revisited: Three American cities", Town Planning Review, part 2,48,3 (1977), pp. 397-418, 400. 32 Mundigo an Crouch, "Three American cities", p. 248. 33 J. de Villiers, "Die Cape Regiment, 1806-1817. 'n Koloniale regiment in Britse diens", Archives Year Book for South African History, 52, I (1989), p. 20. 34 De Villiers, "Cape Regiment", pp. 21-22. 34 De Villiers, "Cape Regiment", pp. 21-22. 32 Mundigo an Crouch, "Three American cities", p. 248. 31 Mundigo and Crouch, "The city planning ordinances of the laws of the Indies revisited American cities", Town Planning Review, part 2,48,3 (1977), pp. 397-418, 400. 35 De Villiers, "Cape Regiment", p. 23. Own translation: "there were also positive developments in the British army of the first decades of the nineteenth century. The most important asped is the multi-faceted contributions which the army made to the general development of various British Colonies; the Cape Colony and Canada being two examples. 36 MP. Conzen, "Town planning analysis in an American setting: Cadastral processes in Boston and Omaha, 1630-1930" in TR. Slater (ed.) The built form of Western cities (Leicester and London, 1990), p. 144. 36 J.W. Reps, The making of urban America: A history of city planning in the United States, (Princeton. 1965) 37 Conzen, "Town Plan analysis", p. 145. 3.5 Military influences By contrast, the British colonial towns were laid out mainly by the Royal Engineers, it is thus, possible that the grid layout was the layout to which they were accustomed because of their standardised training in land surveying and civic design. This argument is supported by J de Villiers in his book on "The Cape Regiment, 1806-1817".33 He states that there were two training centres for junior officers in those times (1801) one at Woolwich and the other at Sandhurst. Woolwich offered outstanding training in artillery and engineering. By 1815 the Board of Ordinance was one of 13 major departments in the British Army Under this department fell the Royal Artillery and the Royal Engineers; therefore, the Ordinance Office through the Secretary of State for Colonies and War, had a major impact on the development of fortifications and other building projects in the Colonies. The scale and range of developmental projects which the Ordinance Department became involved with lead to the lobbyists for the re-structuring of military training to propose that the course be divided into civil and military engineering. This indicates the extent of non-military work which was being handled by the Royal Engineers in the Colonies.34 In fact, De Villiers states: NEW CONTREE 119 ...tog was daar ook positiewe ontwikkelinge in die Britse leer van die eerste dekades van die negentiende eeu. Die belangrikste aspek is die veelsydige bydraes wat die leer tot die algemene vooruitgang van die verskeie Britse koloniale gebiede gemaak het, waarvan Kanada en die Kaapkolonie maar twee voorbeelde was.35 The Royal Engineers carried out the ordinance survey of South Africa which still forms the basis of the land registration system of South Africa. 39 Conzen, "Town plan analysis", p 146. AO R. F. Haswell, An historic townscapes conservation scheme for Natal, Natal Town and Regional planning Commission report, vol 61, (1984), pp. 28-41. 4.2 Estcourt Estcourt originated in 1847 as an inn and ferry point on the fording point of the Bushman's River. Being a strategic location, a cavalry detachment soon established a post there. In 1849 additional settlers arrived under the sponsorship of Thomas Estcourt (a member of the British Parliament). Ten years later the seat of the magistracyforWeenen County was moved to the town. The formal grid plan of the town was added to the original settlement in 1863.40 4 Spatial development I Case studies 4 Spatial development I Case studies Most early British colonial settlements have a formal grid layout. A number of early American towns, for example Philadelphia and Savannah, have clearly structured orthogonal layouts. City planning in the American colonies may have had its beginnings in the seventeenth century fortress-villages (Bastides), which were often surrounded by land farmed on a communal basis. Early colonial settlements that were pre-planned with regard to their physical form commonly used a grid street system laid out on a scale appropriate to the number of inhabitants.36 The medieval bastidal plans of France appear to have been the most common amongst American cities in the late eighteenth century. These plans were, however, to be taken over by the 'speculator's town'. Philadelphia's grid plan was characteristic of this open, democratic cadastre, which was so adaptable to the spread along mercantile alignments throughout the interior of the English colonies.37 Reps notes that nearly all American town plans were speculative in their origins.38 Related to this is the almost universal use of the grid-iron principle for urban planning. Grids offered simplicity in land surveying, recording and subsequent ownership transfer. The grid also allows for a remarkable neutrality towards the positioning of specific urban structures. Furthermore, grids favoured 36 MP. Conzen, "Town planning analysis in an American setting: Cadastral processes in Boston and Omaha, 1630-1930" in TR. Slater (ed.) The built form of Western cities (Leicester and London, 1990), p. 144. 120 NEW CONTREE a fundamental democracy in property market participation. This did not mean that individual wealth could not appropriate considerable urban property, but rather that the basic initial geometry of land parcels bespoke a simple egalitarianism.39 Philadelphia (1682), Detroit (1700), New Orleans (1718) and Savannah (1733) all had basic grid layouts. During the latter half of the seventeenth century these colonial cities were quite small in population and accommodated few specialised land use activities in the modem sense. Figure 1: Philadelphia, 1762 (J. W. Reps, The making of urban America, p.168) Figure 1: Philadelphia, 1762 (J. W. Reps, The making of urban America, p.168) 121 NEW CONTREE Figure. 2: Savannah, 1740 (Reps, Making of urban America, p. 191) Figure. 2: Savannah, 1740 (Reps, Making of urban America, p. 4 Spatial development I Case studies 191) 122 NEW CONTREE In South Africa the British, upon acquisition of the Cape colony from the Dutch East India Company, immediately felt the need for greater control over the isolated frontier settlers and consequently established a number of commercial and administrative centres. Although a number of subtle differences are evident in their layout they all share the common grid layout, with centrally placed administrative structures and little further distinction in the way of land use. The churches tended to be located on the outskirts of town. In South Africa the British, upon acquisition of the Cape colony from the Dutch East India Company, immediately felt the need for greater control over the isolated frontier settlers and consequently established a number of commercial and administrative centres. q y Although a number of subtle differences are evident in their layout they all share the common grid layout, with centrally placed administrative structures and little further distinction in the way of land use. The churches tended to be located on the outskirts of town. Early British settler towns in South Africa were additions to the original Dutch Settlements, such as Cape Town and Grahamstown. The British however, immediately saw the need to establish new settlements on the eastern frontier in order to exert control over the frontier population. A number of administrative settlements were thus, established. For the purposes of this paper, examples of British settlements in Natal have been looked at in order to illustrate the common characteristics. 4.1 Richmond Richmond was laid out in 1850 in order to serve the needs of an agricultural community on the banks of the "Iovo River. The town plan is a formal grid layout with sixty-six blocks of six acres each, subdivided into twelve half acre plots. The streets were named after royalty and colonial officials. By September 1850 the town could already boast three stores and shortly thereafter a number of sports grounds. This was a fundamentally different pattern form the early Dutch settlements in South Africa which were agricultural and religious centres with no permanent commercial activity. 41 Haswell, Historic townscapes , p. 45. 42 Haswell, Historic townscapes , p. 40. 4.3 Pieterrnaritzburg Pietermaritzburg is an interesting case study, in that it started as a Voortrekker (Afrikaans)town and was later taken over by the British. At the time of occupation the town was described by Colenso as "... long white town" but by the 1920's it had changed to 123 NEW CONTREE "...sleepy red-bricked town",41 The change in character came about due to the change of occupant. The original settlers, the Voortrekkers, established a course grain grid with long wide roads and fewer and narrower cross roads. The erven were large as the residents practised subsistence agriculture. The houses lined the street and were built of wattle and daub in the Cape-Dutch vernacular. Once the British took over the town they immediately altered the plot sizes through subdivision and introduced more cross roads. The primary reason for the difference being the commercial nature of the British town as opposed to the subsistence nature of the Afrikaner settlement. The Afrikaner houses were demolished to give way to the double storey brick homes of the British, The British houses were decorated with filigree work and other finishes such as pressed steel ceilings imported from Britain. Figure 4: Pietermaritzburg (Haswell. Historic townscapes .p.22) 4.4 Grahamstown Grahamstown, established as a frontier town in the Eastern Cape, illustrates the military impact on an existing hamlet. The town was at the time of British annexation of the Cape an incipient town, or what the Afrikaners called a "rydorp" (row town- a single street lined with houses). Circa 1814 a British military settlement was superimposed on the hamlet. The odd alignment is still evident in the town centre today. British colonial town planning definitely follows a trend, as Hoskins eloquently says: .there are so many towns to be seen and each ought to be approached on foot: certainly all the smaller towns For only on foot does one detect the subtle rise and fall of the ground to which the earliest settlers were so sensitive, or alignments in the town scene that may throw light on some fundamental change of plan: or the names of streets that set the mind working at once. It would be an interminable occupation were it not for the fact that what one learns about the landscape of one town often throws a flash of light upon a puzzle in another. A pattern begins to form.42 The British towns were characterised by a grid layout, with small square plots and commercial activity. Generally the administrative buildings or cricket ground formed the centre of the town and churches tended to be built on the outskirts of town with associated grave yards. This was fundamentally different from the Dutch settlements which grew around a centrally located church. The locality of the town was also crucial; the towns were commercially based and thus, commanded strategic trade locations such as drift sites, entrances to mountain passes, ports as well as sites of military significance. CONTREE 124 F;g.2.2', J '-'~""""""'" '-"'" ,. ~ 33 33 Figure 3: Richmond (R. F. Haswell, An historic townscapes conservation scheme for Natal, p.33) 125 CONTREE Figure 4: Pietermaritzburg (Haswell. Historic townscapes .p.22) NEW CONTREE 126 Figure 5: Grahamstown 1814 -military structures superimposed on a rydorp (Haswell, Historic townscapes, p.20. Figure 5: Grahamstown 1814 -military structures superimposed on a rydorp (Haswell, Historic townscapes, p.20. 121 NEW CONTREE 5. Conclusi 5 Co c us o Colonial expansion and the establishment of colonial towns was about control and prestige. The expansion of European ideas and settlement forms into America and Africa fundamentally altered the relationship between man and environment. Colonisation lead to the extraction of raw materials on a large scale, the introduction of commercial centres and the opening up of trade routes. The areas had previously been inhabited by largely pastoral groups and trade routes had been limited, whereas the new order linked the colonies to the European markets and hence to their direct influence. The establishment of settlements was seen to be necessary not only as a means of exploiting the regions, but also for military purposes. The introduction of European settlements fundamentally altered the existing trade patterns, the European settlements were related to local settlements but retained separate European strong-holds which cornered the market and thus, forced the locals into a servile position. It is the contention of this paper that the British colonial towns tend to be characterised by a grid layout due to the political, economic, social and imperial influences. The layout has its origins in military control and administration It is a pattern which has been used repeatedly for colonial expansion as it is not only easy to survey and adapt to different environments, but it represents a the formality needed in order to mark the colonial settlements as different form the local settlements. The cities offered a form of administrative control over the local population; all trade occurred through the cities and they were the centres from which missionary work was carried out. A major contention of this paper is that the Royal Engineers played a fundamental role in the planning and establishment of colonial towns. The town's location was of strategic importance, both in terms of trade and to afford the military administrative control over the region.
https://openalex.org/W2942327807	https://peerj.com/articles/6780v0.3/submission	English	null	Peer Review #2 of "Noblella thiuni sp. n., a new (singleton) species of minute terrestrial-breeding frog (Amphibia, Anura, Strabomantidae) from the montane forest of the Amazonian Andes of Puno, Peru (v0.1)"	null	2,019	cc-by	15,275	Manuscript to be reviewed Alessandro Catenazzi Corresp., 1, 2 , Alex Ttito 3, 4 Alessandro Catenazzi Corresp., 1, 2 , Alex Ttito 3, 4 1 Biological Sciences, Florida International University, Miami, FL, United States 2 Centro de Ornitología y Biodiversidad, Lima, Perú 3 Departamento de Ecología, Facultad de Ciencias Biológicas, Pontiﬁcia Universidad Católica de Chile, Santiago, Chile 4 Museo de Historia Natural, Universidad Nacional de San Antonio Abad, Cusco, Perú Corresponding Author: Alessandro Catenazzi Email address: acatenaz@ﬁu.edu Corresponding Author: Alessandro Catenazzi Email address: acatenaz@ﬁu.edu We describe a new species of minute, terrestrial-breeding frog in the genus Noblella. We collected a single specimen in the leaf litter of primary montane forest (2250 m a.s.l.) near Thiuni, in the Provice of Carabaya, Department of Puno, in the upper watershed of a tributary of the Inambari River of southern Peru, the same locality where we found the types of Psychrophrynella glauca Catenazzi & Ttito 2018. We placed the new species within Noblella on the basis of molecular data, minute size, and overall morphological resemblance with the type species N. peruviana and other species of Noblella, including having three phalanges on ﬁnger IV (as in N. coloma, N. heyeri, N. lynchi, N. madreselva, N. peruviana, and N. pygmaea), and terminal phalanges T-shaped and pointed. Noblella thiuni sp. n. is distinguished from all other species of Noblella by having ventral surfaces of legs bright red, and chest and belly copper reddish with a profusion of silvery spots. The new species further diﬀers from known Peruvian species of Noblella by the combination of the following characters: tympanic membrane absent, eyelids lacking tubercles, dorsal skin ﬁnely shagreen, tarsal tubercles or folds absent, three phalanges on Finger IV, tips of digits not expanded, no circumferential grooves on digits, inguinal spots present. The new species has a snout–vent length of 11.0 mm in one adult male. Our new ﬁnding conﬁrms the high levels of endemism and beta diversity of small, terrestrial-breeding frogs inhabiting the moss layers and leaf litter in the montane forests of the Amazonian slopes of the Andes and adjacent moist puna grasslands, and suggests much work remains to be done to properly document this diversity. PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Manuscript to be reviewed Manuscript to be reviewed 1 Noblella thiuni sp. Alessandro Catenazzi Corresp., 1, 2 , Alex Ttito 3, 4 n., a new (singleton) species of 2 minute terrestrial-breeding frog (Amphibia, Anura, 3 Strabomantidae) from the montane forest of the 4 Amazonian Andes of Puno, Peru 5 6 Alessandro Catenazzi1,2, Alex Ttito3,4 7 8 1 Department of Biological Sciences, Florida International University, Miami, Florida, USA 9 2 Centro de Ornitología y Biodiversidad, Lima, Perú 10 3 Departamento de Ecología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de 11 Chile, Santiago, Chile 12 4 Museo de Historia Natural, Universidad Nacional de San Antonio Abad, Cusco, Perú 13 14 15 Corresponding Author: 16 Alessandro Catenazzi1 17 11200 SW 8th Street, Miami, FL 33199, USA 18 Email address: acatenazzi@gmail.com 1 Noblella thiuni sp. n., a new (singleton) species of 2 minute terrestrial-breeding frog (Amphibia, Anura, 3 Strabomantidae) from the montane forest of the 4 Amazonian Andes of Puno, Peru PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) 39 Introduction 66 We wrote the diagnosis and description by following Duellman & Lehr (2009) and Lynch 67 & Duellman (1997), except that we used the term “dentigerous processes of vomers” instead of 68 “vomerine odontophores” (Duellman et al. 2006). We follow Heinicke et al. (2017) and De La 69 Riva et al. (2017) for family placement and taxonomy. We derived meristic traits of similar 70 species from comparisons with museum specimens (Appendix 1), field notes of coloration in live 71 specimens of other species, published photographs, and the original species descriptions 72 (Catenazzi et al. 2015). Abbreviations of collections are: CORBIDI = Herpetology Collection, 73 Centro de Ornitología y Biodiversidad, Lima, Peru; KU = Natural History Museum, The 74 University of Kansas, Lawrence, Kansas, USA; MHNC = Museo de Historia Natural, 75 Universidad San Antonio Abad del Cusco, Cusco, Peru; MHNG = Muséum d’Histoire 76 Naturelle, Genève, Switzerland; MUSM = Museo de Historia Natural, Universidad Nacional 77 Mayor de San Marcos, Lima, Peru. 40 The genus Noblella is distributed from Ecuador to Bolivia (Figure 1; Catenazzi et al. 41 2015), but there is uncertainty regarding the monophyly of the genus and the number of species 42 (Catenazzi & Ttito 2016; Catenazzi & Ttito 2018; De La Riva et al. 2017). The current taxonomy 43 recognizes 12 species of Noblella (AmphibiaWeb 2019; Catenazzi et al. 2015), of which four 44 occur in southern Peru, where several undescribed species have been reported (Figure 2; von 45 May et al. 2017). The small size of these frogs (Lehr & Catenazzi 2009), terrestrial life habits 46 requiring intensive search in the leaf litter (Catenazzi et al. 2011), and micro-endemism of most 47 species of high-elevation, small strabomantid frogs in the Andes (De La Riva et al. 2017) suggest 48 that many additional species remain to be discovered and recognized throughout the tropical 49 Andes (Catenazzi 2015). Here we describe a new species of Noblella on the basis of a singleton 50 (following terminology of Lim et al. 2012) found in the leaf litter of a cloud forest remnant in the 51 Cordillera de Carabaya, in the southern Peruvian department of Puno, along a tributary of the 52 Inambari River. This specimen is unlike any of the previously described species of Noblella or of 53 the morphologically similar Psychrophrynella. 59 Materials & Methods 60 On 14 August 2017 we (AC and AT) conducted a rapid survey (~4 hours) of the leaf 61 litter of a relictual cloud forest along the Macusani-San Gabán road, which connects the Peruvian 62 Altiplano to the Amazon rainforest and the interoceanic highway between Peru and Brazil. We 63 removed the leaf litter by hand and searched opportunistically around fallen logs, rocks, moss- 64 covered soil, etc. We searched within an area of approximately 100 m2. We found a single 65 specimen, which we euthanized with 20% benzocaine. 66 We wrote the diagnosis and description by following Duellman & Lehr (2009) and Lynch 67 & Duellman (1997), except that we used the term “dentigerous processes of vomers” instead of 68 “vomerine odontophores” (Duellman et al. 2006). We follow Heinicke et al. (2017) and De La 69 Riva et al. (2017) for family placement and taxonomy. We derived meristic traits of similar 70 species from comparisons with museum specimens (Appendix 1), field notes of coloration in live 71 specimens of other species, published photographs, and the original species descriptions 72 (Catenazzi et al. 2015). Abbreviations of collections are: CORBIDI = Herpetology Collection, 73 Centro de Ornitología y Biodiversidad, Lima, Peru; KU = Natural History Museum, The 74 University of Kansas, Lawrence, Kansas, USA; MHNC = Museo de Historia Natural, 75 Universidad San Antonio Abad del Cusco, Cusco, Peru; MHNG = Muséum d’Histoire 76 Naturelle, Genève, Switzerland; MUSM = Museo de Historia Natural, Universidad Nacional 77 Mayor de San Marcos, Lima, Peru. 66 We wrote the diagnosis and description by following Duellman & Lehr (2009) and Lynch 67 & Duellman (1997), except that we used the term “dentigerous processes of vomers” instead of 68 “vomerine odontophores” (Duellman et al. 2006). We follow Heinicke et al. (2017) and De La 69 Riva et al. (2017) for family placement and taxonomy. We derived meristic traits of similar 70 species from comparisons with museum specimens (Appendix 1), field notes of coloration in live 71 specimens of other species, published photographs, and the original species descriptions 72 (Catenazzi et al. 2015). Abbreviations of collections are: CORBIDI = Herpetology Collection, 73 Centro de Ornitología y Biodiversidad, Lima, Peru; KU = Natural History Museum, The 75 Universidad San Antonio Abad del Cusco, Cusco, Peru; MHNG = Muséum d’Histoire 76 Naturelle, Genève, Switzerland; MUSM = Museo de Historia Natural, Universidad Nacional 77 Mayor de San Marcos, Lima, Peru. 39 Introduction We formally describe the species after 54 considering the trade-off between a complex integrative approach to delimit the new species, and 55 the need to accelerate the pace of taxonomic descriptions (de Carvalho et al. 2008; Guaysamin et 56 al. 2018; Padial et al. 2010), particularly for micro-endemic taxa inhabiting threatened cloud 57 forests such as many species of small strabomantid frogs. 19 Abstract 20 We describe a new species of minute, terrestrial-breeding frog in the genus Noblella. We 21 collected a single specimen in the leaf litter of primary montane forest (2250 m a.s.l.) near 22 Thiuni, in the Provice of Carabaya, Department of Puno, in the upper watershed of a tributary of 23 the Inambari River of southern Peru, the same locality where we found the types of 24 Psychrophrynella glauca Catenazzi & Ttito 2018. We placed the new species within Noblella on 25 the basis of molecular data, minute size, and overall morphological resemblance with the type 26 species N. peruviana and other species of Noblella, including having three phalanges on finger 27 IV (as in N. coloma, N. heyeri, N. lynchi, N. madreselva, N. peruviana, and N. pygmaea), and 28 terminal phalanges T-shaped and pointed. Noblella thiuni sp. n. is distinguished from all other 29 species of Noblella by having ventral surfaces of legs bright red, and chest and belly copper 30 reddish with a profusion of silvery spots. The new species further differs from known Peruvian 31 species of Noblella by the combination of the following characters: tympanic membrane absent, 32 eyelids lacking tubercles, dorsal skin finely shagreen, tarsal tubercles or folds absent, three 33 phalanges on Finger IV, tips of digits not expanded, no circumferential grooves on digits, 34 inguinal spots present. The new species has a snout–vent length of 11.0 mm in one adult male. 35 Our new finding confirms the high levels of endemism and beta diversity of small, terrestrial- 36 breeding frogs inhabiting the moss layers and leaf litter in the montane forests of the Amazonian 37 slopes of the Andes and adjacent moist puna grasslands, and suggests much work remains to be 38 done to properly document this diversity. PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) 39 Introduction 39 Introduction 40 The genus Noblella is distributed from Ecuador to Bolivia (Figure 1; Catenazzi et al. 41 2015), but there is uncertainty regarding the monophyly of the genus and the number of species 42 (Catenazzi & Ttito 2016; Catenazzi & Ttito 2018; De La Riva et al. 2017). The current taxonomy 43 recognizes 12 species of Noblella (AmphibiaWeb 2019; Catenazzi et al. 2015), of which four 44 occur in southern Peru, where several undescribed species have been reported (Figure 2; von 45 May et al. 2017). The small size of these frogs (Lehr & Catenazzi 2009), terrestrial life habits 46 requiring intensive search in the leaf litter (Catenazzi et al. 2011), and micro-endemism of most 47 species of high-elevation, small strabomantid frogs in the Andes (De La Riva et al. 2017) suggest 48 that many additional species remain to be discovered and recognized throughout the tropical 49 Andes (Catenazzi 2015). Here we describe a new species of Noblella on the basis of a singleton 50 (following terminology of Lim et al. 2012) found in the leaf litter of a cloud forest remnant in the 51 Cordillera de Carabaya, in the southern Peruvian department of Puno, along a tributary of the 52 Inambari River. This specimen is unlike any of the previously described species of Noblella or of 53 the morphologically similar Psychrophrynella. We formally describe the species after 54 considering the trade-off between a complex integrative approach to delimit the new species, and 55 the need to accelerate the pace of taxonomic descriptions (de Carvalho et al. 2008; Guaysamin et 56 al. 2018; Padial et al. 2010), particularly for micro-endemic taxa inhabiting threatened cloud 57 forests such as many species of small strabomantid frogs. 58 59 Materials & Methods 60 On 14 August 2017 we (AC and AT) conducted a rapid survey (~4 hours) of the leaf 61 litter of a relictual cloud forest along the Macusani-San Gabán road, which connects the Peruvian 62 Altiplano to the Amazon rainforest and the interoceanic highway between Peru and Brazil. We 63 removed the leaf litter by hand and searched opportunistically around fallen logs, rocks, moss- 64 covered soil, etc. We searched within an area of approximately 100 m2. We found a single 65 specimen, which we euthanized with 20% benzocaine. Manuscript to be reviewed thiuni sp. n. to obtain DNA sequences for the new species 97 (accession code MK072732; Appendix 2). We also obtained DNA sequences from species of 98 Noblella (Catenazzi et al. 2015) and Psychrophrynella (Catenazzi & Ttito 2016; Catenazzi & 99 Ttito 2018; von May et al. 2017), and downloaded sequences of closely related genera within 100 Holoadenindae (Barycholos, Bryophryne, Holoaden, and Microkayla) from GenBank (Appendix 101 2). Our phylogenetic analyses are preliminary, because there is uncertainty concerning the 102 taxonomic position of Noblella and Psychrophrynella, and because genetic sequences of their 103 type species (N. peruviana and P. bagrecito) are not available (Catenazzi & Ttito 2018). We 104 extracted DNA from liver or skin swab samples (N. madreselva) with a commercial extraction 105 kit (IBI Scientific, Peosta, USA). We followed Hedges et al. (2008) for DNA amplification and 106 sequencing, and used the 16Sar (forward) primer (5'-3' sequence: 107 CGCCTGTTTATCAAAAACAT) and the 16Sbr (reverse) primer (5'-3' sequence: 108 CCGGTCTGAACTCAGATCACGT). The thermocycling conditions during the polymerase 109 chain reaction (PCR) were: one cycle at 96°C/3 min; 35 cycles at 95°C/30 s, 55°C/45 s, 72°C/1.5 110 min; and one cycle at 72°C/7 min. We used a Proflex thermal cycler (Applied Biosystems), 111 purified PCR products with Exosap-IT (ThermoFisher), and shipped purified samples to 112 MCLAB (South San Francisco, CA) for sequencing. We used Geneious, version 11.1.5 113 (Biomatters, http://www.geneious.com/) to align sequences with the MAFFT v7.017 alignment 78 We preserved the holotype in 70% ethanol (without fixing it in formalin). We did not 79 dissect the specimen and we are tentatively identifying it as an adult or subadult male. We 80 measured the following variables to the nearest 0.1 mm with digital calipers under a 81 stereomicroscope: snout–vent length (SVL), tibia length (TL), foot length (FL, distance from 82 proximal margin of inner metatarsal tubercle to tip of Toe IV), head length (HL, from angle of 83 jaw to tip of snout), head width (HW, at level of angle of jaw), eye diameter (ED), tympanum 84 diameter (TY), interorbital distance (IOD), upper eyelid width (EW), internarial distance (IND), 85 eye–nostril distance (E–N, straight line distance between anterior corner of orbit and posterior 86 margin of external nares). We numbered fingers and toes preaxially to postaxially from I–IV and 87 I–V respectively. Manuscript to be reviewed 2015) and Psychrophrynella (Catenazzi & Ttito 2016; Catenazzi & 99 Ttito 2018; von May et al. 2017), and downloaded sequences of closely related genera within 100 Holoadenindae (Barycholos, Bryophryne, Holoaden, and Microkayla) from GenBank (Appendix 101 2). Our phylogenetic analyses are preliminary, because there is uncertainty concerning the 102 taxonomic position of Noblella and Psychrophrynella, and because genetic sequences of their 103 type species (N. peruviana and P. bagrecito) are not available (Catenazzi & Ttito 2018). We 104 extracted DNA from liver or skin swab samples (N. madreselva) with a commercial extraction 105 kit (IBI Scientific, Peosta, USA). We followed Hedges et al. (2008) for DNA amplification and 106 sequencing, and used the 16Sar (forward) primer (5'-3' sequence: 107 CGCCTGTTTATCAAAAACAT) and the 16Sbr (reverse) primer (5'-3' sequence: 107 CGCCTGTTTATCAAAAACAT) and the 16Sbr (reverse) primer (5 -3 sequence: 108 CCGGTCTGAACTCAGATCACGT). The thermocycling conditions during the polymerase 109 chain reaction (PCR) were: one cycle at 96°C/3 min; 35 cycles at 95°C/30 s, 55°C/45 s, 72°C/1.5 110 min; and one cycle at 72°C/7 min. We used a Proflex thermal cycler (Applied Biosystems), 111 purified PCR products with Exosap-IT (ThermoFisher), and shipped purified samples to 112 MCLAB (South San Francisco, CA) for sequencing. We used Geneious, version 11.1.5 113 (Biomatters, http://www.geneious.com/) to align sequences with the MAFFT v7.017 alignment 114 program (Katoh & Standley 2013), and trimmed sequences to a length of 537 bp. Our analysis 115 included 22 terminals. We employed a Maximum Likelihood (ML) to infer a molecular 116 phylogeny generated using MEGA v. 7 (Kumar et al. 2016) based on the General Time 117 Reversible model. Initial tree(s) for the heuristic search were obtained by applying Neighbor- 108 CCGGTCTGAACTCAGATCACGT). The thermocycling conditions during the polymerase 109 chain reaction (PCR) were: one cycle at 96°C/3 min; 35 cycles at 95°C/30 s, 55°C/45 s, 72°C/1.5 110 min; and one cycle at 72°C/7 min. We used a Proflex thermal cycler (Applied Biosystems), 111 purified PCR products with Exosap-IT (ThermoFisher), and shipped purified samples to ( , ) q g , 113 (Biomatters, http://www.geneious.com/) to align sequences with the MAFFT v7.017 alignment 114 program (Katoh & Standley 2013), and trimmed sequences to a length of 537 bp. Our analysis 115 included 22 terminals. We employed a Maximum Likelihood (ML) to infer a molecular 116 phylogeny generated using MEGA v. 7 (Kumar et al. 59 Materials & Methods PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Manuscript to be reviewed 78 We preserved the holotype in 70% ethanol (without fixing it in formalin). We did not 79 dissect the specimen and we are tentatively identifying it as an adult or subadult male. We 80 measured the following variables to the nearest 0.1 mm with digital calipers under a 81 stereomicroscope: snout–vent length (SVL), tibia length (TL), foot length (FL, distance from 82 proximal margin of inner metatarsal tubercle to tip of Toe IV), head length (HL, from angle of 83 jaw to tip of snout), head width (HW, at level of angle of jaw), eye diameter (ED), tympanum 84 diameter (TY), interorbital distance (IOD), upper eyelid width (EW), internarial distance (IND), 85 eye–nostril distance (E–N, straight line distance between anterior corner of orbit and posterior 86 margin of external nares). We numbered fingers and toes preaxially to postaxially from I–IV and 87 I–V respectively. We determined comparative lengths of toes III and V by adpressing both toes 88 against Toe IV; lengths of fingers I and II were determined by adpressing the fingers against 89 each other. We used field notes and photographs we took in the field to describe coloration in 90 life. We have deposited photographs of the live and preserved holotype at the Calphoto online 91 database (http://calphotos.berkeley.edu). 92 We used phylogenetic analyses to confirm generic placement of the new species within 93 Noblella through analysis of the mitochondrial 16S rRNA fragment. This fragment is commonly 94 used for anuran taxonomy (Fouquet et al. 2007; Padial et al. 2009; Vences et al. 2005), and is the 95 sequence most commonly used for species of Holoadeninae (Hedges et al. 2008). We used liver 96 tissues from the holotype of N. thiuni sp. n. to obtain DNA sequences for the new species 97 (accession code MK072732; Appendix 2). We also obtained DNA sequences from species of 98 Noblella (Catenazzi et al. 2015) and Psychrophrynella (Catenazzi & Ttito 2016; Catenazzi & 99 Ttito 2018; von May et al. 2017), and downloaded sequences of closely related genera within 100 Holoadenindae (Barycholos, Bryophryne, Holoaden, and Microkayla) from GenBank (Appendix 101 2). Our phylogenetic analyses are preliminary, because there is uncertainty concerning the 102 taxonomic position of Noblella and Psychrophrynella, and because genetic sequences of their 103 type species (N. peruviana and P. bagrecito) are not available (Catenazzi & Ttito 2018). We 104 extracted DNA from liver or skin swab samples (N. Manuscript to be reviewed madreselva) with a commercial extraction 105 kit (IBI Scientific, Peosta, USA). We followed Hedges et al. (2008) for DNA amplification and 106 sequencing, and used the 16Sar (forward) primer (5'-3' sequence: 107 CGCCTGTTTATCAAAAACAT) and the 16Sbr (reverse) primer (5'-3' sequence: 108 CCGGTCTGAACTCAGATCACGT). The thermocycling conditions during the polymerase 109 chain reaction (PCR) were: one cycle at 96°C/3 min; 35 cycles at 95°C/30 s, 55°C/45 s, 72°C/1.5 110 min; and one cycle at 72°C/7 min. We used a Proflex thermal cycler (Applied Biosystems), 111 purified PCR products with Exosap-IT (ThermoFisher), and shipped purified samples to 112 MCLAB (South San Francisco, CA) for sequencing. We used Geneious, version 11.1.5 113 (Biomatters, http://www.geneious.com/) to align sequences with the MAFFT v7.017 alignment 78 We preserved the holotype in 70% ethanol (without fixing it in formalin). We did not 79 dissect the specimen and we are tentatively identifying it as an adult or subadult male. We 80 measured the following variables to the nearest 0.1 mm with digital calipers under a 81 stereomicroscope: snout–vent length (SVL), tibia length (TL), foot length (FL, distance from 82 proximal margin of inner metatarsal tubercle to tip of Toe IV), head length (HL, from angle of 83 jaw to tip of snout), head width (HW, at level of angle of jaw), eye diameter (ED), tympanum 84 diameter (TY), interorbital distance (IOD), upper eyelid width (EW), internarial distance (IND), 85 eye–nostril distance (E–N, straight line distance between anterior corner of orbit and posterior 86 margin of external nares). We numbered fingers and toes preaxially to postaxially from I–IV and 87 I–V respectively. We determined comparative lengths of toes III and V by adpressing both toes 88 against Toe IV; lengths of fingers I and II were determined by adpressing the fingers against 89 each other. We used field notes and photographs we took in the field to describe coloration in 90 life. We have deposited photographs of the live and preserved holotype at the Calphoto online 91 database (http://calphotos.berkeley.edu). 92 We used phylogenetic analyses to confirm generic placement of the new species within 93 Noblella through analysis of the mitochondrial 16S rRNA fragment. This fragment is commonly 94 used for anuran taxonomy (Fouquet et al. 2007; Padial et al. 2009; Vences et al. 2005), and is the 95 sequence most commonly used for species of Holoadeninae (Hedges et al. 2008). We used liver 96 tissues from the holotype of N. Manuscript to be reviewed We determined comparative lengths of toes III and V by adpressing both toes 88 against Toe IV; lengths of fingers I and II were determined by adpressing the fingers against 89 each other. We used field notes and photographs we took in the field to describe coloration in 90 life. We have deposited photographs of the live and preserved holotype at the Calphoto online 91 database (http://calphotos.berkeley.edu). 92 We used phylogenetic analyses to confirm generic placement of the new species within 93 Noblella through analysis of the mitochondrial 16S rRNA fragment. This fragment is commonly 94 used for anuran taxonomy (Fouquet et al. 2007; Padial et al. 2009; Vences et al. 2005), and is the 95 sequence most commonly used for species of Holoadeninae (Hedges et al. 2008). We used liver 96 tissues from the holotype of N. thiuni sp. n. to obtain DNA sequences for the new species 97 (accession code MK072732; Appendix 2). We also obtained DNA sequences from species of 98 Noblella (Catenazzi et al. 2015) and Psychrophrynella (Catenazzi & Ttito 2016; Catenazzi & 99 Ttito 2018; von May et al. 2017), and downloaded sequences of closely related genera within 100 Holoadenindae (Barycholos, Bryophryne, Holoaden, and Microkayla) from GenBank (Appendix 101 2). Our phylogenetic analyses are preliminary, because there is uncertainty concerning the 102 taxonomic position of Noblella and Psychrophrynella, and because genetic sequences of their 103 type species (N. peruviana and P. bagrecito) are not available (Catenazzi & Ttito 2018). We 104 extracted DNA from liver or skin swab samples (N. madreselva) with a commercial extraction 105 kit (IBI Scientific, Peosta, USA). We followed Hedges et al. (2008) for DNA amplification and 106 sequencing, and used the 16Sar (forward) primer (5'-3' sequence: 92 We used phylogenetic analyses to confirm generic placement of the new species within 93 Noblella through analysis of the mitochondrial 16S rRNA fragment. This fragment is commonly 94 used for anuran taxonomy (Fouquet et al. 2007; Padial et al. 2009; Vences et al. 2005), and is the 95 sequence most commonly used for species of Holoadeninae (Hedges et al. 2008). We used liver 96 tissues from the holotype of N. thiuni sp. n. to obtain DNA sequences for the new species 97 (accession code MK072732; Appendix 2). We also obtained DNA sequences from species of 98 Noblella (Catenazzi et al. Manuscript to be reviewed 118 Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum 119 Composite Likelihood (MCL) approach, and then selecting the topology with superior log 120 likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences 121 among sites (5 categories (+G, parameter = 0.7550)). The rate variation model allowed for some 122 sites to be evolutionarily invariable ([+I], 41.29% sites). We assessed node support using 500 123 bootstrap replicates. We also estimated pairwise, uncorrected genetic distances (p distances) for 124 16S rRNA between the new species and other species of Noblella and Psychrophrynella, as well 125 as species from other genera of Holoadeninae. 118 Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum 119 Composite Likelihood (MCL) approach, and then selecting the topology with superior log 120 likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences 121 among sites (5 categories (+G, parameter = 0.7550)). The rate variation model allowed for some 122 sites to be evolutionarily invariable ([+I], 41.29% sites). We assessed node support using 500 123 bootstrap replicates. We also estimated pairwise, uncorrected genetic distances (p distances) for 124 16S rRNA between the new species and other species of Noblella and Psychrophrynella, as well 125 as species from other genera of Holoadeninae. 126 Our research was approved by the Institutional Animal Care and Use Committee of 127 Southern Illinois University Carbondale (protocol #16-006) and Florida International University 128 (protocol #18-009). The Dirección General Forestal y de Fauna Silvestre, Ministerio de 129 Agricultura y Riego issued the permit authorizing this research (permits #0292-2014-MINAGRI- 130 DGFFS/DGEFFS, #029-2016-SERFOR-DGSPFS). 126 Our research was approved by the Institutional Animal Care and Use Committee of 127 Southern Illinois University Carbondale (protocol #16-006) and Florida International University 128 (protocol #18-009). The Dirección General Forestal y de Fauna Silvestre, Ministerio de 129 Agricultura y Riego issued the permit authorizing this research (permits #0292-2014-MINAGRI- 130 DGFFS/DGEFFS, #029-2016-SERFOR-DGSPFS). 126 Our research was approved by the Institutional Animal Care and Use Committee of 127 Southern Illinois University Carbondale (protocol #16-006) and Florida International University 126 Our research was approved by the Institutional Animal Care and Use Committee of 127 Southern Illinois University Carbondale (protocol #16-006) and Florida International University 128 (protocol #18-009). Manuscript to be reviewed 2016) based on the General Time 117 R ibl d l I i i l ( ) f h h i i h b i d b l i N i hb 117 Reversible model. Initial tree(s) for the heuristic search were obtained by applying Neighbor- PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Manuscript to be reviewed The Dirección General Forestal y de Fauna Silvestre, Ministerio de 129 Agricultura y Riego issued the permit authorizing this research (permits #0292-2014-MINAGRI- 130 DGFFS/DGEFFS, #029-2016-SERFOR-DGSPFS). 131 The electronic version of this article in Portable Document Format (PDF) will represent a 132 published work according to the International Commission on Zoological Nomenclature (ICZ), 133 and hence the new name contained in the electronic version is effectively published under that 134 Code from the electronic edition alone. This published work and the nomenclatural acts it 135 contains have been registered in ZooBank, the online registration system for the ICZN. The 136 ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed 137 through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The 138 LSID for this publication is: urn:lsid:zoobank.org:pub:3F917F8B-5D31-44D4-86E2- 139 389341A21BD1. The online version of this work is archived and available from the following 140 digital repositories: PeerJ, PubMed Central and CLOCKSS. 141 142 Results 143 144 Generic placement 145 Our phylogenetic analysis indicates relatedness of our specimen with species of Noblella from 146 southern Peru (Figure 3), but also with species of Psychrophrynella. As previously discussed 147 (Catenazzi & Ttito 2016; Catenazzi & Ttito 2018; De La Riva et al. 2017; De la Riva et al. 148 2008), the type species of both Noblella and Psychrophrynella, N. peruviana and P. bagrecito, 149 respectively, have not been included in phylogenetic analyses due to lack of DNA sequences. 150 Similarity in meristic traits (i.e., presence of tarsal folds or tubercles), and our present phylogeny 151 showing N. thiuni sp. nov. as the sister taxon to all other species of sampled Noblella and 152 Psychrophrynella, supports the idea that Psychrophrynella species may be nested within a larger 153 Noblella clade. However, the taxonomy cannot be resolved without collecting more information 154 from the type species N. peruviana and P. bagrecito, including DNA sequences, morphometric 155 measurements, and recordings of advertisement calls. Our phylogeny produced from a small 156 dataset of only one mitrochondrial gene fragment (16S) is similar to previously published trees 157 generated using much larger datasets (i.e., De La Riva et al. 2017; Hedges et al. 2008). Our tree 127 Southern Illinois University Carbondale (protocol #16-006) and Florida International University 128 (protocol #18 009). Manuscript to be reviewed The new species differs from species of Microkayla (characters in 183 parentheses) in having T-shaped terminal phalanges (absent), Toe V about the same length as 184 Toe III (Toe V slightly longer than Toe III), elongated tongue (rounded), smooth venter 185 (areolate), tarsal fold (absent), and small size and slender body with longer limbs (larger size 186 with robust body and short extremities). There presently are no meristic traits to differentiate 187 species of Noblella from species of Psychrophrynella, and future work will aim at resolving the 188 taxonomic conundrum posed by the absence of synapomorphies for these two genera. 189 163 The new species is assigned to Noblella, as defined by De la Riva et al. (2008), Duellman 164 & Lehr (2009), and Hedges et al. (2008), on the basis of the frog’s minute size and overall 165 morphological resemblance with the type species Noblella peruviana and other species of 166 Noblella, including having three phalanges on Finger IV (as in N. coloma, N. heyeri, N. lynchi, 167 N. madreselva, N. peruviana, and N. pygmaea), terminal phalanges T-shaped and pointed, 168 tympanic membrane absent (as in N. duellmani, N. peruviana). The species with the lowest 169 uncorrected p-distance for 16S rRNA (Table 1) is N. pygmaea (0.07), followed by N. madreselva 170 (0.08) and N. sp. R (von May et al. 2017) and four species of Psychrophrynella (0.09). Frogs of 171 the genus Noblella are morphologically similar and closely related to Barycholos, Bryophryne, 172 Holoaden, Microkayla and Psychrophrynella (De La Riva et al. 2017; Hedges et al. 2008; 173 Heinicke et al. 2007; Padial et al. 2014). The new species is assigned to Noblella rather than 174 Barycholos (characters in parentheses), because it lacks the dentigerous processes of the vomers 175 (present), has Finger I shorter than Finger II (Finger I > Finger II), and has low, rounded 176 subarticular tubercles (subarticular tubercles elevated). The new species differs from species of 177 Bryophryne (characters in parentheses) in having T-shaped terminal phalanges (knob-shaped), no 178 nuptial pads (present or absent), tarsal fold (absent), small size and slender body with longer 179 limbs (larger size with stubby body and short limbs). Species in the genus Holoaden have 180 prominent dentigerous processes of vomers (absent in N. thiuni sp. Manuscript to be reviewed The Dirección General Forestal y de Fauna Silvestre, Ministerio de 129 Agricultura y Riego issued the permit authorizing this research (permits #0292-2014-MINAGRI- 130 DGFFS/DGEFFS, #029-2016-SERFOR-DGSPFS). 129 Agricultura y Riego issued the permit authorizing this research (permits #0292-2014-MINAGRI- 130 DGFFS/DGEFFS #029 2016 SERFOR DGSPFS) p 145 Our phylogenetic analysis indicates relatedness of our specimen with species of Noblella from 146 southern Peru (Figure 3), but also with species of Psychrophrynella. As previously discussed 147 (Catenazzi & Ttito 2016; Catenazzi & Ttito 2018; De La Riva et al. 2017; De la Riva et al. 148 2008), the type species of both Noblella and Psychrophrynella, N. peruviana and P. bagrecito, 149 respectively, have not been included in phylogenetic analyses due to lack of DNA sequences. 150 Similarity in meristic traits (i.e., presence of tarsal folds or tubercles), and our present phylogeny 151 showing N. thiuni sp. nov. as the sister taxon to all other species of sampled Noblella and 152 Psychrophrynella, supports the idea that Psychrophrynella species may be nested within a larger 153 Noblella clade. However, the taxonomy cannot be resolved without collecting more information 154 from the type species N. peruviana and P. bagrecito, including DNA sequences, morphometric 155 measurements, and recordings of advertisement calls. Our phylogeny produced from a small 156 dataset of only one mitrochondrial gene fragment (16S) is similar to previously published trees 157 generated using much larger datasets (i.e., De La Riva et al. 2017; Hedges et al. 2008). Our tree PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Manuscript to be reviewed 158 however includes a larger number of Noblella species, including undescribed forms from 159 southern Peru (von May et al. 2017). These southern Peruvian species, which occur close to the 160 type locality of N. peruviana and P. bagrecito in the Departments of Puno and Cusco, form a 161 sister clade to Microkayla and Bryophryne, and thus appear not to be closely related to species of 162 Noblella from northern Peru and Ecuador. 163 The new species is assigned to Noblella, as defined by De la Riva et al. (2008), Duellman 164 & Lehr (2009), and Hedges et al. (2008), on the basis of the frog’s minute size and overall 165 morphological resemblance with the type species Noblella peruviana and other species of 166 Noblella, including having three phalanges on Finger IV (as in N. coloma, N. heyeri, N. lynchi, 167 N. madreselva, N. peruviana, and N. pygmaea), terminal phalanges T-shaped and pointed, 168 tympanic membrane absent (as in N. duellmani, N. peruviana). The species with the lowest 169 uncorrected p-distance for 16S rRNA (Table 1) is N. pygmaea (0.07), followed by N. madreselva 170 (0.08) and N. sp. R (von May et al. 2017) and four species of Psychrophrynella (0.09). Frogs of 171 the genus Noblella are morphologically similar and closely related to Barycholos, Bryophryne, 172 Holoaden, Microkayla and Psychrophrynella (De La Riva et al. 2017; Hedges et al. 2008; 173 Heinicke et al. 2007; Padial et al. 2014). The new species is assigned to Noblella rather than 174 Barycholos (characters in parentheses), because it lacks the dentigerous processes of the vomers 175 (present), has Finger I shorter than Finger II (Finger I > Finger II), and has low, rounded 176 subarticular tubercles (subarticular tubercles elevated). The new species differs from species of 177 Bryophryne (characters in parentheses) in having T-shaped terminal phalanges (knob-shaped), no 178 nuptial pads (present or absent), tarsal fold (absent), small size and slender body with longer 179 limbs (larger size with stubby body and short limbs). Species in the genus Holoaden have 180 prominent dentigerous processes of vomers (absent in N. thiuni sp. n.), terminal phalanges knob- 181 shaped (T-shaped), venter areolate (smooth), and much larger size (up to 48 mm SVL) than 182 species of Noblella. 198 Characterization Among the eight species with three 226 phalanges on Finger IV, it differs from N. heyeri, N. lynchi and N. pygmaea by lacking a distinct 227 tympanic membrane, and from N. coloma, N. duellmani, N. madreselva and N. personina by 228 having a tarsal fold. It further differs from N. madreselva by lacking a large white mark on 229 venter, from N. duellmani, N. personina, and N. pygmaea by having inguinal spots, from all 230 species but N. coloma, N. lochites, N. myrmecoides and N. peruviana in having a finely shagreen 231 dorsum, and from all species but N. heyeri, N. lynchi, N. myrmecoides and N. peruviana by 232 lacking tubercles on eyelids. Finally, N. thiuni differs from N. peruviana by having a smaller 233 inner metatarsal tubercle, about the same size as outer metatarsal tubercle (inner tubercle large, 234 about twice the size of outer metatarsal tubercle), Toe V about the same length of Toe III (Toe V 235 shorter than Toe III). 236 The new species is similar to species of Psychrophrynella, including the sympatric P. 237 glauca (Catenazzi Ttito 2018) and the type species P. 198 Characterization bagrecito (Lynch, 1986), which both have 199 A species of Noblella characterized by (1) skin on dorsum finely shagreen; skin on venter 200 smooth, discoidal fold not visible, thin dorsolateral folds visible on anterior half part of body; (2) 201 tympanic membrane not differentiated, tympanic annulus barely visible below skin; (3) snout 202 short, bluntly rounded in dorsal view and in profile; (4) upper eyelid lacking tubercles, narrower 203 than IOD; cranial crests absent; (5) dentigerous process of vomers absent; (6) vocal slits present; 204 nuptial pads absent; (7) Finger I shorter than Finger II; tips of digits bulbous, not expanded 205 laterally; (8) fingers lacking lateral fringes; (9) ulnar tubercles absent; (10) heel lacking 206 tubercles; inner edge of tarsus bearing an elongate, obliquous fold-like tubercle; (11) inner 207 metatarsal tubercle elliptical and about the same size of ovoid, outer metatarsal tubercle; 208 supernumerary plantar tubercles absent; (12) toes lacking lateral fringes; webbing absent; Toe V 209 slightly about the same length as Toe III; tips of digits not expanded, weakly pointed; (13) 210 dorsum tan to brown and gray with dark brown markings; some individuals with a yellow or 211 orange middorsal line extending from tip of snout to cloaca and to posterior surface of thighs; 212 interorbital bar present; chest, venter and ventral parts of arms and legs yellow with brown 213 flecks; throat and palmar and plantar surfaces brown or reddish-brown; (14) SVL 11.0 mm in 214 one male. 217 Noblella thiuni is most similar to N. peruviana but differs from this and other known species in 218 the genus (Catenazzi et al. 2015) by having ventral surfaces of legs bright red, and chest and 219 belly copper reddish with a profusion of silvery spots (Figure 4). Noblella thiuni has three 220 phalanges on Finger IV and differs from N. carrascoicola, N. lochites, N. myrmecoides, and N. 221 ritarasquinae which have two phalanges on Finger IV (De la Riva & Köhler 1998; Duellman & 222 Lehr 2009; Guayasamin & Teran-Valdez 2009; Harvey et al. 2013; Köhler 2000). It further 223 differs from N. myrmecoides in having tips of toes not expanded (slightly expanded in N. 224 duellmani, N. heyeri, and N. lynchi, tips of toes teardrop-shaped in N. myrmecoides) and in 225 lacking circumferential grooves (present in N. myrmecoides). Among the eight species with three 226 phalanges on Finger IV, it differs from N. heyeri, N. Manuscript to be reviewed n.), terminal phalanges knob- 181 shaped (T-shaped), venter areolate (smooth), and much larger size (up to 48 mm SVL) than 182 species of Noblella. The new species differs from species of Microkayla (characters in 183 parentheses) in having T-shaped terminal phalanges (absent), Toe V about the same length as 184 Toe III (Toe V slightly longer than Toe III), elongated tongue (rounded), smooth venter 185 (areolate), tarsal fold (absent), and small size and slender body with longer limbs (larger size 186 with robust body and short extremities). There presently are no meristic traits to differentiate 187 species of Noblella from species of Psychrophrynella, and future work will aim at resolving the 188 taxonomic conundrum posed by the absence of synapomorphies for these two genera. 189 PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) 198 Characterization 198 Characterization 199 A species of Noblella characterized by (1) skin on dorsum finely shagreen; skin on venter 200 smooth, discoidal fold not visible, thin dorsolateral folds visible on anterior half part of body; (2) 201 tympanic membrane not differentiated, tympanic annulus barely visible below skin; (3) snout 202 short, bluntly rounded in dorsal view and in profile; (4) upper eyelid lacking tubercles, narrower 203 than IOD; cranial crests absent; (5) dentigerous process of vomers absent; (6) vocal slits present; 204 nuptial pads absent; (7) Finger I shorter than Finger II; tips of digits bulbous, not expanded 205 laterally; (8) fingers lacking lateral fringes; (9) ulnar tubercles absent; (10) heel lacking 206 tubercles; inner edge of tarsus bearing an elongate, obliquous fold-like tubercle; (11) inner 207 metatarsal tubercle elliptical and about the same size of ovoid, outer metatarsal tubercle; 208 supernumerary plantar tubercles absent; (12) toes lacking lateral fringes; webbing absent; Toe V 209 slightly about the same length as Toe III; tips of digits not expanded, weakly pointed; (13) 210 dorsum tan to brown and gray with dark brown markings; some individuals with a yellow or 211 orange middorsal line extending from tip of snout to cloaca and to posterior surface of thighs; 212 interorbital bar present; chest, venter and ventral parts of arms and legs yellow with brown 213 flecks; throat and palmar and plantar surfaces brown or reddish-brown; (14) SVL 11.0 mm in 214 one male. 215 216 Diagnosis 217 Noblella thiuni is most similar to N. peruviana but differs from this and other known species in 218 the genus (Catenazzi et al. 2015) by having ventral surfaces of legs bright red, and chest and 219 belly copper reddish with a profusion of silvery spots (Figure 4). Noblella thiuni has three 220 phalanges on Finger IV and differs from N. carrascoicola, N. lochites, N. myrmecoides, and N. 221 ritarasquinae which have two phalanges on Finger IV (De la Riva & Köhler 1998; Duellman & 222 Lehr 2009; Guayasamin & Teran-Valdez 2009; Harvey et al. 2013; Köhler 2000). It further 223 differs from N. myrmecoides in having tips of toes not expanded (slightly expanded in N. 224 duellmani, N. heyeri, and N. lynchi, tips of toes teardrop-shaped in N. myrmecoides) and in 225 lacking circumferential grooves (present in N. myrmecoides). Among the eight species with three 226 phalanges on Finger IV, it differs from N. heyeri, N. 198 Characterization lynchi and N. pygmaea by lacking a distinct 227 tympanic membrane, and from N. coloma, N. duellmani, N. madreselva and N. personina by 228 having a tarsal fold. It further differs from N. madreselva by lacking a large white mark on 229 venter, from N. duellmani, N. personina, and N. pygmaea by having inguinal spots, from all 230 species but N. coloma, N. lochites, N. myrmecoides and N. peruviana in having a finely shagreen 231 dorsum, and from all species but N. heyeri, N. lynchi, N. myrmecoides and N. peruviana by 232 lacking tubercles on eyelids. Finally, N. thiuni differs from N. peruviana by having a smaller 233 inner metatarsal tubercle, about the same size as outer metatarsal tubercle (inner tubercle large, 234 about twice the size of outer metatarsal tubercle), Toe V about the same length of Toe III (Toe V 235 shorter than Toe III). 236 The new species is similar to species of Psychrophrynella, including the sympatric P. 237 glauca (Catenazzi Ttito 2018) and the type species P. 198 Characterization bagrecito (Lynch, 1986), which both have 198 Characterization 199 A species of Noblella characterized by (1) skin on dorsum finely shagreen; skin on venter 200 smooth, discoidal fold not visible, thin dorsolateral folds visible on anterior half part of body; (2) 201 tympanic membrane not differentiated, tympanic annulus barely visible below skin; (3) snout 202 short, bluntly rounded in dorsal view and in profile; (4) upper eyelid lacking tubercles, narrower 203 than IOD; cranial crests absent; (5) dentigerous process of vomers absent; (6) vocal slits present; 204 nuptial pads absent; (7) Finger I shorter than Finger II; tips of digits bulbous, not expanded 205 laterally; (8) fingers lacking lateral fringes; (9) ulnar tubercles absent; (10) heel lacking 206 tubercles; inner edge of tarsus bearing an elongate, obliquous fold-like tubercle; (11) inner 207 metatarsal tubercle elliptical and about the same size of ovoid, outer metatarsal tubercle; 208 supernumerary plantar tubercles absent; (12) toes lacking lateral fringes; webbing absent; Toe V 209 slightly about the same length as Toe III; tips of digits not expanded, weakly pointed; (13) 210 dorsum tan to brown and gray with dark brown markings; some individuals with a yellow or 211 orange middorsal line extending from tip of snout to cloaca and to posterior surface of thighs; 212 interorbital bar present; chest, venter and ventral parts of arms and legs yellow with brown 213 flecks; throat and palmar and plantar surfaces brown or reddish-brown; (14) SVL 11.0 mm in 214 one male. 215 216 Diagnosis 217 Noblella thiuni is most similar to N. peruviana but differs from this and other known species in 218 the genus (Catenazzi et al. 2015) by having ventral surfaces of legs bright red, and chest and 219 belly copper reddish with a profusion of silvery spots (Figure 4). Noblella thiuni has three 220 phalanges on Finger IV and differs from N. carrascoicola, N. lochites, N. myrmecoides, and N. 221 ritarasquinae which have two phalanges on Finger IV (De la Riva & Köhler 1998; Duellman & 222 Lehr 2009; Guayasamin & Teran-Valdez 2009; Harvey et al. 2013; Köhler 2000). It further 223 differs from N. myrmecoides in having tips of toes not expanded (slightly expanded in N. 224 duellmani, N. heyeri, and N. lynchi, tips of toes teardrop-shaped in N. myrmecoides) and in 225 lacking circumferential grooves (present in N. myrmecoides). Manuscript to be reviewed 238 a fold-like tubercle on the inner edge of tarsus, small size reaching ∼19 mm, and a prominent 239 ovoid outer metatarsal tubercle (of same or larger size than inner metatarsal tubercle). 240 Furthermore, N. thiuni shares with P. glauca the red coloration on ventral surfaces of legs and 241 venter, and the profusion of silvery or bluish-gray flecks on ventral surfaces of head, body, and 242 legs. Noblella thiuni differs from P. bagrecito in having red coloration extending to all ventral 243 surfaces including chest and throat, relative finger lengths when adpressed, Finger 3 > 2 > 4 > 1 244 (3 > 4 > 2 > 1 in P. glauca), proportionally shorter head, and presumably in having smaller size. 245 When recently preserved, N. thiuni clearly stands out from specimens of P. glauca due to reddish 246 marks turning purple on dorsum and dorsal surfaces of legs; the purple coloration fades to brown 247 after several months of preservation. Noblella thiuni can be distinguished from P. bagrecito 248 (characters in parenthesis for P. bagrecito) by having the snout short and bluntly rounded (snout 249 moderately long, rounded in dorsal view and in profile), skin on venter smooth (areolate), dorsal 250 coloration with broad markings and purple marks middorsally and on legs (longitudinal stripes, 251 no red marks), and ventral coloration in preservative brown with silvery flecks (white to cream 252 with brown mottling). The other two species of Psychrophrynella, P. chirihampatu and P. 253 usurpator, are much larger in size (reaching 27.7 and 30.5 mm SVL, respectively), and have 254 ventral coloration yellow with reddish-brown or gray flecks, and dull brown, gray or black with 255 cream flecks, respectively. 256 257 Description of holotype. Adult or subadult male (11.0 mm SVL); head narrower than body, its 258 length 31% of SVL; head slightly longer than wide; head width 32% of SVL; snout very short, 259 bluntly rounded in dorsal and lateral view (Figure 4), eye large, 44% of head length, its diameter 260 1.87 times as large as its distance from the nostril; nostrils not protuberant, situated close to 261 snout; canthus rostralis slightly curved in dorsal view, rounded in profile; lores flat; lips rounded; 262 upper eyelids lacking tubercles; upper eyelid width 69% of interorbital distance; supratympanic 263 fold short; tympanic membrane absent, tympanic annulus not visible; one long, postrictal ridges 264 barely visible. Manuscript to be reviewed 238 a fold-like tubercle on the inner edge of tarsus, small size reaching ∼19 mm, and a prominent 239 ovoid outer metatarsal tubercle (of same or larger size than inner metatarsal tubercle). 257 Description of holotype. Adult or subadult male (11.0 mm SVL); head narrower than body, its 258 length 31% of SVL; head slightly longer than wide; head width 32% of SVL; snout very short, 259 bluntly rounded in dorsal and lateral view (Figure 4), eye large, 44% of head length, its diameter 260 1.87 times as large as its distance from the nostril; nostrils not protuberant, situated close to 261 snout; canthus rostralis slightly curved in dorsal view, rounded in profile; lores flat; lips rounded; 262 upper eyelids lacking tubercles; upper eyelid width 69% of interorbital distance; supratympanic 263 fold short; tympanic membrane absent, tympanic annulus not visible; one long, postrictal ridges 264 barely visible. Choanae minute, round, positioned anteriorly and laterally, widely separated from 265 each other, slightly concealed by palatal shelf of maxilla; dentigerous processes of vomer and 266 vomerine teeth absent; tongue long and narrow, about 3 three times as long as wide. 257 Description of holotype. Adult or subadult male (11.0 mm SVL); head narrower than body, its 258 length 31% of SVL; head slightly longer than wide; head width 32% of SVL; snout very short, 259 bluntly rounded in dorsal and lateral view (Figure 4), eye large, 44% of head length, its diameter 260 1.87 times as large as its distance from the nostril; nostrils not protuberant, situated close to 261 snout; canthus rostralis slightly curved in dorsal view, rounded in profile; lores flat; lips rounded; 262 upper eyelids lacking tubercles; upper eyelid width 69% of interorbital distance; supratympanic 263 fold short; tympanic membrane absent, tympanic annulus not visible; one long, postrictal ridges 264 barely visible. Choanae minute, round, positioned anteriorly and laterally, widely separated from 265 each other, slightly concealed by palatal shelf of maxilla; dentigerous processes of vomer and 266 vomerine teeth absent; tongue long and narrow, about 3 three times as long as wide. 267 Skin on dorsum finely shagreen, lacking tubercles; thin dorsolateral folds visible on anterior half 268 part of body; skin on flanks smooth; skin on ventral surfaces and gular regions smooth; pectoral 269 fold present, discoidal fold not evident; cloaca protuberant; cloacal region bearing several small 270 tubercles. Manuscript to be reviewed bagrecito) by having the snout short and bluntly rounded (snout 249 moderately long, rounded in dorsal view and in profile), skin on venter smooth (areolate), dorsal 250 coloration with broad markings and purple marks middorsally and on legs (longitudinal stripes, 251 no red marks), and ventral coloration in preservative brown with silvery flecks (white to cream 252 with brown mottling). The other two species of Psychrophrynella, P. chirihampatu and P. 253 usurpator, are much larger in size (reaching 27.7 and 30.5 mm SVL, respectively), and have 254 ventral coloration yellow with reddish-brown or gray flecks, and dull brown, gray or black with 255 cream flecks, respectively. 238 a fold-like tubercle on the inner edge of tarsus, small size reaching ∼19 mm, and a prominent 239 ovoid outer metatarsal tubercle (of same or larger size than inner metatarsal tubercle). 240 Furthermore, N. thiuni shares with P. glauca the red coloration on ventral surfaces of legs and 241 venter, and the profusion of silvery or bluish-gray flecks on ventral surfaces of head, body, and 242 legs. Noblella thiuni differs from P. bagrecito in having red coloration extending to all ventral 243 surfaces including chest and throat, relative finger lengths when adpressed, Finger 3 > 2 > 4 > 1 244 (3 > 4 > 2 > 1 in P. glauca), proportionally shorter head, and presumably in having smaller size. 245 When recently preserved, N. thiuni clearly stands out from specimens of P. glauca due to reddish 246 marks turning purple on dorsum and dorsal surfaces of legs; the purple coloration fades to brown 247 after several months of preservation. Noblella thiuni can be distinguished from P. bagrecito 248 (characters in parenthesis for P. bagrecito) by having the snout short and bluntly rounded (snout 249 moderately long, rounded in dorsal view and in profile), skin on venter smooth (areolate), dorsal 250 coloration with broad markings and purple marks middorsally and on legs (longitudinal stripes, 251 no red marks), and ventral coloration in preservative brown with silvery flecks (white to cream 252 with brown mottling). The other two species of Psychrophrynella, P. chirihampatu and P. 253 usurpator, are much larger in size (reaching 27.7 and 30.5 mm SVL, respectively), and have 254 ventral coloration yellow with reddish-brown or gray flecks, and dull brown, gray or black with 255 cream flecks, respectively. 198 Characterization lynchi and N. pygmaea by lacking a distinct 227 tympanic membrane, and from N. coloma, N. duellmani, N. madreselva and N. personina by 228 having a tarsal fold. It further differs from N. madreselva by lacking a large white mark on 229 venter, from N. duellmani, N. personina, and N. pygmaea by having inguinal spots, from all 230 species but N. coloma, N. lochites, N. myrmecoides and N. peruviana in having a finely shagreen 231 dorsum, and from all species but N. heyeri, N. lynchi, N. myrmecoides and N. peruviana by 232 lacking tubercles on eyelids. Finally, N. thiuni differs from N. peruviana by having a smaller 233 inner metatarsal tubercle, about the same size as outer metatarsal tubercle (inner tubercle large, 234 about twice the size of outer metatarsal tubercle), Toe V about the same length of Toe III (Toe V 235 shorter than Toe III). The new species is similar to species of Psychrophrynella, including the sympatric P 236 The new species is similar to species of Psychrophrynella, including the sympatric P. 237 glauca (Catenazzi Ttito 2018) and the type species P. bagrecito (Lynch, 1986), which both have PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Manuscript to be reviewed Choanae minute, round, positioned anteriorly and laterally, widely separated from 265 each other, slightly concealed by palatal shelf of maxilla; dentigerous processes of vomer and 266 vomerine teeth absent; tongue long and narrow, about 3 three times as long as wide. 267 Skin on dorsum finely shagreen, lacking tubercles; thin dorsolateral folds visible on anterior half 268 part of body; skin on flanks smooth; skin on ventral surfaces and gular regions smooth; pectoral 269 fold present, discoidal fold not evident; cloaca protuberant; cloacal region bearing several small 270 tubercles. Palmar tubercle prominent, oval, approximately 3x the size of elongate, thenar 271 tubercle; low supernumerary palmar tubercles present; subarticular tubercles prominent, ovoid in 272 ventral view, rounded in lateral view, largest at base of fingers; fingers lacking lateral fringes; 273 Finger IV has three phalanges; when adpressed, Finger 3 > 2 > 4 > 1 (Fig. 3); finger tips 274 rounded, circumferential grooves absent (Fig. 3); forearm lacking tubercles. 275 Hindlimb lengths moderate, tibia length 53% of SVL; foot length 50% of SVL; upper and 276 posterior surfaces of hindlimbs finely shagreen to pustulate; heels lacking tubercles; outer 277 surface of tarsus without tubercles; inner metatarsal tubercle, oval, about the same size of 238 a fold-like tubercle on the inner edge of tarsus, small size reaching ∼19 mm, and a prominent 239 ovoid outer metatarsal tubercle (of same or larger size than inner metatarsal tubercle). 240 Furthermore, N. thiuni shares with P. glauca the red coloration on ventral surfaces of legs and 241 venter, and the profusion of silvery or bluish-gray flecks on ventral surfaces of head, body, and 242 legs. Noblella thiuni differs from P. bagrecito in having red coloration extending to all ventral 243 surfaces including chest and throat, relative finger lengths when adpressed, Finger 3 > 2 > 4 > 1 244 (3 > 4 > 2 > 1 in P. glauca), proportionally shorter head, and presumably in having smaller size. 245 When recently preserved, N. thiuni clearly stands out from specimens of P. glauca due to reddish 246 marks turning purple on dorsum and dorsal surfaces of legs; the purple coloration fades to brown 247 after several months of preservation. Noblella thiuni can be distinguished from P. bagrecito 248 (characters in parenthesis for P. Manuscript to be reviewed 278 conical, rounded outer metatarsal tubercle; low plantar supernumerary tubercles present; 279 subarticular tubercles rounded, ovoid in dorsal view; toes with narrow lateral fringes, basal 280 webbing absent; toe tips slightly acuminate, circumferential grooves absent; digital tip of Toe V 281 smaller than tips of Toes III—IV; when adpressed, relative lengths of toes: 4 > 3 > 5 > 2 > 1 282 (Figure 5). 284 Measurements of holotype (in mm): SVL 11.0, TL 5.8, FL 5.5, HL 3.4, HW 3.5, ED 1.5, IOD 285 1.6, EW 1.1, IND 0.8, E–N 0.8. Proportions as follows: TL/SVL 0.53, FL/SVL 0.50, HL/SVL 286 0.31, HW/SVL 0.32, HW/HL 1.03, E-N/ED 0.53, EW/IOD 0.69. 285 1.6, EW 1.1, IND 0.8, E–N 0.8. Proportions as follows: TL/SVL 0.53, FL/SVL 0.50, HL/SVL 286 0.31, HW/SVL 0.32, HW/HL 1.03, E-N/ED 0.53, EW/IOD 0.69. 287 288 Coloration of holotype in alcohol. Dorsal surfaces of head and body tan with a dark brown X- 289 shaped middorsal mark and a dark interorbital bar (not bordered by cream stripe). In the first 290 weeks after preservation, the holotype had red coloration at the center of the X middorsal mark, 291 and on dorsal surfaces of all limbs (Figure 4); this red coloration faded to grayish tan after 292 several months of preservation. Dorsal surfaces of forearms and hindlimbs with transverse dark 293 bars. There are supralabial dark marks separated by transverse cream stripes. The iris is dark 294 gray. Flanks dark tan anteriorly, grayish tan posteriorly. Suprainguinal marks distinct, elongated, 295 reaching the inguinal region. Pericloacal region dark tan, contrasting from grayish tan dorsal 296 coloration anteriorly, fading to yellowish brown coloration with cream marks posterior surfaces 297 of thighs. The throat has dark brown coloration anteriorly, fading into pale grey with cream 298 flecks on posteriorly. Chest and belly grayish tan with numerous cream marks, larger in diameter 299 than flecks on throat. Ventral surface of thighs yellowish brown with cream marks and flecks; 300 ventral surfaces of lower legs and front limbs grayish with darker spots. Plantar and palmar 301 surfaces are brown, but fingers and toes cream. 302 303 Coloration of holotype in life. Manuscript to be reviewed Palmar tubercle prominent, oval, approximately 3x the size of elongate, thenar 271 tubercle; low supernumerary palmar tubercles present; subarticular tubercles prominent, ovoid in 272 ventral view, rounded in lateral view, largest at base of fingers; fingers lacking lateral fringes; 273 Finger IV has three phalanges; when adpressed, Finger 3 > 2 > 4 > 1 (Fig. 3); finger tips 274 rounded, circumferential grooves absent (Fig. 3); forearm lacking tubercles. 275 Hindlimb lengths moderate, tibia length 53% of SVL; foot length 50% of SVL; upper and 276 posterior surfaces of hindlimbs finely shagreen to pustulate; heels lacking tubercles; outer 277 surface of tarsus without tubercles; inner metatarsal tubercle, oval, about the same size of PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Manuscript to be reviewed Overall coloration in life similar to coloration in preservative, 304 except that all ventral surfaces and especially the ventral surface of hindlimbs are suffused with 305 bright red, ventral flecks and marks are silver-bluish, fingers and toes have red marks, including 306 at the tip, and dorsal regions that turned purple red the first week after preservation (around 307 middorsal X mark, and dorsal surfaces of limbs) are beige, and iris is dark tan with bronze 308 reticulations and lining. 309 310 Etymology. The name of the new species refers to the type locality and only known locality in 311 Thiuni, Department of Puno, Peru. 312 313 Distribution, natural history and threats. The cloud forest at the type locality covers a ridge 314 that separates two creeks and is accessible through a power line maintenance trail. We found N. 315 thiuni in the leaf litter along with four specimens of Psychrophrynella glauca, and other frog 316 species (Gastrotheca testudinea, Pristimantis platydactylus, and an unnamed Pristimantis sp.) in 317 the understory vegetation or within bromeliads (Catenazzi & Ttito 2018). Although much of the 288 Coloration of holotype in alcohol. Dorsal surfaces of head and body tan with a dark brown X- 289 shaped middorsal mark and a dark interorbital bar (not bordered by cream stripe). In the first 290 weeks after preservation, the holotype had red coloration at the center of the X middorsal mark, 291 and on dorsal surfaces of all limbs (Figure 4); this red coloration faded to grayish tan after 292 several months of preservation. Dorsal surfaces of forearms and hindlimbs with transverse dark 293 bars. There are supralabial dark marks separated by transverse cream stripes. The iris is dark 294 gray. Flanks dark tan anteriorly, grayish tan posteriorly. Suprainguinal marks distinct, elongated, 295 reaching the inguinal region. Pericloacal region dark tan, contrasting from grayish tan dorsal 296 coloration anteriorly, fading to yellowish brown coloration with cream marks posterior surfaces 297 of thighs. The throat has dark brown coloration anteriorly, fading into pale grey with cream 298 flecks on posteriorly. Chest and belly grayish tan with numerous cream marks, larger in diameter 299 than flecks on throat. Ventral surface of thighs yellowish brown with cream marks and flecks; 300 ventral surfaces of lower legs and front limbs grayish with darker spots. Plantar and palmar 301 surfaces are brown, but fingers and toes cream. Manuscript to be reviewed 303 Coloration of holotype in life. Overall coloration in life similar to coloration in preservative, 304 except that all ventral surfaces and especially the ventral surface of hindlimbs are suffused with 305 bright red, ventral flecks and marks are silver-bluish, fingers and toes have red marks, including 306 at the tip, and dorsal regions that turned purple red the first week after preservation (around 307 middorsal X mark, and dorsal surfaces of limbs) are beige, and iris is dark tan with bronze 308 reticulations and lining. 309 310 Etymology. The name of the new species refers to the type locality and only known locality in 311 Thiuni, Department of Puno, Peru. 312 PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Manuscript to be reviewed 318 Ollachea Valley has been deforested, some relictual forests remain on steep slopes and ridges, 319 and may support more populations of these frogs and of other unreported amphibians. In absence 320 of more details concerning the distribution and population abundance of N. thiuni, and despite 321 the known threats of deforestation, agriculture and hydropower development in the region 322 (Catenazzi & von May 2014), we suggest an IUCN Red List threat assessment category of Data 323 Deficient (IUCN 2013). We recommend to include distribution and population surveys for this 324 species, and the sympatric Psychrophrynella glauca, in any environmental assessment associated 325 with hydroelectric development, power line maintenance, road construction, and similar large 326 scale projects affecting the upper Ollachea Valley. 327 328 Discussion 329 The leaf litter of Andean cloud forests is likely to support a large undescribed diversity of 330 small terrestrial-breeding frogs. Previous studies have documented the high levels of endemism 331 and beta diversity of terrestrial-breeding frogs in moist grasslands of the eastern side of the 332 Andes in southern Peru and Bolivia (Catenazzi & Ttito 2016; De La Riva et al. 2017; Lehr & 333 Catenazzi 2010). The number of highly endemic species could be even higher in the leaf litter of 334 cloud forests, montane scrub and other habitats below the treeline (~3200–3600 m in southern 335 Peru). For example, several species of small terrestrial-breeding frogs inhabit the montane forest 336 along the gradient near Manu National Park, occupying the elevational range from the Amazon 337 lowland to the treeline at 3600 m a.s.l. (Catenazzi et al. 2014; von May et al. 2017). Our short 338 survey at the type locality of Noblella thiuni led to the discovery of another related species, 339 Psychrophrynella glauca (Catenazzi & Ttito 2018). It is likely that additional species of leaf 340 litter terrestrial-breeding frogs will be discovered from the Cordillera de Carabaya and other 341 mountain ranges in southern Peru. Discovering additional species will require targeted searches 342 in the leaf litter, for example by using leaf litter plots (Catenazzi et al. 2011; Lehr & Catenazzi 343 2009). 344 Our description is based on a singleton (following the terminology of Lim et al. 2012) 345 and thus does not include information concerning sexual dimorphism and variation in 346 morphological traits or genetic sequences. 328 Discussion 328 Discussion 329 The leaf litter of Andean cloud forests is likely to support a large undescribed diversity of 330 small terrestrial-breeding frogs. Previous studies have documented the high levels of endemism 331 and beta diversity of terrestrial-breeding frogs in moist grasslands of the eastern side of the 332 Andes in southern Peru and Bolivia (Catenazzi & Ttito 2016; De La Riva et al. 2017; Lehr & 333 Catenazzi 2010). The number of highly endemic species could be even higher in the leaf litter of 334 cloud forests, montane scrub and other habitats below the treeline (~3200–3600 m in southern 335 Peru). For example, several species of small terrestrial-breeding frogs inhabit the montane forest 336 along the gradient near Manu National Park, occupying the elevational range from the Amazon 337 lowland to the treeline at 3600 m a.s.l. (Catenazzi et al. 2014; von May et al. 2017). Our short 338 survey at the type locality of Noblella thiuni led to the discovery of another related species, 339 Psychrophrynella glauca (Catenazzi & Ttito 2018). It is likely that additional species of leaf 340 litter terrestrial-breeding frogs will be discovered from the Cordillera de Carabaya and other 341 mountain ranges in southern Peru. Discovering additional species will require targeted searches 342 in the leaf litter, for example by using leaf litter plots (Catenazzi et al. 2011; Lehr & Catenazzi 343 2009). 344 Our description is based on a singleton (following the terminology of Lim et al. 2012) 345 and thus does not include information concerning sexual dimorphism and variation in 346 morphological traits or genetic sequences. As discussed by previous authors, there is a trade-off 347 between the use of integrative taxonomy to delimit new species and enhance taxonomic stability, 348 and the need to accelerate the pace of taxonomic descriptions (de Carvalho et al. 2008; 349 Guaysamin et al. 2018; Padial et al. 2010). Guayasamin et al. (2018) propose four 350 recommendations for descriptions based on singletons or small type series. These 351 recommendations include the need for a diagnosis relying on traits that present low intraspecific 352 variation among similar genera, congruence among different data sets supporting the validity of 353 the new taxon, use of well-preserved specimens, and precise locality data of type material. 354 In the case of N. Manuscript to be reviewed As discussed by previous authors, there is a trade-off 347 between the use of integrative taxonomy to delimit new species and enhance taxonomic stability, 348 and the need to accelerate the pace of taxonomic descriptions (de Carvalho et al. 2008; 349 Guaysamin et al. 2018; Padial et al. 2010). Guayasamin et al. (2018) propose four 350 recommendations for descriptions based on singletons or small type series. These 351 recommendations include the need for a diagnosis relying on traits that present low intraspecific 352 variation among similar genera, congruence among different data sets supporting the validity of 318 Ollachea Valley has been deforested, some relictual forests remain on steep slopes and ridges, 319 and may support more populations of these frogs and of other unreported amphibians. In absence 320 of more details concerning the distribution and population abundance of N. thiuni, and despite 321 the known threats of deforestation, agriculture and hydropower development in the region 322 (Catenazzi & von May 2014), we suggest an IUCN Red List threat assessment category of Data 323 Deficient (IUCN 2013). We recommend to include distribution and population surveys for this 324 species, and the sympatric Psychrophrynella glauca, in any environmental assessment associated 325 with hydroelectric development, power line maintenance, road construction, and similar large 326 scale projects affecting the upper Ollachea Valley. 327 318 Ollachea Valley has been deforested, some relictual forests remain on steep slopes and ridges, 319 and may support more populations of these frogs and of other unreported amphibians. In absence 320 of more details concerning the distribution and population abundance of N. thiuni, and despite 321 the known threats of deforestation, agriculture and hydropower development in the region 322 (Catenazzi & von May 2014), we suggest an IUCN Red List threat assessment category of Data 323 Deficient (IUCN 2013). We recommend to include distribution and population surveys for this 324 species, and the sympatric Psychrophrynella glauca, in any environmental assessment associated 325 with hydroelectric development, power line maintenance, road construction, and similar large 326 scale projects affecting the upper Ollachea Valley. 327 Manuscript to be reviewed Furthermore, our phylogeny shows that 382 species of Noblella and Psychrophrynella from southern Peru form a distinct and not closely 383 related clade with species of Noblella from northern Peru and Ecuador (N. lochites and N. 384 myrmecoides), suggesting an alternative generic placement for these species. However, we 385 refrain from making any taxonomic decision in absence of material from the type species of 386 Noblella and Psychrophrynella. 387 358 Inca Mine in Santo Domingo”, Provincia Carabaya (the type locality was incorrectly reported in 359 the original description; see De la Riva et al. 2008). Santo Domingo is a cloud forest at 1690 m 360 asl in the upper watershed of a small tributary of the upper Inambari River, ~150 km SE of the 361 type locality of N. thiuni. Furthermore, the type locality of N. thiuni is at higher elevation (2225 362 m asl) in the Ollachea Valley, a tributary of the lower Inambari River. In light of the high 363 endemism of small strabomantid frogs in southern Peru and Bolivia, and the small elevational 364 ranges of many species of Noblella and Psychrophrynella (De la Riva et al. 2017, von May et al. 365 2017), the large geographic distance, difference in habitat types and elevations between N. thiuni 366 and N. peruviana are congruent with the hypothesis that the two species are different. 367 Furthermore, the threat of deforestation and hydroelectric development at the type locality of N. 368 thiuni further encouraged our decision to name the new taxon. The first step of any conservation 369 actions is recognizing the unicity of the organisms and their habitats. We cannot list unnamed 370 species as threatened. The presence of threatened species often triggers the need for conservation 371 actions aimed at preventing or mitigating changes in land use that endanger their populations. 372 Our study does not resolve the taxonomic uncertainty regarding the relationships among 373 species of Noblella and Psychrophrynella (Catenazzi & Ttito 2018; De La Riva et al. 2017; De la 374 Riva et al. 2008). There are no known synapomorphies for these two morphologically similar 375 genera, and there is little information beyond external morphology available for most species. 376 Molecular sequences have been useful in identifying new taxa and providing preliminary 377 hypotheses regarding the evolutionary history in this group. 328 Discussion thiuni, diagnostic traits include presence and relative size of tubercles 355 and toe lengths, which are commonly used diagnostic features in this group. These traits 356 differentiate N. thiuni from the most similar species, N. peruviana. We have precise locality data 357 concerning N. thiuni, and a description of the type locality of N. peruviana as “the vicinity of the 329 The leaf litter of Andean cloud forests is likely to support a large undescribed diversity of 330 small terrestrial-breeding frogs. Previous studies have documented the high levels of endemism 331 and beta diversity of terrestrial-breeding frogs in moist grasslands of the eastern side of the 332 Andes in southern Peru and Bolivia (Catenazzi & Ttito 2016; De La Riva et al. 2017; Lehr & 333 Catenazzi 2010). The number of highly endemic species could be even higher in the leaf litter of 334 cloud forests, montane scrub and other habitats below the treeline (~3200–3600 m in southern 335 Peru). For example, several species of small terrestrial-breeding frogs inhabit the montane forest 336 along the gradient near Manu National Park, occupying the elevational range from the Amazon 337 lowland to the treeline at 3600 m a.s.l. (Catenazzi et al. 2014; von May et al. 2017). Our short 338 survey at the type locality of Noblella thiuni led to the discovery of another related species, 339 Psychrophrynella glauca (Catenazzi & Ttito 2018). It is likely that additional species of leaf 340 litter terrestrial-breeding frogs will be discovered from the Cordillera de Carabaya and other 341 mountain ranges in southern Peru. Discovering additional species will require targeted searches 342 in the leaf litter, for example by using leaf litter plots (Catenazzi et al. 2011; Lehr & Catenazzi 343 2009). PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Manuscript to be reviewed 358 Inca Mine in Santo Domingo”, Provincia Carabaya (the type locality was incorrectly reported in 359 the original description; see De la Riva et al. 2008). Santo Domingo is a cloud forest at 1690 m 360 asl in the upper watershed of a small tributary of the upper Inambari River, ~150 km SE of the 361 type locality of N. thiuni. Furthermore, the type locality of N. thiuni is at higher elevation (2225 362 m asl) in the Ollachea Valley, a tributary of the lower Inambari River. In light of the high 363 endemism of small strabomantid frogs in southern Peru and Bolivia, and the small elevational 364 ranges of many species of Noblella and Psychrophrynella (De la Riva et al. 2017, von May et al. 365 2017), the large geographic distance, difference in habitat types and elevations between N. thiuni 366 and N. peruviana are congruent with the hypothesis that the two species are different. 367 Furthermore, the threat of deforestation and hydroelectric development at the type locality of N. 368 thiuni further encouraged our decision to name the new taxon. The first step of any conservation 369 actions is recognizing the unicity of the organisms and their habitats. We cannot list unnamed 370 species as threatened. The presence of threatened species often triggers the need for conservation 371 actions aimed at preventing or mitigating changes in land use that endanger their populations. 372 Our study does not resolve the taxonomic uncertainty regarding the relationships among 373 species of Noblella and Psychrophrynella (Catenazzi & Ttito 2018; De La Riva et al. 2017; De la 374 Riva et al. 2008). There are no known synapomorphies for these two morphologically similar 375 genera, and there is little information beyond external morphology available for most species. 376 Molecular sequences have been useful in identifying new taxa and providing preliminary 377 hypotheses regarding the evolutionary history in this group. However, the lack of molecular 378 sequences for the type species Noblella peruviana and Psychrophrynella bagrecito prevents a 379 satisfactory resolution of this taxonomic issue. The 16S phylogeny we present here suggests that 380 at least some species of Psychrophrynella may be nested within Noblella, assuming N. thiuni is 381 closely related with the type species N. peruviana. Manuscript to be reviewed 397 of small terrestrial-breeding frogs found at high elevations in the eastern slopes of the Andes of 398 southern Peru and Bolivia. 399 397 of small terrestrial-breeding frogs found at high elevations in the eastern slopes of the Andes of 398 southern Peru and Bolivia. 398 southern Peru and Bolivia. 403 References 403 References 404 AmphibiaWeb. 2019. AmphibiaWeb: Information on amphibian biology and conservation. 405 Available at http://amphibiaweb.org/ (accessed 2 January 2019. 406 Catenazzi A. 2015. State of the world's amphibians. Annual Review of Environment and 407 Resources 40:91–119. 408 Catenazzi A, Lehr E, Rodriguez LO, and Vredenburg VT. 2011. Batrachochytrium 409 dendrobatidis and the collapse of anuran species richness and abundance in the upper 410 Manu National Park, southeastern Peru. Conservation Biology 25:382–391. 411 Catenazzi A, Lehr E, and Vredenburg VT. 2014. Thermal physiology, disease and amphibian 412 declines in the eastern slopes of the Andes. Conservation Biology 28:509–517. 413 Catenazzi A, and Ttito A. 2016. A new species of Psychrophrynella (Amphibia, Anura, 414 Craugastoridae) from the humid montane forests of Cusco, eastern slopes of the Peruvian 415 Andes. Peerj 4:e1807. 416 Catenazzi A, and Ttito A. 2018. Psychrophrynella glauca sp. n., a new species of terrestrial- 417 breeding frogs (Amphibia, Anura, Strabomantidae) from the montane forests of the 418 Amazonian Andes of Puno, Peru. Peerj 6:e444. 419 Catenazzi A, Uscapi V, and von May R. 2015. A new species of Noblella from the humid 420 montane forests of Cusco, Peru. Zookeys 516:71–84. 421 Catenazzi A, and von May R. 2014. Conservation status of amphibians in Peru. Herpetological 422 Monographs 28:1–23. 423 de Carvalho MR, Bockmann FA, Amorim DS, and Brandão CRF. 2008. Systematics must 424 embrace comparative biology and evolution, not speed and automation. Evolutionary 425 Biology 35: 150–157. 426 De La Riva I, Chaparro JC, Castroviejo-Fisher S, and Padial JM. 2017. Underestimated anuran 427 radiations in the high Andes: five new species and a new genus of Holoadeninae, and 428 their phylogenetic relationships (Anura: Craugastoridae). Zoological Journal of the 429 Linnean Society 182:129–172. doi: https://doi.org/10.1093/zoolinnean/zlx1020. 430 De la Riva I, Chaparro JC, and Padial JM. 2008. The taxonomic status of Phyllonastes Heyer and 431 Phrynopus peruvianus (Noble) (Lissamphibia, Anura): resurrection of Noblella Barbour. 432 Zootaxa 1685:67–68. 433 De la Riva I, and Köhler J. 1998. A new minute leptodactylid frog, genus Phyllonastes, from 434 humid montane forests of Bolivia. Journal of Herpetology 32:325–329. 435 Duellman WE, and Lehr E. 2009. Terrestrial-breeding frogs (Strabomantidae) in Peru. Münster: 436 Natur und Tier Verlag. 437 Duellman WE, Lehr E, and Venegas PJ. 2006. Two new species of Eleutherodactylus (Anura: 438 Leptodactylidae) from the Andes of northern Peru. Zootaxa 1285:51–64. Manuscript to be reviewed However, the lack of molecular 378 sequences for the type species Noblella peruviana and Psychrophrynella bagrecito prevents a 379 satisfactory resolution of this taxonomic issue. The 16S phylogeny we present here suggests that 380 at least some species of Psychrophrynella may be nested within Noblella, assuming N. thiuni is 381 closely related with the type species N. peruviana. Furthermore, our phylogeny shows that 382 species of Noblella and Psychrophrynella from southern Peru form a distinct and not closely 383 related clade with species of Noblella from northern Peru and Ecuador (N. lochites and N. 384 myrmecoides), suggesting an alternative generic placement for these species. However, we 385 refrain from making any taxonomic decision in absence of material from the type species of 386 Noblella and Psychrophrynella. 390 We describe a new species of terrestrial-breeding frog in the genus Noblella. We justify 391 generic placement based on morphological similarity and phylogenetic analyses, but we note that 392 such placement is tentative in light of the lack of known synapomorphies distinguishing Noblella 393 from similar genera, particularly Psychrophrynella, and the absence of DNA sequence data for 394 the type species of both Noblella and Psychrophrynella. We discuss limitations for singleton 395 descriptions and adopt recent recommendations justifying our decision to proceed with a formal 396 taxonomic decision for the new species. Our work contributes to documenting the rich diversity PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) 400 Acknowledgements 400 Acknowledgements 401 We thank A. Shepack for lab assistance. Manuscript to be reviewed 441 Guayasamin JM, Arteaga A, and Hutter C. 2018. A new (singleton) rainfrog of the Pristimantis 442 myersi Group (Amphibia: Craugastoridae) from the northern Andes of Ecuador. Zootaxa 443 4527: 323–334. 444 Guayasamin JM, and Teran-Valdez A. 2009. A new species of Noblella (Amphibia: 445 Strabomantidae) from the western slopes of the Andes of Ecuador. Zootaxa:47–59. 446 Harvey MB, Almendáriz A, Brito M J, and Batallas R D. 2013. A new species of Noblella 447 (Anura: Craugastoridae) from the Amazonian slopes of the Ecuadorian Andes with 448 comments on Noblella lochites (Lynch). Zootaxa 3635:1–14. ( y ) 449 Hedges SB, Duellman WE, and Heinicke MP. 2008. New World direct-developing frogs (Anura: 450 Terrarana): molecular phylogeny, classification, biogeography, and conservation. 451 Zootaxa 1737:1–182. 452 Heinicke MP, Duellman WE, and Hedges SB. 2007. Major Caribbean and Central American 453 frog faunas originated by ancient oceanic dispersal. Proceedings of the National Academy 454 of Sciences of the United States of America 104:10092–10097. 455 Heinicke MP, Lemmon AR, Lemmon EM, McGrath K, and Hedges SB. 2017. Phylogenomic 456 support for evolutionary relationships of New World Direct-developing frogs (Anura: 457 Terraranae). Molecular Phylogenetics and Evolution doi: 458 https://doi.org/10.1016/j.ympev.2017.1009.1021. 459 IUCN. 2013. Guidelines for using the IUCN Red List categories and criteria. – Version 10.1. 460 Prepared by the Standards and Petitions Subcommittee. IUCN. 461 Katoh K, and Standley DM. 2013. MAFFT multiple sequence alignment software version 7: 462 improvements in performance and usability. Molecular Biology and Evolution 30:772– 463 780. 464 Köhler J. 2000. A new species of Phyllonastes Heyer from the Chapare region of Bolivia, with 465 notes on Phyllonastes carrascoicola. Spixiana 23:47–53. y 466 Kumar S, Stecher G, and Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis 467 version 7.0 for bigger datasets. Molecular Biology and Evolution 33:1870–1874. 468 Lehr E, and Catenazzi A. 2009. A new species of minute Noblella (Anura: Strabomantidae) from 469 southern Peru: The smallest frog of the Andes. Copeia 2009:148–156. 470 Lehr E, and Catenazzi A. 2010. Two new species of Bryophryne (Anura: Strabomantidae) from 471 high elevations in southern Peru (Region of Cusco). Herpetologica 66:308–319. 472 Lim GS, Balke M, and Meier R. 2012. Determining species boundaries in a world full of rarity: 473 singletons, species delimitation methods. Systematic Biology 61: 165–169.Lynch JD, and 474 Duellman WE. 1997. Frogs of the genus Eleutherodactylus in western Ecuador. 475 Systematics, ecology, and biogeography. 403 References 439 Fouquet A, Gilles A, Vences M, Marty C, Blanc M, and Gemmell NJ. 2007. Underestimation of 440 species richness in Neotropical frogs revealed by mtDNA analyses. PLoS One 2: e1109. PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) 485 Vences M, Thomas M, Bonett R, and Vieites D. 2005. Deciphering amphibian diversity through 486 DNA barcoding: chances and challenges. Philosophical Transactions of the Royal 487 Society of London B 360: 1859–1868. Manuscript to be reviewed The University of Kansas Special Publication 476 23:1–236. 477 Padial JM, Castroviejo-Fisher S, Köhler J, Vilà C, Chaparro JC, and De la Riva I. 2009. 478 Deciphering the products of evolution at the species level: the need for an integrative 479 taxonomy. Zoologica Scripta 38: 431–447. 477 Padial JM, Castroviejo-Fisher S, Köhler J, Vilà C, Chaparro JC, and De la Riva I. 2009. 478 Deciphering the products of evolution at the species level: the need for an integrative 479 taxonomy. Zoologica Scripta 38: 431–447. 480 Padial JM, Grant T, and Frost DR. 2014. Molecular systematics of terraranas (Anura: 481 Brachycephaloidea) with an assessment of the effects of alignment and optimality 482 criteria. Zootaxa 3825:1–132. 480 Padial JM, Grant T, and Frost DR. 2014. Molecular systematics of terraranas (Anura: 481 Brachycephaloidea) with an assessment of the effects of alignment and optimality 482 criteria. Zootaxa 3825:1–132. 483 Padial JM , Miralles A, De la Riva A, and Vences M. 2010. The integrative future of taxonomy. 484 Frontiers in Zoology 7: 16. 483 Padial JM , Miralles A, De la Riva A, and Vences M. 2010. The integrative future of taxonomy. 484 Frontiers in Zoology 7: 16. PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Manuscript to be reviewed 485 Vences M, Thomas M, Bonett R, and Vieites D. 2005. Deciphering amphibian diversity through 486 DNA barcoding: chances and challenges. Philosophical Transactions of the Royal 487 Society of London B 360: 1859–1868. 485 Vences M, Thomas M, Bonett R, and Vieites D. 2005. Deciphering amphibian diversity through 486 DNA barcoding: chances and challenges. Philosophical Transactions of the Royal 487 Society of London B 360: 1859–1868. 485 Vences M, Thomas M, Bonett R, and Vieites D. 2005. Deciphering amphibian diversity through 486 DNA barcoding: chances and challenges. Philosophical Transactions of the Royal 487 Society of London B 360: 1859–1868. 488 von May R, Catenazzi A, Corl A, Santa-Cruz R, Carnaval AC, and Moritz C. 2017. Divergence 489 of thermal physiological traits in terrestrial breeding frogs along a tropical elevational 490 gradient. Ecology and Evolution 7:3257–3267. 488 von May R, Catenazzi A, Corl A, Santa-Cruz R, Carnaval AC, and Moritz C. 2017. Divergence 489 of thermal physiological traits in terrestrial breeding frogs along a tropical elevational 490 gradient. Ecology and Evolution 7:3257–3267. 488 von May R, Catenazzi A, Corl A, Santa-Cruz R, Carnaval AC, and Moritz C. 2017. Divergence 489 of thermal physiological traits in terrestrial breeding frogs along a tropical elevational 490 gradient. Ecology and Evolution 7:3257–3267. 488 von May R, Catenazzi A, Corl A, Santa-Cruz R, Carnaval AC, and Moritz C. 2017. Divergence 489 of thermal physiological traits in terrestrial breeding frogs along a tropical elevational 490 gradient. Ecology and Evolution 7:3257–3267. PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Figure 1 Map showing type localities of species in the genus Noblella (Anura, Strabomantidae). Type localities of N. coloma, N. lochites and N. personina in Ecuador, N. duellmani, N. heyeri, N. lynchi, N. madreselva, N. myrmecoides, N. peruviana, N. pygmaea and N. thiuni sp. n. in Map showing type localities of species in the genus Noblella (Anura, Strabomantidae). Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Figure 2 Species of Noblella (Anura, Strabomantidae) from southern Peru Map showing type localities of species in the genus Noblella (Anura, Strabomantidae). Type localities of N. coloma, N. lochites and N. personina in Ecuador, N. duellmani, N. heyeri, N. lynchi, N. madreselva, N. myrmecoides, N. peruviana, N. pygmaea and N. thiuni sp. n. in Peru, and N. carrascoicola and N. ritarasquinae in Bolivia. PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Manuscript to be reviewed PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Manuscript to be reviewed Species of Noblella (Anura, Strabomantidae) from southern Peru (A) N. madreselva (unvouchered female, SVL 18.9 mm) from Madre Selva, La Convención, Cusco; (B) Noblella sp. "SP" (male AC 95.09, SVL 13.1 mm) from San Pedro, Paucartambo, Cusco; (C) N. pygmaea from Wayqecha, Paucartambo, Cusco (female holotype, MUSM 26320, SVL 12.4 mm); (D) N. thiuni sp. n. from Thiuni, Carabaya, Puno (male holotype, CORBIDI 18723, SVL 11.0 mm). Photographs by A. Catenazzi. PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Phylogenetic analysis of 16S rRNA by using Maximum Likelihood. Phylogenetic analysis of 16S rRNA by using Maximum Likelihood. Maximum likelihood optimal tree with bootrsap node values from the analysis of a dataset of 537 bp of 22 species aligned by MAFFT based on the General Time Reversible model. Initial tree(s) for the heuristic search were obtained by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate diﬀerences among sites (5 categories (+G, parameter = 0.7550)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 41.29% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Figure 4 Photographs of live and preserved specimen of the holotype of Noblella thiuni sp. n. Manuscript to be reviewed Manuscript to be reviewed Photographs of live and preserved specimen of the holotype of Noblella thiuni sp. n. Live (A, C, E) and preserved (B, D, F) holotype of Noblella thiuni sp. n., male CORBIDI 18723 (SVL 11.0 mm) in dorsolateral (A, B), dorsal (C, D) and ventral (E, F) views. Photographs by A. Catenazzi. PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Manuscript to be reviewed PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Manuscript to be reviewed Table 1(on next page) Table 1(on next page) Figure 5 Palmar and plantar surfaces of the holotype of Noblella thiuni sp. n. Palmar and plantar surfaces of the holotype of Noblella thiuni sp Ventral views of hand (A) and foot (B) of holotype, CORBIDI 18723 (hand length 2.2 mm, foot length 5.5 mm) of Noblella thiuni sp. n. Photographs by A. Catenazzi. PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) Table 1(on next page) Pairwise uncorrected p-distance for 16S rRNA between Noblella thiuni sp. n. and related taxa in the subfamily Holadeninae. Lowest genetic distances for N. thiuni are in bold. Specimen codes are listed in Appendix 2. PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019) 1 Barycholos ternetzi Bryophryne bakersfield Bryophryne cophites Bryophryne hanssaueri Bryophryne nubilosus Bryophryne phuyuhampatu Holoaden luederwaldti Microkayla chilina Microkayla iatamasi Microkayla katantika Noblella lochites Noblella madreselva Noblella myrmecoides Noblella pygmaea Noblella spR Noblella spSP Noblella thiuni P. chirihampatu P. glauca P.a spP P. spR P. usurpator Barycholos ternetzi Bryophryne bakersfield 0.19 Bryophryne cophites 0.19 0.02 Bryophryne hanssaueri 0.21 0.04 0.03 Bryophryne nubilosus 0.21 0.04 0.05 0.03 Bryophryne phuyuhampatu 0.20 0.03 0.04 0.04 0.04 Holoaden luederwaldti 0.18 0.16 0.16 0.16 0.16 0.16 Microkayla chilina 0.19 0.14 0.14 0.15 0.15 0.13 0.14 Microkayla iatamasi 0.19 0.13 0.13 0.15 0.15 0.13 0.15 0.03 Microkayla katantika 0.19 0.15 0.15 0.16 0.15 0.14 0.15 0.05 0.04 Noblella lochites 0.16 0.19 0.19 0.19 0.20 0.18 0.13 0.18 0.18 0.17 Noblella madreselva 0.17 0.18 0.16 0.17 0.18 0.18 0.15 0.14 0.13 0.14 0.17 Noblella myrmecoides 0.18 0.21 0.21 0.23 0.22 0.21 0.18 0.20 0.21 0.19 0.11 0.21 Noblella pygmaea 0.18 0.17 0.15 0.16 0.17 0.16 0.14 0.13 0.14 0.14 0.17 0.08 0.19 Noblella spR 0.20 0.17 0.16 0.16 0.18 0.18 0.14 0.15 0.15 0.15 0.17 0.09 0.19 0.07 Noblella spSP 0.18 0.19 0.17 0.18 0.19 0.19 0.16 0.15 0.15 0.15 0.16 0.09 0.20 0.08 0.10 Noblella thiuni sp. n. 0.16 0.15 0.15 0.14 0.15 0.15 0.15 0.12 0.12 0.14 0.18 0.08 0.18 0.07 0.09 0.10 P. chirihampatu 0.16 0.18 0.17 0.18 0.19 0.18 0.17 0.15 0.14 0.13 0.17 0.05 0.19 0.08 0.11 0.10 0.09 P. glauca 0.17 0.18 0.17 0.18 0.19 0.18 0.18 0.14 0.13 0.13 0.18 0.09 0.20 0.10 0.13 0.12 0.09 0.09 P. spP 0.19 0.19 0.17 0.18 0.19 0.19 0.16 0.16 0.16 0.15 0.19 0.10 0.19 0.09 0.10 0.11 0.09 0.10 0.09 P. spR 0.17 0.20 0.19 0.18 0.18 0.18 0.19 0.17 0.16 0.16 0.20 0.10 0.20 0.12 0.15 0.14 0.11 0.11 0.06 0.11 P. Table 1(on next page) usurpator 0.17 0.19 0.18 0.17 0.18 0.19 0.15 0.16 0.15 0.14 0.17 0.04 0.22 0.09 0.11 0.10 0.09 0.05 0.09 0.10 0.10 Manuscript to be reviewed Barycholos ternetzi Bryophryne bakersfield Bryophryne cophites Bryophryne hanssaueri Bryophryne nubilosus Bryophryne phuyuhampatu Holoaden luederwaldti Microkayla chilina Microkayla iatamasi Microkayla katantika Noblella lochites Noblella madreselva Noblella myrmecoides Noblella pygmaea Noblella spR Noblella spSP Noblella thiuni P. chirihampatu P. glauca P.a spP P. spR P. usurpator t i Manuscript to be reviewed Barycholos ternetzi Bryophryne bakersfield Bryophryne cophites Bryophryne hanssaueri Bryophryne nubilosus Bryophryne phuyuhampatu Holoaden luederwaldti Microkayla chilina Microkayla iatamasi Microkayla katantika Noblella lochites Noblella madreselva Noblella myrmecoides Noblella pygmaea Noblella spR Noblella spSP Noblella thiuni P. chirihampatu P. glauca P.a spP P. spR P. usurpator Manuscript to be reviewed 1 PeerJ reviewing PDF \| (2018:10:32099:2:0:NEW 7 Mar 2019)
https://openalex.org/W4362557799	https://discovery.ucl.ac.uk/id/eprint/10167956/1/5.0147291.pdf	English	null	Two-photon electromagnetic induction imaging with an atomic magnetometer	Applied physics letters	2,023	cc-by	4,927	COLLECTIONS This paper was selected as Featured ARTICLES YOU MAY BE INTERESTED IN Perspectives on high-frequency nanomechanics, nanoacoustics, and nanophononics Applied Physics Letters 122, 140501 (2023); https://doi.org/10.1063/5.0142925 K-space interpretation of image-scanning-microscopy Applied Physics Letters 122, 141106 (2023); https://doi.org/10.1063/5.0142000 A vortex-induction underwater energy harvester based on Pb(In1/2Nb1/2)O3– Pb(Mg1/3Nb2/3)O3–PbTiO3 single crystal macro-fiber composites Applied Physics Letters 122, 143903 (2023); https://doi.org/10.1063/5.0144110 Appl. Phys. Lett. 122, 144001 (2023); https://doi.org/10.1063/5.0147291 122, 144001 © 2023 Author(s). Two-photon electromagnetic induction imaging with an atomic magnetometer Benjamin Maddox and Ferruccio Renzoni ABSTRACT Electromagnetic induction imaging (EMI) is a contactless, nondestructive evaluation technique based on sensing the response of a target to oscillating magnetic ﬁelds as they penetrate into materials. Leveraging the enhanced performance of radio frequency atomic magnetometers (RF-AMs) at low frequencies can enable highly sensitive through-barrier EMI measurements, which, for example, can reveal concealed weaponry or inspect subsurface material defects. However, deriving this advantage requires precise control of a well-deﬁned, low bias mag- netic ﬁeld with respect to the background magnetic ﬁeld texture, which presents a cumbersome challenge to stabilize in real-world unshielded scenarios. Here, we implement a two-photon RF-AM scheme in a portable setup to bypass the requirement of a low bias ﬁeld and achieve stable, repeatable resonances in the sub-kHz regime. The improved accessibility to lower primary ﬁeld frequencies offer greater skin-depth in target materials and facilitates an enhancement of a factor of 8 in skin penetration with this portable system, detecting features behind an Al shield of 3.2 mm. The scheme also reduces the need of large compensation coils to stabilize the bias ﬁeld, facilitating the implementation of compact devices. V C 2023 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http:// creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0147291 Electromagnetic induction imaging (EMI) enables nondestructive and noninvasive tomographic conductivity imaging of targets over a wide range of conductivities.1 Exciting a target with a primary oscillat- ing magnetic ﬁeld produces eddy currents in the material, which, in turn, generate secondary oscillating ﬁelds that can be sensed with a probing magnetometer. The skin-effect constrains the maximum pen- etration achievable by the primary ﬁeld, therefore enabling a tomo- graphic image to be formed by varying the primary frequency. Pushing the technique to super-mm penetration depths in conductive targets requires frequencies in the sub-kHz regime, a requirement that makes the choice of the sensor critical for EMI performance. While the signal-to-noise ratio (SNR) of the induction coil weakens with lower frequencies, the fundamental sensitivity of radio frequency atomic magnetometers (RF-AMs) remains ﬂat and presents the opportunity to gain over three orders of magnitude advantage in RF sensitivity at 1 kHz.2 Interest in exploiting this advantage has been increasing, with multiple groups now advancing this technique.3–8 This enhancement comes at the cost of robustness, as RF-AMs demand a well-deﬁned static magnetic ﬁeld, typically called the bias ﬁeld, to produce a stable resonance. a)Author to whom correspondence should be addressed: f.renzoni@ucl.ac.uk a)Author to whom correspondence should be addressed: f.renzoni@ucl.ac.uk Appl. Phys. Lett. 122, 144001 (2023); https://doi.org/10.1063/5.0147291 © 2023 Author(s). 122, 144001 © 2023 Author(s). Applied Physics Letters ARTICLE scitation.org/journal/apl Two-photon electromagnetic induction imaging with an atomic magnetometer Two-photon electromagnetic induction imaging with an atomic magnetometer Cite as: Appl. Phys. Lett. 122, 144001 (2023); doi: 10.1063/5.0147291 Submitted: 21 February 2023 . Accepted: 19 March 2023 . Published Online: 4 April 2023 AFFILIATIONS Department of Physics and Astronomy, University College London, Gower Street, London WC1E 6BT, United Kingdom a)Author to whom correspondence should be addressed: f.renzoni@ucl.ac.uk Department of Physics and Astronomy, University College London, Gower Street, London WC1E 6BT, Un ABSTRACT as no static ﬁeld is applied in the trans- verse direction, the noise on the total ﬁeld seen by the atoms BTOT becomes more and more dominated by transverse ﬁeld noise as BBias reduces. Further complications arise when considering the direction of BTOT with respect to the pump beam propagation. As transverse ﬁeld noise increase, so too does the angular noise, which the atoms experience as a polarization noise on the pump beam, diminishing the atomic polar- ization, which translates to amplitude noise. Active compensation can accommodate for the zeroth- and ﬁrst-order magnetic ﬁeld gradients where d represents the skin depth, which at low frequencies becomes dðxRFÞ ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2 xRFrl r ; (2) (2) where l, e, and r are the magnetic permeability, electrical permittivity, and the electrical conductivity of the target material, respectively. Equation (2) shows the nonlinear payoff in penetration depth by reducing the primary ﬁeld frequency and illustrates the requirement of low-frequency operation for penetration through barriers. Figure 1(a) illustrates the working principle in the conven- tional RF-AM: a vapor of 87Rb is illuminated by a circularly polar- ized pump beam propagating parallel to a bias magnetic ﬁeld BBias in the ^x direction, transferring most of the atomic population into the stretched state of the upper hyperﬁne groundstate. An RF ﬁeld BRF, perpendicular to BBias and resonant with the Zeeman splitting of the magnetic sublevels, creates an oscillating population transfer in the hyperﬁne manifold. This then manifests as an oscillating refractive index to a detuned probe beam propagating perpendicu- larly to BBias and appears as an oscillating polarization rotation when read-off by a polarimeter. BBias generates the groundstate magnetic sublevel splitting via the Zeeman effect and sets the reso- nant frequency x0 as FIG. 2. Surface (a) of the two-photon RF-AM amplitude as a function of BRF and Bc along with cross sections (b) and (c) of the individual parameters. Here, xRF ¼ 2p 3 kHz; xc ¼ 2p 47 kHz, and the bias ﬁeld was set to produce a resonance at x0 ¼ 2p 50 kHz. White dashed lines in (a) show the positions of the cross sections for (b) and (c). x0 ¼ cBBias; (3) (3) x0 ¼ cBBias; FIG. 1. Geometry of the one-photon (a) and two-photon (b) setup along with a reduced level diagram showing adjacent magnetic sublevels in the groundstate and the coupling mechanism in each case. ABSTRACT To produce a resonance in the sub-kHz regime requires BBias < 1:43 mG, which becomes comparable to the order of the earth’s magnetic ﬁeld temporal/spatial ﬂuctuations. Conventionally, this problem is circumvented by attenu- ating the background ﬁeld with magnetic shielding, but many applica- tions require RF-AMs to work in real-world environments where shielding is impossible to achieve or unwanted. Active magnetic ﬁeld cancellation has proved effective at stabilizing the bias ﬁeld in many applications in real-world environments.9–11 However, this method ﬁghts a losing battle to the background ﬁeld as low-frequency opera- tion requires a reduced bias ﬁeld, which increases the inﬂuence of transverse ﬁeld noise and gradient noise, with resulting decrease in sensitivity.12 To combat this, in this work, we drive the RF-AM with a two-photon RF transition.13 As in the proposed system, the required bias ﬁeld strength is determined by the sum of the frequency of the two photons, for a convenient choice of very unequal frequencies of the two photons it is possible to operate the magnetometer at ultra- low frequency while maintaining a large enough bias ﬁeld to guarantee stable operation and high sensitivity in an unshielded environment. The approach affords us the stability to perform ultra-low frequency EMI, and as a result, achieve deeper through-barrier penetration. The requirement of low-frequency operation of EMI systems to achieve penetration through a barrier, as required in security and Appl. Phys. Lett. 122, 144001 (2023); doi: 10.1063/5.0147291 V C Author(s) 2023 122, 144001-1 122, 144001-1 Appl. Phys. Lett. 122, 144001 (2023); doi: 10.1063/5.0147291 Applied Physics Letters ARTICLE scitation.org/journal/apl where c is the gyromagnetic ratio of the valence electron of the probed atom. Since BBias and any spurious transverse magnetic ﬁeld B? sum in quadrature12 surveillance applications, derives from the dependence of the penetra- tion depth of an oscillating magnetic ﬁeld through a material on the ﬁeld frequency. An oscillating magnetic ﬁeld decays evanescently through a target material depth z according to Ref. 14, surveillance applications, derives from the dependence of the penetra- tion depth of an oscillating magnetic ﬁeld through a material on the ﬁeld frequency. An oscillating magnetic ﬁeld decays evanescently through a target material depth z according to Ref. 14, dBTOT ¼ dB2 ? BBias ; (4) (4) BRFðzÞ ¼ BRFð0Þez=dðxRFÞ; (1) (1) epresents the skin depth, which at low frequencies becomes where we assumed B? ’ dB? ABSTRACT 122, 144001 (2023); doi: 10.1063/5.0147291 V C Author(s) 2023 122, 144001-2 Applied Physics Letters scitation.org/journal/apl ARTICLE with Helmholtz and anti-Helmholtz coils, respectively, but the nonlinear texture of the background prevails and is challenging to deal with. and magnetic systems required for an RF-AM are enclosed within a nylon sensor head of dimensions 110 110 145 mm3 (W L H) and of weight 1.49kg. To reduce wall relaxation and increase atomic interaction time, the cubic vapor cell (25mm side length) is ﬁlled with 20Torr of N2 as a buffer gas and held at room temperature. Laser light for pump and probe beams is provided by two VCSEL (vertical-cavity surface-emitting laser) diodes, expanded by lenses to diameters of 10mm. BBias is generated and stabilized by active magnetic stabilization via three pairs of orthogonally orientated square Helmholtz coils, cen- tered on the vapor cell. An additional anti-Helmholtz coil is aligned along ^x to compensate for linear gradients in the bias ﬁeld. Internal ﬂuxgate sensors are used to measure the DC magnetic ﬁelds (band- width 100Hz) in all three axes and are fed to proportional-integral- derivative (PID) controllers to adjust the compensation coil currents. Internal RF coils provide BRF and Bc. An external lock-in ampliﬁer (LIA) demodulates the polarimeter at x=2p and outputs the amplitude R of the magnetic resonance. The resonance is swept with the RF ﬁelds and a linewidth (HWHM) of the resonance C ¼ 2p 680 Hz is typi- cal for the system. The portable RF-AM is raster-scanned across an object using a 2D mechanical translational stage and builds up a con- ductivity image of static targets, which has been able to detect features masked by conductive shields16 and simulated corrosion under insulation.17 In the two-photon scheme shown in Fig. 1(b), two RF photons with different polarizations are used. The scheme relies on the fact that p-polarized RF photons drive Dm ¼ 0 transitions and r6 photons drive Dm ¼ 61 transitions. In the proposed scheme, an additional RF ﬁeld Bc, propagating along BBias, facilitates a two-photon transition at xRF ¼ x0 xc. When added to the r polarized BRF photon, the p polarization of the Bc photon allows the selection rule of Dm ¼ 61 to be satisﬁed and, therefore, couple adjacent magnetic sublevels. Tuning xc effectively shifts the RF-AM resonance with respect to xRF, without the need to alter BBias. ABSTRACT The magenta and red arrows show the pump and probe beam direction as they traverse through the atomic medium (silver spheres), while the copper arrows indicate the light polarization. FIG. 2. Surface (a) of the two-photon RF-AM amplitude as a function of BRF and Bc along with cross sections (b) and (c) of the individual parameters. Here, xRF ¼ 2p 3 kHz; xc ¼ 2p 47 kHz, and the bias ﬁeld was set to produce a resonance at x0 ¼ 2p 50 kHz. White dashed lines in (a) show the positions of the cross sections for (b) and (c). FIG. 2. Surface (a) of the two-photon RF-AM amplitude as a function of BRF and Bc along with cross sections (b) and (c) of the individual parameters. Here, xRF ¼ 2p 3 kHz; xc ¼ 2p 47 kHz, and the bias ﬁeld was set to produce a resonance at x0 ¼ 2p 50 kHz. White dashed lines in (a) show the positions of the cross sections for (b) and (c). FIG. 2. Surface (a) of the two-photon RF-AM amplitude as a function of BRF and Bc along with cross sections (b) and (c) of the individual parameters. Here, xRF ¼ 2p 3 kHz; xc ¼ 2p 47 kHz, and the bias ﬁeld was set to produce a resonance at x0 ¼ 2p 50 kHz. White dashed lines in (a) show the positions of the cross sections for (b) and (c). FIG. 1. Geometry of the one-photon (a) and two-photon (b) setup along with a reduced level diagram showing adjacent magnetic sublevels in the groundstate and the coupling mechanism in each case. The magenta and red arrows show the pump and probe beam direction as they traverse through the atomic medium (silver spheres), while the copper arrows indicate the light polarization. FIG. 1. Geometry of the one-photon (a) and two-photon (b) setup along with a reduced level diagram showing adjacent magnetic sublevels in the groundstate and the coupling mechanism in each case. The magenta and red arrows show the pump and probe beam direction as they traverse through the atomic medium (silver spheres), while the copper arrows indicate the light polarization. Appl. Phys. Lett. ABSTRACT The Rabi frequency of the two-photon transi- tion is given by Geng et al.,13 X ¼ XRFXc 4xc ; (5) (5) which allows the same linearity in XRF as is the case for the standard RF-AM conﬁguration except with an additional factor of Xc=4xc. EMI measurements are generally based on the ratio of the primary BRF and secondary ﬁeld dBRF,1 dBRF BRF / xRFðxRFe0er irÞ; (6) (6) For the two-photon scheme, two identical RF coils (6 mm diame- ter and 10 mm length) are driven by waveform generators, at their respective frequencies of xRF and xc. The BRF coil is positioned under the target 50 mm above the vapor cell in the z axis, while the Bc RF coil is aligned along the x axis and also displaced by 50 mm. The and since XRF / BRF, we require only the linearity of the two-photon RF-AM amplitude on XRF for EMI measurements. The portable RF-AM setup has been described in detail previ- ously.15 Brieﬂy, an isotopically enriched 87Rb vapor cell and the optical FIG. 3. Magnetic resonances for the one-photon (a)–(c) and two-photon scheme (d-f) as the desired resonant primary ﬁeld frequency xRF is lowered. Column titles show the nominal xRF=2p that is desired from the system. The LIA output R1;2 for the one- and two-photon case, respectively, is normalized to the peak measured value at the largest xRF considered. For the one-photon scheme, the bias ﬁeld is reduced to set x0 at the desired xRF, whereas for the two-photon scheme, x0 is ﬁxed and the coupling ﬁeld xc is set such that x0 xc gives the desired xRF. Crosses show the raw data while lines show smoothed averages of the data to guide the eyes. The vertical black dashed line in (a) and (d) represents xRF=2p ¼ 1 kHz. FIG. 3. Magnetic resonances for the one-photon (a)–(c) and two-photon scheme (d-f) as the desired resonant primary ﬁeld frequency xRF is lowered. Column titles show the nominal xRF=2p that is desired from the system. The LIA output R1;2 for the one- and two-photon case, respectively, is normalized to the peak measured value at the largest xRF considered. For the one-photon scheme, the bias ﬁeld is reduced to set x0 at the desired xRF, whereas for the two-photon scheme, x0 is ﬁxed and the coupling ﬁeld xc is set such that x0 xc gives the desired xRF. ABSTRACT Crosses show the raw data while lines show smoothed averages of the data to guide the eyes. The vertical black dashed line in (a) and (d) represents xRF=2p ¼ 1 kHz. ppl. Phys. Lett. 122, 144001 (2023); doi: 10.1063/5.0147291 122, 144001-3 A th ( ) 2023 Appl. Phys. Lett. 122, 144001 (2023); doi: 10.1063/5.0147291 V C Author(s) 2023 122, 144001-3 Applied Physics Letters scitation.org/journal/apl ARTICLE FIG. 4. SNR of the two schemes when matched in Rabi frequency. Crosses show raw data points while lines show a smoothed average to guide the eyes. The black vertical dashed line shows the minimum achievable one-photon resonant frequency of x0=2p ¼ 1:6 kHz, while the dashed magenta line shows the linewidth C=2p ¼ 680 Hz. Figure 3 demonstrates the tunability of the two-photon RF-AM conﬁguration compared to the standard one-photon conﬁguration. For the standard conﬁguration, the bias ﬁeld is reduced to give the res- onance at lower frequencies; however, it becomes impossible to attain a resonance at xRF=2p ¼ 1 kHz despite transverse nulling and linear gradient compensation coils, with the minimum achievable resonance being x0=2p ¼ 1:6 kHz. It is at this point that the bias ﬁeld is compa- rable with the background and the ﬁeld over the cell is dominated by the texture of the background ﬁeld and since the RF-AM measurement will be an average of the bias ﬁeld felt by all the atoms in the pump- probe intersection volume, some non-zero ﬁeld appears due to nonlin- ear background gradients. The two-photon conﬁguration has no such limitation, and it easily fulﬁlls the condition of xRF=2p ¼ 1 kHz just by adjusting xc. The simplicity in tuning the two-photon resonance effectively bypasses the need to adjust transverse ﬁeld/gradient ﬁeld setpoints that would normally be required in bias ﬁeld tuning.18 Reinforcing this is an analysis of the SNR of the two conﬁgura- tions in Fig. 4. With their Rabi coupling matched between the two schemes, both are swept to low frequencies and the SNR is calculated from fast Fourier transform (FFT) traces with BRF on and off at each frequency. Again for the one-photon case, the bias ﬁeld is adjusted to get maximum signal at each point, whereas for two-photon case, x0 stays static and xc is adjusted. ABSTRACT At high frequencies, the SNR is compa- rable between the two schemes, but as the frequency is lowered below the minimum resonance set by the bias ﬁeld stabilization x0=2p ¼ 1:6 kHz (black dashed line) the SNR drops by an order of magni- tude. The two-photon case, however, maintains the SNR through the sub-kHz regime until dropping below 300 Hz. The ultimate decline of the two-photon case is due to xRF approaching the linewidth C of the magnetic resonance, which puts xc in the vicinity of the standard one- photon resonance. Despite the RF coil that produces Bc being aligned FIG. 4. SNR of the two schemes when matched in Rabi frequency. Crosses show raw data points while lines show a smoothed average to guide the eyes. The black vertical dashed line shows the minimum achievable one-photon resonant frequency of x0=2p ¼ 1:6 kHz, while the dashed magenta line shows the linewidth C=2p ¼ 680 Hz. polarimeter signal is then demodulated at x0 by a lock-in ampliﬁer via an internal reference set at x0. Figure 2(a) shows a surface of the two-photon RF-AM amplitude as a function of BRF and Bc, with cross sections in Fig. 2(b) and 2(c). We, thus, veriﬁed the line- arity of the amplitude of the RF-AM on BRF, as predicted by Eq. (5). Such a linearity is required for the two-photon EMI approach introduced in this work. FIG. 5. Two-photon EMI of the double-hole target concealed by 2 mm thick Al skin, at three different values of xRF=2p: (a) 20 kHz, (b) 1 kHz, and (c) 500 Hz along with respective averages through the horizontal axis in (d)–(f). The bias ﬁeld is set to give a resonance at x0=2p ¼ 53 kHz and xc is swept at each pixel to fulﬁll the two-photon resonance condition. Amplitudes for each image are normalized to the corner pixel such that contrast can be compared between the panels. For each pixel, the portable RF- AM is moved by a 2 mm step in the x–y plane and a Gaussian ﬁlter with a width of 1 pixel is used to smooth the image. FIG. 5. Two-photon EMI of the double-hole target concealed by 2 mm thick Al skin, at three different values of xRF=2p: (a) 20 kHz, (b) 1 kHz, and (c) 500 Hz along with respective averages through the horizontal axis in (d)–(f). ABSTRACT The bias ﬁeld is set to give a resonance at x0=2p ¼ 53 kHz and xc is swept at each pixel to fulﬁll the two-photon resonance condition. Amplitudes for each image are normalized to the corner pixel such that contrast can be compared between the panels. For each pixel, the portable RF- AM is moved by a 2 mm step in the x–y plane and a Gaussian ﬁlter with a width of 1 pixel is used to smooth the image. Appl. Phys. Lett. 122, 144001 (2023); doi: 10.1063/5.0147291 V C Author(s) 2023 122, 144001-4 Applied Physics Letters scitation.org/journal/apl ARTICLE measurement to evade the ﬂicker noise (1=f ) of the detection electron- ics, by effectively mixing-up the low frequency xRF to the detected higher frequency x0, within the atomic medium. RF-AM systems that have tighter linewidths will be able to push lower in frequency as one- photon excitation by the coupling ﬁeld will be attenuated and a few unshielded RF-AMs have already demonstrated linewidths on the order of tens of Hz.7,19 along the RF-AM deadzone, which should attenuate any one-photon driving by the coupling ﬁeld,19 the RF coil will have some small trans- verse components, which will couple and inevitably begin to competi- tively interfere with the two-photon process when on resonance. Figure 5 shows a demonstration of through-barrier EMI imaging with the two-photon RF-AM. An Al sheet of thickness dskin ¼ 2 mm conceals a block of Al with two 16 mm diameter holes spaced 36.7 mm apart. The RF-AM is aligned underneath (see Fig. 1) at a standoff of 0:5 mm. For each pixel, the portable RF-AM is mechanically actu- ated in the x–y plane, xRF is held ﬁxed and xc is swept through the two-photon resonance to determine the maximum amplitude. For imaging, we desire only the effect of BRF on the target material, and therefore, it is important to note the geometry of the two RF coils. The Bc coil is not only separated from the target by much larger distance than the BRF coil (greater than 100 times the standoff) but it is also orthogonally aligned to the surface, which is enough to neglect any contribution from the coupling ﬁeld in the EMI measurements. At 20 kHz, BRF fails to penetrate the shielding as dAl < dskin. REFERENCES We have demonstrated the effectiveness of a two-photon excita- tion scheme in RF-AMs for increasing the efﬁcacy of through-barrier EMI detection of holes in Al that are obscured by super-mm thick Al skins. The technique effectively removes the bottleneck of extremely precise and homogeneous magnetic ﬁeld compensation in unshielded environments, which sets limits on miniaturization and presents a demanding engineering challenge. Furthermore, looking at Eq. (4), applying a very high bias ﬁeld could render the transverse ﬁeld noise negligible and remove the need for any transverse compensation at all, allowing for a reduction in complexity. As the technique is pushed to lower and lower operating frequencies, the method allows the 1H. Grifﬁths, “Magnetic induction tomography,” Meas. Sci. Technol. 12, 1126 (2001). 1H. Grifﬁths, “Magnetic induction tomography,” Meas. Sci. Technol. 12, 1126 (2001). 2I. Savukov, S. Seltzer, and M. Romalis, “Detection of NMR signals with a radio-frequency atomic magnetometer,” J. Magn. Resonance 185, 214–220 (2007). ( ) 3C. Deans, L. Marmugi, S. Hussain, and F. Renzoni, “Electromagnetic induction imaging with a radio-frequency atomic magnetometer,” Appl. Phys. Lett. 108, 103503 (2016). 4A. Wickenbrock, N. Leefer, J. W. Blanchard, and D. Budker, “Eddy current imaging with an atomic radio-frequency magnetometer,” Appl. Phys. Lett. 108, 183507 (2016). ( ) 5P. Bevington, L. Wright, R. Gartman, and W. Chalupczak, “Role of the primary radio frequency magnetic ﬁeld distribution in atomic magnetometer based inductive measurements,” J. Appl. Phys. 131, 164502 (2022). 6 FIG. 6. Two-photon EMI imaging of the double-hole target concealed by a 3.2 mm thick Al skin. Here, xRF=2p ¼ 410 Hz with the resonant frequency set at x0=2p ¼ 53 kHz. For each pixel, the portable RF-AM is moved by a 2 mm step in the x–y plane and a Gaussian ﬁlter with a width of 1 pixel is used to smooth the image. 6P. Bevington, R. Gartman, and W. Chalupczak, “Magnetic induction tomogra- phy of structural defects with alkali–metal spin maser,” Appl. Opt. 59, 2276–2282 (2020). 7L. Rushton, T. Pyragius, A. Meraki, L. Elson, and K. Jensen, “Unshielded porta- ble optically pumped magnetometer for the remote detection of conductive objects using eddy current measurements,” arXiv:2206.04631 (2022). 8 8W. Zheng, H. Wang, R. Schmieg, A. Oesterle, and E. S. Polzik, “Entanglement- enhanced magnetic induction tomography,” arXiv:2209.01920v2 (2022). 9 9J. E. Dhombridge, N. R. Claussen, J. Iivanainen, and P. D. AUTHOR DECLARATIONS Conflict of Interest The authors have no conﬂicts to disclose. Author Contributions Benjamin Maddox: Conceptualization (equal); Data curation (lead); Investigation (equal); Methodology (lead); Writing – original draft (lead). Ferruccio Renzoni: Conceptualization (equal); Funding acqui- sition (lead); Investigation (equal); Project administration (lead); Writing – review & editing (equal). DATA AVAILABILITY The data that support the ﬁndings of this study are available from the corresponding author upon reasonable request. The data that support the ﬁndings of this study are available from the corresponding author upon reasonable request. ABSTRACT When dAl dskin the structure of the holes through the skin appears, which matches our expectation from previous studies.16,20–22 Due to eddy current excitation on the plane inside the hole, a portion of the second- ary ﬁeld becomes spatially transverse to the primary ﬁeld and leads to a dispersive-like shape on the amplitude of the RF-AM. As dAl > dskin, the structure becomes clearer and conﬁrms that the two-photon RF- AM can perform feasible EMI measurements. Finally, in Fig. 6, we show the thickest Al skin that can be imaged with two-photon EMI for this particular setup. The features of the double-hole target are clearly revealed from behind an Al skin of 3.2mm thickness, which improves upon our previous study with this system16 by a factor of 8. This work was funded by EPSRC Impact Acceleration Account (Grant No. EP/R511638/1). REFERENCES Schwindt, “High- sensitivity rf detection using an optically pumped comagnetometer based on natural-abundance rubidium with active ambient-ﬁeld cancellation,” Phys. Rev. Appl. 18, 044052 (2022). pp 10C. Deans, L. Marmugi, and F. Renzoni, “Sub-picotesla widely tunable atomic magnetometer operating at room-temperature in unshielded environments,” Rev. Sci. Instrum. 89, 083111 (2018). 11 FIG. 6. Two-photon EMI imaging of the double-hole target concealed by a 3.2 mm thick Al skin. Here, xRF=2p ¼ 410 Hz with the resonant frequency set at x0=2p ¼ 53 kHz. For each pixel, the portable RF-AM is moved by a 2 mm step in the x–y plane and a Gaussian ﬁlter with a width of 1 pixel is used to smooth the image. FIG. 6. Two-photon EMI imaging of the double-hole target concealed by a 3.2 mm thick Al skin. Here, xRF=2p ¼ 410 Hz with the resonant frequency set at x0=2p ¼ 53 kHz. For each pixel, the portable RF-AM is moved by a 2 mm step in the x–y plane and a Gaussian ﬁlter with a width of 1 pixel is used to smooth the image. 11H. Yao, B. Maddox, and F. Renzoni, “High-sensitivity operation of an unshielded single cell radio-frequency atomic magnetometer,” Opt. Express 30, 42015–42025 (2022). Appl. Phys. Lett. 122, 144001 (2023); doi: 10.1063/5.0147291 V C Author(s) 2023 122, 144001-5 122, 144001-5 Applied Physics Letters ARTICLE scitation.org/journal/apl 12R. Zhang, Y. Ding, Y. Yang, Z. Zheng, J. Chen, X. Peng, T. Wu, and H. Guo, Workshop on Metrology for Industry 4.0 & IoT (MetroInd4. 0&IoT) (IEEE, 2022), pp. 104–108. 12R. Zhang, Y. Ding, Y. Yang, Z. Zheng, J. Chen, X. Peng, T. Wu, and H. Guo, “Active magnetic-ﬁeld stabilization with atomic magnetometer,” Sensors 20, 4241 (2020). Workshop on Metrology for Industry 4.0 & IoT (MetroInd4. 0&IoT) (IEEE, 2022), pp. 104–108. “Active magnetic-ﬁeld stabilization with atomic magnetometer,” Sensors 20, 4241 (2020). pp 18P. Bevington, R. Gartman, and W. Chalupczak, “Inductive imaging of the con- cealed defects with radio-frequency atomic magnetometers,” Appl. Sci. 10, 6871 (2020). 13X. Geng, G. Yang, P. Qi, W. Tang, S. Liang, G. Li, and G. Huang, “Laser- detected magnetic resonance induced by radio-frequency two-photon proc- esses,” Phys. Rev. A 103, 053112 (2021). 19P. Bevington, R. Gartman, D. Botelho, R. Crawford, M. Packer, T. Fromhold, and W. Chalupczak, “Object surveillance with radio-frequency atomic magne- tometers,” Rev. Sci. Instrum. 91, 055002 (2020). y 14P. A. Bottomley and E. R. REFERENCES Andrew, “RF magnetic ﬁeld penetration, phase shift and power dissipation in biological tissue: Implications for NMR imaging,” Phys. Med. Biol. 23, 630 (1978). 20P. Bevington, R. Gartman, and W. Chalupczak, “Enhanced material defect imaging with a radio-frequency atomic magnetometer,” J. Appl. Phys. 125, 094503 (2019). y 15C. Deans, Y. Cohen, H. Yao, B. Maddox, A. Vigilante, and F. Renzoni, “Electromagnetic induction imaging with a scanning radio frequency atomic magnetometer,” Appl. Phys. Lett. 119, 014001 (2021). 16 21J. D. Zipfel, S. Santosh, P. Bevington, and W. Chalupczak, “Object composition identiﬁcation by measurement of local radio frequency magnetic ﬁelds with an atomic magnetometer,” Appl. Sci. 12, 8219 (2022). magnetometer,” Appl. Phys. Lett. 119, 014001 (2021). 16 16B. Maddox, Y. Cohen, and F. Renzoni, “Through-skin pilot-hole detection and localization with a mechanically translatable atomic magnetometer,” Appl. Phys. Lett. 120, 014002 (2022). 22P. Bevington, R. Gartman, and W. Chalupczak, “Imaging of material defects with a radio-frequency atomic magnetometer,” Rev. Sci. Instrum. 90, 013103 (2019). y 17B. Maddox, Y. Cohen, and F. Renzoni, “Imaging corrosion under insulation with a mechanically-translatable atomic magnetometer,” in IEEE International 122, 144001-6 122, 144001-6 Appl. Phys. Lett. 122, 144001 (2023); doi: 10.1063/5.0147291 V C Author(s) 2023 Appl. Phys. Lett. 122, 144001 (2023); doi: 10.1063/5.0147291 V C Author(s) 2023
https://openalex.org/W4362521137	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0282854&type=printable	English	null	How firms cope with social crisis: The mediating role of digital transformation as a strategic response to the COVID-19 pandemic	PloS one	2,023	cc-by	17,304	Weilin Wu1, Huanxiang Wang1, Lei LuID2, Guangya Ma2, Xiaoxiao Gao2* 1 School of Economics, Jiaxing University, Jiaxing, Zhejiang, China, 2 School of Business, Macau University of Science and Technology, Avenida WaiLong, Macau, China 1 School of Economics, Jiaxing University, Jiaxing, Zhejiang, China, 2 School of Business, Macau University of Science and Technology, Avenida WaiLong, Macau, China 1 School of Economics, Jiaxing University, Jiaxing, Zhejiang, China, 2 School of Business, Macau University of Science and Technology, Avenida WaiLong, Macau, China * 1194118073@qq.com * 1194118073@qq.com OPEN ACCESS Citation: Wu W, Wang H, Lu L, Ma G, Gao X (2023) How firms cope with social crisis: The mediating role of digital transformation as a strategic response to the COVID-19 pandemic. PLoS ONE 18(4): e0282854. https://doi.org/ 10.1371/journal.pone.0282854 Editor: Vincenzo Basile, University of Naples Federico II: Universita degli Studi di Napoli Federico II, ITALY Received: November 22, 2022 Accepted: February 24, 2023 Published: April 4, 2023 Copyright: © 2023 Wu 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. Abstract The COVID-19 pandemic has drawn attention to the strategic responses of Chinese firms on digital transformation and led to a call for enhancing competitive advantage via accelerat- ing digital transformation. Besides the physical health issue, the pandemic has triggered an extraordinary social and economic crisis in which service industries have been attacked hard. In this situation, firms are meeting increasing competitive pressure, which urges them to achieve better performance with the help of digital transformation. Based on the technol- ogy-organization-environment framework and dynamic capabilities theory, this research proposed two studies with two methods, including a structural equation model and a regres- sion discontinuity design with a fixed-effect model. The findings suggest digital transforma- tion mediates the relationship between competitive pressure and firm performance among Chinese small- and medium-sized enterprises and large firms after the outbreak of COVID- 19, respectively. It confirms that digital transformation is a practical strategic decision for Chinese service firms to respond to increasing competitive pressure in the COVID-19 pan- demic. Besides, the results also illustrate the moderating effects of absorptive, innovative, and adaptive capability on the relationship between digital transformation and firm perfor- mance among large firms. PLOS ONE PLOS ONE RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1. Introduction After the outbreak of COVID-19 at the end of 2019, corporate operations and personal lives have been hit hard by the unprecedented global pandemic, and the service industry is reported to be one of the industries most affected by the COVID-19 pandemic [1]. The service industry requires more professionals to provide services directly, such as consulting, tourism, and edu- cation, instead of machines. In the background of the pandemic, some service companies that offer essential services (e.g., logistics, retailing, and health care) were required to continue to serve in order to maintain the city running, but they need to improve their working model to protect employees and customers. Secondly, due to the necessity of pandemic prevention, some healthy measures were required in corporate operations and daily activities, which How firms cope with social crisis: The mediating role of digital transformation as a strategic response to the COVID-19 pandemic Weilin Wu1, Huanxiang Wang1, Lei LuID2, Guangya Ma2, Xiaoxiao Gao2* Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: The author(s) received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 1 / 32 PLOS ONE How firms cope with social crisis challenged businesses’ development. For example, because of the lockdown policy, some ser- vice providers like hairdressers, hotels and airlines failed to operate [2]; because of the physical distance and quarantine measure, some service providers from the fields of consulting, media and education were unable to engage in offline activities and had to develop their online ser- vice business [1]. They have to seek new ways of connecting with customers and offering their services. For instance, exploiting online office and cloud meetings can avoid physical connec- tions; online orders and sales can decrease firms’ fixed costs. Digital transformation provides these service providers an alternative strategic choice to cope with this social crisis. Also, with the growing scale of the service industry in the national economy, helping enterprises in the service industry cope with a social crisis can reduce the unemployment rate and maintain social stability. Thus, we attempt to determine whether digital transformation can help service enterprises cope with the challenges of the COVID-19 pandemic [3]. On the one hand, some believe it is currently an uncertain and complex business environ- ment in which firms face severe challenges [4]. On the other hand, some argue that proper strategic responses enable firms actively adapt to the volatile environment and perform better [5]. Among many alternative strategies, digital transformation is considered one of the most beneficial business competitive strategies, because it can help firms keep a continuous compet- itive in a rapidly changing business context [6]. Although it has been reported that the COVID-19 pandemic has accelerated firms’ digital transformation [4], it has yet to explore widely whether digital transformation is a practical strategic response to the outbreak of COVID-19 for Chinese firms from the service sector and whether digital transformation can improve firm performance under an environment with more substantial competitive pressure like the environment in the COVID-19 pandemic. This study considers digital transformation a strategic decision that responds to the dynamic environmental change triggered by the COVID-19 pandemic. PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Some necessary pre- vention measures in the pandemic, like No dine-in and home quarantine, decrease business opportunities and exacerbate the competitive pressure among enterprises. According to the technology-organization-environment (TOE) framework, competitive pressure in an environ- mental context is one of the significant antecedents to promote firms’ transformation or adop- tion behavior of innovative technology [7]. However, the influence of increasing competitive pressure from an environmental perspective on firms’ strategy has yet to be considered in the COVID-19 pandemic. Besides, the coming issue is how firms exploited digital transformation to improve their performance, especially during the COVID-19 pandemic. Some reported that firms’ digital transformation hardly brought better performance, but brought economic loss and technolog- ical failures [8]. Thus, it suggests that the Critical for effective digital transformation is the capability to know the usage of technical applications [9]. In this research, we believe firms need digital transformation to improve their performance. Also, they need to have the ability to adopt new technology during the process when digital transformation influences firm per- formance. After coordinating new knowledge and new resources, digital transformation can be integrated better into firms’ operations, routine activities and core tasks [10]. Therefore, based on the dynamic capabilities theory, these capabilities might be boundary conditions that would reinforce the positive effect of digital transformation on firm performance. Prior studies mainly focused on the relationships between dynamic capabilities, organizational behavior, and organizational outcome [11,12] and the moderating role of a single dimension in dynamic capabilities in the process of corporate governance [13–15], but they failed to consider the moderating roles of multiple dimensions in dynamic capabilities, including absorptive capabil- ity, adaptive capability and innovative capability [16], in the process of digital transformation. Therefore, we also test the possibility of dynamic capabilities influencing the effect of digital PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 2 / 32 PLOS ONE How firms cope with social crisis transformation on firm performance in a strong competitive environment like the COVID-19 pandemic. Furthermore, past studies mentioned the differences between small- and medium-sized enterprises (SMEs) and large enterprises when they dealt with public crises and introduced new technology [11,17]. Some suggested that SMEs are more vulnerable to uncertain environ- ments due to limited resources [18]. And, it also was argued that large enterprises need more time executing transformation, because their established operation frameworks in professional fields are too reliable to change [9]. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Thus, this research views firms from the service sector in the light of competitive pressure, digital transformation and firm performance in the COVID- 19 pandemic, in order to examine the details of SMEs compared to large enterprises. And, we also explore the role of dynamic capabilities in the digital transformation process among large companies. To achieve the research objective, we propose the following research questions (RQ): RQ1: Does competitive pressure promote firms’ digital transformation in the COVID-19 pandemic? Does firms’ digital transformation improve their performance? RQ2: Is digital transformation a beneficial strategic response to the COVID-19 pandemic for SMEs and large enterprises? And what effects do dynamic capabilities have on digital trans- formation and firm performance among large companies? To answer these questions, this study conducted two studies with the theoretical help of competitive pressure from the TOE framework [19], and absorptive capability, innovative capability and adaptive capability from dynamic capabilities theory [20]. The first study exploited a set of cross-sectional survey data from Chinese SEMs’ senior managers or owners who have been involved in digital transformation in the service sector collected in 2022. The second study exploited longitudinal data on a sample of Chinese public firms in the service sector from 2018 to 2020. With the help of the structural equation model (SEM) and fixed- effect model, this study attempted to find whether digital transformation is a proper strategic choice for SMEs and large enterprises to respond to increasing competitive pressure after the outbreak of COVID-19. This study makes several contributions. First, prior research confirmed the effect of com- petitive pressure on digital transformation in the manufacturing industry [21]. Still, they have yet to profoundly examine the impact of competitive pressure on digital transformation in the service industry. Under the TOE framework, we identify competitive pressure as the anteced- ent of digital transformation in Chinese SMEs and large firms from the service industry. Sec- ond, focusing on digital transformation in firms from the sector industry, this research contributes to the literature and tests the significant achievement of digital transformation in the service sector as a strategic response to the COVID-19 outbreak. Third, combined with dynamic capabilities theory, this study explores the boundary condition in which firms’ digital transformation under the TOE framework provides a theoretical supplement for understand- ing the TOE framework. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Lastly, this research enriches the comparative investigation of SEMs and large enterprises’ digital transformation under a social crisis via two separate study designs with different contexts and data collection approaches. Given our two studies with two meth- odologies, our reports achieve stronger generalizability and robustness. This study makes several contributions. First, prior research confirmed the effect of com- petitive pressure on digital transformation in the manufacturing industry [21]. Still, they have yet to profoundly examine the impact of competitive pressure on digital transformation in the service industry. Under the TOE framework, we identify competitive pressure as the anteced- ent of digital transformation in Chinese SMEs and large firms from the service industry. Sec- ond, focusing on digital transformation in firms from the sector industry, this research t ib t t th lit t d t t th i ifi t hi t f di it l t f ti i PLOS ONE How firms cope with social crisis Table 1. Research on the TOE framework and competitive pressure. Study Research area Constructs Findings Chen et al., 2022 [26] The factors influencing performance of using artificial intelligence in hospitality industry System quality, perceived risk, management support, innovativeness, competitive pressure, regulatory support The analysis results reveal that perceived AI risk, management support, innovativeness, competitive pressure and regulatory support significantly influence the performance of AI adoption Bag et al., 2022 [27] The antecedents of blockchain technology adoption and its effect on SMEs’ performance Relative advantage, compatibility, complexity, top management support, organizational readiness, competitive pressure, external support from vendors, regulation and legislations, blockchain adoption, financial performance, market performance Relative advantage, compatibility, top management support, organizational readiness, competitive pressures, external support, regulations and legislation significantly impact SMEs’ blockchain adoption Salah et al., 2021 [28] The using of Customer Relationship Management in the Palestinian SMEs Compatibility, IT infrastructure, competitive, relative advantage, security, Top management, competitive pressure, customer pressure, firm size, Customer Relationship Management adoption Firm size moderates the relationship between top management support, compatibility, customer pressure, IT infrastructure, and Customer Relationship Management adoption. Cruz-Jesus et al., 2019 [7] The antecedents that affect Customer Relationship Management adoption stages in firms Technology competence, data quality and integration, top management support, competitive pressure, CRM evaluation, CRM adoption, CRM routinization The intention to adopt CRM in firms is predicted jointly and positively by technology competence, data quality and integration, top management support and CRM evaluation, but competitive pressure negatively influence CRM adoption Zhu, Dong, et al., 2006 [19] The determinants of E-business usage in post-adoption stages of innovation diffusion among European firms Relative advantage, compatibility, costs, security concern, technology competence, organization size, competitive pressure, partner readiness, E-business usage, E-business impact Compatibility, relative advantage, technology competence, partner readiness and competitive pressure significantly drive e-business usage. Security concern, cost, and organization size significantly inhibit e-business usage. Oliveira et al., 2014 [25] The adoption of cloud computing in the manufacturing and services sectors Security concerns, cost savings, relative advantage, complexity, compatibility, cloud computing adoption, technology readiness, top management support, firm size, competitive pressure, regulatory support The relative advantage is positively influenced by cost savings. The adoption is determined by relative advantage, complexity, technological readiness, top management support, and firm size. PLOS ONE Ramakrishnan et al., 2012 [29] The determinants of influencing business intelligence (BI) data collection strategies Institutional isomorphism, competitive pressure, insight, consistency, organizational transformation, problem driven strategy for BI data collection, comprehensive data collection strategy for BI data collection The implementing BI for the purpose of achieving consistency is predicted by institutional isomorphism, and comprehensive data collection strategy for BI data collection is predicted jointly by consistency and organizational transformation. Thong, 1999 [30] The determinants of using Information System (IS) in small businesses CEO’s innovativeness, CEO’s IS knowledge, relative advantage of IS, compatibility of IS, complexity of IS, business size, employees’ IS knowledge, information intensity, competition. CEO’s innovativeness and IS knowledge, relative advantage, compatibility, complexity of IS, business size and level of employees’ IS knowledge collectively influence the likelihood of adoption of IS in small businesses. https://doi org/10 1371/journal pone 0282854 t001 The analysis results reveal that perceived AI risk, management support, innovativeness, competitive pressure and regulatory support significantly influence the performance of AI adoption Firm size moderates the relationship between top management support, compatibility, customer pressure, IT infrastructure, and Customer Relationship Management adoption. The intention to adopt CRM in firms is predicted jointly and positively by technology competence, data quality and integration, top management support and CRM evaluation, but competitive pressure negatively influence CRM adoption Compatibility, relative advantage, technology competence, partner readiness and competitive pressure significantly drive e-business usage. Security concern, cost, and organization size significantly inhibit e-business usage. The relative advantage is positively influenced by cost savings. The adoption is determined by relative advantage, complexity, technological readiness, top management support, and firm size. the technological context, the organizational context and the environmental context [22]. The technological context is related to technology and abilities which are available in the organiza- tion [19]. The organizational context refers to internal criteria, such as organizational size, sales, etc [22]. The environmental context emphasizes the external field where an organization operates, including related industries and competitors [19]. The TOE framework has been applied widely to assess firms’ adoption behavior towards innovative technology, e.g., green information technology [23], customer relationship management [7], electronic data inter- change [24], cloud computing [25], e-business [19], etc. Table 1 lists studies about firms’ inno- vative behaviors under the TOE framework or competitive pressure. Past studies on digital transformation and competitive pressure received the theoretical support of the TOE framework [7,25,31]. 2. Previous research and hypothesis development 2.1 TOE framework and competitive pressure The TOE framework is considered as a significant theoretical perspective for exploring contex- tual determinants of firms’ strategic innovative decisions [19]. The TOE framework declares three factors that affect organizational adoption towards technological innovation, including 3 / 32 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 PLOS ONE Thus, the TOE framework is a widely accepted theo- retical framework investigating the influence of competitive pressure on digital transforma- tion. In this research, we focus on competitive pressure from the environmental context in the TOE framework. Competitive pressure refers to the level of pressure perceived by an PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 4 / 32 PLOS ONE How firms cope with social crisis organization from its competitors in a market [31,32]. Previous studies confirmed the signifi- cance of competitive pressure when an organization adopts innovative technology, as shown in Table 1. Furthermore, firms’ business environment has been plagued by uncertainty during the COVID-19 pandemic, which brings more challenges to the survival and development of firms. The rapid change in the business environment makes the competition between firms more intense, because some pandemic prevention policies, such as keeping distance and higher health standards, reduce business opportunities and increase business costs. In this background, firms’ business environment has undergone a noticeable change. Thus, competi- tion pressure from the environmental context is considered an essential driver of firms’ trans- formation to respond to the new challenges of the COVID-19 pandemic. Competitive pressure makes firms remain vigilant to avoid failure in the industry competi- tion. Fierce competitive pressure forces firms to make change and occupy more market shares [33]. These firms which prefer to adopt innovative technology enhances the flexibility and sta- bility of operations, and integrates differentiation into firms’ services and products [34], which updates the industry standards, elevates industry barriers and establishes new industry bench- mark of being the leader [21]. So, the hypothesis is proposed: Hypothesis 1. Competitive pressure is positively related to firms’ performance during the COVID-19 pandemic. PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 2.2 Digital transformation and competitive pressure Firms’ digital transformation involves utilizing digital technologies to reshape an innovative business model for dealing with the new business environment and outperforming their com- petitors [35]. Digital transformation refers to firms’ transformation process breaking business traditions and seeking innovation and changes based on exploiting digital technologies, including big data, cloud computing, and social platform [36]. For example, past studies have found that digital transformation encourages firms to integrate digital technologies with firms’ operations, in which customers also participate in innovation activities via digital technologies [37]. A social platform named Mi Global Home was built by Xiaomi Technology, which not only promotes communication between users, but also creates an opportunity to understand users’ innovative demands and real feedback for Xiaomi Technology [36]. However, in this context, we focus on the function of digital transformation in coping with the business environment changes caused by the COVID-19 pandemic. In this global crisis, the introduction of digital transformation to overall business activities is more profound than in most of the past [38], because digital transformation plays a crucial role in responding to the COVID-19 pandemic [39]. It helps firms adapt to the changing environment, save costs and own flexibility [40]. In other words, it eliminates the block of space and time, which allows firms to exploit external and internal resources to operate business activities [41]. For instance, digital transformation can improve promotional activities with a falling total expense [21]; dig- ital transformation enables employees to work from home to keep physical a distance when the area where the office is located is temporarily blocked [42]; Microsoft designed a cloud- based platform, known as Azure, that enable healthcare providers to acquire related medical resources quickly [39]; education sector also offers online courses for avoiding potential infec- tion risk among students [43]. Thus, we hypothesize: Hypothesis 2. Digital transformation is positively related to firms’ performance during the COVID-19 pandemic. Competitive pressure has long been seen as a driver for the usage of digital technology, as firms are forced to adapt innovations in order to maintain or seek competitive advantage [19]. Competitive pressure is defined as peer pressure on adopting new technologies [31,32]. Many 5 / 32 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 PLOS ONE How firms cope with social crisis previous studies have confirmed the positive relationship between competitive pressure and digital transformation, like cloud computing [25], Internet-based selling, service, procurement and coordination [19]. 2.2 Digital transformation and competitive pressure However, it should be mentioned that a study about using Customer Relationship Management (CRM) found that firms’ competitive pressure has a significant and negative effect during the CRM adoption stage [7]. Thus, there is still a slight debate between competitive pressure and firms’ digital transformation. According to the TOE framework, environmental contexts were reported as significant drivers of adopting information systems [7]. A study from the Portuguese hospitality industry found that 92% of 51 hotel managers agreed that the pandemic promoted their digital transformation [43]. The outbreak of COVID-19, coming with pandemic prevention policies, created an uncertain environment, decreasing business opportunities and intensifying competition among firms. In order to gain more competitive advantages in the competition, firms introduce digital transformation. So, we propose the following hypothesis: Hypothesis 3. Competitive pressure is positively related to digital transformation during the COVID-19 pandemic. Digital transformation is a support system to help firms respond positively to increasing competitive pressure and gain more revenue [44]. With the growth of competitive pressure in an uncertain environment, firms attempt to exploit digital transformation to lower operational expenses and raise outreach of their products and services [45]. Furthermore, digital transfor- mation enables firms to energetically use existing resources in the pandemic, such as working remotely and in cloud meetings. These firms who adopt digital transformation can outperform their competitors [19], and survive in the uncertain environment of the COVID-19 pandemic. Thus, we present the following hypothesis: Hypothesis 4. Digital transformation will mediate the relationship between competitive pres- sure and firms’ performance during the COVID-19 pandemic. 2.3 Dynamic capabilities theory The dynamic capabilities theory was developed in strategic management as it offered theoreti- cal direction into firms’ distinct capabilities in a shifting environment [46]. Dynamic capabili- ties correspond to the comprehensive capabilities to establish, integrate and reconfigure external and internal resources for dealing with a rapidly changing environment [47]. Firms’ dynamic capabilities are considered strategic operation, which enables firms to adapt to the new environment as the opportunity, public crisis or new demand arises [20]. On the other hand, the emerging complex environment represents a platform for firms to show the full potential of their dynamic capabilities [17]. Prior studies displayed the application of dynamic capabilities theory in exploring firms’ strategic responses under unstable business environ- ments. For example, data collected from 274 international firms confirmed that information technology-enabled dynamic capabilities facilitated firms’ competitive performance in uncer- tain environments [48]; evidence from 339 Chinese firms found that digital transformation helped firms reconstruct internal and external resources under the perspective of dynamic capacity for enhancing organizational resilience [49]; research based on 1162 firms in Nigeria revealed that mobile apps usage enabled firms to maximize opportunity via dynamic capabili- ties in an innovative environment [46]. In the background of the COVID-19 pandemic, dynamic capabilities are viewed from three dimensions: absorptive, adaptive, and innovative capability [16]. Although it is expected that digital transformation can improve firms’ plights in a public crisis [46], firms with dynamic capabilities are more likely to identify and capture the advantages of digital transformation and apply it in a new environment. Firms with more vital dynamic capabilities can absorb new PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 6 / 32 PLOS ONE How firms cope with social crisis technology and skills, adapt to uncertain environments and create new opportunities, gaining an impetus for achieving better performance in the COVID-19 pandemic [17]. 2.4 The moderating effect of absorptive capability The absorptive capability is an ability that helps an organization acquire and assimilate intan- gible resources, including knowledge, technology and expertise, then transform and exploit them to generate unique knowledge, technology and expertise [20]. Past scholars have reported that these firms with access to the same quantity of external knowledge might prog- ress at different levels, because their abilities to identify and transform knowledge are different [50]. As a result, the quantity and amount of external knowledge are unevenly distributed in the whole industry, which means that absorptive capability can be viewed as an organization’s competitive advantage [51]. In this context, digital transformation is considered a firm’s dynamic resource from the perspective of dynamic capabilities theory, being an available pol- icy for reconfiguring firms’ resources [49]. And, according to dynamic capabilities theory, the absorptive ability to integrate external and internal resources is a powerful tool for firms to cope with an uncertain environment [52]. Thus, in this context of the COVID-19 pandemic, the value of digital transformation and its effect on firms’ performance depends on their owned characteristics including absorptive capability [53], because the new knowledge and technology acquired from internal (e.g., within the firm) and external (e.g., market) environ- ment might contain context-dependent features, particular factors or specific prerequisites [54]. In other words, digital transformation might improve firms’ performance when firms possess the proper capabilities to absorb and use the new knowledge and technology. For example, firms with a higher degree of absorptive capability can build an effective learning process and a more diverse knowledge base, increasing the potentiality that firms value and positively use new knowledge and technology [55]. Furthermore, the moderating effect of absorptive capability has been confirmed in the field of innovative activities [53], science-to- industry technology transfer projects [56], new product introduction [13] and international venturing [57]. Therefore, the following hypothesis is proposed: Hypothesis 5. Absorptive capability moderates the relationship between digital transforma- tion and firm performance during the COVID-19 pandemic, such that the relationship will be stronger at a higher level of absorptive capability than at a lower of absorptive capability. PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 2.5 The moderating effect of innovative capability The innovative capability is defined as an ability to produce, accept and execute fresh ideas and new solutions for responding to risks in the market [58,59]. These innovative ideas and solutions are related to new process design, new service or product development and renova- tion, which focuses on dealing with the competition in a dynamic and uncertain environment [60]. Innovation is considered a source where an organization develops a competitive advan- tage, which has been widely explored in large firms across developed countries since 1980 [59]. And, fewer studies paid attention to these firms from emerging economies [61]. Innovative capability development is an investment in firms’ future performance, supplementing firms’ crisis readiness [62]. According to dynamic capabilities theory, both innovative and absorptive capabilities are significant in grasping competitive advantages for firms [63]. Innovative capa- bility can be viewed as complementary to absorptive capability, providing firms with better usage of the knowledge and technologies acquired by absorption [64]. Thus, it is valuable to examine the effect of innovative capability in Chinese firms from the service industry during the COVID-19 pandemic. 7 / 32 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 PLOS ONE How firms cope with social crisis In this context, innovative capability has the potential to help firms to respond rapidly and efficiently to uncertainty in the market [65], especially in this global pandemic. For example, firms with better innovation maintain stronger stability in a changing environment, and they can reliably deliver new quality products and services to the market faster, more frequently, and lower cost than their competitors [66]. Innovation allows firms to improve as the changes in their environment [67]. In other words, innovation is a solution to adapt to the uncertainty of the pandemic for firms, as digital transformation works. Firms with higher innovative capa- bility can better grasp new opportunities and apply new knowledge and technology, which will augment firms’ performance in the COVID-19 pandemic. Based on these arguments, the hypothesis is proposed: Hypothesis 6. Innovative capability moderates the relationship between digital transforma- tion and firm performance during the COVID-19 pandemic, such that the relationship will be stronger at a higher level of innovative capability than at a lower of innovative capability. 2.6 The moderating effect of adaptive capability The adaptive capability refers to an ability to reconfigure resources and coordinate processes for new product and service development [68]. A firm’s adaptive capability involves a dynamic interaction of market, technology and business process, enabling firms to achieve competitive advantages under a turbulent environment [69]. On the other hand, adaptive capability dis- plays the efficacy of problem-solving and the speed of customer response [15]. As dynamic capabilities theory mentioned, dynamic capabilities will bring changes in firms’ resources and capabilities to create sustainable competitive advantage [46]. This capability allows firms to become more flexible in developing beneficial transformations in order to make great progress in a dynamic environment. For example, employees from these firms with adaptive capability can learn various skills and deal with challenges with greater discretion [70]. In this context, adaptive capability is beneficial to firms’ digital transformation. It is an uncertain and highly competitive business environment during the COVID-19 pandemic, which means most employees are unwilling to participate in high-risk activities such as digital transformation in this period [71]. But, a high level of adaptive capability reflects this situation that all employees related to digital transformation can load the required information, knowledge and technology in order to minimize risk in an uncertain environment [14]. Therefore, adaptive capability facilitates the successful conversion of digital transformation into improved firm performance. So, we hypothesize: Hypothesis 7. Adaptive capability moderates the relationship between digital transformation and firm performance during the COVID-19 pandemic, such that the relationship will be stron- ger at a higher level of adaptive capability than at a lower of adaptive capability. Based on the TOE framework, we proposed the research model in Study 1 (see Fig 1) in order to explore the effectiveness of digital transformation in Chinese SMEs from the service industry as a strategic response to the COVID-19 pandemic. Then, combined with the TOE framework and dynamic capabilities theory, we proposed the research model in Study 2 (see Fig 2) in order to examine the roles of absorptive capability, innovative capability and adaptive capability in the process of digital transformation, as the strategic role of digital transformation in large Chinese firms from the service industry in the COVID-19 pandemic. 3. Methodology This study investigated the mediating effect of digital transformation as a strategic response to the COVID-19 pandemic. To expand the generalizability and justification of research results, we sought to execute two empirical analyses based on two data sets collected from small- and 8 / 32 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 PLOS ONE How firms cope with social crisis Fig 1. The research model in Study 1. https://doi.org/10.1371/journal.pone.0282854.g001 Fig 1. The research model in Study 1. Fig 1. The research model in Study 1. https://doi.org/10.1371/journal.pone.0282854.g001 medium-sized enterprises (SMEs) and large enterprises, respectively. Due to the defect of accurate and reliable accounting in Chinese SMEs [72], Study 1 on SMEs relied on senior managers’ or owners’ subjective evaluations and perceptions of performance instead of the objective indicators to measure firms’ characteristics. On the other hand, we lacked the resources to access enough senior managers or owners from large firms and distribute ques- tionnaires, leading to exploiting data from listed firms’ databases, the China Stock Market and Accounting Research (CSMAR), to measure constructs in this research model. Thus, as sug- gested by Tang & Yang [73] and Raithel & Hock [74], we used the data collected from the sur- vey in Study 1, while we exploited the data adopted from CSMAR in Study 2. Even though variables were measured by two data sets, corresponding measurements have received theoretical support from previous studies and ensured the justification of methodology [7,20,21,75–77]. Regarding the ethics issue in Study 1, every respondent was briefed on the purpose of the research and informed that all data would be strictly confidential and used for academic pur- poses only. The relevant information mentioned above was stated at the beginning of the ques- tionnaire to ensure that respondents know their rights and agree with this survey. Thus, this survey obtained written consent from all participants. Finally, this project that collected the data from questionnaires was approved by the Institutional Review Board of the Macau Uni- versity of Science and Technology. An Ethic Issue Form offered by Macau University of Sci- ence and Technology was signed and submitted to promise this article’s authenticity and compliance with academic ethics. Fig 2. The research model in Study 2. https://doi.org/10.1371/journal.pone.0282854.g002 OS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 9 / 32 Fig 2. The research model in Study 2. 3.1 Samples and measures in Study 1 Study 1 explored the effectiveness of digital transformation strategy among Chinese SMEs from the service sector during the COVID-19 pandemic. The data in Study 1 was collected based on a cross-sectional survey executed in China. The construct of competitive pressure was measured by items from Cruz-Jesus et al., Thong, and Zhu et al. (2006) [7,19,30]. The vari- able of digital transformation was measured by questions from Cruz-Jesus et al., Thong, and Zhu et al. [21,36,49]. The construct of firm performance was developed by Singh et al. and Wang et al. [21,78]. These studies explored the influence of competitive pressure on the adop- tion of information systems and the effect of information systems on organizational outcomes, which shared the same research background as our study. In summary, the scale used in this research was developed, examined, accepted, and supported by past studies. All items in this survey were initially shown in English. Through the back-translation approach, they were translated into Chinese to minimize translation bias [79]. A pilot test including 10 question- naires (Chinese edition) was executed among 5 owners of SMEs, 3 professional researchers from the field of organizational behavior and 2 bilingual doctoral students majoring in English and Mandarin, which suggested all Chinese items are clear and understandable in the Chinese context. In April 2022, the survey was distributed to owners or senior managers from SMEs (with less than 500 employees) via social media, including WeChat and E-mail [17,80]. The participants were Chinese students majoring in Executive Master of Business Administration (EMBA), Master of Business Administration (MBA) and Executive Education (EE). 544 valid responses were collected from 1000 SMEs, accounting for 54.4% of the samples. In order to explore the effect of digital transformation in the COVID-19 pandemic, 544 usable samples are qualified as they started their businesses before the pandemic. Estimating the min- imum sample size is one of the most significant problems in PLS-SEM (Partial Least Square- Structural Equation Modelling). The ten times rule for calculating the minimum sample size for PLS-SEM has been widely adopted in past studies, but some experts argued that the ten times rule produced inaccurate estimates [81]. Thus, an inverse square root method was rec- ommended to estimate the minimum sample size in PLS-SEM [82]. 3. Methodology https://doi.org/10.1371/journal.pone.0282854.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 9 / 32 PLOS ONE How firms cope with social crisis PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 PLOS ONE How firms cope with social crisis Table 2. Respondent profiles. Measure Items Frequency Percent (%) Respondent’ level Owner Senior manager 114 430 21.0 79.0 Firm age 3–4 years 5–6 years 7–8 years 9–10 years Over 11 years 147 200 129 12 56 27.0 36.8 23.7 2.2 10.3 Number of employees 1–50 51–250 251–300 301–500 46 208 189 101 8.5 38.2 34.7 18.6 Annual turnover Less than 1 million RMB 1–5 million RMB 5–10 million RMB 10–30 million RMB 30–50 million RMB 50–120 million RMB 120–300 million RMB 136 115 98 68 48 49 30 25.0 21.1 18.0 12.5 8.8 9.0 5.5 Location Eastern China Northern China Northeastern China Central China Southern China Northwestern China Southwestern China 110 103 103 111 41 43 33 20.2 18.9 18.9 20.4 7.5 7.9 6.1 Service industry Catering Tourism Hospitality Education and training Retail trade Advertising and communication media Consulting and insurance Leasing and business services Culture, sports, and entertainment Health and social work Information technology 40 42 52 38 58 49 41 66 52 44 62 7.4 7.7 9.6 7.0 10.7 9.0 7.5 12.1 9.6 8.1 11.4 https://doi.org/10.1371/journal.pone.0282854.t002 3.1 Samples and measures in Study 1 As suggested by Kock and Hadaya’s [81] inverse square root method, the minimum sample size in this study is 56, which is significantly lower than the 544 samples in Study 1. Thus, 544 is a valid sample size for Study 1. As displayed in Table 2, 21.0% of respondents are SME owners, and the rest are senior man- agers of SMEs. Both of them were able to judge their SMEs’ status, including perceived com- petitive pressure, digital transformation and performance [53]. All variables were measured via multiple indicators in a self-reported questionnaire with seven- point Likert scales, ranging from 1 (strongly disagree) to 7 (strongly agree). Table 3 shows that all items in the questionnaire were adapted from prior studies. With the assistance of the statistical package SmartPLS, the PLS-SEM was used to assess and evaluate the research model via the PLS algorithm and bootstrapping method for accurate causal-predictive analysis in Study 1 [83,84]. Regarding the issue of common method bias (CMB), according to the direction of Podsak- off et al. [85], this research adopted some approaches to decrease and test CMB. First, the sur- vey data is strictly confidential, ensuring participants respond honestly. Second, we used the full collinearity assessment method [86,87]. When all constructs were regressed on a random dependent variable, all variance inflation factors of constructs were lower than the threshold of 3.3, supporting the data can be considered free of common method bias. Third, we exploited the correlation matrix procedure to examine common method bias [88]. According to the cor- relation matrix procedure, the values of correlations between variables were 0.782 to 0.832, which were lesser than the threshold of 0.9. Hence, common method bias was not a problem in this study. 10 / 32 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 PLOS ONE PLOS ONE How firms cope with social crisis Table 3. Measurement, indicator loading, reliability and convergent validity. Construct Item Loading t-value CR AVE Competitive pressure [7,19,30] After the Covid-19 pandemic outbreak, your firm is under pressure from competitors to adopt digital transformation. After the Covid-19 pandemic outbreak, some of your competitors have already started digital transformation. After the Covid-19 pandemic outbreak, percentage of competitors in your industry that have conducted digital transformation is high. After the Covid-19 pandemic outbreak, percentage of competitors in your industry that have conducted Internet-based selling or servicing is high. After the Covid-19 pandemic outbreak, the level of rivalry among businesses in the same industry is fierce. 0.784 0.813 0.796 0.753 0.751 49.173 52.841 45.654 37.841 39.211 0.886 0.608 Digital transformation [21,36,49] After the Covid-19 pandemic outbreak, many new business processes built on technologies such as big data, cloud, mobile and social media platform. After the Covid-19 pandemic outbreak, the digital technologies such as social media, big data, cloud and mobile technologies are integrated to drive change. After the Covid-19 pandemic outbreak, the business operations are shifting toward making use of digital technologies such as big data, cloud, mobile and social media platform. After the Covid-19 pandemic outbreak, your firm develops digital products and services. After the Covid-19 pandemic outbreak, supporting communication of commercial and business information through digital technologies (such as big data, cloud computing, mobile and social platforms). 0.764 0.736 0.706 0.757 0.742 36.801 29.317 34.774 35.287 33.000 0.859 0.550 Firm performance [21,78] After the Covid-19 pandemic outbreak, the growth is more as compared to competitors. After the Covid-19 pandemic outbreak, customer satisfaction of your company is better than that of key competitors. After the Covid-19 pandemic outbreak, quality development of your company is better than that of key competitors. After the Covid-19 pandemic outbreak, responsiveness of your company is better than that of key competitors. 0.819 0.800 0.843 0.738 56.017 49.031 73.074 39.623 0.877 0.642 Notes: Loading: Indicator Loading, CR: Composite Reliability, AVE: Average Variance Extracted. Table 3. Measurement, indicator loading, reliability and convergent validity. Notes: Loading: Indicator Loading, CR: Composite Reliability, AVE: Average Variance Extracted. the PLS model were over those from the LM [93]. Thus, it can be concluded that this research model in Study 1 is a satisfactory model, as the PLS-based prediction showed a more accurate out-of-sample predictive power. 3.2 Data analysis and results in Study 1 As shown in Table 3, this study evaluated indicator reliability, composite reliability, convergent validity and discriminant validity in constructs. First, to examine the indicate reliability, all indicators’ outer loading values were calculated and were higher than the threshold number of 0.7 with significance [89]. Then, all constructs’ composite reliability (CR) were greater than 0.7, assuming decent degrees of reliability and internal consistency [90]. Besides, all constructs’ average variance extracted (AVE) values were above 0.5, which also demonstrated convergent validity, as it can be deduced that the latent variable explains over 50% variance of related indi- cators [90]. Fourth, all loadings of indicators were greater than all related cross-loading in Table 4, supporting good levels of discriminant validity between constructs [91]. This study further executed the PLSpredict procedure to evaluate the out-of-sample predic- tive relevance [92]. As displayed in Table 5, all endogenous indicator values of Q2predict were greater than 0, implying the superiority of research model is over than a naïve prediction and owning predictive relevance [93]. Furthermore, most of values of the root mean squared error (RMSE) in the PLS model were less than those in the liner regression model (LM). And, the PLS model produced lower prediction errors, because three of four Q2predict indicators from PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 11 / 32 https://doi.org/10.1371/journal.pone.0282854.t004 PLOS ONE PLOS ONE How firms cope with social crisis Table 5. PLSpredict assessment PLS vs. LM. PLS LM (PLS-LM) RMSE Q2predict RMSE Q2predict RMSE Q2predict Firm performance 1 1.166 0.424 1.173 0.418 -0.007 0.006 Firm performance 2 1.117 0.435 1.123 0.428 -0.006 0.007 Firm performance 3 1.241 0.501 1.249 0.493 -0.008 0.008 Firm performance 4 1.521 0.327 1.517 0.331 0.004 -0.004 Notes: RMSE: Root mean squared error; MAE: Mean absolute error; PLS: Partial least squares path model; LM: Linear regression model; Number of folds = 10 subgroups; Number of repetitions = 10. Table 5. PLSpredict assessment PLS vs. LM. Table 5. PLSpredict assessment PLS vs. LM. MAE: Mean absolute error; PLS: Partial least squares path model; LM: Linear regression model; Number of folds = 10 Notes: RMSE: Root mean squared error; MAE: Mean absolute error; PLS: Partial least squares path model; LM: Linear regression model; Number of folds = 10 subgroups; Number of repetitions = 10. Notes: RMSE: Root mean squared error; MAE: Mean absolute error; PLS: Partial least squares path model; LM: Linear r subgroups; Number of repetitions = 10. https://doi.org/10.1371/journal.pone.0282854.t005 First, the variance inflation factors (VIF) were estimated to exam multicollinearity between constructs. All latent variables’ VIF values were lower than 5, which indicated there was no concerns of multicollinearity among constructs ([89]. Moving forward, we examined path coefficients and t-statistics levels under the bootstrapping method with 5000 samplings. We ran a test of control variables including industry (β = -0.007; bootstrap confidence interval (-0.051, 0.038)), number of employees (β = 0.014; bootstrap confidence interval (-0.032, 0.059)), location (β = -0.009; bootstrap confidence interval (-0.059, 0.040)), firm age (β = -0.015; bootstrap confidence interval (-0.061, 0.027)), and annual turnover (β = 0.007; boot- strap confidence interval (-0.038, 0.053)), which reported control variables have no significant effects [7,17,20]. From Fig 3, it can be viewed that the research model explained 70.1% of the variation in digital transformation, as 69.2% of the variation in firm performance. It also can be seen that their blindfolding-based Q2 metric values were greater than 0, supporting a decent predictive relevance [91]. As shown in Table 6, it demonstrated that competitive pressure positively, directly and significantly influenced digital transformation (ß = 0.832) and firm performance (ß = 0.536). Therefore, H1 and H3 were supported. And also, it illustrated that digital transfor- mation positively and significantly influenced firm performance (ß = 0.334), thus, H2 was supported. PLOS ONE p p p This study executed some assessment procedures to evaluate the outcomes of structural model including multicollinearity between constructs and relevance of the path coefficients. This study executed some assessment procedures to evaluate the outcomes of structural model including multicollinearity between constructs and relevance of the path coefficients. Table 4. Loadings and cross-loadings. Table 4. Loadings and cross-loadings. Competitive pressure (CP) Digital transformation (DT) Firm performance (FP) CP1 0.784 0.631 0.664 CP2 0.813 0.670 0.656 CP3 0.796 0.723 0.647 CP4 0.753 0.600 0.574 CP5 0.751 0.613 0.636 DT1 0.639 0.764 0.587 DT2 0.625 0.736 0.582 DT3 0.597 0.706 0.583 DT4 0.621 0.757 0.579 DT5 0.600 0.742 0.567 FP1 0.655 0.636 0.819 FP2 0.662 0.658 0.800 FP3 0.712 0.672 0.843 FP4 0.577 0.527 0.738 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 12 / 32 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 PLOS ONE And, the year of 2020 was viewed as the ending point, because CSMAR database currently discloses data up to 2020. This Study 2 excluded B-share firms because they only disclosed financial data in their annual reports without relative data required by Study 2. Besides, it excluded firms that have been listed for one year or less in order to show the influence of the COVID-19 pandemic and reduce the uncertainty from newly listed firms. Moving forward, it dropped firm-year observa- tions with missing values and obtained a final sample of 31859 firm-year observations from 4341 listed companies. 3.4 Dependent variable in Study 2 Tobin’s Q served as the dependent variable in this study. Tobin’s Q as a metric has won wide- spread acceptance because it was claimed to be forward-looking and comparable across diverse Table 6. The assessment of structural model. Relationship Std Beta Std error t-value p-value Bootstrap confidence interval f 2 Competitive pressure -> Firm performance 0.536 0.041 12.994 0.000 0.453 0.615 0.292 Competitive pressure -> Digital transformation 0.832 0.015 53.925 0.000 0.801 0.860 2.250 Digital transformation -> Firm performance 0.334 0.041 8.112 0.000 0.255 0.416 0.114 Competitive pressure -> Digital transformation -> Firm performance 0.278 0.035 7.895 0.000 0.212 0.349 ---- Notes: Bootstrapping based on n = 5,000 bootstrap samples; *Significant at p<0.001, Significant at p<0.01, Significant at p<0.05 (two-tailed test). https://doi.org/10.1371/journal.pone.0282854.t006 Table 7. The mediating effect test. Total effect Direct effect Indirect effects on performance Path p value Path p value Point estimate Confidence interval Sig. VAF CP -> FP 0.814 0.000 CP -> FP 0.536 0.000 CP -> DT -> FP 0.278 0.212 0.349 Yes 34.15% Table 6. The assessment of structural model. Relationship Std Beta Std error t-value p-value Bootstrap confidence interval f 2 Competitive pressure -> Firm performance 0.536 0.041 12.994 0.000 0.453 0.615 0.292 Competitive pressure -> Digital transformation 0.832 0.015 53.925 0.000 0.801 0.860 2.250 Digital transformation -> Firm performance 0.334 0.041 8.112 0.000 0.255 0.416 0.114 Competitive pressure -> Digital transformation -> Firm performance 0.278 0.035 7.895 0.000 0.212 0.349 ---- Notes: Bootstrapping based on n = 5,000 bootstrap samples; Significant at p<0.001, Significant at p<0.01, Significant at p<0.05 (two-tailed test). https://doi org/10 1371/journal pone 0282854 t006 Table 6. The assessment of structural model. Relationship 3.3 Samples and measures in Study 2 Study 1 confirmed the effectiveness of the digital transformation strategy among Chinese SMEs during the COVID-19 pandemic. According to Tang et al. [73], Study 1 was beneficial in building the baseline relationship between competitive pressure and firm performance via digital transformation. Therefore, in Study 2, we confirmed the mediating effect of digital transformation in the relationship between competitive pressure and firm performance. Also, in Study 2, based on the baseline relationship, we explored the moderating effect of dynamic capabilities on the relationship between digital transformation and firm performance to enhance the theoretical contributions of our findings. Our study 2 panel data sample covered Chinese listed firms on the Shanghai and Shenzhen Stock Exchanges from 2007–2020. In this study, we exploited the data of listed companies from service industry as the data of large companies from service industry (more than 500 employees) collected from the China Stock Market and Accounting Research (CSMAR) data- base [95]. Data from CSMAR contains comprehensive historical information of listed firms on the Shanghai and Shenzhen Stock Exchanges like financial statements and corporate gover- nance reports, which have been applied widely in studies related to corporate strategy [95,96]. The year of 2007 was considered as the starting point, because Chinese listed firms have been required to adopt new accounting standards since 2006. And, the year of 2020 was viewed as the ending point, because CSMAR database currently discloses data up to 2020. g y This Study 2 excluded B-share firms because they only disclosed financial data in their annual reports without relative data required by Study 2. Besides, it excluded firms that have been listed for one year or less in order to show the influence of the COVID-19 pandemic and reduce the uncertainty from newly listed firms. Moving forward, it dropped firm-year observa- tions with missing values and obtained a final sample of 31859 firm-year observations from 4341 listed companies. PLOS ONE Then, a mediating effect test was executed (see Table 7). Followed a formal mediation anal- ysis [93,94], the indirect effect between competitive pressure and firm performance was tested and was significant. The second step was that the direct effect of competitive pressure on digi- tal transformation was estimated and showed significance. Lastly, the direct effect shared the same direction as the indirect effect. Thus, it can be conducted that digital transformation had a partial mediating effect between competitive pressure and firm performance, which meant H4 was supported. Fig 3. Structural model results in Study 1. Notes: Significant at p<0.001, Significant at p<0.01, Significant at p<0.05. https://doi.org/10.1371/journal.pone.0282854.g003 Fig 3. Structural model results in Study 1. Notes: Significant at p<0.001, Significant at p<0.01, Significant at p<0.05. https://doi.org/10.1371/journal.pone.0282854.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 13 / 32 PLOS ONE Relationship Std Beta Std error t-value p-value Bootstrap confidence interval f 2 Competitive pressure -> Firm performance 0.536 0.041 12.994 0.000 0.453 0.615 0.292 Competitive pressure -> Digital transformation 0.832 0.015 53.925 0.000 0.801 0.860 2.250 Digital transformation -> Firm performance 0.334 0.041 8.112 0.000 0.255 0.416 0.114 Competitive pressure -> Digital transformation -> Firm performance 0.278 0.035 7.895 0.000 0.212 0.349 ---- Notes: Bootstrapping based on n = 5,000 bootstrap samples; Significant at p<0.001, Significant at p<0.01, Significant at p<0.05 (two-tailed test). https://doi.org/10.1371/journal.pone.0282854.t006 Table 7. The mediating effect test. Total effect Direct effect Indirect effects on performance Path p value Path p value Point estimate Confidence interval Sig. VAF CP -> FP 0.814 0.000 CP -> FP 0.536 0.000 CP -> DT -> FP 0.278 0.212 0.349 Yes 34.15% Notes: CP -> FP: Competitive pressure -> Firm performance; CP -> DT -> FP: Competitive pressure -> Digital transformation -> Firm performance; Sig.: Significance; VAF: Variance Accounted For. https://doi.org/10.1371/journal.pone.0282854.t007 3.3 Samples and measures in Study 2 Study 1 confirmed the effectiveness of the digital transformation strategy among Chinese SMEs during the COVID-19 pandemic. According to Tang et al. [73], Study 1 was beneficial in building the baseline relationship between competitive pressure and firm performance via digital transformation. Therefore, in Study 2, we confirmed the mediating effect of digital transformation in the relationship between competitive pressure and firm performance. Also, in Study 2, based on the baseline relationship, we explored the moderating effect of dynamic capabilities on the relationship between digital transformation and firm performance to enhance the theoretical contributions of our findings. Our study 2 panel data sample covered Chinese listed firms on the Shanghai and Shenzhen Stock Exchanges from 2007–2020. In this study, we exploited the data of listed companies from service industry as the data of large companies from service industry (more than 500 employees) collected from the China Stock Market and Accounting Research (CSMAR) data- base [95]. Data from CSMAR contains comprehensive historical information of listed firms on the Shanghai and Shenzhen Stock Exchanges like financial statements and corporate gover- nance reports, which have been applied widely in studies related to corporate strategy [95,96]. The year of 2007 was considered as the starting point, because Chinese listed firms have been required to adopt new accounting standards since 2006. PLOS ONE How firms cope with social crisis 3.3 Samples and measures in Study 2 Study 1 confirmed the effectiveness of the digital transformation strategy among Chinese SMEs during the COVID-19 pandemic. According to Tang et al. [73], Study 1 was beneficial in building the baseline relationship between competitive pressure and firm performance via digital transformation. Therefore, in Study 2, we confirmed the mediating effect of digital transformation in the relationship between competitive pressure and firm performance. Also, in Study 2, based on the baseline relationship, we explored the moderating effect of dynamic capabilities on the relationship between digital transformation and firm performance to enhance the theoretical contributions of our findings. Our study 2 panel data sample covered Chinese listed firms on the Shanghai and Shenzhen Stock Exchanges from 2007–2020. In this study, we exploited the data of listed companies from service industry as the data of large companies from service industry (more than 500 employees) collected from the China Stock Market and Accounting Research (CSMAR) data- base [95]. Data from CSMAR contains comprehensive historical information of listed firms on the Shanghai and Shenzhen Stock Exchanges like financial statements and corporate gover- nance reports, which have been applied widely in studies related to corporate strategy [95,96]. The year of 2007 was considered as the starting point, because Chinese listed firms have been required to adopt new accounting standards since 2006. And, the year of 2020 was viewed as the ending point, because CSMAR database currently discloses data up to 2020. This Study 2 excluded B-share firms because they only disclosed financial data in their annual reports without relative data required by Study 2. Besides, it excluded firms that have been listed for one year or less in order to show the influence of the COVID-19 pandemic and reduce the uncertainty from newly listed firms. Moving forward, it dropped firm-year observa- tions with missing values and obtained a final sample of 31859 firm-year observations from 4341 listed companies. 3.4 Dependent variable in Study 2 Tobin’s Q served as the dependent variable in this study. Tobin’s Q as a metric has won wide- spread acceptance because it was claimed to be forward-looking and comparable across diverse Table 6. The assessment of structural model. 3.4 Dependent variable in Study 2 Tobin’s Q served as the dependent variable in this study. Tobin’s Q as a metric has won wide- spread acceptance because it was claimed to be forward-looking and comparable across diverse Table 7. The mediating effect test. Total effect Direct effect Indirect effects on performance Path p value Path p value Point estimate Confidence interval Sig. VAF CP -> FP 0.814 0.000 CP -> FP 0.536 0.000 CP -> DT -> FP 0.278 0.212 0.349 Yes 34.15% Notes: CP -> FP: Competitive pressure -> Firm performance; CP -> DT -> FP: Competitive pressure -> Digital transformation -> Firm performance; Sig.: Significance; VAF: Variance Accounted For. Table 7. The mediating effect test. Table 7. The mediating effect test. Total effect Direct effect Indirect effects on performance Path p value Path p value Point estimate Confidence interval Sig. VAF CP -> FP 0.814 0.000 CP -> FP 0.536 0.000 CP -> DT -> FP 0.278 0.212 0.349 Yes 34.15% Notes: CP -> FP: Competitive pressure -> Firm performance; CP -> DT -> FP: Competitive pressure -> Digital transformation -> Firm performance; Sig.: Significance; VAF: Variance Accounted For. PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 14 / 32 PLOS ONE How firms cope with social crisis industries. Tobin’s Q has been widely used to measure firms’ financial performance. It is defined the Q ratio as a firm’s market value relative to its assets’ replacement cost [97]. Researchers have exploited the Q ratio to study the effects of market power on performance [75,76]. In this study, we used Chung and Pruitt’s method to calculate Q ratio [97]. The advan- tage of this approach is that the financial and accounting information required by this formula is available in the CSMAR database. 3.6 Mediating variable in Study 2 Prior studies have established various measures on firms’ digital transformation (DT), which represented the degree of firms’ digital transformation [75]. With the rapid development of machine learning and text mining technology, Study 2 attempted to construct the DT index of listed firms by text mining. During the process of collecting data about the DT index, this study firstly retrieved and collated the annual reports of sample firms via the Python crawler function and secondly extracted all textual contexts via Python program to establish a data pool for subsequent feature word screening [75]. After that, this study identified the feature words of digital transformation from the academic and industrial perspective. On the one hand, the feature words from the academic perspective were absorbed from influential litera- ture on the field of digital transformation [99,100]. On the other hand, the feature words were grasped from firms’ website. Then the Study 2 created a pool of feature words including digital transformation, big data, cloud computing, blockchain, artificial intelligence and etc. Lastly, based on the pool of feature words of digital transformation, the Study 2 searched, matched and counted the frequency of words after extracting text content from sample firms’ annual reports under the assistant of these tools including Jieba package and the word list of deactiva- tions. The ultimate characteristics of feature words were summarized after all steps above. As the typical characteristic of right deviation owned by the set of data, the Study 2 executed a log- arithmic process using In (‘the total frequency’ +1) to measure the digital transformation of firms. 3.5 Explanatory variable in Study 2 The Herfindahl-Hirschman Index (HHI) is a measure of the industry’s competitiveness in a given year. Drawing on the measures of Haveman et al. and Jia et al., the HHI among service industry segment for the year was used to measure the degree of competitiveness [77,98]. The HHI is calculated by classifying each service industry segment based on the industry code (two digits) of the Securities and Futures Commission, then calculating the market share of each company according to the business revenue of all companies in each segment, and finally cal- culating the squared sum of the market shares of all companies in the segment. Whit less the HHI, relative service industry presents a stronger competitive pressure. For a more intuitive interpretation of the subsequent empirical results, the index is processed (1-HHI), to obtain the industry competitiveness index (IH) for the model test in this paper. 3.8 Control variables in Study 2 We controlled several factors that might influence firms’ digital transformation including firm size (Size, the natural logarithm of a firm’s total asset), firm age (Age, counting from the start of firm), the number of board directors (Board, the logarithm of the number of board mem- bers), the ratio of independent directors in the whole board directors (Inde, the proportion of independent directors), the CEO and the chairman of board are held by the same person (Dua- liy, 1 for the CEO and he chairman of board are held by the same person and zero otherwise), the ownership of the largest shareholder (Top1, the share of the largest shareholder), some financial indicators (Lev, asset liability ratio; Cash, cash flow ratio; Tangiblity, fixed assets ratio), firms’ dummy and years’ dummy. It can be viewed the measurement scales in Table 8 and reported the means and standard deviations of the variables. In the regression models, this Study 2 computed variance inflation factors (VIFs), which ranged from 1.05 to 1.74 and were far below the cutoff of 10. Thus, multi- collinearity was not a major concern. This Study 2 also tested these hypotheses using panel analysis with standard errors clustered at firm level. It took several initiatives to address potential endogeneity in this analysis. First, the Study 2 lagged dependent variables by one year to minimize the possibility of reverse cau- sality. Second, the Study 2 controlled a set of executive-, board-, and company-level variables that may affect firm performance, simultaneously. Third, the Study 2 included year and indus- try fixed effect (i.e., 14 year-dummies, 19 industry-dummies, and 4341 firm-dummies) in the regression models to account for within-group variation over time and limit the potential bias caused by omitted variables. This empirical approach allowed the predicted mean of the dependent variable to vary across groups and thus controlled unobserved heterogeneity [103]. Table 8. Descriptive statistics. 3.7 Moderating variables in Study 2 The Study 2 constructed a panel data structure to measure dynamic capabilities in three dimensions: innovation capability, absorptive capability and adaptive capability [101,102]. The specific indicators are as follows. Innovation capability (IC) in Study 2 used the two indicators of sample firms’ annual R&D investment intensity and the proportion of technical employees for comprehensive evaluation. The values of two indicators were standardized separately and summed to the combined value PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 15 / 32 PLOS ONE How firms cope with social crisis of innovation capability: of innovation capability: IC ¼ xRD minRD maxRD minRD þ xIT minIT maxIT minIT Absorptive capacity (Absor) was measured using R&D expenditure intensity, i.e., the ratio of annual R&D expenditure to operating revenue of sample firms. Adaptive capability (Adapt) was measured by the coefficient of variation of three types of expenditure on R&D, capital and advertising reflecting the flexibility of resource allocation and showing firms’ adaptive capabilities. 3.9 Data analysis and results in Study 2 Table 9 reported the results of fixed-effect regression and logit regression. Model 1 displayed the main effect of IH on firm performance. The results of Model 2 showed that the estimated coefficient of IH was positive and significant at 5% level, indicating that IH was positively related with firms’ digital transformation. In other words, competitive pressure enhanced the degree of digital transformation among Chinese listed firms. And, the results of Model 3 showed that the IH coefficient was significantly negative, indicating that external industry competitive pressure reduced the level of firm performance, and suggesting that the mediating effect of digital transformation between competitive pressure and firm performance was signif- icant. Thus, H1 was not supported and H2, H3 and H4 were supported. Table 10 reported the results of estimating the interaction effects of dynamic capabilities on the relationship between digital transformation and firm performance. In Model 4, we found a positive and significant interaction between digital transformation and innovation capacity (b = 0.0575 p<0.05). We graphed the moderating effect of innovation capacity on the relation- ship between digital transformation and firm performance in Fig 4. As it shown, high levels of innovation capacity (the dotted line) strengthened the positive relationship between digital transformation and firm performance. Model 5 revealed a positive and significant coefficient estimate for the interaction between digital transformation and absorptive capability (b = 0.4719, p<0.1). We graphed the moderating effect of absorptive capacity on the relation- ship between digital transformation and firm performance in Fig 5. Similarly, the results of Model 6 showed that the estimated coefficients of digital transformation and adaptive capabil- ity were significantly positive (b = 0.0770, p<0.05). We graphed the moderating effect of adap- tive capability on the relationship between digital transformation and firm performance in Fig 6. In summary, the relationship between digital transformation and firm performance were positively moderated by absorptive capability, innovative capability and adaptive capability, thus H5, H6 and H7 were supported. 3.8 Control variables in Study 2 Variables Obs Mean SD Min Med Max TobinQ 31859 2.2468 1.4542 0.9189 1.7756 9.5098 DT 31859 1.1222 1.3389 0.0000 0.6931 4.9053 IH 31859 0.9426 0.0909 0.5343 0.9826 0.9919 Size 31859 22.0112 1.2482 19.6582 21.8339 25.9283 Age 31859 2.7861 0.3672 1.6094 2.8332 3.4657 Board 31859 2.2525 0.1787 1.7918 2.3026 2.7726 Dualiy 31859 0.2722 0.4451 0.0000 0.0000 1.0000 Inde 31859 0.3730 0.0528 0.3077 0.3333 0.5714 Top1 31859 0.3677 0.1497 0.0842 0.3544 0.7572 Lev 31859 0.4176 0.2047 0.0500 0.4108 0.8919 Cash 31859 0.9674 1.6980 0.0205 0.4032 11.2034 Tangiblity 31859 0.2259 0.1625 0.0037 0.1917 0.7146 https://doi org/10 1371/journal pone 0282854 t008 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 16 / 32 PLOS ONE How firms cope with social crisis To test research hypotheses, we proposed the regression models as follow: esearch hypotheses, we proposed the regression models as follow: Tobinqi;tþ1 ¼ a0 þ a1IHi;t þ ak X Controli;tþεi;t ð1Þ DTi;tþ1 ¼ b0 þ b1IHi;t þ bk X Controli;tþεi;t ð2Þ Tobinqi;tþ1 ¼ g0 þ g1IHi;t þ g1DTi;t þ ak X Controli;tþεi;t ð3Þ Tobinqi;tþ1 ¼ a0 þ a1IHi;t þ ak X Controli;tþεi;t ð1Þ ð1Þ ð1Þ ð2Þ Tobinqi;tþ1 ¼ g0 þ g1IHi;t þ g1DTi;t þ ak X Controli;tþεi;t ð3Þ ð3Þ In addition, the Study 2 tested these hypotheses with fixed-effect models estimating the influence of independent variables on firm performance and the integration effect of explana- tory variables. To examine the moderating hypotheses, this study executed a main model within moderates and related results were presented and tested via graphs, confidence intervals and robust standard errors. PLOS ONE PLOS ONE How firms cope with social crisis Table 9. The mediating mechanism of relationship between competitive pressure and firm performance. Model 1 Model 2 Model 3 Tobinq DT Tobinq IH -0.9271 0.3958 -0.9659* (-3.8618) (2.1368) (-4.0170) DT 0.0489* (3.6585) Size -0.5165* 0.2083* -0.5282* (-15.3695) (9.3110) (-15.6472) Age 0.7784* -0.0217 0.7732* (5.2059) (-0.1461) (5.1874) Board -0.1151 0.2298* -0.1300 (-0.9857) (2.7185) (-1.1133) Dualiy -0.0358 0.0164 -0.0358 (-1.2578) (0.6739) (-1.2604) Inde 0.2879 -0.0258 0.2845 (0.9769) (-0.1141) (0.9655) Top1 -0.6427* -0.3247 -0.6217* (-4.0323) (-2.5431) (-3.9132) Lev 0.0722 -0.0872 0.0767 (0.6420) (-1.0687) (0.6830) Cash -0.0831* -0.0292* -0.0812* (-8.5072) (-4.3319) (-8.2860) Tangiblity -0.1223 -0.4944* -0.0982 (-0.8777) (-5.3183) (-0.7084) Constant 11.9605* -4.8606* 12.2686* (12.7308) (-7.3140) (13.0785) Industry Yes Yes Yes Year Yes Yes Yes N 26846 26846 26846 Adj.R2 0.2667 0.3639 0.2676 Notes: *Significant at p<0.01, Significant at p<0.05, Significant at p<0.10, T-value shown in parentheses. Table 9. The mediating mechanism of relationship between competitive pressure and firm performance. It confirmed digital transformation improved the negative influence of competitive pressure on firm performance for Chinese listed firms from service industry in the COVID-19 pan- demic. Specifically, Fig 7 illustrated that the relationship between the driver variable of the COVID-19 pandemic and IH, which indicated a clear downward jump in the IH ratio to the right of the breakpoint. In Fig 8, it represented the relationship between the COVID-19 pan- demic and digital transformation, which showed a clear upward jump in digital transforma- tion near the discontinuity. Fig 9 reported the relationship between the COVID-19 pandemic and Tobin’s Q, and it demonstrated that there was also a clear upward jump in firm perfor- mance near the discontinuity. Combined with these figures, the initial indication disclosed the impact caused by the COVID-19 pandemic has an impact on performance by raising competi- tive pressures and promoting digital transformation. In Study 2, the optimal bandwidth at minimum mean square error was calculated following the specific method [104] and the regression results for the regression discontinuity were esti- mated. As shown in Table 11, the optimal bandwidth was 1.1602 years when the dependent variable was IH. The regression discontinuity treatment effect was not significant. When the Notes: Significant at p<0.01, Significant at p<0.05, Significant at p<0.10, T-value shown in parentheses. PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 3.10 Further research in Study 2 The Study 2 used the Regression Discontinuity Design (RDD) to examine the impact of exter- nal competitive pressures on firm performance using the breakpoint time of the outbreak of COVID-19 at the end of 2019. Due to the limitation of data, the Study 2 intercepted data from 2017–2020. The results were displayed by plotting the regression discontinuity. As shown in Figs 7, 8 and 9, there was a significant jump in competitive pressure, digital transformation and firm performance around the year of 2019 under the impact of the COVID-19 pandemic. PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 17 / 32 dependent variable was digital transformation, the optimal bandwidth was 1.8911 years. The regression discontinuity treatment effect was significantly positive at the 1% level. When the dependent variable was Tobin’s Q, the optimal bandwidth was 1.239 years. The regression PLOS ONE https://doi.org/10.1371/journal.pone.0282854.t009 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 18 / 32 It confirmed digital transformation improved the negative influence of competitive pressure on firm performance for Chinese listed firms from service industry in the COVID-19 pan- demic. Specifically, Fig 7 illustrated that the relationship between the driver variable of the COVID-19 pandemic and IH, which indicated a clear downward jump in the IH ratio to the right of the breakpoint. In Fig 8, it represented the relationship between the COVID-19 pan- demic and digital transformation, which showed a clear upward jump in digital transforma- tion near the discontinuity. Fig 9 reported the relationship between the COVID-19 pandemic and Tobin’s Q, and it demonstrated that there was also a clear upward jump in firm perfor- mance near the discontinuity. Combined with these figures, the initial indication disclosed the impact caused by the COVID-19 pandemic has an impact on performance by raising competi- tive pressures and promoting digital transformation. In Study 2, the optimal bandwidth at minimum mean square error was calculated following the specific method [104] and the regression results for the regression discontinuity were esti- mated. As shown in Table 11, the optimal bandwidth was 1.1602 years when the dependent variable was IH. The regression discontinuity treatment effect was not significant. When the PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 18 / 32 PLOS ONE PLOS ONE How firms cope with social crisis Table 10. The moderating effect of dynamic capability. Model 4 Model 5 Model 6 Tobinq Tobinq Tobinq DT 0.0173 0.0183 0.0411 (1.2710) (1.3251) (3.1403) IC 0.1010 (1.5771) DT×IC 0.0575 (2.0593) Absor 1.1913** (1.9868) DT×Absor 0.4719* (1.9083) Adapt 0.1466* (4.3025) DT×Adapt 0.0770 (1.9933) Size -0.4629* -0.4596* -0.6396* (-12.6114) (-12.5364) (-15.5548) Age 0.7068* 0.7133* 0.7397* (4.5532) (4.5991) (5.0373) Board -0.0631 -0.0668 -0.1473 (-0.5170) (-0.5472) (-1.2686) Dualiy -0.0539* -0.0534* -0.0362 (-1.7703) (-1.7572) (-1.2762) Inde 0.5156 0.5059 0.2405 (1.6150) (1.5859) (0.8289) Top1 -0.6399* -0.6455* -0.6042* (-3.5519) (-3.5799) (-3.8242) Lev 0.1658 0.1707 0.0815 (1.3337) (1.3691) (0.7267) Cash -0.0766* -0.0784* -0.0731* (-7.7149) (-7.9493) (-7.3627) Tangiblity -0.2031 -0.2257 -0.1354 (-1.3356) (-1.4764) (-0.9769) Constant 9.5935* 9.5397* 14.0899* (9.5304) (9.4717) (13.2794) Industry Yes Yes Yes Year Yes Yes Yes N 20989 20989 26846 Adj.R2 0.2249 0.2251 0.2682 Notes Table 10. The moderating effect of dynamic capability. dependent variable was digital transformation, the optimal bandwidth was 1.8911 years. The regression discontinuity treatment effect was significantly positive at the 1% level. When the dependent variable was Tobin’s Q, the optimal bandwidth was 1.239 years. The regression PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 19 / 32 PLOS ONE How firms cope with social crisis Fig 4. The moderating effect of innovation capability on firm performance. https://doi.org/10.1371/journal.pone.0282854.g004 Fig 4. The moderating effect of innovation capability on firm performance. htt //d i /10 1371/j l 0282854 004 Fig 4. The moderating effect of innovation capability on firm performance. Fig 4. The moderating effect of innovation capability on firm performance. https://doi.org/10.1371/journal.pone.0282854.g004 https://doi.org/10.1371/journal.pone.0282854.g004 discontinuity treatment effect was significantly positive at the 1% level. Given the results of the fixed-effect regression and regression discontinuity design, it can be concluded that digital transformation relieved the impact of the COVID-19 pandemic and improved firm perfor- mance, which means digital transformation was an effective strategic response to the COVID- 19 pandemic for Chinese listed firms from service industry. PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 4. Discussion and implications The COVID-19 pandemic creates huge challenges for most service businesses, as the uncertain social distancing restrictions and cities’ lockdowns decrease people’s consumption demand and consumption opportunities [5]. And, digital transformation has been widely considered a critical path to achieving competitive advantage in response to the public crisis for firms [17]. For example, during the COVID-19 pandemic in China, evidence from catering displayed that the delivery chain, including ordering online and takeaway, avoids physical contact between humans [39]. Evidence from hospitality and tourism exhibited that they mark safe attractions and safe hotels on related mobile applications via an online-offline integration featured func- tion and invest in new high-tech facilities to avoid physical contact between guests and employees [105,106]. Evidence from service employees showed that a virtual work environ- ment built by digital technology guarantees service businesses running when they are advised PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 20 / 32 PLOS ONE How firms cope with social crisis Fig 5. The moderating effect of absorptive capability on firm performance. https://doi.org/10.1371/journal.pone.0282854.g005 Fig 5. The moderating effect of absorptive capability on firm performance. https://doi.org/10.1371/journal.pone.0282854.g005 https://doi.org/10.1371/journal.pone.0282854.g005 to adopt safety measures [1]. However, most studies in the field of digital transformation in response to the COVID-19 outbreak have only focused on its outcomes, such as organizational efficiency [107], individual work performance [1] and organizational resilience [49], and ignored the antecedent of digital transformation caused by the COVID-19 outbreak. This quantitative research within two studies comprehensively demonstrates digital transforma- tion’s antecedent and outcome in the service industry. Based on the TOE framework, the results from Study 1 and Study 2 presented that intense competitive pressure significantly affects firm performance. Interestingly, the directions between competitive pressure and firm performance among SMEs and listed firms differ. In Study 1, the effect size f 2 value indicated that competitive pressure had a large and positive influence on firm performance among SEMs [89], while in Study 2, the result of fixed-effect regression indicated that competitive pressure had a negative influence on firm performance among listed firms. Both two studies found that digital transformation had a positive influence on firms, including SMEs and listed firms, thus, supporting H2. Similarly, both two studies confirmed that competitive pressure had a positive influence on digital transformation, thus H3 was supported. After testing these direct relationships, Study 1 and Study 2 reported digital transformation mediated the relationship between competitive pressure and firm perfor- mance. PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 4. Discussion and implications In other words, competitive pressure positively impacted firm performance via digital 21 / 32 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 PLOS ONE How firms cope with social crisis Fig 6. The moderating effect of adaptive capability on firm performance. https://doi.org/10.1371/journal.pone.0282854.g006 Fig 6. The moderating effect of adaptive capability on firm performance. https://doi org/10 1371/journal pone 0282854 g006 Fig 6. The moderating effect of adaptive capability on firm performance. Fig 6. The moderating effect of adaptive capability on firm performance. https://doi.org/10.1371/journal.pone.0282854.g006 https://doi.org/10.1371/journal.pone.0282854.g006 transformation for SMEs and listed firms in the COVID-19 pandemic, supporting H4. Fur- thermore, based on dynamic capabilities theory, Study 2 also deeply explored how digital transformation influence firm performance among listed firms. The results of Study 2 demon- strated that the effect of digital transformation on firm performance was moderated by firms’ absorptive capability, innovative capability and adaptive capability, so H5, H6 and H7 were supported. PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 4.1 Implications for research This research within two studies is grounded in and contributes to the theoretical perspectives of the TOE framework and the digital transformation literature. It is one of the first empirical studies to understand the antecedent and outcome of digital transformation among Chinese firms from the service industry. First, we identify competitive pressure as a significant anteced- ent of digital transformation for SMEs and listed firms in the COVID-19 pandemic. Prior studies affiliated with digital transformation during the COVID-19 pandemic have paid con- siderable attention to the outcomes of firms’ digital transformation [1,17,38,107]. Perceptive as these studies have been, the straightforward question of what factor may promote Chinese ser- vice firms’ digital transformation remains understudied in the COVID-19 pandemic. The TOE framework predicted firms’ environmental characteristics are critical determinants of 22 / 32 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 PLOS ONE How firms cope with social crisis Fig 7. The COVID-19 pandemic and IH. https://doi org/10 1371/journal pone 0282854 g007 Fig 7. The COVID-19 pandemic and IH. https://doi.org/10.1371/journal.pone.0282854.g007 https://doi.org/10.1371/journal.pone.0282854.g007 firm behaviors in adopting innovative strategies [22]. Combined with the TOE framework, we supplement the digital transformation research in the COVID-19 pandemic and extend the applied field of the TOE framework in a public crisis by confirming a direct relationship between competitive pressure and digital transformation. This research also extends our understanding of the relationship between competitive pres- sure and firm performance. The results from Study 1 and Study 2 reported an opposite and sig- nificant relationship between competitive pressure and firm performance. Study 1 presented that competitive pressure positively affects firm performance, which is in line with most past studies [21,108,109]. And, a few studies reported there is no significant relationship between competitive pressure and firm performance [110]. However, Study 2 found that competitive pressure negatively and significantly affected firm performance among Chinese listed firms from the service sector. A study on the adoption behavior of cloud computing in Germany exhibited that compared to SMEs, large firms thought cloud computing was more complex and more challenging to implement [111]. Similarly, a study also found that large firms paid more attention to potential risks than SMEs [112]. Thus, in the COVID-19 pandemic, large firms might more easily perceive stronger competitive pressure than SMEs, harming their 23 / 32 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 PLOS ONE How firms cope with social crisis Fig 8. The COVID-19 pandemic and digital transformation. PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 4.1 Implications for research Fig 8. The COVID-19 pandemic and digital transformation. https://doi.org/10.1371/journal.pone.0282854.g008 performance. Joining the above discussion, this study attempts to answer the debate about the relationship between competitive pressure and firm performance. Third, our theoretical predictions in Study 1 and Study 2 are confirmed through SEM and the fixed-effect regression. Both two studies demonstrated digital transformation improved firm performance, and there was a positive mediation effect of digital transformation between competitive pressure and firm performance. So, it can be concluded that the digital transfor- mation strategy is a practical strategic response to the COVID-19 pandemic for Chinese SMEs and large firms in the service industry. Two studies with two methods in this research boost our conclusions’ generalizability and guarantee the robustness of our theoretical predictions [73]. Finally, we are grounded in and extend to the theoretical application of the TOE and dynamic capabilities theory, providing evidence of the moderation effects of absorptive capa- bility, innovative capability and adaptive capability in the relationship between digital transfor- mation and firm performance among large Chinese firms from the service industry in Study 2. Most previous studies related to digital transformation have explored the mediation effect of dynamic capabilities [9,17,46]. Insightful as these scholars have been, they failed to test the moderation effects of dynamic capabilities theory on firms’ transformation process. This 24 / 32 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 PLOS ONE How firms cope with social crisis research further advances the literature by exploring three organizational abilities based on dynamic capabilities theory as three boundary conditions to the effect of digital transformation on organizational outcome. This also distinguishes the present research from studies on dynamic capabilities viewed as the mediating roles or antecedents of organizational behavior [12,48,49]. Thus, as absorptive capability, innovative capability and adaptive capability have Fig 9. The COVID-19 pandemic and firm performance. https://doi.org/10.1371/journal.pone.0282854.g009 Table 11. Regression discontinuity results. IH DT Tobinq Is the COVID-19 pandemic 0.0019 0.1001* 0.2816* bandwidth 1.1602 1.8911 1.239 N 13247 13247 13247 Notes Significant at p<0.01 Significant at p<0.05 Significant at p<0.10. https://doi.org/10.1371/journal.pone.0282854.t011 NE \| https://doi org/10 1371/journal pone 0282854 April 4 2023 25 / 32 Fig 9. The COVID-19 pandemic and firm performance. https://doi.org/10.1371/journal.pone.0282854.g009 Fig 9. The COVID-19 pandemic and firm performance. https://doi.org/10.1371/journal.pone.0282854.g009 research further advances the literature by exploring three organizational abilities based on dynamic capabilities theory as three boundary conditions to the effect of digital transformation on organizational outcome. 4.1 Implications for research This also distinguishes the present research from studies on dynamic capabilities viewed as the mediating roles or antecedents of organizational behavior [12,48,49]. Thus, as absorptive capability, innovative capability and adaptive capability have research further advances the literature by exploring three organizational abilities based on dynamic capabilities theory as three boundary conditions to the effect of digital transformation on organizational outcome. This also distinguishes the present research from studies on dynamic capabilities viewed as the mediating roles or antecedents of organizational behavior [12,48,49]. Thus, as absorptive capability, innovative capability and adaptive capability have Table 11. Regression discontinuity results. IH DT Tobinq Is the COVID-19 pandemic 0.0019 0.1001* 0.2816* bandwidth 1.1602 1.8911 1.239 N 13247 13247 13247 Notes *Significant at p<0.01 Significant at p<0.05 *Significant at p<0.10. https://doi.org/10.1371/journal.pone.0282854.t011 Table 11. Regression discontinuity results. 25 / 32 PLOS ONE \| https://doi.org/10.1371/journal.pone.0282854 April 4, 2023 PLOS ONE How firms cope with social crisis been found to influence the relationship between digital transformation and firm perfor- mance, Study 2 extends the application of dynamic capabilities theory. 4.2 Implications for practice The results of this research have some practical implications for both SMEs and large firms. First, senior managers and owners in SMEs should have confidence in the storm of the COVID-19 pandemic, and moderate optimism can help them perform better under strong competitive pressure [73]. The results in Study 1 confirmed competitive pressure can promote SMEs to generate better firm performance. Furthermore, according to the results of Study 1 and Study 2, SMEs and large firms should invest more resources in digital transformation, including big data, application usage, cloud technology, etc., to achieve better performance during the COVID-19 pandemic. Third, large firms should effectively develop their dynamic capabilities, enabling them to exploit digital technology better to improve their performance. Supporting information Supporting information S1 File. (PDF) S2 File. (XLSB) 4.3 Limitations and further research Certain fields of the outcomes displayed should be interpreted in light of their limitations. First, we collected SMEs’ data using a survey approach and collected large firms’ data using panel data. The operationalization of collecting data from SMEs and large firms should be con- sistent in future research. Due to the defect of accurate accounting in Chinese SMEs [72], Study 1 on SMEs relied on senior managers’ or owners’ subjective evaluations and perceptions of performance instead of the objective indicators to measure firms’ characteristics. Due to the defect of available resources, Study 2 on large firms adopted data from listed firms’ database CSMAR. Thus, further studies should adopt the same approach. For instance, scholars can ask CEOs or top management from large firms or listed firms directly about their perceived com- petitive pressure; scholars can find enough SMEs with accurate accounting processes to estab- lish statistical power. 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https://openalex.org/W3132955285	https://www.eurosurveillance.org/deliver/fulltext/eurosurveillance/26/15/eurosurv-26-15-1.pdf?itemId=%2Fcontent%2F10.2807%2F1560-7917.ES.2021.26.15.2100272&mimeType=pdf&containerItemId=content/eurosurveillance	English	null	Impact of January 2021 curfew measures on SARS-CoV-2 B.1.1.7 circulation in France	medRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	4,500	Modelling SARS-CoV-2 two-strain transmission dynamics y We extended a previously developed age-stratified transmission model that was used to assess the impact of interventions against the coronavirus dis- ease (COVID-19) pandemic in France in 2020 [9-11], fit- ted to hospital admission data and validated against the estimates from serological studies [9]. The model is discrete, stochastic, and integrates demography, age profile, social contacts and mobility data over time to account for social distancing measures. Details are provided in [9] and in the Supplement. The new B.1.1.7 variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (20I/501Y.V1, also called variant of concern (VOC) 202012/01) ini- tially detected in the United Kingdom [1,2] has rapidly expanded its geographical range across European countries [3]. A large-scale genome sequencing initia- tive was conducted in France on 7–8 January (Flash1 survey [4], the first of a set of surveys), reporting that 3.3% of all SARS-CoV-2 detections were B.1.1.7 viruses. To limit SARS-CoV-2 spread, strengthened social dis- tancing measures were implemented in the country in the month of January. Starting from a curfew at 20:00 in place since mid-December, the national authorities set a curfew at 18:00 from 2 January in several depart- ments with deteriorating indicators. This was extended nationwide on 16 January, with renewed recommenda- tions on teleworking and preventive measures. On 31 January, stricter controls of the compliance with the measures and closure of large commercial centres were applied. The model was extended to describe the circulation of two SARS-CoV-2 variants – the historical strains and B.1.1.7. Variant circulation was initialised on Flash1 data [4]: France (3.3%), the Île-de-France region report- ing the highest penetration (6.9%) and the Nouvelle Aquitaine region reporting one of the lowest penetra- tions (1.7%). We considered a 59% higher transmissi- bility (95% confidence interval (CI): 54–65) for B.1.1.7 estimated for France on Flash1 and Flash2 survey data [4] in line with previous estimates [1,2] and assumed complete cross-immunity [1,2]. The model was fitted to daily hospital admission data in each territory to evaluate the impact of curfew in January (weeks 2–5) and of curfew and school holidays in February (weeks 6–9, with regional calendars: weeks 6–7 in Nouvelle Aquitaine, weeks 7–8 in Île-de-France). We projected future trends in hospitalisations at the end of the holidays, assuming the estimated curfew conditions. Impact of January 2021 curfew measures on SARS- CoV-2 B.1.1.7 circulation in France Laura Di Domenico1 , Chiara E Sabbatini1 , Giulia Pullano1,2, Daniel Lévy-Bruhl³ , Vittoria Colizza1,4 1. INSERM, Sorbonne Université, Pierre Louis Institute of Epidemiology and Public Health, Paris, France 2. Orange Labs, Sociology and Economics of Networks and Services (SENSE), Chatillon, France 3. Santé publique France, Saint-Maurice, France 3 a p q a , a a , a 4. Tokyo Tech World Research Hub Initiative, Institute of Innovative Research, Tokyo Institute of Technology, Tokyo, Japan C d Vitt i C li ( itt i li i f ) 3 p q , , 4. Tokyo Tech World Research Hub Initiative, Institute of Innovative Research, Tokyo Institute of Technolog 3 p q 4. Tokyo Tech World Research Hub Initiative, Institute of Innovative Research, To Correspondence: Vittoria Colizza (vittoria.colizza@inserm.fr) Citation style for this article: Di D i L S bb ti i Citation style for this article: Di Domenico Laura, Sabbatini Chiara E, Pullano Giulia, Lévy-Bruhl Daniel, Colizza Vittoria. Impact of January 2021 curfew measures on circulation in France. Euro Surveill. 2021;26(15):pii=2100272. https://doi.org/10.2807/1560-7917.ES.2021.26.15.2100272 cle: atini Chiara E, Pullano Giulia, Lévy-Bruhl Daniel, Colizza Vittoria. Impact of January 2021 curfew measures on SARS-CoV-2 B.1.1.7 Surveill. 2021;26(15):pii=2100272. https://doi.org/10.2807/1560-7917.ES.2021.26.15.2100272 Article submitted on 09 Mar 2021 / accepted on 14 Apr 2021 / published on 15 Apr 2021 Following the spread of the SARS-CoV-2 B.1.1.7 vari- ant, social distancing was strengthened in France in January 2021. Using a two-strain mathematical model calibrated on genomic surveillance, we estimated that curfew measures allowed hospitalisations to plateau by decreasing transmission of the historical strains while B.1.1.7 continued to grow. School holidays appear to have further slowed down progression in February. Without progressively strengthened social distancing, a rapid surge of hospitalisations is expected, despite the foreseen increase in vaccination rhythm. that monitored variant frequency over time. Assessing the impact of implemented measures on the two strains through modelling is key for epidemic management. Rapid communication Rapid communication www.eurosurveillance.org Modelling SARS-CoV-2 two-strain transmission dynamics We also considered two scenarios corre- sponding to the strengthening and relaxation of social distancing measures, obtained with, respectively, a The presence of the B.1.1.7 variant on the territory, however, poses critical challenges to epidemic control. Its higher transmissibility represents a strong selective advantage that makes it prone to rapidly becoming the dominant strain [1,2,4-8]. Social distancing has a differ- ential impact on the variant and the historical strains, not visible before the implementation of surveillance Figure 1 Figure 1 Projected weekly hospital admissions due to SARS-CoV-2 historical strains and B.1.1.7 variant in France and two French regions, October 2020–April 2021 Projected weekly hospital admissions due to SARS-CoV-2 historical strains and B.1.1.7 variant in France and two French regions, October 2020–April 2021 Projected weekly hospital admissions due to SARS-CoV-2 historical strains and B.1.1.7 variant in France and two French regions, October 2020–April 2021 Projected weekly hospital admissions due to SARS-CoV-2 historical strains and B.1.1.7 variant in France and two French regions, October 2020–April 2021 O Week W O O O O O O O W W W O O W W week 4 Week Week O O D H B (100,000-200,000 rhythm) Oct Nov Dec Jan Feb Mar Apr Oct Nov Dec Jan Feb Mar Apr Oct Nov Dec Jan Feb Mar Apr (100,000-300,000 rhythm) (100,000 rhythm) (100,000-200,000 rhythm) (100,000-200,000 rhythm) O O O W W W W week 4 W O O W W O O Week Oct Nov Dec Jan Feb Mar Apr Week W O Oct Nov Dec Jan Feb Mar Apr O Week Oct Nov Dec Jan Feb Mar Apr O O D (100,000-300,000 rhythm) (100,000 rhythm) O H B (100,000-200,000 rhythm) (100,000-200,000 rhythm) (100,000-200,000 rhythm) Île-de-France; NAQ: Nouvelle Aquitaine; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SD: social distancing. From left to right, different scenarios after the winter school holidays are considered: strengthening of SD measures scenario, equivalent to the second lockdown; curfew scenario, estimated in week 4 and assuming no additional changes; relaxation of SD measures scenario, compatible with the situation at the start of the year before increased restrictions. www.eurosurveillance.org Figure 2 Projected prevalence of SARS-CoV-2 variant B.1.1.7 over time and estimated week when B.1.1.7 becomes the dominant strain in France and two French regions, 11 January–26 April 2021 Figure 2 Projected prevalence of SARS-CoV-2 variant B.1.1.7 over time and estimated week when B.1.1.7 becomes the dominant strain in France and two French regions, 11 January–26 April 2021 Projected prevalence of SARS-CoV-2 variant B.1.1.7 over time and estimated week when B.1.1.7 becomes the dominant strain in France and two French regions, 11 January–26 April 2021 I E V , D CI: confidence interval; IDF: Île-de-France; NAQ: Nouvelle Aquitaine; R: reproductive number; SARS-CoV-2: severe acute respiratory syndrome CI: confidence interval; IDF: Île-de-France; NAQ: Nouvelle Aquitaine; R: reproductive number; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2. Top: estimated percentage of B.1.1.7 cases over time, considering a 59% (95% CI: 54–65) higher transmissibility for t right: the different regions considered. Circles represent the estimates from the genomic surveillance in the Flash surveys (Flash1 on 7–8 January, Flash2 on 27 January, Flash3 on 16 February). The Flash2 estimate was not available for Nouvelle Aquitaine, as sequencing failed or was not performed on the majority of samples, preventing a reliable estimate. Squares represent results from weekly virological surveillance screening allowing the detection of the N501Y mutation specific to the B.1.1.7 variant (Supplement). We estimated 95% CI assuming a normal distribution. Flash3 survey estimates have larger CI as sequencing was performed on a smaller sample of viruses. Horizontal bars in weekly virological surveillance correspond to the week of reference. Bottom left: percentage increase in the overall effective reproductive number at the population level following an increased penetration of the variant, assuming a 59% higher transmissibility of the variant. Curves represent median values; shaded areas around the curves represent 95% probability ranges obtained from 500 stochastic simulations. Bottom right: estimated week of B.1.1.7 dominance, assuming a 59% (95% CI: 54–65) higher transmissibility for the variant, and considering the curfew scenario (middle point) and the scenarios with strengthening (lighter colour, left point) and relaxation (darker colour, right) of social distancing measures. Error bars represent 95% probability ranges. Grey diamonds correspond to the last week when the reported frequency was < 50% (open symbol) and to the first week with reported frequency > 50% (filled symbol). from week 10, and with a stable rhythm maintaining the administration of 100,000 doses per day over time. 15% reduction and increase of the effective reproduc- tive number estimated for the curfew. Figure 2 Projected prevalence of SARS-CoV-2 variant B.1.1.7 over time and estimated week when B.1.1.7 becomes the dominant strain in France and two French regions, 11 January–26 April 2021 Vaccination prioritised to older age groups was simu- lated according to the daily rhythm of 100,000 vaccine doses administered per day as recorded in February [12] and then increased to 200,000 (first) doses per day (accelerated rhythm) from week 10 following gov- ernment announcements (Supplement) [13]. This vac- cination roll-out was compared for sensitivity with an optimistic rhythm of 300,000 (first) doses per day Modelling SARS-CoV-2 two-strain transmission dynamics b h diff i id d The solid grey curve refers to the median overall trajectory, obtained under the accelerated vaccination roll-out (100,000–200,000 doses/day) and because of the concurrent circulation of the historical strains (dashed green curve) and the B.1.1.7 variant (solid green curve) assumed to have a 59% higher transmissibility (median value is estimated for France; increases corresponding to the 95% confidence intervals of this estimate are reported in the Supplement). A slower (100,000, dotted curve) and an optimistic (100,000–300,000, dot-dashed curve) vaccination rhythm are also shown (only median curves of the overall trajectories are shown, for the sake of visualisation). The shaded area around the curves corresponds to the 95% probability range obtained from 500 stochastic simulations. Dots correspond to weekly hospital admission data. The model is fitted to daily hospital admissions since the start of the epidemic, propagating uncertainty over time; the figure shows weekly data to simplify the visualisation. The second wave is shown for reference, together with indications of the timing of social distancing measures; the shaded rectangle around the second wave corresponds to the second lockdown. Months are indicated at the bottom of the x-axis (from October 2020 to April 2021). The results do not integrate the effect of more stringent measures recently implemented to curb the third wave, and do not include Easter school holidays, nor seasonal effects www.eurosurveillance.org 2 www.eurosurveillance.org Estimated impact of social distancing measures and resulting B.1.1.7 trends The estimated Re for the B.1.1.7 variant was largely above 1 in all regions: Re FR = 1.53 (95% CI: 1.51– 1.54), Re IDF = 1.43 (95% CI: 1.37–1.48), Re NAQ = 1.34 (95% CI: 1.22–1.43). School holidays further slowed down the historical strains, with Re FR = 0.78 (95% CI: 0.77–0.79) and Re IDF = 0.64 (95% CI: 0.62–0.67) in week 8 and Re NAQ = 0.65 (95% CI: 0.62–0.68) in week 7, but their effect was still insufficient against the variant (median Re FR  > 1 for the B.1.1.7 variant in all territories). Estimated impact of social distancing measures and resulting B.1.1.7 trends The variant was expected to increase by more than 55% the overall effective reproductive number by 18 March in Île-de-France, by 30 March in France and by 4 April in Nouvelle Aquitaine, compared with a situation without the variant. on the estimated prevalence of the B.1.1.7 variant on 7–8 January yielded by the Flash survey and on the reported hospitalisations in weeks 2–5, the model explains this plateau as the counterbalance between two opposing dynamics: a decreasing circulation of the historical strains (with effective reproductive num- bers Re FR = 0.96 (95% CI: 0.95–0.97), Re IDF = 0.90 (95% CI: 0.86–0.93) and Re NAQ = 0.84 (95% CI: 0.77–0.90) in week 4 for France (FR), Île-de-France (IDF) and Nouvelle Aquitaine (NAQ), respectively) vs the expo- nential increase of the variant (Figure 1). Curfew and other social distancing measures reduced the repro- ductive number of the historical strains below 1, but they were not enough to prevent the increasing B.1.1.7 dynamics. The estimated Re for the B.1.1.7 variant was largely above 1 in all regions: Re FR = 1.53 (95% CI: 1.51– 1.54), Re IDF = 1.43 (95% CI: 1.37–1.48), Re NAQ = 1.34 (95% CI: 1.22–1.43). School holidays further slowed down the historical strains, with Re FR = 0.78 (95% CI: 0.77–0.79) and Re IDF = 0.64 (95% CI: 0.62–0.67) in week 8 and Re NAQ = 0.65 (95% CI: 0.62–0.68) in week 7, but their effect was still insufficient against the variant (median Re FR  > 1 for the B.1.1.7 variant in all territories). on the estimated prevalence of the B.1.1.7 variant on 7–8 January yielded by the Flash survey and on the reported hospitalisations in weeks 2–5, the model explains this plateau as the counterbalance between two opposing dynamics: a decreasing circulation of the historical strains (with effective reproductive num- bers Re FR = 0.96 (95% CI: 0.95–0.97), Re IDF = 0.90 (95% CI: 0.86–0.93) and Re NAQ = 0.84 (95% CI: 0.77–0.90) in week 4 for France (FR), Île-de-France (IDF) and Nouvelle Aquitaine (NAQ), respectively) vs the expo- nential increase of the variant (Figure 1). Curfew and other social distancing measures reduced the repro- ductive number of the historical strains below 1, but they were not enough to prevent the increasing B.1.1.7 dynamics. Estimated impact of social distancing measures and resulting B.1.1.7 trends After an increase in registered hospital admissions from December (average 6,700 weekly hospitalisa- tions at national level) to early January (ca 9,000 in week 2), the epidemic plateaued in the second half of January, following increased restrictions. Based 3 Estimated week when COVID-19 hospitalisations will exceed the peak of the second wave in France and two French i M h M 2021 Estimated week when COVID-19 hospitalisations will exceed the peak of the second wave in France and two French regions, March–May 2021 Peak weekly hospitalisations in the second wave B.1.1.7 transmissibility advantage Strengthening of SD measures Curfew (as in week 4 of 2021) Relaxation of SD measures France 16,000 weekly hospitalisations 54% NR Week 13 (12–14) Week 12 (11–12) 59% After week 15 Week 13 (12–13) Week 12 (11–12) 65% Week 15 (13–16) Week 12 (11–13) Week 12 (11–12) Île-de-France 3,000 weekly hospitalisations 54% NR Week 12 (11–12) Week 11 (11–12) 59% NR Week 12 (11–12) Week 11 (11–12) 65% NR Week 11 (11–12) Week 11 (11–12) Nouvelle Aquitaine 800 weekly hospitalisations 54% NR Week 16 (14–20) Week 12 (12–14) 59% NR Week 15 (13–19) Week 12 (11–13) 65% Week 15 (12–17) Week 13 (12–15) Week 11 (11–12) COVID-19: coronavirus disease; NR: not reached; SD: social distancing. Projections after winter school holidays consider three scenarios. From left to right: strengthening of SD measures scenario, equivalent to the second lockdown in November 2020; curfew scenario, estimated in week 4 and assuming no additional changes; relaxation of SD measures scenario, compatible with the situation at the start of 2021 before increased restrictions. Results correspond to a 59%, 54% and 65% higher transmissibility of the variant compared with the previously circulating virus (median and values of the 95% confidence interval of the estimate for France) and to the 100,000–200,000 daily doses rhythm (see Supplement for details and results from other daily rhythms). Uncertainties (in brackets) refer to 95% probability ranges; NR indicates that the peak level is not predicted to be reached before week 16, the end of the time period under study. The results do not integrate the effect of more stringent measures recently implemented to curb the third wave, and do not include Easter school holidays, nor seasonal effects. www.eurosurveillance.org Projected hospitalisations under different scenarios Assuming that the epidemic progressed under the esti- mated epidemiological conditions of the curfew, and if vaccination was accelerated as announced, the model predicted that hospitalisation levels similar to the peak in November 2020 (close to hospital capacity in a num- ber of regions) would be reached around week 13 in France, week 12 in Île-de-France and week 15 in Nouvelle Aquitaine (Table). This was later confirmed by data, reporting that COVID-19 hospitalisations exceeded the peak of the second wave in week 12 in Île-de-France [15], then triggering more stringent interventions to curb the third wave. Under a partial relaxation of social distancing – approximately corresponding to the situ- ation at the turn of the year before stricter measures were implemented in January 2021 – these hospitali- sation levels were expected to be reached at least 1 week sooner. Implementation of stronger social dis- tancing immediately after school holidays, equivalent to the second lockdown, were predicted to maintain hospitalisations below the peak of the second wave in Île-de-France and Nouvelle Aquitaine when assuming the median estimate for the transmissibility advantage of the variant. However, this scenario predicted that a rise of hospitalisations comparable to the second wave was possible in France even under the acceler- ated vaccination rhythm (100,000–200,000 doses/ The projected increase in B.1.1.7 prevalence over time was confirmed by sequence data in the Flash2 and Flash3 surveys (conducted on 27 January and 16 February, respectively [4,14]) and by weekly virological surveillance data available starting week 6 detecting mutations specific to the variants of concern (Figure 2; Supplement). The data also matched the esti- mated date of B.1.1.7 dominance, showing that B.1.1.7 accounted for the majority of cases by week 8 in France and Nouvelle Aquitaine and by week 7 in Île-de-France. 4 www.eurosurveillance.org projections of the national model, unable to account for geographically targeted interventions put in place on 20 March. Our analysis based on the estimated trans- missibility advantage of B.1.1.7 at the national level [4] identified differences between the two regions Île-de- France and Nouvelle Aquitaine. These could be partly due to biases affecting Flash survey data and linked to reinforced tracing around suspected or confirmed variants. These biases are expected to be stronger in regions with small epidemics (Nouvelle Aquitaine) than in regions with higher incidence levels and variant penetration (Île-de-France). Also, small sample sizes in Nouvelle Aquitaine increase uncertainty around the estimates. Projected hospitalisations under different scenarios We did not consider in the main analysis additional differences between the variant and the historical strains beyond the transmissibility advan- tage. The recently estimated increased hospitalisation rate associated with B.1.1.7 infection [18] would lead to a higher peak in projected hospitalisations at the end of April 2021, after the period under study here (Supplement). We did not consider other variants that were estimated to have a lower penetration, but their circulation is likely to contribute to the expected surge in cases [19]. day). Accelerated and optimistic vaccination roll-out would reduce weekly hospitalisations by, respectively, ca 20% and 35% in week 16 compared with a stable vaccination campaign without acceleration. Conflict of interest None declared. Discussion We estimated that social distancing progressively implemented at the start of January 2021 was able to bring the effective reproductive number of the histori- cal SARS-CoV-2 strains below 1, leading to its decline, while B.1.1.7 cases increased exponentially. School holidays in February slowed down the dynamics fur- ther. The predicted growth in this variant’s frequency and the date when it became the dominant strain matched recent data. Social distancing was the combined effect of imposed restrictions [16] and individual responses to renewed recommendations on teleworking and risk reduction. Teleworking, estimated from mobility data [9,17], was maintained in January at the levels reached before releasing the second lockdown. Measures, however, were not enough to lead to a decline in the variant spread, not even under the additional impact of holidays, owing to this variant’s more efficient transmission. Accelerating vaccination roll-out is key [20], but even optimistic roll-out plans would require more rigorous and intensified social distancing than curfew alone to curb the B.1.1.7 epidemic. Strengthening social distancing through a mild lock- down, such as the one implemented to curb the second wave in November 2020, was predicted to allow certain regions to avoid a third wave of the same magnitude of the second, supported by acquired immunity (Île-de- France) or lower incidence levels (Nouvelle Aquitaine, having achieved a marked decrease in hospitalisations in February). The lockdown in November 2020 included restrictions on mobility, closure of non-essential shops, while school at all levels remained open. In our model, however, the strengthening of social distanc- ing measures was optimistically implemented imme- diately after school holidays in February and with a duration longer than a month. In the absence of these early measures, the model predicted that curfew alone would not be sufficient to prevent a rapid resurgence of hospitalisations, as was later confirmed by the rising third wave in France in March 2021. Projections on the week exceeding hospitalisation levels of the second peak in Île-de-France matched observations [15] before the authorities applied more stringent measures in the region and other territories on 20 March and extended them nationwide at the end of the month. Acknowledgements Acknowledgements Acknowledgements We thank Santé publique France for the access to Flash sur- vey preliminary estimates. Funding: This study was partially funded by: ANR projects DATAREDUX (ANR-19-CE46-0008-03), EVALCOVID-19 (ANR- 20-COVI-0007); EU H2020 grants MOOD (H2020-874850; pa- per MOOD 007) and RECOVER (H2020-101003589); REACTing COVID-19 modeling grant. www.eurosurveillance.org 2. Volz E, Mishra S, Chand M, Barrett JC, Johnson R, Geidelberg L, et al. Assessing transmissibility of SARS-CoV-2 lineage B.1.1.7 in England. Nature. 2021. https://doi.org/10.1038/s41586-021- 03470-x PMID: 33767447 Authors’ contributions VC conceived and designed the analysis. LDD, CES, GP per- formed the analysis. LDD, CES, GP, DLB, VC interpreted the results. VC wrote the manuscript. LDD, CES, GP, DLB, VC criti- cally revised the manuscript and approved its final version. Our study has limitations. Results are based on the estimated impact of curfew and scenarios anticipating a possible strengthening or relaxation of social distanc- ing. We did not consider changes in behaviour such as a progressive abandoning of teleworking because of fatigue or increased risk prevention triggered by grow- ing concern. We could not yet include the impact of more stringent measures recently put in place to curb the third wave that will inevitably alter the projected dynamics from the end of March. This will also affect License, supplementary material and copyright 5. Sabbatini CE, Di Domenico L, Pullano G, Colizza V. Estimated date of dominance of VOC-202012/01 strain in France and projected scenarios. Paris: EPIcx lab; 2021. 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https://openalex.org/W4310507030	https://www.research.ed.ac.uk/files/307996098/Language_Integrated_FOWLER_DOA10102022_AFV.pdf	English	null	Language-Integrated Query for Temporal Data	null	2,022	cc-by	14,488	Edinburgh Research Explorer Digital Object Identifier (DOI): 10.1145/3564719.3568690 Document Version: Peer reviewed version Published In: Proceedings of the 21st ACM SIGPLAN International Conference on Generative Programming: Concepts and Experiences General rights C i h f h General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Language-Integrated Query for Temporal Data Citation for published version: Fowler, S, Galpin, V & Cheney, J 2022, Language-Integrated Query for Temporal Data. in B Scholz & Y Kameyama (eds), Proceedings of the 21st ACM SIGPLAN International Conference on Generative Programming: Concepts and Experiences . ACM Association for Computing Machinery, pp. 5-19, The 21st International Conference on Generative Programming: Concepts & Experiences, 2022, Auckland, New Zealand, 6/12/22. https://doi.org/10.1145/3564719.3568690 Citation for published version: Digital Object Identifier (DOI): 10.1145/3564719.3568690 Link: Link to publication record in Edinburgh Research Explorer Document Version: Peer reviewed version Published In: Proceedings of the 21st ACM SIGPLAN International Conference on Generative Programming: Concepts and Experiences 1 Introduction Most interesting programs access or query data stored persis- tently, often in a database. Relational database management systems (RDBMSs) are the most popular option and provide a standard domain-specific language, SQL, for querying and modifying the data. Ideally, programmers can express the desired queries or updates declaratively in SQL and leave the database to decide how to answer queries or perform up- dates efficiently and safely (e.g. in the presence of concurrent accesses), but there are many pitfalls arising from interfacing with SQL from a general-purpose language, leading to the well-known impedance mismatch problem [10]. These diffi- culties range from run-time failures due to the generation of queries as unchecked SQL strings at runtime, to serious security vulnerabilities like SQL injection attacks [32]. Modern applications often manage time-varying data. De- spite decades of research on temporal databases, which cul- minated in the addition of temporal data operations into the SQL:2011 standard, temporal data query and manipula- tion operations are unavailable in most mainstream database management systems, leaving developers with the unenvi- able task of implementing such functionality from scratch. In this paper, we extend language-integrated query to sup- port writing temporal queries and updates in a uniform host language, with the language performing the required rewriting to emulate temporal capabilities automatically on any standard relational database. We introduce two core languages, 𝜆TLINQ and 𝜆VLINQ, for manipulating transaction time and valid time data respectively, and formalise exist- ing implementation strategies by giving provably correct semantics-preserving translations into a non-temporal core language, 𝜆LINQ. We show how existing work on query nor- malisation supports a surprisingly simple implementation strategy for sequenced joins. We implement our approach in the Links programming language, and describe a non-trivial case study based on curating COVID-19 statistics. Among the most successful approaches to overcome the above challenges, and the approach we build upon in this pa- per, is language-integrated query, exemplified by Microsoft’s popular LINQ for .NET [25, 36] and in a number of other language designs such as Links [9, 23] and libraries such as Quill [29]. Within this design space we focus on a family of techniques derived from foundational work by Buneman et al. Language-Integrated Query for Temporal Data Simon Fowler University of Glasgow UK simon.fowler@glasgow.ac.uk Vashti Galpin University of Edinburgh UK vashti.galpin@ed.ac.uk James Cheney University of Edinburgh UK jcheney@inf.ed.ac.uk Vashti Galpin University of Edinburgh UK vashti.galpin@ed.ac.uk Simon Fowler University of Glasgow UK simon.fowler@glasgow.ac.uk James Cheney University of Edinburgh UK jcheney@inf.ed.ac.uk 1 Introduction [3] on the nested relational calculus (NRC), a core query language with monadic collection types; work by Wong [37] on rewriting NRC expressions to normal forms that can be translated to SQL, which forms the basis of the approach taken in Links [8, 23] and has been adapted to F# [5]. CCS Concepts: • Software and its engineering →Do- main specific languages; • Information systems →Struc- tured Query Language; Temporal data. CCS Concepts: • Software and its engineering →Do- main specific languages; • Information systems →Struc- tured Query Language; Temporal data. Keywords: language-integrated query, temporal databases, domain-specific languages, multi-tier programming Many interesting database applications involve data that changes over time. Perhaps inevitably, temporal data man- agement [19] has a long history. Temporal databases pro- vide powerful features for querying and modifying data that changes over time, and are particularly suitable for mod- eling time-varying phenomena, such as enterprise data or evolving scientific knowledge, and supporting auditing and transparency about how the current state of the data was achieved, for example in financial or scientific settings. ACM ISBN 978-1-4503-9920-3/22/12...$15.00 https://doi.org/10.1145/3564719.3568690 Take down policy Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 © 2022 Copyright held by the owner/author(s). Publication rights licensed to ACM. ACM Reference Format: Simon Fowler, Vashti Galpin, and James Cheney. 2022. Language- Integrated Query for Temporal Data. In Proceedings of the 21st ACM SIGPLAN International Conference on Generative Program- ming: Concepts and Experiences (GPCE ’22), December 06–07, 2022, Auckland, New Zealand. ACM, New York, NY, USA, 15 pages. https: //doi.org/10.1145/3564719.3568690 Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page. Copyrights for components of this work owned by others than the author(s) must be honored. Abstracting with credit is permitted. To copy otherwise, or republish, to post on servers or to redistribute to lists, requires prior specific permission and/or a fee. Request permissions from permissions@acm.org. GPCE ’22, December 06–07, 2022, Auckland, New Zealand To illustrate how temporal databases work and why they are useful, consider the following toy example: a temporal to- do list. A temporal database can be conceptualised abstractly as a function mapping each possible time instant (e.g. times of day) to a database state [20]. For efficiency in the common case where most of the data is unchanged most of the time, temporal databases are often represented by augmenting each row with an interval timestamp indicating the time © 2022 Copyright held by the owner/author(s). Publication rights licensed to ACM. GPCE ’22, December 06–07, 2022, Auckland, New Zealand Simon Fowler, Vashti Galpin, and James Cheney Types 𝐴, 𝐵 ::= 𝐶\| 𝐴→𝐸𝐵\| Bag(𝐴) \| ( g ℓ: 𝐴) \| Table(𝐴) Base types 𝐶 ::= String \| Int \| Bool \| Time Effects 𝑒 ::= read \| write Effect sets 𝐸 Terms 𝐿, 𝑀, 𝑁 ::= 𝑥\| 𝑐\| 𝑡 \| 𝜆𝑥.𝑀\| 𝑀𝑁\| ⊙{−→ 𝑀} \| if 𝐿then 𝑀else 𝑁 \| * + \| 𝑀+ \| 𝑀⊎𝑁\| for (𝑥←𝑀) 𝑁 \| ( ℓ= 𝑀) \| 𝑀.ℓ\| now \| query 𝑀\| get 𝑀\| insert 𝑀values 𝑁 \| update (𝑥⇐𝐿) where 𝑀set ( ℓ= 𝑁) \| delete (𝑥⇐𝑀) where 𝑁 Figure 1. ACM Reference Format: Concretely, we make three main contributions: 1. Based on 𝜆LINQ (§2), a formalism based on the Nested Relational Calculus (NRC) [31], we introduce typed 𝜆-calculi to model queries and modifications on trans- action time (§3) and valid time (§4) databases. We give semantics-preserving translations to 𝜆LINQ for both. The problems of querying and updating temporal databases have been well-studied, leading to the landmark language de- sign TSQL2 [33] extending SQL. However, despite decades of effort, only limited elements of TSQL2 were eventually incor- porated into the SQL:2011 standard [22] and these features have not yet been widely adopted. Directly implementing temporal queries in SQL is possible, but painful: a TSQL2- style query or update operation may grow by a factor of 10 or more when translated to plain SQL, which leaves plenty of scope for error, and thus these powerful capabilities remain outside the grasp of non-experts. In this paper we take first steps towards reconciling temporal data management with language-integrated query based on query normalisation. We propose supporting temporal capabilities by translation to ordinary language-integrated query and hypothesise that this approach can make temporal data management safer, easier to use and more accessible to non-experts than the current state of affairs. As an initial test of this hypothesis we present a high-level design, a working implementation, and detail our experience with a nontrivial case study. 2. We show how existing work on query normalisation allows a surprisingly straightforward implementation strategy for sequenced joins (§5). 3. We implement our constructs in the Links functional web programming language, and describe a case study based on curating COVID-19 data (§6). Although the concepts behind translating temporal queries and updates into non-temporal core languages are well known [34], our core calculi 𝜆TLINQ and 𝜆VLINQ are novel and aid us in showing (to the best of our knowledge) the first correctness results for the translations. We relegate several details and all proofs to the extended version of the paper [14]. ACM Reference Format: Syntax of 𝜆LINQ Types 𝐴, 𝐵 ::= 𝐶\| 𝐴→𝐸𝐵\| Bag(𝐴) \| ( g ℓ: 𝐴) \| Table(𝐴) Base types 𝐶 ::= String \| Int \| Bool \| Time Effects 𝑒 ::= read \| write Effect sets 𝐸 Terms 𝐿, 𝑀, 𝑁 ::= 𝑥\| 𝑐\| 𝑡 \| 𝜆𝑥.𝑀\| 𝑀𝑁\| ⊙{−→ 𝑀} \| if 𝐿then 𝑀else 𝑁 \| + \| 𝑀+ \| 𝑀⊎𝑁\| for (𝑥←𝑀) 𝑁 \| ( ℓ= 𝑀) \| 𝑀.ℓ\| now \| query 𝑀\| get 𝑀\| insert 𝑀values 𝑁 \| update (𝑥⇐𝐿) where 𝑀set ( ℓ= 𝑁) \| delete (𝑥⇐𝑀) where 𝑁 Figure 1. Syntax of 𝜆LINQ period when the row is present. For technical reasons, closed- open intervals [𝑠𝑡𝑎𝑟𝑡,𝑒𝑛𝑑) representing the times start ≤𝑡< end are typically used [34]. In our temporal to-do list, the table at each time instant has fields “task”, a string field, and “done”, a Boolean field. Additional fields “start” and “end” record the endpoints of the time interval during which each row is to be considered part of the table. An end time of ∞(“forever”) reflects that there is no (currently known) end time and in the absence of other changes, the row is considered present from the start time onwards. For example, the table: task done start end Go shopping true 11:00 ∞ Cook dinner false 11:00 17:30 Walk the dog false 11:00 ∞ Cook dinner true 17:30 ∞ Watch TV false 11:00 19:00 Figure 1. Syntax of 𝜆LINQ of scientific database development, where data versioning for accountability and research integrity are very important needs that are not well-supported by conventional database systems [4]. Temporal data management has the potential to become a “killer app” for language-integrated query, and this paper takes a first but significant step towards this goal.i represents a temporal table where all four tasks were added at 11:00, with “Go shopping” being complete and the others incomplete; at 17:30 “Cook dinner” was marked “done” from then onwards, and at 19:00 “Watch TV” was removed from the table without being completed. Technically, note that this example interprets the time annotations as transaction time, that is, the times indicate when certain data was in the database; there is another dimension, valid time, and we will discuss both dimensions in greater detail later on. The overarching contribution of this paper is the first ex- tension of language-integrated query to support transaction time and valid time temporal data. 2 Background: Language-Integrated Query We begin by introducing a basic 𝜆-calculus, called 𝜆LINQ, to model language-integrated query in non-temporal databases. Our calculus is based heavily on the Nested Relational Cal- culus [31], with support for database modifications heavily inspired by the calculus of Fehrenbach and Cheney [13]. The calculus uses a type-and-effect system to ensure database accesses can occur in ‘safe’ places, i.e., that we do not attempt to perform a modification operation in the middle of a query. Effects include read (denoting a read from a database) and write (denoting an update to the database). Although both language-integrated query and temporal databases are now well-studied topics, we believe that their combination has never been considered before. Doing so has a number of potential benefits, including making the power of well-studied language designs such as TSQL2 more acces- sible to non-expert programmers, and providing a high-level abstraction that can be implemented efficiently in different ways. Our interest is particularly motivated by the needs Language-Integrated Query for Temporal Data GPCE ’22, December 06–07, 2022, Auckland, New Zealand insert 𝑀values 𝑁, requiring 𝑀to be a table reference of type Table(𝐴), and the inserted values 𝑁to be a bag of type Bag(𝐴). T-Update ensures the predicate and update terms are typable under an environment extended with the row type, and ensures that all updated values match the type expected by the row. Rule T-Delete is similar. All subterms used as predicates or used to calculate updated terms must be pure (that is, side-effect free), and all modifications have the write effect. Types 𝐴, 𝐵include base types 𝐶, effect-annotated function types 𝐴→𝐸𝐵, unordered collection types Bag(𝐴), record types (ℓ𝑖: 𝐴𝑖)𝑖denoting a record with labels ℓ𝑖and types 𝐴𝑖, and handles Table(𝐴) for tables containing records of type 𝐴. A record is a base record if it contains only fields of base type. We assume that the base types includes at least Bool and the Time type, which denotes (abstract) timestamps. Basic terms include variables 𝑥, constants 𝑐, table handles 𝑡, functions 𝜆𝑥.𝑀, application 𝑀𝑁, n-ary operations ⊙{−→ 𝑀}, and conditionals if 𝐿then 𝑀else 𝑁. We assume that the set of operations contains the usual unary and binary rela- tion operators, as well as the 𝑛-ary operations greatest and least on timestamps which return their largest and smallest arguments respectively. 2 Background: Language-Integrated Query We assume that the set of constants contains timestamps 𝜄of type Time, and two distinguished timestamps −∞and ∞of type Time, which denote the mini- mum and maximum timestamps respectively. The calculus also includes the empty multiset constructor +; the single- ton multiset constructor 𝑀+; multiset union 𝑀⊎𝑁; and comprehensions for (𝑥←𝑀) 𝑁. We write 𝑀1, . . . , 𝑀𝑛+ as sugar for 𝑀1 +⊎. . . ⊎𝑀𝑛+. We also have records (ℓ𝑖= 𝑀𝑖)𝑖 and projection 𝑀.ℓ. Term now retrieves the current time. 2.2 Semantics Figure 3 shows the syntax and typing rules of values, and the big-step semantics of 𝜆LINQ. Most rules are standard, and presented in the extended version. Values 𝑉,𝑊include func- tions, constants, tables, fully-evaluated records, and fully- evaluated bags e𝑉+. Unlike the unary bag constructor 𝑀+, fully-evaluated bags contain an unordered collection of val- ues. All values are pure. We write ⊕for record extension, e.g., (ℓ1 = 𝑀) ⊕(ℓ2 = 𝑁) = (ℓ1 = 𝑀, ℓ2 = 𝑁).f Since evaluation is effectful (as database operations can update the database), the evaluation judgement has the shape 𝑀⇓Δ,𝜄(𝑉, Δ′); this can be read “term 𝑀with current data- base Δ at time 𝜄evaluates to value 𝑉with updated database Δ′”. A database is a mapping from table names to bags of base records. To avoid additional complexity, we assume evaluation is atomic and does not update the time; one could straightforwardly update the semantics with a tick opera- tion without affecting any results. We write let 𝑥= 𝑀in 𝑁as the usual syntactic sugar for (𝜆𝑥.𝑁) 𝑀, and 𝑀; 𝑁as sugar for (𝜆𝑥.𝑁)𝑀for some fresh 𝑥. We also define where𝑀𝑁as sugar for if 𝑀then 𝑁else * +. We denote unordered collections with a tilde (e.g., e 𝑀), and ordered sequences with an arrow (e.g., −→ 𝑀). The get 𝑀term retrieves the contents of a table into a bag; insert 𝑀values 𝑁inserts values 𝑁into table 𝑀; update (𝑥⇐𝐿) where 𝑀set (ℓ𝑖= 𝑁𝑖)𝑖updates table 𝐿, updating the fields ℓ𝑖to 𝑁𝑖of each record 𝑥satisfying predicate 𝑀. The delete (𝑥⇐𝑀) where 𝑁term removes those records 𝑥in table 𝑀satisfying predicate 𝑁. We use two further evaluation relations for terms which do not write to the database: 𝑀⇓★ 𝜄𝑉states that a pure term 𝑀 (i.e., a term typable under an empty effect set) evaluates to 𝑉. Similarly, 𝑀⇓★ Δ,𝜄𝑉states that a term 𝑀which only performs the read effect evaluates to𝑉. We omit the definitions, which are similar to the evaluation relation but do not propagate database changes (since no changes can occur).u Syntax of values, operations on values, and value typing Syntax of values, operations on values, and value typing T-BagV (Γ ⊢𝑉𝑖:𝐴! ∅)𝑖 Γ ⊢e𝑉+ :Bag(𝐴) ! ∅ Big-step reduction rules 𝑀⇓Δ,𝜄(𝑉, Δ′) update (𝑥⇐𝐿) where 𝑀set (ℓ= 𝑁) ⇓Δ,𝜄((), Δ2) Figure 3. Semantics of 𝜆LINQ (selected) optimiser can takes advantage of any available integrity con- straints or statistics about the data. optimiser can takes advantage of any available integrity con- straints or statistics about the data. However, since updates and deletions in 𝜆LINQ are destruc- tive, we have lost the original data. Instead, let us see how this could be handled by a transaction-time database: task done start end Go shopping true 11:00 ∞ Cook dinner false 11:00 ∞ Walk the dog false 11:00 ∞ Watch TV false 11:00 ∞ 2.1 Typing Rules {write} ∪𝐸 T-Update Γ ⊢𝐿:Table(𝐴) ! 𝐸 𝐴= (ℓ𝑖: 𝐵𝑖)𝑖∈𝐼 Γ,𝑥: 𝐴⊢𝑀:Bool ! ∅ (𝑗∈𝐼∧Γ,𝑥: 𝐴⊢𝑁𝑗:𝐵𝑗! ∅)𝑗∈𝐽 Γ ⊢update (𝑥⇐𝐿) where 𝑀set (ℓ𝑗= 𝑁𝑗)𝑗∈𝐽:() ! {write} ∪𝐸 T-Delete Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ,𝑥: 𝐴⊢𝑁:Bool ! ∅ Γ ⊢delete (𝑥⇐𝑀) where 𝑁:() ! {write} ∪𝐸 Query typing A :: QType 𝐶:: QType (𝐴𝑖:: QType)𝑖 (ℓ𝑖: 𝐴𝑖)𝑖:: QType 𝐴:: QType Bag(𝐴) :: QType Figure 2. Typing rules for 𝜆LINQ (selected) Syntax of values, operations on values, and value typing Values 𝑉,𝑊 ::= 𝜆𝑥.𝑀\| 𝑐\| 𝑡\| (ℓ𝑖= 𝑉𝑖)𝑖\| e𝑉+ e𝑉+ ˆ⊎f 𝑊+ ≜ e𝑉· f 𝑊+ ((ℓ𝑖= 𝑉𝑖)𝑖∈𝐼with (ℓ𝑗= 𝑊𝑗)) ≜ (ℓ𝑖= 𝑉𝑖)𝑖∈𝐼\𝐽⊕(ℓ𝑗= 𝑊𝑗)𝑗∈𝐽 T-BagV (Γ ⊢𝑉𝑖:𝐴! ∅)𝑖 Γ ⊢e𝑉+ :Bag(𝐴) ! ∅ Big-step reduction rules 𝑀⇓Δ,𝜄(𝑉, Δ′) E-Now now ⇓Δ,𝜄(𝜄, Δ) E-Query 𝑀⇓★ Δ,𝜄𝑉 query 𝑀⇓Δ,𝜄(𝑉, Δ) E-Get 𝑀⇓Δ,𝜄(𝑡, Δ′) get 𝑀⇓Δ,𝜄(Δ′(𝑡), Δ′) E-Insert 𝑀⇓Δ,𝜄(𝑡, Δ′) 𝑁⇓★ 𝜄𝑉 insert 𝑀values 𝑁⇓Δ,𝜄((), Δ′[𝑡↦→Δ′(𝑡) ˆ⊎𝑉]) E-Update 𝐿⇓Δ,𝜄(𝑡, Δ1) Δ2 = Δ1[𝑡↦→upd(v) \| v ∈Δ1(𝑡)+] upd(v) = ( (v with ℓ= 𝑊) if 𝑀{v/𝑥} ⇓★ 𝜄true and (𝑁𝑖{v/𝑥} ⇓★ 𝜄𝑊𝑖)𝑖 v if 𝑀{v/𝑥} ⇓★ 𝜄false update (𝑥⇐𝐿) where 𝑀set ( ℓ= 𝑁) ⇓Δ,𝜄((), Δ2) E-Delete 𝑀⇓Δ,𝜄(𝑡, Δ1) Δ2 = Δ1[𝑡↦→v ∈Δ(𝑡) \| 𝑁{v/𝑥} ⇓★ 𝜄false+] delete (𝑥⇐𝑀) where 𝑁⇓Δ,𝜄((), Δ2) Figure 3. Semantics of 𝜆LINQ (selected) Γ ⊢𝑀:𝐴! 𝐸 T-Get Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ ⊢get 𝑀:Bag(𝐴) ! {read} ∪𝐸 T-Insert Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ ⊢𝑁:Bag(𝐴) ! ∅ Γ ⊢insert 𝑀values 𝑁:() ! {write} ∪𝐸 T-Delete Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ,𝑥: 𝐴⊢𝑁:Bool ! ∅ Γ ⊢delete (𝑥⇐𝑀) where 𝑁:() ! {write} ∪𝐸 Query typing A :: QType Figure 2. Typing rules for 𝜆LINQ (selected) Figure 2. Typing rules for 𝜆LINQ (selected) 2.1 Typing Rules 𝐸 T-Now Γ ⊢now:Time ! ∅ T-Get Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ ⊢get 𝑀:Bag(𝐴) ! {read} ∪𝐸 T-Insert Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ ⊢𝑁:Bag(𝐴) ! ∅ Γ ⊢insert 𝑀values 𝑁:() ! {write} ∪𝐸 T-Update Γ ⊢𝐿:Table(𝐴) ! 𝐸 𝐴= (ℓ𝑖: 𝐵𝑖)𝑖∈𝐼 Γ,𝑥: 𝐴⊢𝑀:Bool ! ∅ (𝑗∈𝐼∧Γ,𝑥: 𝐴⊢𝑁𝑗:𝐵𝑗! ∅)𝑗∈𝐽 Γ ⊢update (𝑥⇐𝐿) where 𝑀set (ℓ𝑗= 𝑁𝑗)𝑗∈𝐽:() ! {write} ∪𝐸 T-Delete Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ,𝑥: 𝐴⊢𝑁:Bool ! ∅ Γ ⊢delete (𝑥⇐𝑀) where 𝑁:() ! {write} ∪𝐸 Query typing A :: QType 𝐶:: QType (𝐴𝑖:: QType)𝑖 (ℓ𝑖: 𝐴𝑖)𝑖:: QType 𝐴:: QType Bag(𝐴) :: QType Figure 2. Typing rules for 𝜆LINQ (selected) Term typing Γ ⊢𝑀:𝐴! 𝐸 T-Query Γ ⊢𝑀:Bag(𝐴) ! 𝐸 𝐴:: QType 𝐸⊆{read} Γ ⊢query 𝑀:Bag(𝐴) ! 𝐸 T-Now Γ ⊢now:Time ! ∅ T-Get Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ ⊢get 𝑀:Bag(𝐴) ! {read} ∪𝐸 T-Insert Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ ⊢𝑁:Bag(𝐴) ! ∅ Γ ⊢insert 𝑀values 𝑁:() ! {write} ∪𝐸 T-Update Γ ⊢𝐿:Table(𝐴) ! 𝐸 𝐴= (ℓ𝑖: 𝐵𝑖)𝑖∈𝐼 Γ,𝑥: 𝐴⊢𝑀:Bool ! ∅ (𝑗∈𝐼∧Γ,𝑥: 𝐴⊢𝑁𝑗:𝐵𝑗! ∅)𝑗∈𝐽 Γ ⊢update (𝑥⇐𝐿) where 𝑀set (ℓ𝑗= 𝑁𝑗)𝑗∈𝐽:() ! {write} ∪𝐸 T-Delete Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ,𝑥: 𝐴⊢𝑁:Bool ! ∅ Γ ⊢delete (𝑥⇐𝑀) where 𝑁:() ! {write} ∪𝐸 Query typing A :: QType 𝐶:: QType (𝐴𝑖:: QType)𝑖 (ℓ𝑖: 𝐴𝑖)𝑖:: QType 𝐴:: QType Bag(𝐴) :: QType Figure 2. Typing rules for 𝜆LINQ (selected) Syntax of values, operations on values, and value typing Values 𝑉,𝑊 ::= 𝜆𝑥.𝑀\| 𝑐\| 𝑡\| (ℓ𝑖= 𝑉𝑖)𝑖\| e𝑉+ e𝑉+ ˆ⊎f 𝑊+ ≜ e𝑉· f 𝑊+ ((ℓ𝑖= 𝑉𝑖)𝑖∈𝐼with (ℓ𝑗= 𝑊𝑗)) ≜ (ℓ𝑖= 𝑉𝑖)𝑖∈𝐼\𝐽⊕(ℓ𝑗= 𝑊𝑗)𝑗∈𝐽 T-BagV (Γ ⊢𝑉𝑖:𝐴! ∅)𝑖 Γ ⊢e𝑉+ :Bag(𝐴) ! ∅ Big-step reduction rules 𝑀⇓Δ,𝜄(𝑉, Δ′) E-Now now ⇓Δ,𝜄(𝜄, Δ) E-Query 𝑀⇓★ Δ,𝜄𝑉 query 𝑀⇓Δ,𝜄(𝑉, Δ) E-Get 𝑀⇓Δ,𝜄(𝑡, Δ′) get 𝑀⇓Δ,𝜄(Δ′(𝑡), Δ′) E-Insert 𝑀⇓Δ,𝜄(𝑡, Δ′) 𝑁⇓★ 𝜄𝑉 insert 𝑀values 𝑁⇓Δ,𝜄((), Δ′[𝑡↦→Δ′(𝑡) ˆ⊎𝑉]) E-Update 𝐿⇓Δ,𝜄(𝑡, Δ1) Δ2 = Δ1[𝑡↦→upd(v) \| v ∈Δ1(𝑡)+] upd(v) = ( (v with ℓ= 𝑊) if 𝑀{v/𝑥} ⇓★ 𝜄true and (𝑁𝑖{v/𝑥} ⇓★ 𝜄𝑊𝑖)𝑖 v if 𝑀{v/𝑥} ⇓★ 𝜄false update (𝑥⇐𝐿) where 𝑀set ( ℓ= 𝑁) ⇓Δ,𝜄((), Δ2) E-Delete 𝑀⇓Δ,𝜄(𝑡, Δ1) Δ2 = Δ1[𝑡↦→v ∈Δ(𝑡) \| 𝑁{v/𝑥} ⇓★ 𝜄false+] delete (𝑥⇐𝑀) where 𝑁⇓Δ,𝜄((), Δ2) Figure 3. Semantics of 𝜆LINQ (selected) Term typing Γ ⊢𝑀:𝐴! 𝐸 T-Query Γ ⊢𝑀:Bag(𝐴) ! 𝐸 𝐴:: QType 𝐸⊆{read} Γ ⊢query 𝑀:Bag(𝐴) ! 𝐸 T-Now Γ ⊢now:Time ! ∅ T-Get Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ ⊢get 𝑀:Bag(𝐴) ! {read} ∪𝐸 T-Insert Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ ⊢𝑁:Bag(𝐴) ! ∅ Γ ⊢insert 𝑀values 𝑁:() ! 2.1 Typing Rules Rule E-Now returns the current timestamp. Rule E-Query evaluates the body of a query using the read-only evaluation relation. Rule E-Get evaluates its subject to a table reference, and then returns the contents of a table. Rule E-Insert does similar, evaluating the values to insert, and then appending them to the contents of the table. Rule E-Update iterates over a table, updating a record if the predicate matches, and leaving it unmodified if not. Finally, E-Delete deletes those rows satisfying the deletion predicate. Figure 2 shows the typing rules for 𝜆LINQ; the typing judge- ment has the shape Γ ⊢𝑀:𝐴! 𝐸, which can be read, “Under type environment Γ, term 𝑀has type 𝐴and produces ef- fects 𝐸”. The rules are implicitly parameterised by a data- base schema Σ mapping table names to types of the form Bag((ℓ𝑖: 𝐶𝑖)𝑖). Many rules are similar to those of the simply- typed 𝜆-calculus extended with monadic collection opera- tions [3] and a set-based effect system [24], and such standard rules are relegated to the extended version.u 𝜆LINQ enjoys a standard type soundness property. Rule T-Query states that a term query 𝑀is well-typed if 𝑀is of a query type: either a base type, a record type whose fields are query types, or a bag whose elements are query types. The term 𝑀must also only have read effects. These restrictions allow efficient compilation to SQL [6, 8]. Proposition 2.1 (Type soundness). If · ⊢𝑀:𝐴! 𝐸then there exists some 𝑉and Δ′ such that 𝑀⇓Δ,𝜄(𝑉, Δ′) and · ⊢𝑉:𝐴! ∅. Proposition 2.1 (Type soundness). If · ⊢𝑀:𝐴! 𝐸then there exists some 𝑉and Δ′ such that 𝑀⇓Δ,𝜄(𝑉, Δ′) and · ⊢𝑉:𝐴! ∅. More importantly, the type-and-effect system ensures that query and update expressions in 𝜆LINQ can be translated to SQL equivalent, even in the presence of higher-order func- tions and nested query results [5, 6, 8, 23]. This alternative implementation is equivalent to the semantics given here but usually much more efficient since the database query Rule T-Get states that get 𝑀has type Bag(𝐴) if 𝑀 has type Table(𝐴), and produces the read effect. Rule T- Table states that a table reference follows the type of the table in the schema. T-Insert types a database insert Simon Fowler, Vashti Galpin, and James Cheney GPCE ’22, December 06–07, 2022, Auckland, New Zealand Term typing Γ ⊢𝑀:𝐴! 𝐸 T-Query Γ ⊢𝑀:Bag(𝐴) ! 𝐸 𝐴:: QType 𝐸⊆{read} Γ ⊢query 𝑀:Bag(𝐴) ! 3.1 Calculus 𝜆TLINQ extends 𝜆LINQ with native support for transaction time operations; instead of performing destructive updates, we adjust the end timestamp of affected rows and, if necessary, insert updated rows. 𝜆TLINQ database entries are therefore of the form 𝑉[𝑉2,𝑉3) 1 , where 𝑉1 is the record data and 𝑉2 and 𝑉3 are the start and end timestamps. The (omitted) translation of 𝜆TLINQ types into 𝜆LINQ types is straightforward, save for TransactionTime(𝐴) which is translated into a record (data:J𝐴K, start:Time, end:Time). The same is true for the basic 𝜆-calculus terms. Timestamped rows 𝑉[𝑉start,𝑉end ) data are translated to fit the above record type; specifically, (data = J𝑉dataK, start = J𝑉startK, end = J𝑉endK). Figure 4 shows the syntax, typing rules, and semantics of 𝜆TLINQ; for brevity, we show the main differences to 𝜆LINQ. Period-stamped database rows are represented as triples data[start,end) with type TransactionTime(𝐴), where data has type 𝐴(the type of each record), and both start and end have type Time. A row is currently present in the database if its end value is ∞. We introduce three accessors: data 𝑀 extracts the data record from a transaction-time row; start𝑀 extracts the start time; and end𝑀extracts the end time. The get construct has an updated type to show that it returns a bag of TransactionTime(𝐴) values, rather than the records directly. The typing rules for the other constructs remain the same as in 𝜆LINQ. Remark 3.1. We have chosen to represent a 𝜆TLINQ period- stamped record as a nested record in 𝜆LINQ, but we could equally adopt a flat representation. Since we build on the Nested Relational Calculus, we take advantage of the abil- ity to return nested results; previous work on query shred- ding [6] allows us to flatten nested results in a later transla- tion pass. A nested representation is more convenient for our implementation and makes the translation simpler and more compositional, so we mirror this choice in the formalism. The accessor rules ET-Data, ET-Start, and ET-End project the expected component of the transaction-time row. Rule ET-Insert period-stamps each record to begin at the current time, and sets the end time to be ∞. Rule ET-Delete records deletions for current rows satisfying the deletion predicate. Instead of being removed from the database, the end times of the affected rows are set to the current times- tamp. Finally, rule ET-Update performs updates for current rows satisfying the update predicate. 3.2 Translation Since timestamps are either ∞or only refer to the past; users do not modify period stamps directly; and the informa- tion in the database grows monotonically, we can reconstruct the state of the database at any given time. We can implement the native transaction-time semantics for 𝜆TLINQ database operations by translation to 𝜆LINQ. Our translation adapts the SQL implementations of temporal operations by Snodgrass [34] to a language-integrated query setting. We prove correctness relative to the semantics. 𝜆TLINQ has a native representation of period-stamped data, whereas 𝜆LINQ requires table types to be flat. Consequently, the translations require knowledge of the types of each record. We therefore annotate each 𝜆TLINQ database term with the type of table on which it operates (this can be achieved through a standard type-directed translation pass). The (omitted) translation of 𝜆TLINQ types into 𝜆LINQ types is straightforward, save for TransactionTime(𝐴) which is translated into a record (data:J𝐴K, start:Time, end:Time). The same is true for the basic 𝜆-calculus terms. Timestamped rows 𝑉[𝑉start,𝑉end ) data are translated to fit the above record type; specifically, (data = J𝑉dataK, start = J𝑉startK, end = J𝑉endK). 𝜆TLINQ has a native representation of period-stamped data, whereas 𝜆LINQ requires table types to be flat. Consequently, the translations require knowledge of the types of each record. We therefore annotate each 𝜆TLINQ database term with the type of table on which it operates (this can be achieved through a standard type-directed translation pass). 3 Transaction Timei The first dimension of time we investigate is transaction time [35], which records how the state of the database changes over time. The key idea behind transaction time databases is that update operations are non-destructive, so we can always view a database as it stood at a particular point in time. There are several methods by which we can maintain the temporal information in the database: for example we could maintain a tracking log which records each entry, or we could use various temporal partitioning strategies [34]. In this paper, we use a period-stamped representation, where each record in the database is augmented with fields delimiting the interval when the record was present in the database. Let us illustrate with the to-do list example from the in- troduction. The original table is on the left. The table after making some changes is shown on the right. The time period is a closed-open representation, meaning that each row is in the database from (and including) the time stated in the start column, and is in the database up to (but not including) the time stated in the end column. We also assume that 𝑠𝑡𝑎𝑟𝑡< 𝑒𝑛𝑑always holds. making some changes is shown on the right. task done Go shopping true Cook dinner false Walk the dog false Watch TV false task done Go shopping true Cook dinner true Walk the dog false GPCE ’22, December 06–07, 2022, Auckland, New Zealand Language-Integrated Query for Temporal Data current(tbl) = task done Go shopping true Cook dinner true Walk the dog false Here, our database states that all four tasks were entered into the database at 11:00. However, if we then decide to check off “Cook dinner” at 17:30 and delete “Watch TV” at 19:00, we obtain the table shown in the introduction. 3.1 Calculus Instead of changing a record directly, the upd definition generates two records: the previous record, closed off at the current timestamp, and the new record with updated values, starting from the current timestamp and with end field ∞. Returning to our running example, define at(tbl, time) to return all records in tbl start- ing before time and ending after time. We can then query the database as it stood at 18:00: We define the flattening of a 𝜆TLINQ row and database as: We define the flattening of a 𝜆TLINQ row and database as: We define the flattening of a 𝜆TLINQ row and database as: ↓((ℓ𝑖= 𝑉𝑖)[𝑊1,𝑊2) 𝑖 ) ≜ (ℓ𝑖= 𝑉𝑖)𝑖⊕(start = 𝑊1, end = 𝑊2) ↓Δ ≜ [𝑡↦→f ↓𝐷+ \| 𝑡↦→e𝐷+ ∈Δ] ↓((ℓ𝑖= 𝑉𝑖)[𝑊1,𝑊2) 𝑖 ) ≜ (ℓ𝑖= 𝑉𝑖)𝑖⊕(start = 𝑊1, end = 𝑊2) ↓Δ ≜ [𝑡↦→f ↓𝐷+ \| 𝑡↦→e𝐷+ ∈Δ] Figure 5 shows the translation of 𝜆TLINQ terms into 𝜆LINQ. The translation makes use of three auxiliary definitions. Eta-expansion 𝜂(𝑥, eℓ) eta-expands a variable of record type with respect to a sequence of labels, and restrict(𝑥, eℓ, 𝑀) ap- plies an eta-expanded record to a 𝑀under a 𝜆-binder for 𝑥(required since the 𝜆TLINQ predicates expect just the data from the record, and not the period-stamping fields). Finally, isCurrent(𝑀) tests whether the end field of 𝑀is ∞. Since timestamped rows are translated to records, the tem- poral accessor functions are translated to record projection. The get function is translated to retrieve the flattened 𝜆LINQ representation of the table and pack it via 𝜂-expansion into a bag of nested records. The translation of insert extends the provided values with a start field referring to the current timestamp and an end field set to ∞, before inserting them into the database. the database as it stood at 18:00: at(t, time) ≜ for (𝑥←get t) where (start 𝑥≤time ∧time < end 𝑥) data 𝑥+ at(tbl, 18:00) task done Go shopping true Cook dinner true Walk the dog false Watch TV false Let current(t) = at(t, ∞) return the current snapshot of 𝑡. We can then query the current snapshot of the database: at(t, time) ≜ for (𝑥←get t) where (start 𝑥≤time ∧time < end 𝑥) data 𝑥+ at(tbl, 18:00) task done Go shopping true Cook dinner true Walk the dog false Watch TV false Let current(t) = at(t, ∞) return the current snapshot of 𝑡. We can then query the current snapshot of the database: Simon Fowler, Vashti Galpin, and James Cheney GPCE ’22, December 06–07, 2022, Auckland, New Zealand dditional Syntax for 𝜆TLINQ Types 𝐴, 𝐵 ::= · · · \| TransactionTime(𝐴) Timestamped rows 𝐷 ::= 𝑉[𝑉2,𝑉3) 1 Terms 𝐿, 𝑀, 𝑁 ::= · · · \| data 𝑀\| start 𝑀\| end 𝑀 Values 𝑉,𝑊 ::= · · · \| 𝐷 odified Typing Rules for 𝜆TLINQ Γ ⊢𝑉:𝐴! 𝐸 Γ ⊢𝑀:𝐴! 𝐸 T-Row Γ ⊢𝑉1:𝐴! ∅ Γ ⊢𝑉2:Time ! ∅ Γ ⊢𝑉3:Time ! ∅ Γ ⊢𝑉[𝑉2,𝑉3) 1 :TransactionTime(𝐴) ! ∅ T-Data Γ ⊢𝑀:TransactionTime(𝐴) ! 3.3 Metatheory We restrict our attention to well formed rows and databases, where the start timestamp is less than the end timestamp. Unlike in a transaction time database, the database does not necessarily grow monotonically since we can apply destructive updates and deletions. Furthermore, whereas in a transaction time database timestamps can only refer to the past (or ∞), in a valid time database we may state that a row is valid until some specific point in the future (for example, the end of a fixed-term employment contract). A further difference from transaction time databases is that users can modify timestamps directly, and can also apply updates and deletions over a time period. Let us illustrate with the ‘employees’ table of an HR database: Definition 3.1 (Well formed rows and databases). A data- base Δ is well formed, written wf(Δ), if for each timestamped row 𝑉[𝑉start,𝑉end ) data in Δ we have that 𝑉start < 𝑉end. Definition 3.2 (Maximum timestamp). The maximum times- tamp of a collection of records e𝐷is defined as the maximum timestamp in the set {𝑉end \| 𝑉[𝑉start,𝑉end ) data ∈e𝐷,𝑉end ≠∞}, or −∞if the set is empty. The maximum timestamp of a database Δ, written max(Δ), is the maximum timestamp of all its constituent tables. The maximum timestamp of a database Δ, written max(Δ), is the maximum timestamp of all its constituent tables. 4 Valid Time The other dimension of time we will look at is valid time, which tracks when something is true in the domain being modelled. Each timestamp therefore defines the period of validity (PV) of each record. We can now show that the translation is correct: We can now show that the translation is correct: A delete is translated as a 𝜆LINQ update operation, which sets the end record of each affected row to the current times- tamp. An update is translated in three steps: querying the database to obtain the affected current records, with updated values and timestamps; updating the database to close off the existing affected rows; and materialising the insertion. We can now show that the translation is correct: Theorem 3.1. If · ⊢𝑀:𝐴! 𝐸and 𝑀⇓T Δ,𝜄(𝑉, Δ′) where wf(Δ) and max(Δ) ≤𝜄, then J𝑀K ⇓↓Δ,𝜄(J𝑉K, ↓Δ′). We define the flattening of a 𝜆TLINQ row and database as: 𝐸 Γ ⊢data 𝑀:𝐴! 𝐸 T-Start Γ ⊢𝑀:TransactionTime(𝐴) ! 𝐸 Γ ⊢start 𝑀:Time ! 𝐸 T-End Γ ⊢𝑀:TransactionTime(𝐴) ! 𝐸 Γ ⊢end 𝑀:Time ! 𝐸 T-Get Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ ⊢get 𝑀:Bag(TransactionTime(𝐴)) ! {read} ∪𝐸 mantics for 𝜆TLINQ database operations 𝑀⇓T Δ,𝜄(𝑉, Δ′) ET-Data 𝑀⇓T Δ,𝜄(𝑉[𝑉2,𝑉3) 1 , Δ′) data 𝑀⇓T Δ,𝜄(𝑉1, Δ′) ET-Start 𝑀⇓T Δ,𝜄(𝑉[𝑉2,𝑉3) 1 , Δ′) start 𝑀⇓T Δ,𝜄(𝑉2, Δ′) ET-End 𝑀⇓T Δ,𝜄(𝑉[𝑉2,𝑉3) 1 , Δ′) end 𝑀⇓T Δ,𝜄(𝑉3, Δ′) ET-Insert 𝑀⇓T Δ,𝜄(𝑡, Δ1) 𝑁⇓★ 𝜄e𝑉+ vs = v [𝜄,∞) \| v ∈e𝑉+ Δ2 = Δ′[𝑡↦→Δ1(𝑡) ˆ⊎vs] insert 𝑀values 𝑁⇓T Δ,𝜄((), Δ2) ET-Delete 𝑀⇓T Δ,𝜄(𝑡, Δ1) Δ2 = Δ1[𝑡↦→del(v) \| v ∈Δ1(𝑡)+] del(data[start,end) ) = ( data[start,𝜄) if end = ∞and 𝑁{data/𝑥} ⇓★ 𝜄true data[start,end) otherwise delete (𝑥⇐𝑀) where 𝑁⇓T Δ,𝜄((), Δ2) ET-Update 𝐿⇓T Δ,𝜄(𝑡, Δ1) Δ2 = Δ1[𝑡↦→ˆ Ú * upd(v) \| v ∈Δ1(𝑡)+] upd(data[start,end) ) =   data[start,𝜄), (data with ℓ= 𝑉) [𝜄,∞)+ if 𝑀{data/𝑥} ⇓★ 𝜄true, (𝑁𝑖{data/𝑥} ⇓★ 𝜄𝑉𝑖)𝑖, and end = ∞ data[start,end) + otherwise update (𝑥⇐𝐿) where 𝑀set ( ℓ= 𝑁) ⇓T Δ,𝜄((), Δ2) Figure 4. Syntax, typing rules, and semantics of 𝜆TLINQ Semantics for 𝜆TLINQ database operations 𝑀⇓T Δ,𝜄(𝑉, Δ′) Figure 4. Syntax, typing rules, and semantics of 𝜆TLINQ Again, 𝜆TLINQ enjoys type soundness. We can record this using a sequenced update query: update sequenced (𝑥⇐employees) between 2023 and 2028 where (𝑥.name = “Dolores”) set (position = “Head of School”) with the resulting table being: name position salary start end Dolores Professor 70000 2022 2023 Dolores Head of School 70000 2023 2028 Dolores Professor 70000 2028 ∞ · · · · · · · · · · · · · · · update sequenced (𝑥⇐employees) between 2023 and 2028 where (𝑥.name = “Dolores”) set (position = “Head of School”) update sequenced (𝑥⇐employees) between 2023 and 2028 where (𝑥.name = “Dolores”) set (position = “Head of School”) with the resulting table being: g g name position salary start end Dolores Professor 70000 2022 2023 Dolores Head of School 70000 2023 2028 Dolores Professor 70000 2028 ∞ · · · · · · · · · · · · · · · Since the period of applicability of the sequenced update is entirely contained within the period of validity of Dolores’s row, we end up with three rows: the unchanged record before and after the PA, and the updated record during the PA. We also allow a sequenced deletion, and a sequenced insertion, where each record’s period of validity is given explicitly. Additionally, suppose that all PhD students are to be given a 1-year extension due to the disruption caused by the pan- demic; in this case we want to change the period of validity directly. This is known as a nonsequenced update. We cannot express this modification using either current or sequenced modifications since we must calculate the each row’s new end date from its previous end date. We can write the modi- fication as follows, noting that we can both read from, and write to, the period of validity directly: update nonsequenced (𝑥⇐employees) where ((data 𝑥).position = “PhD student”) set () valid from (start 𝑥) to (end 𝑥+ 1)i update nonsequenced (𝑥⇐employees) where ((data 𝑥).position = “PhD student”) set () valid from (start 𝑥) to (end 𝑥+ 1)i update nonsequenced (𝑥⇐employees) where ((data 𝑥).position = “PhD student”) set () valid from (start 𝑥) to (end 𝑥+ 1)i The resulting table shows that the ‘end’ field of Bob’s and Charles’ records are updated to 2024 and 2023 respectively: Figure 5. Again, 𝜆TLINQ enjoys type soundness. Translation from 𝜆TLINQ into 𝜆LINQ g g gu y p Auxiliary Definitions 𝜂(𝑥, eℓ) ≜ (ℓ𝑖= 𝑥.ℓ𝑖)𝑖 restrict(𝑥, eℓ, 𝑀) ≜ (𝜆𝑥.𝑀) 𝜂(eℓ,𝑥) isCurrent(𝑀) ≜ 𝑀.end = ∞ Translation on database terms Jdata 𝑀K = J𝑀K.data Jstart 𝑀K = J𝑀K.start Jend 𝑀K = J𝑀K.end Jget(ℓ𝑖:𝐴𝑖)𝑖𝑀K = query for (𝑥←get J𝑀K) (data = 𝜂(𝑥, eℓ), start = 𝑥.start, end = 𝑥.end)+ Jinsert(ℓ𝑖:𝐴𝑖)𝑖𝑀values 𝑁K = let rows = for (𝑥←J𝑁K) 𝜂(𝑥, eℓ) ⊕(start = now, end = ∞)+ in insert J𝑀K values rows Jdelete(ℓ𝑖:𝐴𝑖)𝑖(𝑥⇐𝑀) where 𝑁K = update (𝑥⇐J𝑀K) where (restrict(𝑥, eℓ, J𝑁K) ∧isCurrent(𝑥)) set (end = now) Jupdate(ℓ𝑖:𝐴𝑖)𝑖∈𝐼(𝑥⇐𝐿) where 𝑀set (ℓ= 𝑁𝑗)𝑗∈𝐽K = let tbl = J𝐿K in let affected = query for (𝑥←get tbl) where ((restrict(𝑥, {ℓ𝑖}𝑖∈𝐼, J𝑀K) ∧isCurrent(𝑥))) H (ℓ𝑖= 𝑥.ℓ𝑖)𝑖∈𝐼\𝐽⊕ (ℓ𝑗= restrict(𝑥, {ℓ𝑖}𝑖∈𝐼, J𝑁𝑗K))𝑗∈𝐽⊕ (start = now, end = ∞) I in update (𝑥⇐tbl) where (restrict(𝑥, eℓ, J𝑀K) ∧isCurrent(𝑥)) set (end = now); insert tbl values affected Figure 5. Translation from 𝜆TLINQ into 𝜆LINQ Auxiliary Definitions 𝜂(𝑥, eℓ) ≜ (ℓ𝑖= 𝑥.ℓ𝑖)𝑖 restrict(𝑥, eℓ, 𝑀) ≜ (𝜆𝑥.𝑀) 𝜂(eℓ,𝑥) isCurrent(𝑀) ≜ 𝑀.end = ∞ Translation on database terms Jdata 𝑀K = J𝑀K.data Jstart 𝑀K = J𝑀K.start Jend 𝑀K = J𝑀K.end Jget(ℓ𝑖:𝐴𝑖)𝑖𝑀K = query for (𝑥←get J𝑀K) (data = 𝜂(𝑥, eℓ), start = 𝑥.start, end = 𝑥.end)+ Jinsert(ℓ𝑖:𝐴𝑖)𝑖𝑀values 𝑁K = let rows = for (𝑥←J𝑁K) 𝜂(𝑥, eℓ) ⊕(start = now, end = ∞)+ in insert J𝑀K values rows Jdelete(ℓ𝑖:𝐴𝑖)𝑖(𝑥⇐𝑀) where 𝑁K = update (𝑥⇐J𝑀K) where (restrict(𝑥, eℓ, J𝑁K) ∧isCurrent(𝑥)) set (end = now) Jupdate(ℓ𝑖:𝐴𝑖)𝑖∈𝐼(𝑥⇐𝐿) where 𝑀set (ℓ= 𝑁𝑗)𝑗∈𝐽K = let tbl = J𝐿K in let affected = query for (𝑥←get tbl) where ((restrict(𝑥, {ℓ𝑖}𝑖∈𝐼, J𝑀K) ∧isCurrent(𝑥))) H (ℓ𝑖= 𝑥.ℓ𝑖)𝑖∈𝐼\𝐽⊕ (ℓ𝑗= restrict(𝑥, {ℓ𝑖}𝑖∈𝐼, J𝑁𝑗K))𝑗∈𝐽⊕ (start = now, end = ∞) I in update (𝑥⇐tbl) where (restrict(𝑥, eℓ, J𝑀K) ∧isCurrent(𝑥)) set (end = now); insert tbl values affected Figure 5. Translation from 𝜆TLINQ into 𝜆LINQ Auxiliary Definitions A powerful feature of valid-time databases is the ability to perform sequenced modifications, which apply an update or deletion over a particular period of applicability (PA). In fact, current modifications are a special case of sequenced modifications applied from now until ∞. Suppose that Do- lores has agreed to act as Head of School between 2023 and 2028. Again, 𝜆TLINQ enjoys type soundness. Translation from 𝜆TLINQ into 𝜆LINQ name position salary start end Bob PhD Student 15000 2019 2024 Charles PhD Student 15000 2018 2023 · · · · · · · · · · · · · · · The first modification is to hire Dolores as a professor, on an open-ended contract. As this is an insertion operation on the database at the current moment in time, it is known as a current insertion. We can write the following query: insert employees values The 𝜆VLINQ calculus gives a direct semantics to valid time op- erations. Like 𝜆TLINQ, 𝜆VLINQ has a native notion of a period- stamped database row, with accessors for the data and each timestamp; the typing rules, reduction rules, and translations are straightforward adaptations of those in 𝜆TLINQ. Next, we want to record that Alice has resigned. We can write the following current deletion query: Next, we want to record that Alice has resigned. We can write the following current deletion query: 4.1 Calculus insert employees values (name = “Dolores”, position = “Professor”, salary = 70000) Again, 𝜆TLINQ enjoys type soundness. name position salary start end Alice Lecturer 40000 2010 2018 Alice Senior Lecturer 50000 2018 ∞ Bob PhD Student 15000 2019 2023 Charles PhD Student 15000 2018 2022 Proposition 3.2 (Type soundness (𝜆TLINQ)). If · ⊢𝑀:𝐴! 𝐸, then given a wf(Δ) and 𝜄such that max(Δ) ≤𝜄, then there exists some 𝑉and well formed Δ′ such that 𝑀⇓T Δ,𝜄(𝑉, Δ′). Proposition 3.2 (Type soundness (𝜆TLINQ)). If · ⊢𝑀:𝐴! 𝐸, then given a wf(Δ) and 𝜄such that max(Δ) ≤𝜄, then there exists some 𝑉and well formed Δ′ such that 𝑀⇓T Δ,𝜄(𝑉, Δ′). Language-Integrated Query for Temporal Data GPCE ’22, December 06–07, 2022, Auckland, New Zealand g g g Qu y p Auxiliary Definitions 𝜂(𝑥, eℓ) ≜ (ℓ𝑖= 𝑥.ℓ𝑖)𝑖 restrict(𝑥, eℓ, 𝑀) ≜ (𝜆𝑥.𝑀) 𝜂(eℓ,𝑥) isCurrent(𝑀) ≜ 𝑀.end = ∞ Translation on database terms Jdata 𝑀K = J𝑀K.data Jstart 𝑀K = J𝑀K.start Jend 𝑀K = J𝑀K.end Jget(ℓ𝑖:𝐴𝑖)𝑖𝑀K = query for (𝑥←get J𝑀K) (data = 𝜂(𝑥, eℓ), start = 𝑥.start, end = 𝑥.end)+ Jinsert(ℓ𝑖:𝐴𝑖)𝑖𝑀values 𝑁K = let rows = for (𝑥←J𝑁K) 𝜂(𝑥, eℓ) ⊕(start = now, end = ∞)+ in insert J𝑀K values rows Jdelete(ℓ𝑖:𝐴𝑖)𝑖(𝑥⇐𝑀) where 𝑁K = update (𝑥⇐J𝑀K) where (restrict(𝑥, eℓ, J𝑁K) ∧isCurrent(𝑥)) set (end = now) Jupdate(ℓ𝑖:𝐴𝑖)𝑖∈𝐼(𝑥⇐𝐿) where 𝑀set (ℓ= 𝑁𝑗)𝑗∈𝐽K = let tbl = J𝐿K in let affected = query for (𝑥←get tbl) where ((restrict(𝑥, {ℓ𝑖}𝑖∈𝐼, J𝑀K) ∧isCurrent(𝑥))) H (ℓ𝑖= 𝑥.ℓ𝑖)𝑖∈𝐼\𝐽⊕ (ℓ𝑗= restrict(𝑥, {ℓ𝑖}𝑖∈𝐼, J𝑁𝑗K))𝑗∈𝐽⊕ (start = now, end = ∞) I in update (𝑥⇐tbl) where (restrict(𝑥, eℓ, J𝑀K) ∧isCurrent(𝑥)) set (end = now); insert tbl values affected Figure 5. insert employees values (name = “Dolores”, position = “Professor”, salary = 70000) insert employees values (name = “Dolores”, position = “Professor”, salary = 70000) delete (𝑥⇐employees) where 𝑥.name = “Alice” ∅ Γ ⊢delete nonsequenced (𝑥⇐𝑀) where 𝑁:() ! {write} ∪𝐸 TV-NonseqDelete Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ,𝑥: ValidTime(𝐴) ⊢𝑁:Bool ! ∅ Γ ⊢delete nonsequenced (𝑥⇐𝑀) where 𝑁:() ! {write} ∪𝐸 Figure 6. Syntax and typing rules for 𝜆VLINQ Current updates and deletions can be implemented as se- quenced updates and deletions where the period of applica- bility spans from now until ∞: update sequenced (𝑥⇐𝐿) between 𝑀1 and 𝑀2 where 𝑀3 set ( ℓ= 𝑁) Terms 𝑀1 and 𝑀2 must be of type Time, referring to the pe- riod of applicability of the sequenced update. Nonsequenced updates are described by the term: update sequenced (𝑥⇐𝐿) between 𝑀1 and 𝑀2 where 𝑀3 set ( ℓ= 𝑁) Terms 𝑀1 and 𝑀2 must be of type Time, referring to the pe- riod of applicability of the sequenced update. Nonsequenced updates are described by the term: update (𝑥⇐𝐿) where 𝑀set (ℓ𝑖= 𝑁𝑖)𝑖{ update sequenced (𝑥⇐𝐿) between now and ∞where 𝑀set (ℓ𝑖= 𝑁𝑖)𝑖 delete (𝑥⇐𝑀) where 𝑁{ delete sequenced (𝑥⇐𝑀) between now and ∞where 𝑁 update (𝑥⇐𝐿) where 𝑀set (ℓ𝑖= 𝑁𝑖)𝑖{ update sequenced (𝑥⇐𝐿) between now and ∞where 𝑀set (ℓ𝑖= 𝑁𝑖)𝑖 update nonsequenced (𝑥⇐𝐿) where 𝑀set ( ℓ= 𝑁) valid from 𝑁′ 1 to 𝑁′ 2 where 𝑀set ( ℓ= 𝑁) valid from 𝑁′ 1 t with TV-NonseqUpdate stating that the database row (in- cluding period information) is bound as 𝑥in the predicate 𝑀, update terms 𝑁𝑗, and new time periods 𝑁′ 1 and 𝑁′ 2. Finally, the term: delete (𝑥⇐𝑀) where 𝑁{ delete sequenced (𝑥⇐𝑀) between now and ∞where 𝑁 delete (𝑥⇐𝑀) where 𝑁{ delete sequenced (𝑥⇐𝑀) between now and ∞where 𝑁 Fig. 7 shows selected reduction rules: we show sequenced inserts and updates, and nonsequenced updates; the rules for other cases employ similar ideas and are included in the extended version. Nonsequenced updates and deletes are similar to their analogues in 𝜆LINQ but allow access to, and modification of, row timestamps. For sequenced insertions, EV-SeqInsert checks that the period of validity for each row is correct (i.e., that the start field is less than the end field) and appends the provided bag to the table. Sequenced updates and deletions must account for the various ways that the period of applicability can overlap the period of validity. There are five main cases, corresponding to the five ways two closed-open intervals can overlap (or fail to do so): insert 𝑀values 𝑁{ let rows = for (𝑥←𝑁)𝑥[now,∞)+ in insert sequenced 𝑀values rows delete (𝑥⇐employees) where 𝑥.name = “Alice” The resulting table state shows that Dolores is a Professor from the current time onwards, and that the ‘end’ field of Alice’s current row is updated to the current year: Figure 6 shows how the syntax and typing rules for 𝜆VLINQ differ from those of 𝜆LINQ. Unlike in 𝜆TLINQ, we can use the term 𝑀[𝑀2,𝑀3) 1 to construct a valid-time row. Sequenced insertions are described by the term insert sequenced 𝑀values 𝑁where TV-SeqInsert en- sures that 𝑁is a bag of timestamped records. Sequenced updates are described by: name position salary start end Alice Lecturer 40000 2010 2018 Alice Senior Lecturer 50000 2018 2022 Dolores Professor 70000 2022 ∞ · · · · · · · · · · · · · · · GPCE ’22, December 06–07, 2022, Auckland, New Zealand Simon Fowler, Vashti Galpin, and James Cheney Syntax Types 𝐴, 𝐵 ::= ValidTime(𝐴) Terms 𝐿, 𝑀, 𝑁 ::= · · · \| 𝐿[𝑀,𝑁) \| data 𝑀\| start 𝑀\| end 𝑀\| insert sequenced 𝑀values 𝑁 \| update sequenced (𝑥⇐𝐿) between 𝑀1 and 𝑀2 where 𝑀3 set ( ℓ= 𝑁) \| update nonsequenced (𝑥⇐𝐿) where 𝑀set ( ℓ= 𝑁) valid from 𝑁′ 1 to 𝑁′ 2 \| delete sequenced (𝑥⇐𝐿) between 𝑀1 and 𝑀2 where 𝑁 \| delete nonsequenced (𝑥⇐𝑀) where 𝑁 Values 𝑉,𝑊 ::= · · · \| 𝑉[𝑉2,𝑉3) 1 Typing rules Γ ⊢𝑀: 𝐴! 𝐸 TV-Get Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ ⊢get 𝑀:Bag(ValidTime(𝐴)) ! {read} ∪𝐸 TV-SeqInsert Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ ⊢𝑁:Bag(ValidTime(𝐴)) ! ∅ Γ ⊢insert sequenced 𝑀values 𝑁:() ! {write} ∪𝐸 TV-SeqUpdate Γ ⊢𝐿:Table(𝐴) ! 𝐸 𝐴= (ℓ𝑖: 𝐵𝑖)𝑖∈𝐼 Γ ⊢𝑀1:Time ! ∅ Γ ⊢𝑀2:Time ! ∅ Γ,𝑥: 𝐴⊢𝑀3:Bool ! ∅ (𝑗∈𝐼∧Γ,𝑥: 𝐴⊢𝑁𝑗:𝐵𝑗! ∅)𝑗∈𝐽 Γ ⊢update sequenced (𝑥⇐𝐿) between 𝑀1 and 𝑀2 where 𝑀3 set (ℓ𝑗= 𝑁𝑗)𝑗∈𝐽:() ! {write} ∪𝐸 TV-NonseqUpdate Γ ⊢𝐿:Table(𝐴) ! 𝐸 𝐴= (ℓ𝑖: 𝐵𝑖)𝑖∈𝐼 Γ,𝑥: ValidTime(𝐴) ⊢𝑀:Bool ! ∅ (𝑗∈𝐼∧Γ,𝑥: ValidTime(𝐴) ⊢𝑁𝑗:𝐵𝑗! ∅)𝑗∈𝐽 Γ,𝑥: ValidTime(𝐴) ⊢𝑁′ 1:Time ! ∅ Γ,𝑥: ValidTime(𝐴) ⊢𝑁′ 2:Time ! ∅ Γ ⊢update nonsequenced (𝑥⇐𝐿) where 𝑀set (ℓ𝑗=𝑁𝑗)𝑗∈𝐽valid from 𝑁′ 1 to 𝑁′ 2:() ! {write} ∪𝐸 TV-SeqDelete Γ ⊢𝐿:Table(𝐴) ! 𝐸1 Γ ⊢𝑀1:Time ! 𝐸2 Γ ⊢𝑀2:Time ! 𝐸3 Γ,𝑥:𝐴⊢𝑁:Bool ! ∅ Γ ⊢delete sequenced (𝑥⇐𝐿) between 𝑀1 and 𝑀2 where 𝑁:() ! {write} ∪𝐸1 ∪𝐸2 ∪𝐸3 TV-NonseqDelete Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ,𝑥: ValidTime(𝐴) ⊢𝑁:Bool ! ∅ Γ ⊢delete nonsequenced (𝑥⇐𝑀) where 𝑁:() ! {write} ∪𝐸 TV-NonseqDelete Γ ⊢𝑀:Table(𝐴) ! 𝐸 Γ,𝑥: ValidTime(𝐴) ⊢𝑁:Bool ! delete sequenced (𝑥⇐𝐿) between 𝑀1 and 𝑀2 where 𝑁 describes a sequenced deletion which removes the portion of each record satisfying 𝑁between times 𝑀1 and 𝑀2. Since current insertions, updates, and deletions are special cases of sequenced operations, we need not consider them explicitly; for completeness, direct semantics can be found in the extended version. Instead, we show macro translations to the sequenced constructs. Current insertions can be imple- mented by desugaring to sequenced insertions, annotating each row with [now, ∞): insert 𝑀values 𝑁{ let rows = for (𝑥←𝑁)𝑥[now,∞)+ in insert sequenced 𝑀values rows Language-Integrated Query for Temporal Data Language-Integrated Query for Temporal Data GPCE ’22, December 06–07, 2022, Auckland, New Zealand Language-Integrated Query for Temporal Data tion rules 𝑀⇓V Δ,𝜄(𝑉, Δ′) EV-Row 𝑀1 ⇓V Δ,𝜄(𝑉1, Δ1) 𝑀2 ⇓V 𝜄,Δ1 (𝑉2, Δ2) 𝑀3 ⇓V 𝜄,Δ2 (𝑉3, Δ3) 𝑀[𝑀2,𝑀3) 1 ⇓V Δ,𝜄(𝑉[𝑉2,𝑉3) 1 , Δ3) EV-SeqInsert 𝑀⇓V Δ,𝜄(𝑡, Δ1) 𝑁⇓★ 𝜄e𝑉+ ∀data[start,end) ∈e𝑉.start < end Δ2 = Δ1[𝑡↦→Δ1(𝑡) ˆ⊎e𝑉+] insert sequenced 𝑀values 𝑁⇓V Δ,𝜄((), Δ2) EV-SeqUpdate 𝐿⇓V Δ,𝜄(𝑡, Δ1) 𝑀1 ⇓★ 𝜄𝑉start 𝑀2 ⇓★ 𝜄𝑉end 𝑉start < 𝑉end Δ2 = Δ1[𝑡↦→ˆ Ú * upd(𝑑) \| 𝑑∈Δ1(𝑡)+] upd(v [start,end) ) =   𝑊[start,end)+ if 𝑀3{v/𝑥} ⇓★ 𝜄true and 𝑉start ≤start and 𝑉end ≥end (Case 1) 𝑊[start,𝑉end ), v [𝑉end,end)+ if 𝑀3{v/𝑥} ⇓★ 𝜄true and 𝑉start ≤start and 𝑉end < end (Case 2) v [start,𝑉start ),𝑊[𝑉start,𝑉end ), v [𝑉end,end)+ if 𝑀3{v/𝑥} ⇓★ 𝜄true and 𝑉start > start and 𝑉end < end (Case 3) v [start,𝑉start ),𝑊[𝑉start,end)+ if 𝑀3{v/𝑥} ⇓★ 𝜄true and 𝑉start > start and 𝑉end ≥end (Case 4) v [start,end)+ otherwise (Case 5) where for all cases, 𝑊= (v with ℓ= 𝑊′) given (𝑁𝑖⇓★ 𝜄𝑊′ 𝑖)𝑖 update sequenced (𝑥⇐𝐿) between 𝑀1 and 𝑀2 where 𝑀3 set (ℓ𝑖= 𝑁𝑖)𝑖⇓Δ,𝜄((), Δ2) EV-NonseqUpdate 𝐿⇓Δ,𝜄(𝑡, Δ1) Δ2 = Δ1[𝑡↦→upd(𝑑) \| 𝑑∈Δ1(𝑡)+] upd(𝐷= v [start,end) ) =   (v with ℓ= 𝑊) [𝑊start,𝑊end ) if 𝑀{𝐷/𝑥} ⇓★ 𝜄true and (𝑁𝑖{𝐷/𝑥} ⇓★ 𝜄𝑊𝑖)𝑖and 𝑁′ 1 {𝐷/𝑥} ⇓★ 𝜄𝑊start and 𝑁′ 2 {𝐷/𝑥} ⇓★ 𝜄𝑊end and 𝑊start < 𝑊end 𝐷 if 𝑀{𝐷/𝑥} ⇓★ 𝜄false update nonsequenced (𝑥⇐𝐿) where 𝑀set (ℓ𝑖= 𝑁𝑖)𝑖valid from 𝑁′ 1 to 𝑁′ 2 ⇓Δ,𝜄((), Δ2) Figure 7. Reduction rules for 𝜆VLINQ (selected) 𝑀⇓V Δ,𝜄(𝑉, Δ′) 𝑀⇓V Δ,𝜄(𝑉, Δ′) Reduction rules EV-NonseqUpdate Figure 7. Reduction rules for 𝜆VLINQ (selected) Figure 7. delete sequenced (𝑥⇐𝐿) between 𝑀1 and 𝑀2 where 𝑁 Reduction rules for 𝜆VLINQ (selected) Case 1: PA overlaps PV entirely after the end of the PA will contain the original values, and the overlapping portion will contain the updated values. Case 4: PA overlaps PV on the right PV PA Similar to case 2, but at the end of the PV. Case 5: PA entirely before or after PV PV PA PA In the case of either a sequenced deletion or a sequenced update, the row will be unaffected. after the end of the PA will contain the original values, and the overlapping portion will contain the updated values. Case 4: PA overlaps PV on the right after the end of the PA will contain the original values, and the overlapping portion will contain the updated values. Case 4: PA overlaps PV on the right In the case of a sequenced deletion, the entire row will be deleted. In the case of a sequenced update, the entire row will be updated. PA PA Similar to case 2, but at the end of the PV. Case 5: PA entirely before or after PV Case 2: PA overlaps PV on the left In the case of a sequenced deletion, the overlapping por- tion will be deleted; in the case of a sequenced update, the overlapping portion will contain the updated values and the remaining portion of the PV will contain the previous values. In the case of a sequenced deletion, the overlapping por- tion will be deleted; in the case of a sequenced update, the overlapping portion will contain the updated values and the remaining portion of the PV will contain the previous values. PA PA In the case of either a sequenced deletion or a sequenced update, the row will be unaffected. PA PA In the case of either a sequenced deletion or a sequenced update, the row will be unaffected. 4.2 Translation remaining portion of the PV will contain the previous values. Case 3: PA contained within PV PV PA In the case of a sequenced deletion, there will be two records: the portion of the PV before the start of the PA, and the portion of the PV after the end of the PA. In the case of a sequenced update, there will be three records: the portion of the PV before the start of the PA, and the portion of the PV Case 3: PA contained within PV PV PA Case 3: PA contained within PV PV Figure 8 illustrates the translation from 𝜆VLINQ into 𝜆LINQ. We discuss the translations for sequenced inserts and both se- quenced and nonsequenced updates; the other modifications are similar and included in the extended version. As before, we require annotations on each of the database update terms. Figure 8 illustrates the translation from 𝜆VLINQ into 𝜆LINQ. We discuss the translations for sequenced inserts and both se- quenced and nonsequenced updates; the other modifications are similar and included in the extended version. As before, we require annotations on each of the database update terms. Nonsequenced updates and deletions can be updated di- rectly by their corresponding 𝜆LINQ operation; we use an auxiliary definition, lift, which lifts the flat representation into the nested representations expected by the predicate and update fields. Sequenced inserts flatten the contents of the provided bag and map directly to an insert. PA In the case of a sequenced deletion, there will be two records: the portion of the PV before the start of the PA, and the portion of the PV after the end of the PA. In the case of a sequenced update, there will be three records: the portion of the PV before the start of the PA, and the portion of the PV In the case of a sequenced deletion, there will be two records: the portion of the PV before the start of the PA, and the portion of the PV after the end of the PA. 4.2 Translation GPCE ’22, December 06–07, 2022, Auckland, New Zealand Simon Fowler, Vashti Galpin, and James Cheney Linsert(ℓ𝑖:𝐴𝑖)𝑖sequenced 𝑀values 𝑁M = let tbl = L𝑀M in let rows = for (𝑥←L𝑁M) 𝜂(𝑥.data, eℓ) ⊕(start = 𝑥.start, end = 𝑥.end)+ in insert tbl values rows L update(ℓ𝑖:𝐴𝑖)𝑖∈𝐼sequenced (𝑥⇐𝐿) between 𝑀1 and 𝑀2 where 𝑀3 set (ℓ𝑗= 𝑁𝑗)𝑗∈𝐽 M = let tbl = L𝐿M in let aStart = L𝑀1M in let aEnd = L𝑀2M in let lRows = startRows(tbl, pred, aStart) in let rRows = endRows(tbl, pred, aEnd) in update (𝑥⇐tbl) where (pred ∧(𝑥.start < aEnd) ∧(𝑥.end > aStart)) set © « (ℓ𝑗= restrict(𝑥, {ℓ𝑖}𝑖∈𝐼, L𝑁𝑗M))𝑗∈𝐽, start = greatest(𝑥.start, aStart), end = least(𝑥.end, aEnd) ª® ¬ ; insert tbl values lRows; insert tbl values rRows where pred ≜restrict(𝑥, {ℓ𝑖}𝑖∈𝐼, L𝑀3M) startRows(tbl, pred, aStart) ≜query for (𝑥←get tbl) where (pred ∧(𝑥.start < aStart) ∧(𝑥.end > aStart)) 𝜂(𝑥, {ℓ𝑖}𝑖∈𝐼) ⊕(start = 𝑥.start, end = aStart)+ endRows(tbl, pred, aEnd) ≜(symmetric) L update(ℓ𝑖:𝐴𝑖)𝑖∈𝐼nonsequenced (𝑥⇐𝐿) where 𝑀 set (ℓ𝑗= 𝑁𝑗)𝑗∈𝐽valid from 𝑁′ 1 to 𝑁′ 2 M = update(ℓ𝑖:𝐴𝑖)𝑖∈𝐼(𝑥⇐L𝐿M) where (lift(𝑥, L𝑀M)) set © « ((ℓ𝑗= lift(𝑥, L𝑁𝑗M))𝑗∈𝐽, start = lift(𝑥, L𝑁′ 1M), end = lift(𝑥, L𝑁′ 2M)) ª® ¬ where lift(𝑥, 𝑓) ≜ (𝜆𝑥.𝑓) (data = 𝜂(𝑥, {ℓ𝑖}𝑖∈𝐼), start = 𝑥.start, end = 𝑥.end) Linsert(ℓ𝑖:𝐴𝑖)𝑖sequenced 𝑀values 𝑁M = let tbl = L𝑀M in let rows = for (𝑥←L𝑁M) 𝜂(𝑥.data, eℓ) ⊕(start = 𝑥.start, end = 𝑥.end)+ in insert tbl values rows L update(ℓ𝑖:𝐴𝑖)𝑖∈𝐼sequenced (𝑥⇐𝐿) between 𝑀1 and 𝑀2 where 𝑀3 set (ℓ𝑗= 𝑁𝑗)𝑗∈𝐽 M = let tbl = L𝐿M in let aStart = L𝑀1M in let aEnd = L𝑀2M in let lRows = startRows(tbl, pred, aStart) in let rRows = endRows(tbl, pred, aEnd) in update (𝑥⇐tbl) where (pred ∧(𝑥.start < aEnd) ∧(𝑥.end > aStart)) set © « (ℓ𝑗= restrict(𝑥, {ℓ𝑖}𝑖∈𝐼, L𝑁𝑗M))𝑗∈𝐽, start = greatest(𝑥.start, aStart), end = least(𝑥.end, aEnd) ª® ¬ ; insert tbl values lRows; insert tbl values rRows where pred ≜restrict(𝑥, {ℓ𝑖}𝑖∈𝐼, L𝑀3M) startRows(tbl, pred, aStart) ≜query for (𝑥←get tbl) where (pred ∧(𝑥.start < aStart) ∧(𝑥.end > aStart)) 𝜂(𝑥, {ℓ𝑖}𝑖∈𝐼) ⊕(start = 𝑥.start, end = aStart)+ endRows(tbl, pred, aEnd) ≜(symmetric) L update(ℓ𝑖:𝐴𝑖)𝑖∈𝐼nonsequenced (𝑥⇐𝐿) where 𝑀 set (ℓ𝑗= 𝑁𝑗)𝑗∈𝐽valid from 𝑁′ 1 to 𝑁′ 2 M = update(ℓ𝑖:𝐴𝑖)𝑖∈𝐼(𝑥⇐L𝐿M) where (lift(𝑥, L𝑀M)) set © « ((ℓ𝑗= lift(𝑥, L𝑁𝑗M))𝑗∈𝐽, start = lift(𝑥, L𝑁′ 1M), end = lift(𝑥, L𝑁′ 2M)) ª® ¬ where lift(𝑥, 𝑓) ≜ (𝜆𝑥.𝑓) (data = 𝜂(𝑥, {ℓ𝑖}𝑖∈𝐼), start = 𝑥.start, end = 𝑥.end) Unlike 𝜆LINQ and 𝜆TLINQ, evaluation in 𝜆VLINQ is partial in order to reflect the need for dynamic checks that start times precede end times. 4.2 Translation In practice, our implementation evalu- ates temporal updates as single transactions and raises an exception (aborting the transaction) when a well-formedness check fails, but our formalisation assumes updates preserve well-formedness in order to avoid clutter.i Our translation from 𝜆VLINQ into 𝜆TLINQ satisfies the fol- lowing correctness property: Theorem 4.1. If · ⊢𝑀:𝐴! 𝐸and 𝑀⇓V Δ,𝜄(𝑉, Δ′) for some wf(Δ), then L𝑀M ⇓↓Δ,𝜄(L𝑉M, ↓Δ′) Theorem 4.1. If · ⊢𝑀:𝐴! 𝐸and 𝑀⇓V Δ,𝜄(𝑉, Δ′) for some wf(Δ), then L𝑀M ⇓↓Δ,𝜄(L𝑉M, ↓Δ′) Theorem 4.1. If · ⊢𝑀:𝐴! 𝐸and 𝑀⇓V Δ,𝜄(𝑉, Δ′) for some wf(Δ), then L𝑀M ⇓↓Δ,𝜄(L𝑉M, ↓Δ′) Figure 8. Translation from 𝜆VLINQ into 𝜆LINQ (selected cases) The remaining sequenced operations are the most complex to translate. Since a sequenced modification may partition a row, the startRows and endRows functions calculate the records which must be inserted before and after the period of applicability. To translate a sequenced update, we calcu- late the rows to insert, perform an update to set the new values and set the new period of applicability to the over- lap between the PA and PV using the greatest and least functions, and finally materialise the insertions. Sequenced deletions (shown in the extended version) are similar but delete the rows that overlap the PA instead of updating them. We can join this table with the non-temporal salaries table as follows; for clarity, we denote valid-time get as getV: query for (𝑒←getv employees) for (𝑠←get salaries) where ((data 𝑒).band = 𝑠.band) (name = 𝑒.name, salary = 𝑠.salary) [start 𝑒,end 𝑒)+ giving us the corresponding table in Section 4. giving us the corresponding table in Section 4. Things get more interesting when both tables are temporal.l 5 Sequenced Joins Queries that join multiple tables are straightforward to en- code using language integrated query. Keeping with our employee database, say we wish to separate out the salary into a separate table. The non-temporal employee database might look as follows: insert tbl values lRows; insert tbl values rRows where g employees name position band Alice Senior Lecturer A08 Bob PhD Student B01 Charles PhD Student B01 Dolores Professor A10 salaries band salary A08 40000 A09 50000 A10 70000 B01 15000 We can get the salary for each employee as follows: employees employees name position band Alice Senior Lecturer A08 Bob PhD Student B01 Charles PhD Student B01 Dolores Professor A10 salaries band salary A08 40000 A09 50000 A10 70000 B01 15000 We can get the salary for each employee as follows: query for (𝑒←get employees) for (𝑠←get salaries) where (𝑒.band = 𝑠.band) (name = 𝑒.name, salary = 𝑠.salary)+ name salary Alice 40000 Bob 15000 Charles 15000 Dolores 70000 J i i t l t bl ith t l t bl i l We can get the salary for each employee as follows: g query for (𝑒←get employees) for (𝑠←get salaries) where (𝑒.band = 𝑠.band) (name = 𝑒.name, salary = 𝑠.salary)+ name salary Alice 40000 Bob 15000 Charles 15000 Dolores 70000 Joining a temporal table with a non-temporal table is also eas- ily expressible. Consider a version of our previous temporal employees table from just after when Dolores joined: (𝜆𝑥.𝑓) (data = 𝜂(𝑥, {ℓ𝑖}𝑖∈𝐼), start = 𝑥.start, end = 𝑥.end) name position band start end Alice Lecturer A08 2010 2018 Alice Senior Lecturer A09 2018 ∞ Bob PhD Student B01 2019 2023 Charles PhD Student B01 2018 2022 Dolores Professor A10 2022 ∞ Figure 8. Translation from 𝜆VLINQ into 𝜆LINQ (selected cases) 4.2 Translation In the case of a sequenced update, there will be three records: the portion of the PV before the start of the PA, and the portion of the PV Nonsequenced updates and deletions can be updated di- rectly by their corresponding 𝜆LINQ operation; we use an auxiliary definition, lift, which lifts the flat representation into the nested representations expected by the predicate and update fields. Sequenced inserts flatten the contents of the provided bag and map directly to an insert. Proposition 4.1 (Preservation (𝜆VLINQ)). If · ⊢𝑀:𝐴! 𝐸and 𝑀⇓V Δ,𝜄(𝑉, Δ′) for some wf(Δ), then · ⊢𝑉:𝐴! ∅and wf(Δ′). Language-Integrated Query for Temporal Data Con- cretely, joining the above two temporal tables would give: Normal forms Queries 𝑄 ::= 𝐾1 ⊎· · · ⊎𝐾𝑛 Comprehensions 𝐾 ::= for ( e 𝐺) where 𝑃 𝑆+ Generators 𝐺 ::= 𝑥←get 𝑡\| 𝑥←getv 𝑡 Normalised terms 𝑆 ::= 𝑃\| 𝑅 Base terms 𝑃 ::= 𝑐\| 𝑥.ℓ\| ⊙{−→ 𝑃} Record terms 𝑅 ::= (ℓ𝑖= 𝑃𝑖)𝑖 name salary start end Alice 38000 2010 2015 Alice 40000 2015 2018 Alice 50000 2018 ∞ · · · · · · · · · · · ·f Translation on normal forms ∥join 𝑄∥= query ∥𝑄∥ ∥𝐾1 ⊎· · · ⊎𝐾𝑛∥= ∥𝐾1∥⊎· · · ⊎∥𝐾𝑛∥ ∥join 𝑄∥= query ∥𝑄∥ ∥𝐾1 ⊎· · · ⊎𝐾𝑛∥= ∥𝐾1∥⊎· · · ⊎∥𝐾𝑛∥ Now there are records for Alice forthree different periods:i • The first when Alice was on salary band A08, confer- ring a salary of £38000. g y • The second when band A08 increased to £40000. • The second when band A08 increased to £40000. • The third when Alice was promoted to band A09. • The third when Alice was promoted to band A09. Such joins are called sequenced because they (conceptually) evaluate the join on the whole sequence of states encoded by each table. Manually writing the sequenced joins in SQL is error-prone. We instead introduce a construct, join, which allows us to write the following: join for (𝑒←getv employees) for (𝑠←getv salaries) where ((data 𝑒).band = (data 𝑠).band) (name = (data 𝑒).name, salary = (data 𝑠).salary)+ join for (𝑒←getv employees) for (𝑠←getv salaries) where ((data 𝑒).band = (data 𝑠).band) (name = (data 𝑒).name, salary = (data 𝑠).salary)+ join Figure 9. Sequenced joins Figure 9. Sequenced joins joins as described in Section 5. In this section, we describe a case study based on curating COVID-19 data. (name = (data 𝑒).name, salary = (data 𝑠).salary)+ Note that we do not need to calculate the period of validity for each resulting row; this is computed automatically. Note that we do not need to calculate the period of validity for each resulting row; this is computed automatically. Our translations from 𝜆VLINQ into 𝜆LINQ are trivially real- isable in SQL. Queries can be compiled using known tech- niques (e.g., [8]). The startRows and endRows functions can be compiled using an SQL WITH statement, and there is a direct correspondence between 𝜆LINQ modification opera- tions and their SQL equivalents. 1Links code is available at https://github.com/vcgalpin/links-covid-curation [16] Language-Integrated Query for Temporal Data Each translated temporal modification is executed as an SQL transaction, with primary key and referential integrity constraint checking deferred until the end of the transaction. Figure 9 shows how sequenced joins can be implemented; we show the constructs for valid time, but the same technique can be used for transaction time. The typing rule requires that the result of a join query is flat (nested sequenced queries are conceptually nontrivial). As mentioned earlier, queries can be rewritten to normal forms for conversion to SQL, as shown in Figure 9. The structure of these normal forms allows sequenced joins to be implemented through a simple rewrite: the greatest and least functions are used to calculate the intersections of the periods of validity for each combination of records from each generator, with the modified predicate ensuring that the periods of overlap make sense. The calculated overlapping periods of validity are then returned in the resulting row. Case study. We have used the temporal features of Links in two prototypes based on curated scientific databases: cu- ration of publicly available COVID-19 data, and storage and curation of XML documents [18] using the Dynamic Dewey labelling algorithm [38].i We concentrate on the first application1; a previous proto- type which used a preliminary version of the language design has previously been presented as a short demo paper [17]. In 2020, the Scottish Government began releasing various data about the COVID-19 pandemic [26], which included weekly data of fatalities in Scotland in various categories (such as Language-Integrated Query for Temporal Data Language-Integrated Query for Temporal Data GPCE ’22, December 06–07, 2022, Auckland, New Zealand Language-Integrated Query for Temporal Data Typing rules A :: FQType Γ ⊢𝑀:𝐴! 𝐸 𝐶:: FQType (ℓ𝑖: 𝐶𝑖)𝑖:: FQType Γ ⊢𝑀:Bag(𝐴) ! 𝐸 𝐴:: FQType 𝐸⊆{read} Γ ⊢join 𝑀:Bag(ValidTime(𝐴)) ! 𝐸 Normal forms Queries 𝑄 ::= 𝐾1 ⊎· · · ⊎𝐾𝑛 Comprehensions 𝐾 ::= for ( e 𝐺) where 𝑃 𝑆+ Generators 𝐺 ::= 𝑥←get 𝑡\| 𝑥←getv 𝑡 Normalised terms 𝑆 ::= 𝑃\| 𝑅 Base terms 𝑃 ::= 𝑐\| 𝑥.ℓ\| ⊙{−→ 𝑃} Record terms 𝑅 ::= (ℓ𝑖= 𝑃𝑖)𝑖 Translation on normal forms ∥join 𝑄∥= query ∥𝑄∥ ∥𝐾1 ⊎· · · ⊎𝐾𝑛∥= ∥𝐾1∥⊎· · · ⊎∥𝐾𝑛∥ ∥𝑃∥= 𝑃 ∥𝑅∥= 𝑅 ∥ for 𝑥1 ←getv 𝑡1, . . . , 𝑥𝑚←getv 𝑡𝑚, 𝑦1 ←get 𝑡′ 1, . . . , 𝑦𝑛←get 𝑡′𝑛 where (𝑃) 𝑅+ ∥ = for 𝑥1 ←getv 𝑡1, . . . , 𝑥𝑚←getv 𝑡𝑚, 𝑦1 ←get 𝑡′ 1, . . . , 𝑦𝑛←get 𝑡′𝑛 where (𝑃∧joinStart < joinEnd) 𝑅[joinStart,joinEnd)+ where joinStart ≜greatest(𝑥1.start, . . . ,𝑥𝑚.start) joinEnd ≜least(𝑥1.end, . . . ,𝑥𝑚.end) Figure 9. Sequenced joins band salary start end A08 38000 2000 2015 A09 48000 2000 2015 A08 40000 2015 ∞ A09 50000 2015 ∞ · · · · · · · · · · · · Typing rules A :: FQType Γ ⊢𝑀:𝐴! 𝐸 𝐶:: FQType (ℓ𝑖: 𝐶𝑖)𝑖:: FQType Γ ⊢𝑀:Bag(𝐴) ! 𝐸 𝐴:: FQType 𝐸⊆{read} Γ ⊢join 𝑀:Bag(ValidTime(𝐴)) ! 𝐸 What does it mean to join two temporal tables? In essence, we want to record all configurations of a particular joined record, creating new records with shorter periods of validity whenever data from either underlying table changes. 4.3 Metatheory Things get more interesting when both tables are temporal. Salaries are not static over time; bands go up with inflation, for example. Suppose we now have two temporal tables. Consider the above table along with a temporal salaries table showing a pay increase in 2015: Salaries are not static over time; bands go up with inflation, for example. Suppose we now have two temporal tables. Evaluation preserves typing and well-formedness. Proposition 4.1 (Preservation (𝜆VLINQ)). If · ⊢𝑀:𝐴! 𝐸and 𝑀⇓V Δ,𝜄(𝑉, Δ′) for some wf(Δ), then · ⊢𝑉:𝐴! ∅and wf(Δ′). Consider the above table along with a temporal salaries table showing a pay increase in 2015: 6 Implementation and Case Study From a provenance point of view, this data is interesting because a column for an earlier week may contain updated data. We developed a web application for the querying of the data ("How do the Male and Female sub- categories compare in terms of the change in fatalities from last week to this week?") as well as querying the changes in the data ("How do the Male and Female subcategories compare in terms of number of updates to existing values?"). fun getCurrentData (category) { fun getCurrentData (category) { query nested { for (x <-- subcategory) where (x.cat == category) [(subcat_name = x.subcat_name, cat = x.cat, results = for (y <- vtCurrent(covid_data)) for (z <-- week) where (y.subcat == x.subcat && y.weekdate == z.weekdate && z.all_zero == false) [(count = y.count, weekdate = y.weekdate)])]} } Considering the non-temporal data, an entry in a database table would be a row consisting of the key fields subcat and weekdate and a value field giving the corresponding count. In the case of the temporal data, the key fields are insufficient to uniquely identify the value of the count because it may have different values over time. Thus the time validity fields are necessary to provide a key for the value. Instead of an explicit get construct, Links uses the ‘double ar- row’ comprehension <-- to represent a nontemporal database query, with <-t- and <-v- supporting transaction time and valid time queries respectively. The ‘single arrow’ compre- hension <- denotes a list comprehension. Finally, vtCurrent is a standard library function which performs a valid time query to obtain the values valid at the current time. The prototype uses a valid time table for fatality data to capture the notion that a count value, either brand new or an update, becomes valid as soon as the CSV is uploaded into the interface2 (this can be a different time from when the new value is accepted and written to the database). In Links it is possible to specify the names of the period stamping fields, which have the built-in type DateTime. This table is defined using the following Links code; we have omitted some details in the code snippets for brevity. 2Other possibilities for the start time of validity are the date of the release of the CSV file or the start of the new week. 6 Implementation and Case Study The Links programming language [9] is a statically-typed functional web programming language which allows client, server, and database code to be written in a uniform language. We have extended Links with support for the constructs described in Sections 3 and 4, as well as support for temporal Simon Fowler, Vashti Galpin, and James Cheney GPCE ’22, December 06–07, 2022, Auckland, New Zealand ‘Sex’). Each weekly release was a CSV file, with a row for each subcategory (e.g., ‘Sex’ has the subcategories ‘Male’ and ‘Female’) and a column for each week for which data was available. Each release included an additional week column with the latest data (see Figure 10). Importantly, each release could include revisions to data for previous weeks. accepted, it is added using a sequenced update. Figure 10 il- lustrates how the table changes as a result of a single update. The prototype also provides functionality to query data, both as current data, and as data with informa- tion about changes. The current data is obtained using a current query. The result is a list of weeks and counts grouped by subcategory. This is repeated for each category. Information about the changes to the data over time is often desired to understand its provenance and assess its trustworthiness [4]. From a provenance point of view, this data is interesting because a column for an earlier week may contain updated data. We developed a web application for the querying of the data ("How do the Male and Female sub- categories compare in terms of the change in fatalities from last week to this week?") as well as querying the changes in the data ("How do the Male and Female subcategories compare in terms of number of updates to existing values?"). Considering the non-temporal data, an entry in a database table would be a row consisting of the key fields subcat and weekdate and a value field giving the corresponding count. In the case of the temporal data, the key fields are insufficient to uniquely identify the value of the count because it may have different values over time. Thus the time validity fields are necessary to provide a key for the value. Information about the changes to the data over time is often desired to understand its provenance and assess its trustworthiness [4]. 6 Implementation and Case Study For update provenance queries of individual counts, a self join is computed over the subcategory and week fields of the valid time table to provide a nested result table where each count is associated with a list of count values and their associ- ated start and end time information. This is a nonsequenced query because the time period information is explicitly added to the result table. The user can specify the subcategory and week they are interested in, and obtain details of modifi- cations. The interface also supports update provenance by week and by category. This is illustrated in Figure 11. var covid_data = table "covid_data" with (subcat: Int, weekdate: String, count: Int) using valid_time(valid_from, valid_to) from database "covid_curation"; var covid_data = table "covid_data" with (subcat: Int, weekdate: String, count: Int) using valid_time(valid_from, valid_to) from database "covid_curation"; The prototype’s upload workflow is as follows: the user up- loads a new CSV file, and the count values for the new week are added to the database. For counts that pertain to earlier weeks and that now have different values, the user is shown these counts and can accept them, reject them or move them to a pending list for a later decision. In terms of implemen- tation, brand new count values are added to the table with a sequenced insert, using the upload time as the start time. The Links code for this and other examples can be found in the extended version. The process is more complex for updated count values, because the interface shows the user previous value, to support decision making. This requires a conditional join over the current state of the covid_data table and the count values from the CSV file. If a modification is 7 Discussionfi Efficiency. Our main focus has been on portability: by dis- tilling a language design and formalising and implementing a translation from temporal calculi to non-temporal calculi, we allow temporal functionality to be used on a mainstream DBMS. The cost of portability is that our translations will inevitably not perform as well as a native implementation. Although we do not make any specific claims about effi- ciency, we have no reason to believe that the performance is any different to hand-translated SQL. In particular, as discussed in §6, all translated queries can be run directly on the database and do not require in-memory processing. Previous work on language-integrated query (e.g., [6, 9]) shows how the “nested loop” style of query is translated into efficient SQL, and our translation of queries Language-Integrated Query for Temporal Data Language-Integrated Query for Temporal Data GPCE ’22, December 06–07, 2022, Auckland, New Zealand Language-Integrated Query for Temporal Data Week Subcat Count Valid-from Valid-to 30-Mar-2020 Female 126 <first CSV upload> <later CSV upload> 30-Mar-2020 Female 127 <later CSV upload> <forever> 30-Mar-2020 Male 156 <first CSV upload> <forever> Week Subcat Count Valid-from Valid-to 30-Mar-2020 Female 126 <first CSV upload> <forever> 30-Mar-2020 Male 156 <first CSV upload> <forever> Updates from the CSV of 13-Apr-2020 New data from the CSV of 30-Mar-2020 Figure 10. Example of data uploads, sequenced insertion and sequenced update Figure 10. Example of data uploads, sequenced insertion and sequenced update Figure 11. Interface screenshot: history of a count Moving between the two DBMSs would not require any changes to application source code. However, not all oper- ations can be as straightforwardly translated: in particular, SQL:2011 does not natively support sequenced joins. 8 Related and Future Workf Most of the focus of effort on language-integrated query has been (perhaps unsurprisingly) on queries rather than updates, beginning with the foundational work on nested relational calculus by Buneman et al. [3] and on rewrit- ing queries for translation to SQL by Wong [37]. Lindley and Cheney [23] presented a calculus including both query and update capabilities and our type and effect system for tracking database read and write access is loosely based on theirs. More recently a number of contributions extending the formal foundations of language-integrated query have appeared, including to handle higher-order functions [8], nested query results [6], sorting/ordering [21], grouping and aggregation [27, 28], and deduplication [30]. Our core cal- culus 𝜆LINQ only incorporates the first two of these, and developing a core calculus that handles more features, as well as translating temporal queries involving them, is an obvious future direction. To the best of our knowledge no previous work on language-integrated query has considered temporal data specifically. Figure 11. Interface screenshot: history of a count happens prior to normalisation. Further optimisation is sub- sequently performed by the DBMS, and anecdotally we have not observed any performance issues in any applications we have written using the temporal extensions to Links. Supporting existing native implementations. Some (mainly proprietary) DBMSs, for example Teradata [1], have native support for some temporal features inspired by TSQL2 or SQL:2011. Although we are yet to explore this, an advan- tage of the LINQ approach is that temporal SQL syntax could be generated for backends which support temporal opera- tions directly, while maintaining functionality in mainstream backends without native temporal support.i The ubiquity and importance of time in applications of databases was appreciated from an early stage [7] and led to a significant community effort to standardise temporal extensions to SQL based on the TSQL2 language design in the 1990s and early 2000s [33]. This effort ultimately re- sulted in standardisation of a relatively limited subset of the original proposal in SQL:2011 [22]. Since then temporal database research has progressed steadily, including recent contributions showing how to implement temporal data man- agement as a layer on top of a standard RDBMS [11], and establishing connections between temporal querying and data provenance and annotation models [12]. The translation between our temporal modification con- structs and SQL:2011 (and similarly, TSQL2) would be fairly direct. References [1] Mohammed Al-Kateb, Ahmad Ghazal, Alain Crolotte, Ramesh Bhashyam, Jaiprakash Chimanchode, and Sai Pavan Pakala. 2013. Tem- poral query processing in Teradata. In EDBT. ACM, 573–578. p q y p g [2] J.F. Armstrong, E. Faccenda, S.D. Harding, A.J. Pawson, C. Southan, [2] J.F. Armstrong, E. Faccenda, S.D. Harding, A.J. Pawson, C. Southan, J.L. Sharman, B. Campo, D.R. Cavanagh, S.P.H. Alexander, A.P. Daven- port, M. Spedding, and J.A. Davies. 2020. The IUPHAR/BPS Guide to PHARMACOLOGY in 2020: extending immunopharmacology content and introducing the IUPHAR/MMV Guide to MALARIA PHARMA- COLOGY. Nucleic Acids Research 48 (2020), D1006–D1021. https: //doi.org/10.1093/nar/gkz951 [3] Peter Buneman, Shamim Naqvi, Val Tannen, and Limsoon Wong. 1995. Principles of programming with complex objects and collection types. Theor. Comput. Sci. 149, 1 (1995). https://doi.org/10.1016/0304-3975(95) 00024-Q One important future application is to retrofit temporal aspects to expert-curated databases, an example being the Guide to Pharmacology Database (GtoPdb) that summarises pharmacological targets and interactions [2]. Links has been used to implement a workalike version of GtoPdb [15] and we hope to build on this to provide a fully versioned imple- mentation of GtoPdb. An important requirement here is to minimise changes to the existing system. [4] Peter Buneman and Wang-Chiew Tan. 2018. Data Provenance: What next? SIGMOD Rec. 47, 3 (2018), 5–16. https://doi.org/10.1145/3316416. 3316418 [5] James Cheney, Sam Lindley, and Philip Wadler. 2013. A practical theory of language-integrated query. In ICFP. https://doi.org/10.1145/ 2500365.2500586 [6] James Cheney, Sam Lindley, and Philip Wadler. 2014. Query shred- ding: efficient relational evaluation of queries over nested multisets. In SIGMOD. ACM, 1027–1038. https://doi.org/10.1145/2588555.2612186f Finally we mention two immediate next steps. First, we plan to investigate bitemporal databases [35], which allow transaction and valid time to be used together, in turn allow- ing us to write queries such as “when was it recorded that Bob’s contract length was extended?”. Bitemporal databases are considerably more difficult to formalise and reason about, so we aim to investigate how bitemporal support can be added in a compositional manner. Second, at present, the result of a sequenced join must be a flat record; further work is required to understand the semantics and implementation techniques for joins that produce nested results. [7] James Clifford and David S. Warren. 1983. Formal Semantics for Time in Databases. ACM Trans. Database Syst. 8, 2 (1983), 214–254. https://doi.org/10.1145/319983.319986 [8] Ezra Cooper. 2009. 8 Related and Future Workf Consider a sequenced update in 𝜆VLINQ: update sequenced (𝑥⇐employees) between 2022−01−1 and 2022−10−29 where (𝑥.salary < 30000) set (salary = 𝑥.salary + 1000) update sequenced (𝑥⇐employees) between 2022−01−1 and 2022−10−29 where (𝑥.salary < 30000) set (salary = 𝑥.salary + 1000) This could be implemented in SQL:2011 as follows: UPDATE employees FOR PORTION OF EPeriod FROM DATE '2022-01-01' TO DATE '2022-10-29' WHERE salary < 30000 SET salary = salary + 1000 UPDATE employees FOR PORTION OF EPeriod FROM DATE '2022-01-01' TO DATE '2022-10-29' WHERE salary < 30000 SET salary = salary + 1000 Snodgrass [34] describes how to implement TSQL2-style updates and queries by translation to SQL, but we are not aware of previous detailed formal proofs of correctness of Simon Fowler, Vashti Galpin, and James Cheney GPCE ’22, December 06–07, 2022, Auckland, New Zealand study. Our work is a first but significant step towards fully supporting temporal data management at the language level. translations for transaction time and valid time updates. Al- though timestamping rows with time intervals is among the most popular ways to represent temporal databases as flat relational tables, it is not the only possibility. Jensen et al. [20] proposed a bitemporal conceptual data model that captures the abstract meaning of a temporal table and used it to compare different representation strategies. References The Script-Writer’s Dream: How to Write Great SQL in Your Own Language, and Be Sure It Will Succeed. In DBPL (Lecture Notes in Computer Science, Vol. 5708). Springer, 36–51. https: //doi.org/10.1007/978-3-642-03793-1_3 [9] Ezra Cooper, Sam Lindley, Philip Wadler, and Jeremy Yallop. 2006. Links: Web Programming Without Tiers. In FMCO (Lecture Notes in Computer Science, Vol. 4709). 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Some of these issues appear orthogonal to the high-level language design and could be incorporated “under the hood” into the implementation or even performed directly on the database.i Language-Integrated Query for Temporal Data Language-Integrated Query for Temporal Data GPCE ’22, December 06–07, 2022, Auckland, New Zealand by-theme/vital-events/general-publications/weekly-and-monthly- data-on-births-and-deaths/deaths-involving-coronavirus-covid-19- in-scotland/archive Last accessed: 23 April 2021. by-theme/vital-events/general-publications/weekly-and-monthly- data-on-births-and-deaths/deaths-involving-coronavirus-covid-19- in-scotland/archive Last accessed: 23 April 2021. [15] Simon Fowler, Simon Harding, Joanna Sharman, and James Cheney. 2020. Cross-tier Web Programming for Curated Databases: A Case Study. International Journal of Digital Curation 16, 1 (2020). https: //doi.org/10.2218/ijdc.v15i1.717 [27] Rui Okura and Yukiyoshi Kameyama. 2020. Language-Integrated Query with Nested Data Structures and Grouping. In FLOPS. 139–158. https://doi.org/10.1007/978-3-030-59025-3_9 [16] Vashti Galpin. 2022. vcgalpin/links-covid-curation: Initial release (v0.1.0). Zenodo. https://doi.org/10.5281/zenodo.7199221. https://doi.org/10.1007/978-3-030-59025-3_9 [28] Rui Okura and Yukiyoshi Kameyama. 2020. Reorganizing Queries with Grouping. In GPCE (Virtual, USA). ACM, 13 pages. https://doi.org/10. 1145/3425898.3426960 [17] Vashti Galpin and James Cheney. 2021. Curating Covid-19 Data in Links. In IPAW 2020 + IPAW 2021 (Lecture Notes in Computer Science, Vol. 12839). Springer, 237–243. https://doi.org/10.1007/978-3-030- 80960-7_19 [29] Quill team. 2022. Quill: Compile-Time Language Integrated Queries for Scala. Open source project. https://github.com/getquill/quill. [18] Vashti Galpin, Ian Smith, and Jean-Laurent Hippolyte. 2022. Sup- porting provenance of digital calibration certificates with temporal databases. In IMEKO TC6 M4Dconf. 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Database Syst. 41, 4, Article 26 (2016), 46 pages. https://doi.org/10. 1145/2967608 In spite of decades of work on temporal databases and even an extension to the SQL standard, mainstream support for temporal data remains limited, requiring developers to im- plement temporal functionality from scratch. In this paper, we have shown how to extend language-integrated query to support transaction time and valid time data, making tem- poral data management accessible without explicit DBMS support. We have formalised our constructs and translational implementation strategies based on those proposed by Snod- grass [34], and proved that the translations are semantics- preserving. We have implemented our approach in the Links programming language and assessed its value through a case [12] Anton Dignös, Boris Glavic, Xing Niu, Michael Böhlen, and Johann Gamper. 2019. Snapshot Semantics for Temporal Multiset Relations. Proc. 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Sci. 52, 3 (1996). https://doi.org/10.1006/jcss.1996.0037 [25] Erik Meijer, Brian Beckman, and Gavin M. Bierman. 2006. LINQ: rec- onciling object, relations and XML in the .NET framework. In SIGMOD. https://doi.org/10.1145/1142473.1142552 [38] Liang Xu, Tok Wang Ling, Huayu Wu, and Zhifeng Bao. 2009. DDE: from dewey to a fully dynamic XML labeling scheme. In SIGMOD. ACM, 719–730. https://doi.org/10.1145/1559845.1559921 [26] National Records of Scotland. 2021. Deaths involving coronavirus (COVID-19) in Scotland: archive. https: //www.nrscotland.gov.uk/statistics-and-data/statistics/statistics- Received 2022-08-12; accepted 2022-10-10
https://openalex.org/W4200191421	https://www.frontiersin.org/articles/10.3389/fcimb.2021.781790/pdf	English	null	Table_1.doc	null	2,021	cc-by	16,358	Keywords: quorum-sensing molecules, farnesol, C. albicans, diketopiperazines, dimorphism, antihyphal, in silico docking and molecular dynamic simulation ORIGINAL RESEARCH published: 03 December 2021 doi: 10.3389/fcimb.2021.781790 ORIGINAL RESEARCH published: 03 December 2021 doi: 10.3389/fcimb.2021.781790 ORIGINAL RESEARCH published: 03 December 2021 doi: 10.3389/fcimb.2021.781790 Correspondence: Shanmugaraj Gowrishankar gowrishankar.alu@gmail.com; gowrishankars@ alagappauniversity.ac.in Shunmugiah Karutha Pandian sk_pandian@rediffmail.com; pandiansk@alagappauniversity.ac.in †These authors have contributed equally to this work Specialty section: This article was submitted to Clinical Microbiology, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 23 September 2021 Accepted: 12 November 2021 Published: 03 December 2021 Specialty section: This article was submitted to Clinical Microbiology, a section of the journal Frontiers in Cellular and Infection Microbiology Edited by: Raja Veerapandian, Texas Tech University Health Sciences Center El Paso, United States Edited by: Raja Veerapandian, Texas Tech University Health Sciences Center El Paso, United States Center El Paso, United States Reviewed by: Thiruselvam Viswanathan, The University of Texas Health Science Center at San Antonio, United States Chandrajit Lahiri, Sunway University, Malaysia Correspondence: Shanmugaraj Gowrishankar gowrishankar.alu@gmail.com; gowrishankars@ alagappauniversity.ac.in Shunmugiah Karutha Pandian sk_pandian@rediffmail.com; pandiansk@alagappauniversity.ac.in †These authors have contributed equally to this work Reviewed by: Thiruselvam Viswanathan, The University of Texas Health Science Center at San Antonio, United States Chandrajit Lahiri, Sunway University, Malaysia Department of Biotechnology, Alagappa University, Karaikudi, India Farnesol, a self-secreted quorum-sensing molecule (QSM) of Candida albicans, has been known to limit yeast-to-hyphal transition by blocking the RAS1–cAMP–PKA pathway. In a similar fashion, certain bacterial QSMs have also been reported to be successful in attenuating C. albicans bioﬁlm and hyphal formation at relatively high cell density. This prompted us to investigate the antihyphal efﬁcacy of certain bacterial QSMs through virtual docking against seminal drug targets, viz., CYCc and RAS1, that have been reported to be the hallmark players in C. albicans dimorphic virulence cascade. Against this backdrop, 64 QSMs belonging to ﬁve different bacterial QS signaling systems were subjected to initial virtual screening with farnesol as reference. Data of the virtual screening unveiled QSMs belonging to diketopiperazines (DKPs), i.e., 3-benzyl-6-isobutylidene-2,5-piperazinedione (QSSM 1157) and cyclo(L-Pro-L-Leu) (QSSM 1112), as potential inhibitors of CYCc and RAS1 with binding energies of −8.2 and −7.3 kcal mol−1, respectively. Further, the molecular dynamics simulations (for 50 ns) of CYCc-QSSM 1157 and RAS1-QSSM 1112 complexes revealed the mean ligand root mean square deviation (RMSD) values of 0.35 and 0.27 Å, respectively, which endorsed the rigid nature, less ﬂuctuation in binding stiffness, and conformation of binding complexes. Furthermore, the identiﬁed two QSMs were found to be good in solubility, absorption, and permeation and less toxic in nature, as revealed by pharmacokinetics and toxicity analyses. In addition, the in vitro antihyphal assays using liquid and solid media, germ-tube experiment, and microscopic analysis strongly validated DKP-QSSM 1112 as a promising inhibitor of hyphal transition. Taken together, the present study unequivocally proves that DKPs can be used as potent inhibitors of C. albicans virulence dimorphism. ORIGINAL RESEARCH published: 03 December 2021 doi: 10.3389/fcimb.2021.781790 1 INTRODUCTION albicans yeast-to-hyphal transition by mimicking the function of farnesol. The other two QSMs, namely, BDSF and SDSF, have been reported to follow a distinct mode of action unlike 3OC12HSL (Hogan et al., 2004; Grainha et al., 2020). Naturally, such antagonistic interactions between bacteria and fungi occur in human microbiota wherein the bacterial QSMs play an indispensable role in controlling fungal growth to maintain the ecological balance. Therefore, understanding the mighty role of bacterial QSMs, it is envisaged that these bacterial QSMs could be an effective agent to probe against fungal ﬁlamentation by mimicking the action mechanism of the farnesol molecule. p p To date, numerous natural/synthetic compounds have been reported for their remarkable antihyphal efﬁcacy against C. albicans (Manoharan et al., 2017; Priya and Pandian, 2020). However, farnesol, being a self-produced quorum-sensing molecule (QSM) of C. albicans, creates a high level of curiosity due to its unique mode of action. The presence of QSM in C. albicans was ﬁrst reported by Hornby et al. (Hornby et al., 2001), and they made it clear that besides external signal, internally secreted farnesol also modulates the regulation of yeast-to- hyphal transition and subsequent bioﬁlm formation in accordance with cell density. After the discovery of farnesol, few other research groups have tried to ﬁnd out its accurate mode of action in inhibiting yeast-to-hyphal transitions (Nickerson and Atkin, 2017; Chen et al., 2018), but a clear understanding is still lacking. It has been well studied and reported that farnesol negatively regulates the RAS1–cAMP–PKA pathway by hindering the functions of CYR1 by binding with one of its active domains called CYCc (Davis-Hanna et al., 2008; Hall et al., 2011). The GTP-bound RAS1 (activated) directly interacts with CYR1 to catalyze the synthesis of cAMP that eventually activates PKA leading to the expression of several virulence genes in response to different environmental cues (Wang, 2013). Thus, the RAS1–cAMP–PKA pathway seems to be responsible for the expression of several genes associated with yeast-to-hyphal transition as well as other virulence traits and establishes infection (Wang, 2013). Furthermore, farnesol produced by in situ planktonic cells also limits the C. albicans bioﬁlm formation Against this backdrop, the present study was focused to screen and envisage bacterial QSMs with potency to inhibit the seminal protein targets of C. albicans ﬁlamentation through in silico and in vitro analyses. 1 INTRODUCTION owing to the strong association between hyphal morphology and bioﬁlm development (Ramage et al., 2002). Apart from the fungal world, farnesol has also been shown to thwart the virulence such as bioﬁlm and lipase productions in bacteria, viz., Staphylococcus aureus, and sensitizes the drug-resistant S. aureus to gentamicin (Jabra-Rizk et al., 2006). Despite the advances in modern medicine, the management of infectious diseases has become more challenging, as microbial pathogens consistently break down every antimicrobial wall through a phenomenon called “antimicrobial resistance” (Llor and Bjerrum, 2014; Dadgostar, 2019). At times, the situation becomes worse with fungus-associated infections owing to the limitation of therapeutic options (Roemer and Krysan, 2014). Among the various fungal species, Candida albicans is the most representative pathogen at clinical setup, which causes severe contagious infections in humans (Jha and Kumar, 2018). C. albicans is a diploid polymorphic fungus that asymptomatically colonizes various niches of healthy humans as commensal. However, given the opportunity, C. albicans turns into a pathogen and causes clinically diverse infections, especially in individuals with disturbed immune surveillance or other debilitating conditions (Garcia et al., 2021). Various endogenous and exogenous signals facilitate C. albicans to manifest its devastating pathogenesis. Nevertheless, a plethora of studies on C. albicans pathogenesis at the molecular level have reinforced the morphological transition from yeast to hyphae as the hallmark event, which in turn triggers all other virulence traits (at any level of pathogenic circumstances) in a cascade manner (Calderone and Fonzi, 2001; Banerjee et al., 2019). Therefore, identifying molecules with potency to target dimorphic switching has been considered as one of the promising alternatives to effectively combat the infections associated with antifungal-resistant C. albicans by nullifying the phenomenon of selection pressure. Given the prominence of farnesol in effectively interfering with the yeast-to-hyphal switch, several natural and synthetic farnesol analogs have been investigated for their improved inhibitory efﬁcacy against C. albicans dimorphic switching (Shchepin et al., 2003; Lee et al., 2020). Following the same paradigm, certain QSMs of bacteria have been envisaged and well demonstrated to greatly inﬂuence the morphogenesis of C. albicans. For instance, the QSMs N-(3-oxododecanoyl)-L- homoserine lactone (3OC12HSL), cis-2-dodecenoic acid (BDSF), and trans-2-decenoic acid (SDSF) produced by Pseudomonas aeruginosa, Burkholderia cenocepacia, and Streptococcus mutans, respectively, have been reported to substantially limit C. albicans hyphae without altering its basic metabolism (Grainha et al., 2020). Besides, it has also been deciphered that 3OC12HSL interferes with the C. Citation: Jothi R, Hari Prasath N, Gowrishankar S and Pandian SK (2021) Bacterial Quorum-Sensing Molecules as Promising Natural Inhibitors of Candida albicans Virulence Dimorphism: An In Silico and In Vitro Study. Front. Cell. Infect. Microbiol. 11:781790. doi: 10.3389/fcimb.2021.781790 December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 1 Bacterial QS Molecules as Anti-Hyphal Inhibitors Jothi et al. 1 INTRODUCTION Owing to the essentiality of CYR1 and RAS1 in farnesol-mediated hyphal inhibition, they were chosen as potential drug targets in the current study (Wang, 2013; Boutet et al., 2016). Overall, the current study is the ﬁrst of its kind that provides potential insights into bacterial QSMs mediated inter-microbial cross-talk. Further, the outcome of the study will prompt the researchers working in the arena of alternative medicine to refocus/revisit the bacterial QSMs as a novel hyphal inhibitor against pathogenic fungi. 2.2 Validation of Modeled Protein The stereochemical qualities and reliability of modeled structures were veriﬁed by PROCHECK (Program to check the stereochemical quality of protein structures), a program that relies on the Ramachandran plot for structure validation. The number of factors such as overall G-factor, the total number of amino acid residues in the core, and allowed, generously allowed, and disallowed regions were considered for choosing the best model (Laskowski et al., 2005). 2.1 Homology Modeling The experimentally solved crystal structure of CYCc and RAS1 was not publicly available in the Protein Data Bank (PDB). Therefore, three-dimensional (3D) structures of these proteins were modeled on the basis of homology modeling using SWISS- Model (https://swissmodel.expasy.org/). Initially, the amino acid sequences of these target proteins, i.e., CYCc (UniProt ID. December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 2 Bacterial QS Molecules as Anti-Hyphal Inhibitors Jothi et al. P0CY32-1) and RAS1 (UniProt ID. A0A1D8PR83-1; aa length. 1,329–1,466), were retrieved from UniProt KnowledgeBase (UniProtKB) in FASTA format. The resulting sequence was submitted to PDB-BLAST in order to identify the appropriate templates for structural modeling. Finally, the 3D structures of CYCs and RAS1 were generated using SWISS-Model (Boutet et al., 2016). P0CY32-1) and RAS1 (UniProt ID. A0A1D8PR83-1; aa length. 1,329–1,466), were retrieved from UniProt KnowledgeBase (UniProtKB) in FASTA format. The resulting sequence was submitted to PDB-BLAST in order to identify the appropriate templates for structural modeling. Finally, the 3D structures of CYCs and RAS1 were generated using SWISS-Model (Boutet et al., 2016). respectively. Initially, docked complexes were kept in a cubic box surrounded by SPC, a water molecule (water density 1.0) using SPC/E water models, and further, the whole system was neutralized by the addition of Na+ ions. The solvated structures were energy minimized using GROMOS54a7 force ﬁeld and equilibrated by running simulations for 100 ps under constant NVT (300 K temperature) and NPT (1 atm pressure) ensemble. The equilibrated complexes were further extended to run production MD simulation for 50 ns at constant temperature and pressure. The GROMACS package was used to calculate the structural properties such as root mean square deviation (RMSD), root mean square ﬂuctuation (RMSF), hydrogen bond radius of gyration, and solvent accessible surface area. Finally, the resulting values were plotted as graphs using Origin Pro. 2.9 In Vitro Filamentation Assay 2.9 In Vitro Filamentation Assay In order to substantiate the in silico ﬁnding, in vitro antihyphal assay was performed using farnesol as the positive control (Priya and Pandian, 2020). For this experiment, one of the predicted ligands, i.e., QSSM 1112, was employed to assess its inhibitory propensity on C. albicans dimorphic switching. The QSSM 1112 (purity >98% based on high-performance liquid chromatograph) used in the current study was reported in our previous study (Gowrishankar et al., 2014) and deployed for assays by dissolving in methanol at the ﬁnal concentration of 100 mg/ml. Brieﬂy, C. albicans cells (1 × 106 CFU/ml) were dispensed into 24-well microtiter plates (MTPs) containing 1 ml of spider medium supplemented with QSSM 1112 and farnesol at various concentrations (0–1,024 µg/ml) along with 10% of fetal bovine serum (FBS). After incubation at 37°C for 24 h, the pellets were resuspended in 10 µl of phosphate-buffered saline (PBS), and the 2.4 Ligand Selection and Preparation 2.4 Ligand Selection and Preparation A total of 64 bacterial QSMs screened in this study were retrieved from the SigMol database created by Rajput et al. (2016) (http:// bioinfo.imtech.res.in/manojk/sigmol) (Rajput et al., 2016). Basic information about the ligands such as molecular weight and 3D structure (.SDF) was retrieved from PubChem and ChemSpider databases, respectively. Furthermore, the AutoDock formats (.pdbqt) were generated through AutoDock Vina in PyRx 0.8 software for the molecular docking simulation (Rolta et al., 2020). 2.8 Strain and Culture Condition The test organism C. albicans (ATCC 90028) used in the present study was procured from HiMedia, India. The fungus was maintained in Sabouraud Dextrose Agar (SDA) plates and routinely cultured in yeast extract peptone dextrose (YEPD) broth at 37°C until used for tests. The culture with 0.1 optical density (OD) (1 × 106 CFU/ml) was used as the inoculum to perform antihyphal assays. The hyphal assay was performed in spider medium (consisting of mannitol 1%, K2HPO4 0.2%, and nutrient broth 1%) to allow hyphal elongation. 2.5 Docking-Based Virtual Screening In order to identify the potential inhibitors against putative active sites of target proteins CYCc and RAS1, virtual screening was performed using PyRx, 0.8. The minimized proteins and ligand molecules were imported to PyRx, and blind docking was performed (Schuttelkopf and van Aalten, 2004). The full grid map was constructed around the complete target proteins. The resulting docking conformations were ranked based on their binding afﬁnities in comparison with the reference molecule (farnesol). Finally, the best-docked complexes were visualized using Discovery studio visualize and ligplot+. 2.3 Protein Preparation The antihyphal inhibitors screened via molecular docking and molecular simulations were checked for various pharmacokinetic parameters using SwissADME online tool (http://www. swissadme.ch) (Rodrigues et al., 2020). The smileys of both ligands were collected from SigMol database and added into SwissADME as an input to analyze the principles of ADMET (absorption, distribution, metabolism, and elimination), pharmacokinetics, drug likeliness, and medical chemistry. In order to predict the toxicity dosage and toxicity class of the ligands, ProTox-II server (https://tox-new.charite.de/protox_II/) was utilized (Banerjee et al., 2018). p Further, the validated modeled structures of CYCs and RAS1 were prepared for docking using AutoDock. In order to minimize the energy, the solvated structures were reﬁned through the computation of Gasteiger charges with the addition of the polar hydrogens. Finally, reﬁned protein structures were subjected to further analysis. 2.11 Fluorescence Microscopic Analysis 2.11 Fluorescence Microscopic Analysis To further ascertain the QSSM 1112 mediated hyphal and germ- tube inhibition, ﬂuorescence microscopic analysis was performed. For hyphal assay, the C. albicans cells were allowed to form hyphal formation on a 1-cm2 glass slide in 24-well MTPs containing spider medium supplemented with farnesol and QSSM 1112 at their HIC. After incubation, non-adherent planktonic cells were removed, and adhered hyphal cells on the slides were stained with 1% acridine orange for 20 min. For germ-tube inhibition assay, C. albicans cells were grown in test tubes containing spider medium supplemented with farnesol and QSSM 1112 at their HIC for 24 h. Subsequent to incubation, cell pellets were collected by centrifugation at 8,000 rpm for 10 min. Then, pellets were resuspended in 100 µl of PBS and followed by staining with 20 µl of 1% acridine orange for 20 min. After incubation, the difference in cell morphology was visualized under ﬂuorescence microscopy (Ts2R, ECLIPSE, and Japan). q y y Since the experimentally solved 3D structure of selected target proteins, i.e., CYCc and RAS1, were not available in freely accessible public databases, homology modeling was done using SWISS-MODEL to obtain the good models. The primary sequences of target proteins CYCc (Q9P977_CANAX) and RAS1 (P0CY32_CANAX) were retrieved from UNIPROTKB database. Based on the results of SWISS-MODEL’s Critical Assessment of Structure Prediction (CASP), adenylate cyclase from Trypanosoma brucei (PDB: 1fx2) and GTPase KRas from Homo sapiens (PDB: 6god) were identiﬁed to have high sequence similarity, i.e., 64.71% and 34.78% for CYCc and RAS1, respectively. In general, a protein sequence with a minimum 30% identity with target protein is adequate to be considered as an appropriate template for accurate homology modeling (Fiser, 2010). Correspondingly, 1fx2 and 6god were selected as templates for building CYCc and RAS1 models. The 3D structures of modeled proteins are shown in Figures 1A, B. Subsequently, the qualities of generated protein models were 2.6 Molecular Dynamics Simulation y The best interactive ligand molecules with their target proteins were subjected to 50 ns of molecular dynamics (MD) simulation by Gromacs 5.1.4 simulation package (http://www.gromacs.org/) using all-atom optimized potentials for liquid simulations (OPLS-AA) force ﬁeld (Abdulazeez, 2019). The topological parameter ﬁles of proteins and ligand molecules were prepared from GROMOS96 force ﬁeld and PRODRG2 web server, December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 3 Bacterial QS Molecules as Anti-Hyphal Inhibitors Jothi et al. ratio of yeast to hyphal cells was further conﬁrmed by a light microscope at ×400 magniﬁcation (Nikon Eclipse 80i, Japan). ratio of yeast to hyphal cells was further conﬁrmed by a light microscope at ×400 magniﬁcation (Nikon Eclipse 80i, Japan). commensal yeasts as opportunistic pathogens, blocking this morphological switch is imperative to control infections associated with C. albicans (Roemer and Krysan, 2014; Banerjee et al., 2019). So far, many molecules with antihyphal proﬁciency have been reported from natural and organic sources against C. albicans (Manoharan et al., 2017; Priya and Pandian, 2020). However, the QSMs of bacteria remain unexplored for antihyphal propensity. Few reports have been focused on the competitive antagonistic interactions between bacteria and C. albicans, which signify that bacterial QSMs could play a key role in targeting and blocking yeast-to-hyphal transition (Hogan et al., 2004; Nickerson and Atkin, 2017). This intertaxon chemical communication between bacteria and fungi prompted us to investigate the effect of bacterial QSMs as natural hyphal inhibitors against C. albicans through in silico approach. Therefore, in the current study, 64 QSMs belonging to ﬁve different bacterial QS signaling systems such as acylated homoserine lactones (AHLs), diketopiperazines (DKPs), diffusible signal factors (DSFs), autoinducer-2 (AI-2), and 4- hydroxy-2-alkylquinolines (HAQs) were selected and subjected to virtual docking against the two identiﬁed seminal drug targets (CYCc and RAS1) of C. albicans. It was anticipated that the outcome of the present study would render a basic understanding of the interaction of bacterial QSMs against important hyphal proteins of C. albicans. 3.1 Homology Modeling 3.1 Homology Modeling Primarily, homology modeling of target protein structures was performed. Protein crystal structure is important for understanding the mechanisms of biological systems and protein complexes to manipulate or inhibit protein function (Vyas et al., 2012). Owing to the unavailability of resolved crystal structure for the target protein in PDB, computational-based techniques were used for 3D structure prediction. Homology modeling offers 3D models of protein structures when only sequence data are available (Satyanarayana et al., 2018). 2.10 Germ-Tube Inhibition Assay The efﬁciency of QSSM 1112 in inhibiting the C. albicans germ- tube formation was evaluated through germ-tube assay as described by Zhang et al. (2011) (Zhang et al., 2011). Here again, farnesol was used as a positive control for the comparative analysis. In brief, C. albicans cells (1 OD) were used to inoculate into the 200 µl of FBS along with QSSM 1112 at its hyphal inhibitory concentration (HIC), i.e., 64 µg/ml. The tubes were incubated at 37°C for 5 h to allow germ-tube induction. At different time intervals, i.e., 0, 2, 4, and 6 h, the cells were removed aseptically and examined under a light microscope at ×400 magniﬁcation in order to assess the germ-tube induction (Nikon Eclipse 80i, Japan). Additionally, the inhibitory propensity of QSSM 1112 on preformed germ-tube formation was evaluated. Initially, the C. albicans cells were allowed for germ-tube induction following the protocol mentioned above. Then, the QSSM 1112 (at HIC) was manifested on germinated C. albicans cells and incubated at 37°C for 3 h. The cells without the active agent were considered as untreated control. Subsequent to incubation, the morphological changes between QSSM 1112-treated and untreated control cells were observed under a light microscope at ×400 magniﬁcation (Nikon Eclipse 80i, Japan). 3 Structure-Based Virtual Screening 3.3 Structure-Based Virtual Screening As the virtual screening workﬂow offers selective ﬁltration of ligands (lead drug) that are capable of interacting with protein macromolecules (drug target) with good binding energy, it has been an imperative tool in drug discovery (Maia et al., 2020). In the current study, virtual screening was done for 64 QSMs belonging to ﬁve different bacterial QS signaling systems, and 3 RESULTS AND DISCUSSION Clinical complications associated with the conventional antifungals immensely demand a new treatment strategy against the dimorphic fungus C. albicans. One of the proven strategies that propel in the arena of alternative medicine to get rid of drug resistance is “disarming the virulence without disturbing the metabolism” of any fungus (Charro and Mota, 2015). As hyphal transition is a hallmark event that lionizes Subsequently, the qualities of generated protein models were validated through the Ramachandran plot constructed using PROCHECK module from SAVES Server (https://saves.mbi. December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Bacterial QS Molecules as Anti-Hyphal Inhibitors Jothi et al. ucla.edu/). As can be seen from Figure 1C, the predicted 3D structure of CYCc has 95.0%, 12.5%, 0.0%, and 0.0% of residues in the most favored, additional allowed, generously allowed, and disallowed regions, respectively. Similarly, the RAS1 structure has 94.8%, 5.2%, 0.0%, and 0.0% of residues in the most favored, additional allowed, generously allowed, and disallowed regions, respectively (Figure 1D). According to the Ramachandran plot, a model structure upholding more than 90% of residues in the most favored region is deemed to be as accurate as like the experimentally solved crystal structure (2-Å resolution) (Sobolev et al., 2020). Interestingly, both the generated models showed ≥90% residues [i.e., CYCc (95.0%) and RAS1 (94.8)] in the most favored region. Besides, both the models did not cover any residue in the disallowed region, which ascertained that the SWISS-MODEL predicted models as reasonable and reliable for further molecular docking studies. and to remove close contacts in the geometric chain for the predicted proteins. Initially, the reﬁned proteins were energy minimized using AMBER ff14SB and Gasteiger charges for standard and non-standard amino acid residues, respectively. Finally, the minimized protein structures were saved in “.pdb” extension and used for further molecular docking. The 3D structures of 64 bacterial QSMs were retrieved from SigMoL database (Rajput et al., 2016). Open Babel platform was used to equalize the ligand format to “.sdf” extension, which was collected from different primary databases integrated with SigMoL database. The reformatted ligands were energy minimized with uff force ﬁeld using Open Babel wizard on PYRX 0.8. The ligands were saved in “.pdbqt” extension for further molecular docking and protein–ligand simulation analyses. 3.2 Protein Preparation Generally, the 3D structures built through homology modeling used to have unfavorable bond lengths, bond angles, torsion angles, and contacts. Therefore, energy minimization was done using UCSF Chimera to normalize local bond length and angle A B D C FIGURE 1 \| The three-dimensional structure of CYCc (A) and RAS1 (B) built using SWISS-MODEL. Ramachandran plot generated portraying the stereochemical qualities of the modeled CYCc (C) and RAS1 (D) proteins. C A B D B D FIGURE 1 \| The three-dimensional structure of CYCc (A) and RAS1 (B) built using SWISS-MODEL. Ramachandran plot generated portraying the stereochemical qualities of the modeled CYCc (C) and RAS1 (D) proteins. December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Bacterial QS Molecules as Anti-Hyphal Inhibitors Jothi et al. the best hits are listed (Tables 1 and 2) based on their binding energy toward the selected two seminal drug targets, viz., CYCc and RAS1. The obtained binding energies and hydrogen bond interactions of docked ligands with target proteins were summarized in Tables 1 and 2. the best hits are listed (Tables 1 and 2) based on their binding energy toward the selected two seminal drug targets, viz., CYCc and RAS1. The obtained binding energies and hydrogen bond interactions of docked ligands with target proteins were summarized in Tables 1 and 2. has been reported against C. albicans (Srivastava and Dubey, 2016). Taken together, the present docking result with other existing investigations suggests the plausible existence of intercommunication cross-talk between Streptomyces species and C. albicans, which in a deeper sense certainly requires further investigation. In this study, a well-known antihyphal QSM of C. albicans, viz., farnesol, was used as the positive control. It showed a binding afﬁnity of −6.4 and −7.0 kcal mol−1 toward CYCc and RAS1, respectively. Two ﬁltration processes were done for 64 QSMs to select one best hit against each of the two target proteins. Initially, the top three lead molecules were selected. However, on comparing the binding afﬁnity and binding proﬁle with reference molecule farnesol, the best hit was identiﬁed. The second top hit molecule QSSM 1141 belonging to DKP signaling system has been documented to be produced by Gram- negative proteobacterium Ruegeria spp. It formed two hydrogen bonds with Asp76 and Ser138 at 2.92 and 2.75 distances, respectively. 3.2 Protein Preparation It also formed 10 hydrophobic interactions with the amino acid residues of Phe4, Thr 5, Asp6, Lys45, Glu47, Ala50, Met52, Thr74, Arg105, and Ser135 (Figure 2Ab). QSSM 1153, also belonging to DKP signaling system and isolated from soil-dwelling Gram-positive bacterium Saccharothrix espanaensis, has the required structure to interact through two hydrogen bonds with Asp76 and Ser138 at 2.96 and 2.73 distances, respectively. Similar to QSSM 1141, it can also form 10 hydrophobic bonds with the amino acid residues of Phe4, Thr 5, Asp6, Lys45, Glu47, Ala50, Met52, Thr74, Arg105, and Ser135. It is noteworthy to mention here that a new class of antibiotics, namely, saccharomicins produced by S. espanaensis, has already been reported to have potent antimicrobial efﬁcacy against certain pathogenic bacteria and yeast (Figure 2Ac) (Singh et al., 2000). 3.3.1 Quorum-Sensing Molecules’ Interaction Mode Against CYCc As shown in Table 1, the binding afﬁnity of docked QSMs against the target protein CYCc ranged from −4.9 to −8.2 kcal mol−1. The maximum binding afﬁnity of −8.2 kcal mol−1 was displayed by a QSM named cyclo(L-Phe-L-4-OH-Pro), which belongs to DKP signaling system. The initial selection was based on the binding afﬁnity toward the target protein in which out of 31 AHLs, 15 DKPs, 14 DSFs, 2 HAQs and 2 AI-2, 7 AHLs, 9 DKPs, and 2 HAQs were considered for selecting the top three hits, as they exhibited higher binding afﬁnity than the farnesol. On the other hand, the QSMs belonging to AI-2 and DSFs displayed considerably lower binding energy than the positive control farnesol, and hence, these were excluded for the selection of the top three hits. Based on the farnesol pattern of hydrogen bond formation and non-hydrophobic interactions with the target proteins CYCc and RAS1, the top three QSMs were examined, and one best ligand molecule was selected. Accordingly, QSSM 1157 was selected as the best ligand molecule among the three DKPs. It can establish two hydrogen bonds (at Asp6 and Asp76) and ﬁve non- hydrophobic bonds (at Phe4, Glu47, Ala50, Met52, and Thr74) at the same position as like farnesol with a greater binding afﬁnity score. Hence, this ligand molecule was taken forward for MD analysis to understand its interaction with CYCc protein. Apart from the binding energy, the binding proﬁle of QSMs that closely resembles farnesol’s binding proﬁle was considered as the chief criterion to select the top three lead molecules. Based on this, QSMs 3-benzyl-6-(2-methylpropylidene)-2,5- piperazinedione (QSSM 1157), cyclo(L-Phe-L-4-OH-Pro) (QSSM 1141), and cyclo(L-Phe-cis-4-OH-D-Pro) (QSSM 1153) were selected as the top three hits, which showed the binding afﬁnity of −8.2, −8.1, and −7.9 kcal mol−1, respectively (Figure 2A). 3.3.2 Quorum-Sensing Molecules’ Interaction Mode Against RAS1 p y g The positive control (farnesol) exhibited a strong binding afﬁnity toward CYCc with the docking score of −6.4 kcal mol−1. Farnesol interacted with CYCc by forming three hydrogen bonds with amino acid residues of Asp6, Asp76, and Arg105 at 3.05, 3.07, and 2.80 distances, respectively. Furthermore, it showcased 10 hydrophobic interactions with Phe4, Thr 5, Lys45, Glu47, Ala50, Met52, Thr74, Tyr126, Phe127, and Val131 (Figure 2Ad). First, the ligand QSSM 1157 isolated from bacterium Streptomyces albulus has shown two hydrogen bonds with the residues of Asp6 and Asp76 at 3.02 and 3.12 distances, respectively. It also showed nine hydrophobic bonds with the residues of Phe4, Glu47, Ala50, Met52, Thr74, Ala75, Arg105, Ser135, and Ser138. Notably, S. albulus has already been reported to be a good producer of the fungicide cycloheximide (Figure 2Aa) (Yin et al., 2014). The antifungal effect of S. albulus synthesized dipeptides; i.e., e-polylysine was demonstrated against C. albicans (Shima et al., 1984). In addition, the antibioﬁlm activity of a metabolite produced by other Streptomyces species, i.e., Streptomyces chrestomyceticus, The positive control (farnesol) exhibited a strong binding afﬁnity toward CYCc with the docking score of −6.4 kcal mol−1. Farnesol interacted with CYCc by forming three hydrogen bonds with amino acid residues of Asp6, Asp76, and Arg105 at 3.05, 3.07, and 2.80 distances, respectively. Furthermore, it showcased 10 hydrophobic interactions with Phe4, Thr 5, Lys45, Glu47, Ala50, Met52, Thr74, Tyr126, Phe127, and Val131 (Figure 2Ad). As tabulated (Table 2), the 64 docked QS ligand molecules displayed a range of binding afﬁnity with a maximum of −8.5 kcal mol−1 and a minimum of −5.4 kcal mol−1 against the modeled crystal structure of RAS1 protein. In particular, a QSM named p-coumaroyl-HSL belonging to the AHL system demonstrated the highest binding afﬁnity (−8.5 kcal mol−1). In parallel, the reference molecule (farnesol) exhibited strong binding interactions toward RAS1 with a binding energy of −7 kcal mol−1 (Figure 2B). First, the ligand QSSM 1157 isolated from bacterium Streptomyces albulus has shown two hydrogen bonds with the residues of Asp6 and Asp76 at 3.02 and 3.12 distances, respectively. It also showed nine hydrophobic bonds with the residues of Phe4, Glu47, Ala50, Met52, Thr74, Ala75, Arg105, Ser135, and Ser138. Notably, S. albulus has already been reported to be a good producer of the fungicide cycloheximide (Figure 2Aa) (Yin et al., 2014). The antifungal effect of S. 3.3.2 Quorum-Sensing Molecules’ Interaction Mode Against RAS1 albulus synthesized dipeptides; i.e., e-polylysine was demonstrated against C. albicans (Shima et al., 1984). In addition, the antibioﬁlm activity of a metabolite produced by other Streptomyces species, i.e., Streptomyces chrestomyceticus, For the identiﬁcation of the top three hits against RAS1, the same selection criterion used for the CYC1 target protein was employed. Accordingly, the QSMs belonging to DKP (9), AHL (18), and HAQ (2) QS signaling systems were selected and considered for the next level of screening, as they displayed better binding energy than the reference molecule. On the other hand, the QSMs of AI-2 and DSFs showcased marginal binding energy than the biomolecules of other QS systems and reference molecules, and hence they were excluded from further consideration. December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 6 Bacterial QS Molecules as Anti-Hyphal Inhibitors Jothi et al. TABLE 1 \| List of the 64 screened QSMs with their binding energy and interaction residues against CYCc as predicted through virtual docking. S. No SigMolQSSM ID Ligand name ID Energy (kcal mol−1) H-bonding Signaling system 1. QSSM 1141 Cyclo(L-Phe-L-4-OH-Pro) 8643197* −8.2 Asp76, Ser138 DKPs 2. QSSM 1111 Cyclo(L-Pro-L-Tyr) 106647* −8.1 Ser138 DKPs 3. QSSM 1153 Cyclo(L-Phe-cis-4-OH-D-Pro) 8976612* −8.1 Asp76, Ser138 AHLs 4. QSSM 1157 3-Benzyl-6-isobutylidene-2,5-Piperazinedione 8666841* −7.9 Asp6, Asp76 DKPs 5. QSSM 1133 Cyclo(L-Pro-D-Phe) 391657* −7.6 Ser135 DKPs 6. QSSM 0615 Cyclo(L-Phe-L-Pro) 90257* −7.4 Ser138 DKPs 7. QSSM 0614 p-Coumaroyl-HSL 30790745* −7.3 Lys45, Arg105, Ser135, Ser138 AHL 8. QSSM 1156 3-Benzylidene-6-isobutyl-2,5-piperazinedione 24725401* −7.2 Ser135 DKPs 9. QSSM 0615 Cinnamoyl-HSL 9797494* −7.2 Arg105, Ser135, Ser138 AHLs 10. QSSM 0005 N-Tetradec-7-enoyl-L-homoserine lactone 28589930* −7.1 Ser10, Asp49 AHLs 11. QSSM 1144 Cyclo(Gly-L-Tyr) 19927129* −7 Lys45, Ser138 DKPs 12. QSSM 0271 N-(3-Oxononanoyl)-L-homoserine lactone 9768814* −7 Ser10, Thr11 AHLs 13. QSSM 1132 Cyclo(L-Pro-L-isoLeu) 8166289* −6.9 Asp76 DKPs 14. QSSM 1262 2-Heptylquinolin-4(1H)-one 164974# −6.8 Thr74, Ser138 HAQs 15. QSSM 0619 N-Isovaleryl-L-homoserine lactone 29368455* −6.6 Ser10, Thr11 AHLs 16. QSSM 0042 N-(3-Hydroxydecanoyl)-L-homoserine lactone 9696680 −6.6 Ser10, Thr11, Asp76 AHLs 17. QSSM 0012 2-Heptyl-3-hydroxy-4-quinolone 2763159# −6.5 Thr74 HAQs 18. QSSM 1151 Cyclo(L-Leu-trans-4-OH-L-Pro) 10184045* −6.4 Ala50 DKPs QSSM Farnesol 445070# −6.4 Asp6, Asp76, Arg105 Fungal QSM 19. QSSM 1112 Cyclo(L-Pro-L-Leu) 5428292* −6.4 Thr74 DKPs 20. QSSM 0739 N-(3-Oxotetradec-7-enoyl)-L-homoserine lactone 9057115* −6.4 Asp76, Ser135 AHLs 21. QSSM 0891 N-Pentanoyl-L-homoserine lactone 9688673* −6.4 Ser10, Thr11 AHLs 22. QSSM 0012 N-(3-Oxododecanoyl)-L-homoserine lactone 10221060# −6.2 Arg100 AHLs 23. 3.3.2 Quorum-Sensing Molecules’ Interaction Mode Against RAS1 QSSM 0368 N-(3-Oxohexadec-9-enoyl)-L-homoserine lactone 29341818* −6.2 Arg100, Ser135 AHLs 24. QSSM 1148 Cyclo(D-4-OH-Pro-L-Val) 34017887* −6.2 Lys45 DKPs 25. QSSM 0041 N-(3-Hydroxydodecanoyl)-L-homoserine lactone (OH- dDHL) 9067166* −6.2 Thr74, Asp76 AHLs 26. QSSM 1114 Cyclo(L-Val-L-Leu) 122161* −6.1 Asp6, Thr11, Asp76 DKPs 27. QSSM 0234 N-Tetradec-2,7-dienoyl-L-homoserine lactone 28589928* −6.1 Asp76, Ser135 AHLs 28. QSSM 1194 (2E,6E)-Farnesoic acid 4439611* −6.1 Ser135 DSFs 29. QSSM 0389 N-(3-Hydroxytetradec-7-enoyl)-L-homoserine lactone 4445245* −6.1 Thr74, Asp76, Ser135 AHLs 30. QSSM 1177 (2E,4E)-11-Methyl-2,4-dodecadienoic acid 8031542* −6.1 Ile7, Asn9, Arg105 DSFs 31. QSSM 0573 3-Oxo-C16-HSL 9683431* −6.1 Thr74, Ser135 AHLs 32. QSSM 0046 N-(3-Oxooctanoyl) homoserine lactone 127293# −6 Asp76 AHLs 33. QSSM 0987 (1S,4S,5R)-7,7-dihydroxy-1-methyl-2,6,8-trioxa-7- boranuidabicyclo[3.3.0]octane-4,5-diol 395434* −6 Asp6, Thr11, Asp72, Arg105 AI-2 34. QSSM 0104 N-(3-Hydroxyoctanoyl)-L-homoserine lactone 9761556* −6 Asp76 AHLs 35. QSSM 0038 N-(3-Hydroxybutanoyl)-L-homoserine lactone 115719* −5.9 Ser138 AHLs 36. QSSM 0026 3-Hydroxy-N-[(3S)-2-oxotetrahydro-3-furanyl] tetradecanamide 9078807* −5.9 Thr74, Asp76 AHLs 37. QSSM 0020 N-Hexanoyl-L-homoserine lactone 10058590# −5.8 Ser138 AHLs 38. QSSM 0013 N-Butyryl-L-homoserine lactone 10130163# −5.8 Asp76 AHLs 39. QSSM 0048 N-Heptanoylhomoserine lactone 443437# −5.8 Asp76 AHLs 40. QSSM 0003 N-Octanoyl-L-homoserinelactone 6914579# −5.8 Ser135 AHLs 41. QSSM 0369 N-(Tetrahydro-2-oxo-3-furanyl)-octadecanamide 69757690# −5.8 Ser135 AHLs 42. QSSM 1129 Cyclo(Pro-Val) 8997025* −5.8 Asp6, Arg105 DKPs 43. QSSM 1129 Cyclo(Pro-Val) 8997025* −5.8 Asp6, Arg105 DKPs 44. QSSM 0107 N-3-Oxo-tetradecanoyl homoserine lactone 11688418# −5.7 Arg100, Ser135 AHLs 45. QSSM 0025 N-(3-Hydroxyhexanoyl)-L-homoserine lactone 9754171* −5.7 Asp76 AHLs 46. QSSM 1202 Decanoic acid 2863# −5.6 Asn9, Ser10, Thr11, Arg105 DSFs 47. QSSM 1191 2-Pentadecenoic acid 4445869* −5.6 Ser135 DSFs 48. QSSM 0001 N-(beta-Ketocaproyl)-L-homoserine lactone 688505# −5.6 Ser135 AHLs 49. QSSM 0764 N-(3-Hydroxyhexadecanoyl)-L-homoserine lactone 9741349* −5.6 Arg100, Ser135 AHLs 50. QSSM 1175 Decenoic acid 4445851* −5.5 Asn9, Ser10, Thr11, Arg105 DSFs 51. QSSM 1190 2-Tetradecenoic acid 4445865* −5.5 Thr5, Ser138 DSFs 52. QSSM 1167 11-Methyl-2-dodecenoic acid 9644750* −5.5 Asp6 DSFs 53. QSSM 1204 Tridecylic acid 12013* −5.4 Ser135, Ser138 DSFs 54. QSSM 0367 N-Hexadec-9-enoyl-L-homoserine lactone 28589932* −5.4 Ser135 AHLs 55. QSSM 1181 Undecenoic acid 4445855* −5.4 Lys45, Asp125, Tyr126 DSFs 56. QSSM 0016 N-Hexadecanoyl-homoserine lactone 11609787# −5.3 Arg100 AHLs 57. QSSM 1192 Lauric acid 3756* −5.3 Ser135, Ser138 DSFs (Contin of the 64 screened QSMs with their binding energy and interaction residues against CYCc as predicted through virtual docking. December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Bacterial QS Molecules as Anti-Hyphal Inhibitors Jothi et al. TABLE 1 \| Continued S. 3.3.2 Quorum-Sensing Molecules’ Interaction Mode Against RAS1 No SigMolQSSM ID Ligand name ID Energy (kcal mol−1) H-bonding Signaling system 58. QSSM 0990 4,5-Dihydroxy-2,3-pentanedione 9346083* −5.3 Asp6, Ser10, Thr11, Asp49, Arg105 AI-2 59. QSSM 1205 Pentadecanoic acid 13249* −5.2 Ser134 DSFs 60. QSSM 1145 Cyclo(Gly-L-Val) 644035* −5.2 Gln67, Asp118, Asp125 DKPs 61. QSSM 0289 N-(3-Oxononanoyl)-L-homoserine lactone 289 −5.1 Asp6, Ser10, Glu47, Asp49 AHLs 62. QSSM 1183 2-Tridecenoic acid 4445862* −5 Ile7, Asn9, Thr11 DSFs 63. QSSM 1159 2-Dodecenoic acid 4471801* −5 Thr74, Asp76 DSFs 64. QSSM 1179 2-Octenoic acid 4445841# −4.9 Asn9, Thr11, Arg105 DSFs QSMs, quorum-sensing molecules; DKPs, diketopiperazines; AHLs, acylated homoserine lactones. From the analysis, it was evident that QSMs cyclo(L-Pro-L- Leu) (QSSM 1112), cyclo(L-Leu-trans-4-OH-L-Pro) (QSSM 1151), and cyclo(D-4-OH-Pro-L-Val) (QSSM 1148) with binding energies of −7, −7.3, and −7.3 kcal mol−1 occupy the top three hit positions, as they showcased accurate binding poses in comparison with farnesol (Figure 2Bd). The positive control (farnesol) interacts with the RAS1 protein by establishing four hydrogen bonds with the amino acid residues of Asp120, Ser147, Ala148, and Lys149 at the distances of 2.91, 2.86, 2.97, and 2.89, respectively. selected as the best ligand, as it established four strong hydrogen Q18 bonds (at Asp120, Ser147, Ala148 and Lys 149) and six non- hydrophilic bonds (Gly14, Gly16, Ala19, Phe29, Tyr33 and Asn117 ) with RAS1 and taken forward to investigate the QSSM 1112-RAS1 complex stability. Overall, out of 64 QSMs belonging to ﬁve different QS signaling systems used in the virtual docking analysis, two QSMs, viz., QSSM 1157 and QSSM 1112, belonging to DKPs were selected as the best hits against CYCc and RAS1, respectively. The 3D interaction of QSSM 1157 and QSSM 1112 against CYCc and RAS1 is shown in Figure 3. Moreover, the data of virtual screening obviously demonstrated the phenomenal binding efﬁcacy of QSMs from DKP QS system than other docked signaling systems. In contrast, a couple of earlier studies have reported the inhibitory efﬁcacy of AHLs as well as DSFs toward C. albicans hyphal dimorphism in a dose- dependent manner (Zhang et al., 2011; Grainha et al., 2020). QSSM 1112 isolated from soil-dwelling Gram-positive bacterium S. espanaensis could establish three hydrogen bonds with the amino acid residues of Asp120, Ser147, and Ala148 at the distances of 2.97, 3.06 and 3.19, respectively. In addition, it formed six hydrophobic bonds with Gly14, Gly16, Ala19, Phe29, Tyr33, Asn117, and Lys118 (Figure 2Ba). 3.3.2 Quorum-Sensing Molecules’ Interaction Mode Against RAS1 y , , y ( g ) The QS molecule QSSM 1151 has been identiﬁed from various bacterial sources, namely, Pseudomonas putida, Burkholderia cepacia, Shewanella baltica, and Streptomyces spp. It stabilized the interaction with RAS1 through three hydrogen bonds with Ala148, Lys149, and Asp120 (at binding distances of 3.10, 3.10, and 3.05, respectively) and eight hydrophobic bonds with amino acid residues of Gly16, Ala19, Phe29, Asp31, Asn117, Lys118, Leu121, and Ser147 (Figure 2Bb). A study conducted by Li et al. (2007) revealed the antifungal efﬁcacy of a novel compound from B. cepacia. Further, B. cepacia has been reported as a source of production of antibiotics such as pyrrolnitrin, altericidins, and cepacin. Furthermore, another study signiﬁed the antifungal efﬁcacy of extract from Streptomyces spp. against C. albicans (Human et al., 2016). Notably, the antagonistic mechanism of B. cepacia through its signaling molecules, i.e., BDSF, toward C. albicans has already been well investigated by Boon et al. (2008), since the QSM of B. cepacia showed a better binding interaction with RAS1, which further signiﬁes the possible interspecies communication-mediated regulation of C. albicans virulence dimorphism by B. cepacia QSM. Similar to QSSM 1141, QSSM 1148 is synthesized by the Gram-negative proteobacterium Ruegeria spp. It interacts with RAS1 by forming three hydrogen bonds with Ser147, Alu148, and Lys49 and 10 hydrophobic bonds with Gly14, Gly16, Ala19, Phe29, Glu32, Tyr33, Asn117, Lys118, and Leu121 (Figure 2Bc). Out of the top three hits, the best one QSM against RAS1 was selected based on the same interaction criteria followed for CYCc. As a result, the QS ligand molecule QSSM 1112 was y y ( g ) The QS molecule QSSM 1151 has been identiﬁed from various bacterial sources, namely, Pseudomonas putida, Burkholderia cepacia, Shewanella baltica, and Streptomyces spp. It stabilized the interaction with RAS1 through three hydrogen bonds with Ala148, Lys149, and Asp120 (at binding distances of 3.10, 3.10, and 3.05, respectively) and eight hydrophobic bonds with amino acid residues of Gly16, Ala19, Phe29, Asp31, Asn117, Lys118, Leu121, and Ser147 (Figure 2Bb). A study conducted by Li et al. (2007) revealed the antifungal efﬁcacy of a novel compound from B. cepacia. Further, B. cepacia has been reported as a source of production of antibiotics such as pyrrolnitrin, altericidins, and cepacin. Furthermore, another study signiﬁed the antifungal efﬁcacy of extract from Streptomyces spp. against C. albicans (Human et al., 2016). Notably, the antagonistic mechanism of B. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 3.3.2 Quorum-Sensing Molecules’ Interaction Mode Against RAS1 cepacia through its signaling molecules, i.e., BDSF, toward C. albicans has already been well investigated by Boon et al. (2008), since the QSM of B. cepacia showed a better binding interaction with RAS1, which further signiﬁes the possible interspecies communication-mediated regulation of C. albicans virulence dimorphism by B. cepacia QSM. Despite having better binding energy, AHL could not occupy the top hit list owing to its weak binding proﬁle when compared with farnesol. On the other hand, the well-reported structural and functional farnesol analogs of DSFs have displayed a very weak binding energy among the used signaling systems. The weak binding proﬁle of DSFs toward target proteins (CYCc and RAS1) could be because of a distinct antihyphal mode of action different from that of the action mechanism of farnesol. In line with this, a previous ﬁnding has also demonstrated that, unlike AHLs and farnesol, the antihyphal mechanism of BDSFs (a B. cenocepacia QSM) follows a different mode of action (Hall et al., 2011). The data of the current study unveiled the excellent binding mode of DKPs over essential hyphal proteins. DKPs are the simplest cyclic dipeptides that have been utilized for myriads of pharmaceutical propensities over the past few decades (de Carvalho and Abraham, 2012). The drug likeliness properties of DKPs attracted the attention of researchers to use them for a wide range of biological activities including antitumor, antiviral, antifungal, antibacterial, and antihyperglycemic efﬁcacies (Rhee, 2004). Besides, the QS intervening efﬁcacy of DKPs by acting as a QS signaling molecule during bacterial cross-talk has been reviewed critically by Bellezza et al. (2014). Similar to QSSM 1141, QSSM 1148 is synthesized by the Gram-negative proteobacterium Ruegeria spp. It interacts with RAS1 by forming three hydrogen bonds with Ser147, Alu148, and Lys49 and 10 hydrophobic bonds with Gly14, Gly16, Ala19, Phe29, Glu32, Tyr33, Asn117, Lys118, and Leu121 (Figure 2Bc). 3.4 Molecular Dynamics and Simulation In order to understand the stability and interatomic motions of CYCc-QSSM 1157 and RAS1-QSSM 1112 complexes, MD Out of the top three hits, the best one QSM against RAS1 was selected based on the same interaction criteria followed for CYCc. As a result, the QS ligand molecule QSSM 1112 was December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 8 Bacterial QS Molecules as Anti-Hyphal Inhibitors Jothi et al. 3.3.2 Quorum-Sensing Molecules’ Interaction Mode Against RAS1 TABLE 2 \| List of the 64 screened QSMs with their binding energy and interaction residues against RAS1 as predicted through virtual docking. S. no. SigMol Ligand name ID Energy (kcal mol−1) H-bonding Signa syste QSSM ID 1. QSSM 0614 p-Coumaroyl-HSL 30790745* −8.5 Gly16, Lys17, Asp120 AHLs 2. QSSM 1111 Cyclo(L-Pro-L-Tyr) 106647* −8.2 Ser18, Asn117 DKPs 3. QSSM 1141 Cyclo(L-Phe-L-4-OH-Pro) 8643197* −8.2 Gly14, Glu32, Asp34 DKPs 4. QSSM 1153 Cyclo(L-Phe-cis-4-OH-D-Pro) 8976612* −8.2 Gly14, Glu32, Asp34 AHLs 5. QSSM 0615 Cinnamoyl-HSL 9797494* −8.2 Ser18 AHLs 6. QSSM 1113 Cyclo(L-Phe-L-Pro) 90257* −8.1 Asn117 DKPs 7. QSSM 0234 N-Tetradec-2,7-dienoyl-L-homoserine lactone 28589928* −8 Gly16, Lys17 AHLs 8. QSSM 1194 (2E,6E)-Farnesoic acid 4439611* −8 Val15, Gly14, Lys17 DSFs 9. QSSM 0389 N-(3-Hydroxytetradec-7-enoyl)-L-homoserine lactone 4445245* −7.9 Val15, Gly16, Lys17, Ser18, Glu32 AHLs 10. QSSM 0041 N-(3-Hydroxydodecanoyl)-L-homoserine lactone (OH-dDHL) 9067166* −7.9 Gly16, Lys17, Ser18, Val30 AHLs 11. QSSM 0042 N-(3-Hydroxydecanoyl)-L-homoserine lactone 9696680 −7.8 Val15, Gly16, Lys17, Val30, Glu32 AHLs 12. QSSM 1262 2-Heptylquinolin-4(1H)-one 164974# −7.6 Asn117 HAQs 13. QSSM 0026 N-(3-Hydroxytetradecanoyl)-L-homoserine lactone 9078807* −7.6 Val15, Gly16, Lys17, Ala19, Glu32 AHLs 14. QSSM 0104 N-(3-Hydroxyoctanoyl)-L-homoserine lactone 9761556* −7.5 Val15, Gly16, Lys17, Ser18 AHLs 15. QSSM 1156 3-Benzylidene-6-isobutyl-2,5-piperazinedione 24725401* −7.4 DKPs 16. QSSM 0368 N-(3-Oxohexadec-9-enoyl)-L-homoserine lactone 29341818* −7.4 Gly16, Lys17 AHLs 17. QSSM 1133 Cyclo(L-Pro-D-Phe) 391657* −7.4 Val30, Lys118 DKPs 18. QSSM 0764 N-(3-Hydroxyhexadecanoyl)-L-homoserine lactone 9741349* −7.4 Val15, Gly16, Lys17, Glu32 AHLs 19. QSSM 1151 Cyclo(L-Leu-trans-4-OH-L-Pro) 10184045* −7.3 Asp120, Ser147, Ala148 DKPs 20. QSSM 0012 N-(3-Oxododecanoyl)-L-homoserine lactone 10221060# −7.3 Gly16, Lys17 AHLs 21. QSSM 0107 N-3-Oxo-tetradecanoyl homoserine lactone 11688418# −7.3 Gly16, Lys17, Ser18 AHLs 22. QSSM 0012 2-Heptyl-3-hydroxy-4-quinolone 2763159# −7.3 HAQs 23. QSSM 1148 Cyclo(D-4-OH-Pro-L-Val) 34017887* −7.3 Ser147, Alu148, Lys49 DKPs 24. QSSM 1157 (Z)-3-Benzyl-6-isobutylidene-2,5-piperazinedione 8666841* −7.3 DKPs 25. QSSM 0573 3-Oxo-C16-HSL 9683431* −7.3 Lys17, Ser18 AHLs 26. QSSM 1144 Cyclo(Gly-L-Tyr) 19927129* −7.2 Gly14, Lys17, Asp31, Asp34 DKPs 27. QSSM 0369 N-(Tetrahydro-2-oxo-3-furanyl)-octadecanamide 69757690# −7.2 Gly16, Lys17 AHLs 28. QSSM 0016 N-Hexadecanoyl-homoserine lactone 11609787# −7.1 Val15, Gly16, Lys17, Ser18 AHLs 29. QSSM 0046 N-(3-Oxooctanoyl) homoserine lactone 127293# −7.1 Gly16, Lys17 AHLs 30. QSSM 0271 N-(3-Oxononanoyl)-L-homoserine lactone 9768814* −7.1 Lys17, Ser18, Ala19 AHLs QSSM Farnesol 445070# −7 Asp120, Ser147, Ala148, Lys149 Fungal Q (Conti December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Bacterial QS Molecules as Anti-Hyphal Inhibitors Jothi et al. TABLE 2 \| Continued S. no. SigMol Ligand name ID Energy (kcal mol−1) H-bonding Signalin system QSSM ID 31. 3.3.2 Quorum-Sensing Molecules’ Interaction Mode Against RAS1 QSSM 1112 Cyclo(L-Pro-L-Leu) 5428292** −7 Ala148, Lys149, Asp120 DKPs 32. QSSM 0003 N-Octanoyl-L-homoserinelactone 6914579# −7 Gly16, Lys17, Ser18 AHLs 33. QSSM 0367 N-Hexadec-9-enoyl-L-homoserine lactone 28589932* −6.8 Ser18, Thr36 AHLs 34. QSSM 0048 N-Heptanoylhomoserine lactone 443437# −6.8 Gly16, Lys17 AHLs 35. QSSM 1177 11-Methyl-2,4-dodecadienoic acid 8031542* −6.8 Val15, Gly16, Lys17, Ser18 DSFs 36. QSSM 0038 N-(3-Hydroxybutanoyl)-L-homoserine lactone 115719* −6.7 Glu14, Val15, Gly16, Lys17, Ser18 AHLs 37. QSSM 1205 Pentadecanoic acid 13249* −6.7 Val15, Gly16, Lys17, Ser18 DSFs 38. QSSM 0001 N-(beta-Ketocaproyl)-L-homoserine lactone 688505# −6.7 Gly16, Lys17 AHLs 39. QSSM 1183 2-Tridecenoic acid 4445862* −6.6 Val15, Gly16, Lys17, Ser18 DSFs 40. QSSM 1191 2-Pentadecenoic acid 4445869* −6.6 Val15, Gly16, Lys17, Ser18 DSFs 41. QSSM 0270 N-(3-Oxoheptanoyl)-L-homoserine lactone 9710716* −6.6 Ser18, Ala19, Asp117 AHLs 42. QSSM 0619 N-Isovaleryl-L-homoserine lactone 29368455* −6.5 Gly16, Lys17, Ser18 AHLs 43. QSSM 1190 2-Tetradecenoic acid 4445865* −6.5 Val15, Gly16, Lys17, Ser18 DSFs 44. QSSM 1132 Cyclo(L-Pro-L-isoLeu) 8166289* −6.5 Asn117 DKPs 45. QSSM 1167 11-Methyl-2-dodecenoic acid 9644750* −6.4 Glu14, Val15, Gly16, Lys17 DSFs 46. QSSM 0739 N-(3-Oxotetradec-7-enoyl)-L-homoserine lactone 9057115* −6.3 Gly14, Gly16, Lys17 AHLs 47. QSSM 0891 N-Pentanoyl-L-homoserine lactone 9688673* −6.3 Gly16, Lys17 AHLs 48. QSSM 0020 N-Hexanoyl-L-homoserine lactone 10058590# −6.2 Gly16, Lys17, Ser18 AHLs 49. QSSM 0005 N-Tetradec-7-enoyl-L-homoserine lactone 28589930* −6.2 Ser18 AHLs 50. QSSM 1181 Undecenoic acid 4445855* −6.2 Val15, Gly16, Lys17, Ser18 DSFs 51. QSSM 0025 N-(3-Hydroxyhexanoyl)-L-homoserine lactone 9754171* −6.2 Asn117, Lys118, Ser147, Ala148, Lys149 AHLs 52. QSSM 1204 Tridecylic acid 12013* −6.1 Val15, Gly16, Lys17 DSFs 53. QSSM 1192 Lauric acid 3756* −6.1 Gly14, Val15, Lys17, Ser18 DSFs 54. QSSM 1145 Cyclo(Gly-L-Val) 644035* −6 Gly14, Gly16, Asp34 DKPs 55. QSSM 1114 Cyclo(L-Val-L-Leu) 122161* −5.9 Asp31 DKPs 56. QSSM 0987 (1S,4S,5R)-7,7-Dihydroxy-1-methyl-2,6,8-trioxa-7- boranuidabicyclo[3.3.0]octane-4,5-diol 395434* −5.9 Ser18, Thr36, Asp34 AI-2 57. QSSM 1159 2-Dodecenoic acid 4471801* −5.9 Val15, Gly16, Lys17, Ser18 DSFs 58. QSSM 1175 Decenoic acid 4445851* −5.8 Val15, Gly16, Lys17, Ser18 DSFs 59. QSSM 1129 Cyclo(Pro-Val) 8997025* −5.8 DKPs 60. QSSM 0990 4,5-Dihydroxy-2,3-pentanedione 9346083* −5.8 Gly14, Lys17, Ser18, Asp34, Thr36, Thr59, Gly61 AI-2 (Continu TABLE 2 \| Continued S. no. SigMol Ligand name QSSM ID 31. QSSM 1112 Cyclo(L-Pro-L-Leu) 32. QSSM 0003 N-Octanoyl-L-homoserinelactone 33. QSSM 0367 N-Hexadec-9-enoyl-L-homoserine lactone 34. QSSM 0048 N-Heptanoylhomoserine lactone 35. QSSM 1177 11-Methyl-2,4-dodecadienoic acid 36. QSSM 0038 N-(3-Hydroxybutanoyl)-L-homoserine lacton 37. QSSM 1205 Pentadecanoic acid 38. QSSM 0001 N-(beta-Ketocaproyl)-L-homoserine lactone 39. QSSM 1183 2-Tridecenoic acid 40. QSSM 1191 2-Pentadecenoic acid 41. QSSM 0270 N-(3-Oxoheptanoyl)-L-homoserine lactone 42. QSSM 0619 N-Isovaleryl-L-homoserine lactone 43. 3.3.2 Quorum-Sensing Molecules’ Interaction Mode Against RAS1 QSSM 1190 2-Tetradecenoic acid 44. QSSM 1132 Cyclo(L-Pro-L-isoLeu) 45. QSSM 1167 11-Methyl-2-dodecenoic acid 46. QSSM 0739 N-(3-Oxotetradec-7-enoyl)-L-homoserine lac 47. QSSM 0891 N-Pentanoyl-L-homoserine lactone 48. QSSM 0020 N-Hexanoyl-L-homoserine lactone 49. QSSM 0005 N-Tetradec-7-enoyl-L-homoserine lactone 50. QSSM 1181 Undecenoic acid 51. QSSM 0025 N-(3-Hydroxyhexanoyl)-L-homoserine lacton 52. QSSM 1204 Tridecylic acid 53. QSSM 1192 Lauric acid 54. QSSM 1145 Cyclo(Gly-L-Val) 55. QSSM 1114 Cyclo(L-Val-L-Leu) 56. QSSM 0987 (1S,4S,5R)-7,7-Dihydroxy-1-methyl-2,6,8-tr boranuidabicyclo[3.3.0]octane-4,5-diol 57. QSSM 1159 2-Dodecenoic acid 58. QSSM 1175 Decenoic acid 59. QSSM 1129 Cyclo(Pro-Val) 60. QSSM 0990 4,5-Dihydroxy-2,3-pentanedione ame ID Energy (kcal mol−1) H-bonding Signalin system 5428292** −7 Ala148, Lys149, Asp120 DKPs 6914579# −7 Gly16, Lys17, Ser18 AHLs actone 28589932* −6.8 Ser18, Thr36 AHLs 443437# −6.8 Gly16, Lys17 AHLs 8031542* −6.8 Val15, Gly16, Lys17, Ser18 DSFs ne lactone 115719* −6.7 Glu14, Val15, Gly16, Lys17, Ser18 AHLs 13249* −6.7 Val15, Gly16, Lys17, Ser18 DSFs lactone 688505# −6.7 Gly16, Lys17 AHLs 4445862* −6.6 Val15, Gly16, Lys17, Ser18 DSFs 4445869* −6.6 Val15, Gly16, Lys17, Ser18 DSFs actone 9710716* −6.6 Ser18, Ala19, Asp117 AHLs 29368455* −6.5 Gly16, Lys17, Ser18 AHLs 4445865* −6.5 Val15, Gly16, Lys17, Ser18 DSFs 8166289* −6.5 Asn117 DKPs 9644750* −6.4 Glu14, Val15, Gly16, Lys17 DSFs serine lactone 9057115* −6.3 Gly14, Gly16, Lys17 AHLs 9688673* −6.3 Gly16, Lys17 AHLs 10058590# −6.2 Gly16, Lys17, Ser18 AHLs actone 28589930* −6.2 Ser18 AHLs 4445855* −6.2 Val15, Gly16, Lys17, Ser18 DSFs ne lactone 9754171* −6.2 Asn117, Lys118, Ser147, Ala148, Lys149 AHLs 12013* −6.1 Val15, Gly16, Lys17 DSFs 3756* −6.1 Gly14, Val15, Lys17, Ser18 DSFs 644035* −6 Gly14, Gly16, Asp34 DKPs 122161* −5.9 Asp31 DKPs -2,6,8-trioxa-7- diol 395434* −5.9 Ser18, Thr36, Asp34 AI-2 4471801* −5.9 Val15, Gly16, Lys17, Ser18 DSFs 4445851* −5.8 Val15, Gly16, Lys17, Ser18 DSFs 8997025* −5.8 DKPs 9346083* −5.8 Gly14, Lys17, Ser18, Asp34, Thr36, Thr59, Gly61 AI-2 (Continu December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 10 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 10 Bacterial QS Molecules as Anti-Hyphal Inhibitors Jothi et al. TABLE 2 \| Continued S. no. SigMol Ligand name ID Energy (kcal mol−1) H-bonding Signaling system QSSM ID 61. QSSM 1202 Decanoic acid 2863# −5.7 Val15, Gly16, Lys17, Ser18 DSFs 62. QSSM 0289 N-(3-Oxononanoyl)-L-homoserine lactone 289 −5.7 Asp120, Ser147, Ala148, Lys149 AHLs 63. QSSM 0013 N-Butyryl-L-homoserine lactone 10130163# −5.5 Gly16, Lys17 AHLs 64. QSSM 1179 2-Octenoic acid 4445841# −5.4 Val15, Gly16, Lys17, Ser18 DSFs QSMs, quorum-sensing molecules; DKPs, diketopiperazines; AHLs, acylated homoserine lactones. 3.3.2 Quorum-Sensing Molecules’ Interaction Mode Against RAS1 with a higher RMSD value against its respective protein–ligand complexes would suggest the insufﬁcient binding of the ligand to the target protein (Opo et al., 2021). Thereby, RMSD analysis would reveal how often a protein–ligand interaction is integrated and stabilized. As shown in Figures 4C, D, the protein maintained a steady RMSD throughout the simulation run. The CYCc-QSSM 1157 and RAS1-QSSM 1112 protein complexes begin to stabilize after 5 and 25 ns of MD simulation run, respectively. The average protein backbone RMSD for the CYCc-QSSM 1157 and RAS1- QSSM 1112 protein complexes was determined to be 0.42 and 0.31 Å, respectively. The average ligand RMSD for the CYCc-QSSM 1157 and RAS1-QSSM 1112 protein complexes was determined to be 0.35 and 0.27 Å, respectively. The plot in Figures 4C, D indicates that the ligand RMSD is not substantially greater than the protein RMSD, which signiﬁes that the ligand does not diffuse away from the original binding site. The RMSD of MD simulation and molecular docking revealed that the attained complexes CYCc- simulation analysis was performed using GROMACS, which would offer essential information regarding the physical movement of atoms and molecules in a biophysical system using computational approaches (Barnes and Williams, 1996). Therefore, the predicted protein–ligand complexes, i.e., CYCc- QSSM 1157 and RAS1-QSSM 1112 complexes, were subjected to 50-ns MD simulation. The subsequent MD run trajectories were examined for protein and ligand RMSDs, protein RMSF, and hydrogen bond interactions. 3.4.2 H-Bond Interaction Analysis As the effective binding of ligand to the target protein is mainly attributed to H-bond formation, the QSSM 1157 and QSSM 1112 were monitored for H-bond formation toward CYCc and RAS1 proteins throughout the simulation run. To identify the system stability during the simulation procedure, the number of hydrogen bonds formed vs. simulation duration was calculated. The hydrogen bonds stability was evaluated by means of 50-ns molecular simulation (Figures 4E, F). The maximum number of hydrogen bonds formed for CYCc-QSSM 1157 and RAS1-QSSM 1112 was 9 and 7, respectively, indicating the greater afﬁnity of ligand attachment to the protein binding pockets. The MD calculated hydrogen bond formation was in agreement with the result of molecular docking analysis. The number of hydrogen bonds formed by the QSSM 1157 and QSSM 1112 over CYCc and RAS1 throughout the simulation showed enough ligand binding strength to the proteins. Furthermore, the hydrogen bond ensures greater afﬁnity and stability of ligand to the proteins. SwissADME, the drugability of the ligands was evaluated by comparing various properties with pre-developed drug likeliness modules such as Lipinski’s rule of ﬁve, Ghose’s rule, Veber’s rule, Egan’s rule, and Muegge’s rule. Each of the mentioned modules contains different parameters that estimate ligand drug likeliness. Molecular weight, hydrogen donors, hydrogen acceptors, rotatable bonds, and PSA are various ligand properties that have been taken into account to evaluate a ligand drug’s transport characteristics. 3.5 Pharmacokinetic Prediction Using SwissADME Server As can be seen in Table 3, the structures of QSSM 1157 and QSSM 1112 possess a better drug likeliness without any deviation in Lipinski’s rule of ﬁve, Ghose’s rule, Veber’s rule, Egan’s rule, and Muegge’s rule as their values fall in the desirable range. To be precise, all the parameters, viz., molecular weight, rotatable bonds, number of hydrogen acceptors, number of hydrogen donors, PSA, molar refractivity, log P, and clogP, have fallen within the acceptable range. Furthermore, the QSSM 1157 and QSSM 1112 showed better lipophilicity (XLOGP3 between −0.7 and +5.0) and good water solubility scores. This result conﬁrms that the chemical structure and orientation of both ligands will not cause any irrelevant metabolic interactions during drug formulation. In the de novo drug development process, most of the drugs have failed in the ﬁnal clinical phase due to their inappropriate pharmacokinetics and toxicity proﬁle. To overcome this issue, pharmacokinetic and ADME prediction of the new drug candidate has to be conducted at the initial stages (Lee et al., 2003). To attain a desirable in vivo response, there should be an equilibrium between pharmacokinetic and pharmacodynamic qualities (Tuntland et al., 2014). Presently, in silico-based ADME screening is being utilized for the identiﬁcation of the most promising compounds devoid of drug attrition. In the present study, numerous parameters such as cell permeability, drug solubility, partition coefﬁcient, gastrointestinal (GI) absorption, polar surface area (PSA), and drug homology were evaluated through virtual screening approaches provided by SwissADME interface. The results obtained in SwissADME are listed in Table 3, Supplementary Table 1, which contain ADMET, pharmacokinetics, drug likeliness, and medical chemistry of predicted ligands QSSM 1157 and QSSM 1112. 3.4.1 Root Mean Square Deviation and Root Mean Square Fluctuation Analyses TABLE 3 \| Pharmacokinetics, drug likeness, and medicinal chemistry properties of QSSM 1157 and QSSM 1112 as predicted through SwissADME. TABLE 3 \| Pharmacokinetics, drug likeness, and medicinal chemistry properties of QSSM 1157 and QSSM 1112 as predicted through SwissADME. QSSM 1157 and RAS1-QSSM 1112l are stable in their conformations. of QSSM 1157 and QSSM 1112 as predicted through SwissADME. Parameters QSSM 1112 QSSM 1157 Pharmacokinetics GI absorption High Low BBB permeation No No P-gp substrate No No CYP1A2 inhibitor No Yes CYP2C19 inhibitor No No CYP2C9 inhibitor No No CYP2D6 inhibitor No No CYP3A4 inhibitor No No Drug likeness Lipinski Yes; 0 violation Yes; 0 violation Ghose Yes; 0 violation Yes; 0 violation Veber Yes Yes Egan Yes Yes Muegge Yes Yes Bioavailability Score 0.55 0.55 Medicinal chemistry PAINS 0 alert 0 alert Brenk 0 alert 1 alert: michael_acceptor_1 Leadlikeness Yes; 0 violation Yes; 0 violation GI, gastrointestinal; BBB, blood–brain barrier; P-gp, permeability glycoprotein; PAINS, Pan-Assay Interference compounds. To gain more insights into the stability of modeled proteins, the RMSF was estimated during the MD simulation. The RMSF of the Ca-atoms demonstrated the residual ﬂexibility of each protein. Further, the results also showed the amino acid oscillations in catalytic and non-catalytic sites. For CYCc- QSSM 1157 and RAS1-QSSM 1112, the average protein RMSF was in the range of 0.1–0.73 and 0.06–0.47 Å, respectively (Figures 4A, B). Further, negligible ﬂuctuations were observed in disallowed and looping regions of CYCc and RAS1 proteins, which obviously signiﬁes the stability of both predicted proteins throughout the simulation reactions. 3.5.2 Pharmacokinetics Prediction The SwissADME pharmacokinetics evaluation revealed that both the ligands have no blood–brain barrier (BBB) permeation and no substrate of permeability glycoprotein (P-gp) (Table 3). Furthermore, the QSSM 1157 and QSSM 1112 showed high and low GI absorption, respectively. The cytochrome P450 (CYP) is a hemeprotein that is responsible for the metabolism of drugs at various stages. Usually, CYP will have exhibited as ﬁve major isomers such as CYP1A2, CYP3A4, CYP2C9, CYP2C19, and CYP2D6. 3.4.1 Root Mean Square Deviation and Root Mean Square Fluctuation Analyses The stabilized interaction of protein–ligand complexes during MD simulation was evaluated by estimating their respective RMSD. Generally, the higher RMSD value signiﬁes the instability of inspected molecules due to the high level of conformational changes within the complex. Additionally, the ligand molecule A B FIGURE 2 \| (A) The two-dimensional interaction image showcasing the binding pattern of selected top three ligands such as QSSM 1157 (a), QSSM 1141 (b), and QSSM 1153 (c) and the positive control farnesol (d) against CYCc. (B) The two-dimensional interaction pattern of selected top three ligands such as QSSM 1151 (a), QSSM 1148 (b), and QSSM 1112 (c) and the positive control farnesol (d) against RAS1. FIGURE 2 \| (A) The two-dimensional interaction image showcasing the binding pattern of selected top three ligands such as QSSM 1157 (a), QSSM 1141 (b), and QSSM 1153 (c) and the positive control farnesol (d) against CYCc. (B) The two-dimensional interaction pattern of selected top three ligands such as QSSM 1151 (a), QSSM 1148 (b), and QSSM 1112 (c) and the positive control farnesol (d) against RAS1. December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 11 Jothi et al. Bacterial QS Molecules as Anti-Hyphal Inhibitors A B D C FIGURE 3 \| The three-dimensional binding pattern of top hits QSSM 1157 (C) QSSM 1112 (D) and positive control farnesol (A, B) against CYCc and RAS1. FIGURE 3 \| The three-dimensional binding pattern of top hits QSSM 1157 (C) QSSM 1112 (D) and positive control farnesol (A, nsional binding pattern of top hits QSSM 1157 (C) QSSM 1112 (D) and positive control farnesol (A, B) against CYCc and RAS1. A B D E F C FIGURE 4 \| The RMSF, RMSD, and hydrogen bond values plotted for CYCc-QSSM 1157 (A–C) and RAS1-QSSM 1112 complexes (D–F) during 50-ns MD simulation. RMSF, root mean square ﬂuctuation; RMSD, root mean square deviation; MD, molecular dynamics. E A C B D F B D F FIGURE 4 \| The RMSF, RMSD, and hydrogen bond values plotted for CYCc-QSSM 1157 (A–C) and RAS1-QSSM 1112 complexes (D–F) during 50-ns MD simulation. RMSF, root mean square ﬂuctuation; RMSD, root mean square deviation; MD, molecular dynamics. December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 12 Bacterial QS Molecules as Anti-Hyphal Inhibitors Jothi et al. 3.5.1 Drug Likeliness Prediction Drug likeness of a ligand molecule is predicted by comparing the structure or other merit parameters with known drugs. In December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 13 Jothi et al. Bacterial QS Molecules as Anti-Hyphal Inhibitors 3.6 Toxicity Prediction via ProTox-II Server The prediction of chemical toxicity is a crucial step in the drug development process. Computational-based toxicity prediction plays a signiﬁcant role in fairly reducing the time and the number of animals to be used in in vivo studies for toxicity prediction (Raies and Bajic, 2016). ProTox-II uses 33 various models for predicting molecular similarity, fragment propensities, and machine learning to predict various toxicity endpoints including acute toxicity, hepatotoxicity, cytotoxicity, carcinogenicity, mutagenicity, and immunotoxicity. Inhibition of CYP pathway may lead to unwanted toxic effects due to reduced drug metabolism (Lynch and Price, 2007). Hence, the property of QSSM 1157 and QSSM 1112 to inhibit CYP was evaluated. The CYP interaction results showed that the QSSM 1112 is not a CYP inhibitor, whereas QSSM 1157 is an inhibitor of one of the isomers of CYP1A2. The obtained result clearly evidences that both QSSM 1157 and QSSM 1112 have a lesser possibility to interfere with CYP, which guarantees the drug selectivity and bioavailability. Inhibition of CYP pathway may lead to unwanted toxic effects due to reduced drug metabolism (Lynch and Price, 2007). Hence, the property of QSSM 1157 and QSSM 1112 to inhibit CYP was evaluated. The CYP interaction results showed that the QSSM 1112 is not a CYP inhibitor, whereas QSSM 1157 is an inhibitor of one of the isomers of CYP1A2. The obtained result clearly evidences that both QSSM 1157 and QSSM 1112 have a lesser possibility to interfere with CYP, which guarantees the drug selectivity and bioavailability. The Pan-Assay Interference compounds (PAINS) and Brenk technique were used to identify potentially toxic and unstable fragments present in the intact structure. In many biochemical and pharmacological trials, the PAINS evaluate the misleading hit compounds showing false-positive biological yields (Baell and Nissink, 2018). As expected, both QSSM 1157 and QSSM 1112 had “zero alerts” in PAINS prediction. The molecular weight distribution proﬁle of both ligands was estimated by the ProTox-II server, and it was in the range of 258.32 to 319.67 for QSSM 1157 and 222.24 to 319.67 for QSSM 1112 (Figures 5A, 6A). 3.5.1 Drug Likeliness Prediction (C) Radar chart demonstrating various predicted toxicity proﬁles of QSSM 1112 through ProTox-II The radar chart shows the bioavailability of the selected hit ligands. The pink area of the radar indicates the ideal area for properties such as ﬂexibility (FLEX), insolubility (INSOLU), unsaturation (INSATU), lipophilicity (LIPO), polarity (POLAR), and SIZE. Both the selected ligands match with the recommended size of 500 g mol−1 as indicated by Lipinski for successful medication candidates. The POLAR was assessed for the selected ligands using their total area of polarity (TPSA). The TPSA value of a polar molecule should be 20 and 130 Å. The TPSA of QSSM 1157 and 1112 falls within the allowed range. Through INSOLU of the target ligands depicted with their respective ESOL and ESOL Class, it was found that QSSM 1157 and QSSM 1112 displayed high and moderate water- soluble properties, respectively. within the recommended range. Taken together, the data of pharmacokinetic and pharmacodynamic analyses of the selected hit molecules underline their highest plausibility for drug development. Both the active agents were analyzed for various toxicity proﬁles and are listed in Tables 4, 5. QSSM 1157 showed mild toxicity possibility rates in hepatotoxicity, carcinogenicity, and cytotoxicity proﬁles, whereas QSSM 1112 demonstrated mild toxicity rates in carcinogenicity and cytotoxicity proﬁles. As listed in Tables 4, 5, both ligands showed no toxicity in Tox21 receptor signaling pathway proﬁles. The overall toxicity class was predicted to be 4 out of 6 for QSSM 1112 and 5 out of 6 for QSSM 1157, which indicates the lesser toxicity of ligands. Overall, the analyses unambiguously predict that the identiﬁed QSM-based hyphal inhibitors would be safe for biological use. The carbon fraction Sp3 (CSP3) and the rotatable bond count should not be greater than 9 and should be within the range of 0.5–1 (CSP3). It has been used to assess the INSATU and FLEX of the identiﬁed hit ligands. Both the selected hits QSSM 1157 (CSP3:0.33) and QSSM 1112 (CSP3: 0.45) showed CSP3 values fairly near csp3 range. In general, the numbers of rotatable bonds should not exceed nine; data showed that QSSM 1157 and QSSM 1112 possess 3 and 2 rotatable bonds, respectively. XLogP3 and ESOL (Log S) with the suggested range of −0.7 to + 5.0 and 0 to 6, respectively, were used to access the LIPO and INSOLU proﬁle of the selected ligands. Interestingly, both the selected ligands fall 3.5.1 Drug Likeliness Prediction The toxicity was estimated by recognizing the ligand fragments and structure. The various toxicity proﬁles of both ligands estimated through ProTox-II server are given in Tables 4, 5 and Figures 5, 6. The toxicity radar chart in Figures 5C, 6C was intended to quickly illustrate the conﬁdence of positive toxicity results compared with the average of its class. The LD50 values of QSSM 1112 and QSSM 1157 were predicted to be 1,600 and 2,287 mg/kg, respectively. The distribution of dose values for QSSM 1112 and QSSM 1157 with its mean dose value is represented in Figures 5A, 6A, respectively. Although the QSSM 1112 does not have any violation in the medicinal chemistry proﬁle, the Brenk list predicted QSSM 1157 as a Michael acceptor, indicating the possibility for a hazardous interaction of its fragments with other biological molecules. Since the QSSM 1157 showed inadequate toxicity in the Brenks– Michael acceptor alert, it is a necessity to evaluate the complete toxicity proﬁle of the screened ligands. Hence, it is necessary to evaluate the toxicity proﬁle of the screened ligands. A B C FIGURE 5 \| (A) Graphical representation of molecular weight and dose value distribution of QSSM 1157. (B) Swiss-ADME generated bioavailability radar chart for QSSM 1157. Pink area indicates the oral bioavailability of QSSM 1157. (C) Radar chart showcasing various toxicity proﬁles of QSSM 1157 predicted through ProTox-II. A B C B C FIGURE 5 \| (A) Graphical representation of molecular weight and dose value distribution of QSSM 1157. (B) Swiss-ADME generated bioavailability radar chart for QSSM 1157. Pink area indicates the oral bioavailability of QSSM 1157. (C) Radar chart showcasing various toxicity proﬁles of QSSM 1157 predicted through ProTox-II. December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Bacterial QS Molecules as Anti-Hyphal Inhibitors Jothi et al. A B C FIGURE 6 \| (A) Graphical representation of molecular weight and dose value distribution of QSSM 1112. (B) Swiss-ADME generated bioavailability radar chart for QSSM 1112. Pink area indicates the oral bioavailability of QSSM 1112. (C) Radar chart demonstrating various predicted toxicity proﬁles of QSSM 1112 through ProTox-II A B C B C FIGURE 6 \| (A) Graphical representation of molecular weight and dose value distribution of QSSM 1112. (B) Swiss-ADME generated bioavailability radar chart for QSSM 1112. Pink area indicates the oral bioavailability of QSSM 1112. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 3.7 In Vitro Antihyphal Assay Revealed Dose-Dependent Inhibitory Potency of QSSM 1112 Against Candida albicans Dimorphism At the end of the virtual screening, the obtained result clearly showed that QSSM 1157 and QSSM 1112 have a similar kind of interaction like farnesol against CYCc and RAS1, respectively. Among the two predicted hits, biomolecule QSSM 1112 (active against RAS1) has been well studied and documented by our December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 15 Bacterial QS Molecules as Anti-Hyphal Inhibitors Jothi et al. TABLE 4 \| Different toxicity proﬁle of QSSM 1157 predicted through ProTox-II server. TABLE 4 \| Different toxicity proﬁle of QSSM 1157 predicted through ProTox-II server. Classiﬁcation Target Prediction Probability Organ toxicity Hepatotoxicity Inactive 0.71 Toxicity endpoints Carcinogenicity Inactive 0.69 Toxicity endpoints Immunotoxicity Inactive 0.98 Toxicity endpoints Mutagenicity Inactive 0.70 Toxicity endpoints Cytotoxicity Inactive 0.67 Tox21-Nuclear receptor signaling pathways Aryl hydrocarbon receptor (AhR) Inactive 0.81 Tox21-Nuclear receptor signaling pathways Androgen receptor (AR) Inactive 0.96 Tox21-Nuclear receptor signaling pathways Androgen receptor ligand binding domain (AR-LBD) Inactive 0.99 Tox21-Nuclear receptor signaling pathways Aromatase Inactive 0.95 Tox21-Nuclear receptor signaling pathways Estrogen receptor alpha (ER) Inactive 0.93 Tox21-Nuclear receptor signaling pathways Estrogen receptor ligand binding domain (ER-LBD) Inactive 0.96 Tox21-Nuclear receptor signaling pathways Peroxisome proliferator-activated receptor gamma (PPAR-Gamma) Inactive 0.97 Tox21-Stress response pathways Nuclear factor (erythroid-derived 2)-like 2/antioxidant responsive element (nrf2/ARE) Inactive 0.97 Tox21-Stress response pathways Heat shock factor response element (HSE) Inactive 0.97 Tox21-Stress response pathways Mitochondrial Membrane Potential (MMP) Inactive 0.89 Tox21-Stress response pathways Phosphoprotein (tumor suppressor) p53 Inactive 0.87 Tox21-Stress response pathways ATPase family AAA domain-containing protein 5 (ATAD5) Inactive 0.96 in vitro antihyphal assay was performed using QSSM 1112, with farnesol as the positive control. research group for its remarkable quorum quelling, antibioﬁlm, and antivirulence efﬁcacies toward various multidrug-resistant pathogens, viz., S. mutans (Gowrishankar et al., 2014), S. aureus (Gowrishankar et al., 2016a), Listeria monocytogenes (Gowrishankar et al., 2016b), Staphylococcus epidermidis, and Serratia marcescens (Gowrishankar and Pandian, 2017; Gowrishankar et al., 2019). This information led us to be more curious to validate the in vitro efﬁciency of QSSM 1112 in inhibiting yeast-to-hyphal transition due to its in-house availability. Quite a few earlier studies on QSSM 1112 have already been reported for its synthesis (from Achromobacter xylosoxidans and Streptomyces sp.) and exhibition of antagonistic activity against aﬂatoxins of Aspergillus parasiticus and rice blast fungus Pyricularia oryzae (Yan et al., 2004; Hall et al., 2011). 3.7 In Vitro Antihyphal Assay Revealed Dose-Dependent Inhibitory Potency of QSSM 1112 Against Candida albicans Dimorphism A B C FIGURE 7 \| (A) Inhibitory efﬁcacy of QSSM 1112 (0–1,024 µg/ml) on yeast-to-hyphal transition of Candida albicans in liquid spider media. After 24-h manifestation with QSSM 1112, C. albicans cells were photographed under a light microscope at ×400 magniﬁcation. Micrograph of the control group portrays very dense and lengthy ﬁlamentous cells with few yeast cells; the QSSM 1112-treated groups display more number of evenly distributed yeast cells even from the lowest concentration (64 µg/ml). (B) Conformation of yeast-to-hyphal inhibition by QSSM 1112 using solid spider medium. The fungal colonies were imaged after 7 days of incubation at 37°C. The light micrograph and agar plate images clearly portray the remarkable reduction of hyphal protrusion in QSSM 1112-treated samples compared with untreated control (which bares deep and densely elongated hyphal network). (C) The impact of positive control farnesol (0–1,024 µg/ml) on C. albicans yeast-to-hyphal transition. The last panel in the ﬁgure showcases the effect of solvent (methanol) on yeast-to-hyphal transition. B C FIGURE 7 \| (A) Inhibitory efﬁcacy of QSSM 1112 (0–1,024 µg/ml) on yeast-to-hyphal transition of Candida albicans in liquid spider media. After 24-h manifestation with QSSM 1112, C. albicans cells were photographed under a light microscope at ×400 magniﬁcation. Micrograph of the control group portrays very dense and lengthy ﬁlamentous cells with few yeast cells; the QSSM 1112-treated groups display more number of evenly distributed yeast cells even from the lowest concentration (64 µg/ml). (B) Conformation of yeast-to-hyphal inhibition by QSSM 1112 using solid spider medium. The fungal colonies were imaged after 7 days of incubation at 37°C. The light micrograph and agar plate images clearly portray the remarkable reduction of hyphal protrusion in QSSM 1112-treated samples compared with untreated control (which bares deep and densely elongated hyphal network). (C) The impact of positive control farnesol (0–1,024 µg/ml) on C. albicans yeast-to-hyphal transition. The last panel in the ﬁgure showcases the effect of solvent (methanol) on yeast-to-hyphal transition. Mosel et al. (2005) reported that the concentration of farnesol required for the inhibition of yeast-to-hyphal transition has varied depending on the medium (Mosel et al., 2005). They found that 2 µM (0.44 µg/ml) and 250 µM (55.29 µg/ml) of farnesol were required to inhibit 50% of C. albicans hyphal formation in deﬁned media and serum-containing media, respectively (Mosel et al., 2005). 3.7 In Vitro Antihyphal Assay Revealed Dose-Dependent Inhibitory Potency of QSSM 1112 Against Candida albicans Dimorphism Except for the anticandidal effect of QSSM 1112 (Rhee, 2004), so far, none of the studies has been focused on its inhibitory potency toward C. albicans virulence dimorphism. Hence, in order to accomplish our curiosity in validating the in silico docking result, For this, C. albicans cells were cultured with and without the active agent (QSSM 1112) in liquid spider medium supplemented with 10% of hyphal inducer (FBS). As expected, the result of the liquid antihyphal assay signiﬁed that QSSM 1112 was potent enough to inhibit the yeast-to-hyphal transition in a concentration-dependent manner (Figure 7A). Of the various concentrations (0–1,024 µg/ml) used, it was found that QSSM 1112 successfully thwarts the hyphal formation from the maximum (1,024 µg/ml) to minimum (64 µg/ml) concentrations. Therefore, the lowest concentration that brings a substantial hyphal inhibitory condition was considered to be the minimal HIC. Consequently, a concentration of 64 µg/ml was considered as HIC of QSSM 1112, as it demonstrated a phenomenal antihyphal efﬁcacy in a similar fashion to higher concentration (1,024 µg/ml) as well. As depicted in Figure 7A, the cells devoid of active compound (untreated control) have TABLE 5 \| Different toxicity proﬁle of QSSM 1112 predicted through ProTox-II server. Classiﬁcation Target Prediction Probability Organ toxicity Hepatotoxicity Inactive 0.68 Toxicity endpoints Carcinogenicity Inactive 0.63 Toxicity endpoints Immunotoxicity Inactive 0.97 Toxicity endpoints Mutagenicity Inactive 0.75 Toxicity endpoints Cytotoxicity Inactive 0.69 Tox21-Nuclear receptor signaling pathways Aryl hydrocarbon receptor (AhR) Inactive 0.87 Tox21-Nuclear receptor signaling pathways Androgen receptor (AR) Inactive 0.95 Tox21-Nuclear receptor signaling pathways Androgen receptor ligand binding domain (AR-LBD) Inactive 0.95 Tox21-Nuclear receptor signaling pathways Aromatase Inactive 0.88 Tox21-Nuclear receptor signaling pathways Estrogen receptor alpha (ER) Inactive 0.94 Tox21-Nuclear receptor signaling pathways Estrogen receptor ligand binding domain (ER-LBD) Inactive 0.96 Tox21-Nuclear receptor signaling pathways Peroxisome proliferator-activated receptor gamma (PPAR-Gamma) Inactive 0.92 Tox21-Stress response pathways Nuclear factor (erythroid-derived 2)-like 2/antioxidant responsive element (nrf2/ARE) Inactive 0.86 Tox21-Stress response pathways Heat shock factor response element (HSE) Inactive 0.86 Tox21-Stress response pathways Mitochondrial membrane potential (MMP) Inactive 0.84 Tox21-Stress response pathways Phosphoprotein (tumor suppressor) p53 Inactive 0.75 Tox21-Stress response pathways ATPase family AAA domain-containing protein 5 (ATAD5) Inactive 0.92 TABLE 5 \| Different toxicity proﬁle of QSSM 1112 predicted through ProTox-II server. December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 16 Bacterial QS Molecules as Anti-Hyphal Inhibitors Jothi et al. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 3.7 In Vitro Antihyphal Assay Revealed Dose-Dependent Inhibitory Potency of QSSM 1112 Against Candida albicans Dimorphism However, our study demonstrated that 32 µg/ml of farnesol was potent enough to inhibit the ﬁlamentation of C. albicans in both spider medium and serum. To further conﬁrm the propensity of QSSM 1112 in inhibiting the C. albicans dimorphic behavior, solid spider agar assay was done. For that, overnight C. albicans culture was spotted on the spider agar medium supplemented with and without QSSM 1112 at its HIC (Figure 7B). After 2 days of incubation, the agar plates were directly visualized under the light microscope to observe the change in colony morphology upon QSSM 1112 treatment. The micrograph of untreated control has shown rough colony morphology with densely elongated hyphal cells. At the same time, the micrographs of displayed a highly dense criss-cross architecture of hyphal cell population along with few yeast cells. On the other hand, the cells exposed with QSSM 1112 have showcased only yeast cell population even under strong hyphae-induced conditions, which clearly proves that the existence of QSSM 1112 disarmed the C. albicans cells to undergo hyphae induction. Moreover, the consistency of QSSM 1112 in expressing the sustained hyphal inhibitory potency even in the presence of serum would make the QSSM 1112 an interesting drug candidate in the treatment against invasive C. albicans infection. Farnesol was used as the positive control in all the in vitro assays. In a similar fashion to QSSM 1112, farnesol also exhibited a profound concentration-dependent inhibition on ﬁlamentous growth of C. albicans. At the lowest concentration of 32 µg/ml, the farnesol has shown complete hyphal inhibition (0% hyphal) (Figure 7C). Although the inﬂuence of farnesol on the C. albicans dimorphic switching has already been demonstrated, the exact concentration required for yeast-to-hyphal inhibition varied from study to study owing to multiple associated factors. December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 17 Bacterial QS Molecules as Anti-Hyphal Inhibitors Jothi et al. into true hyphae. Hence, the efﬁcacy of QSSM 1112 (HIC) on C. albicans germ-tube formation was evaluated by culturing the C. albicans cells in FBS. In parallel, the effect of farnesol on germ- tube formation was evaluated for comparative purposes. The C. albicans cells were imaged in different time intervals (from 0 to 6 h) for comparison of germ-tube formation with and without QSSM 1112. 3.7 In Vitro Antihyphal Assay Revealed Dose-Dependent Inhibitory Potency of QSSM 1112 Against Candida albicans Dimorphism As can be seen in Figure 8A, both QSSM 1112- treated and untreated control cells appeared as yeast form at the 0-h time interval. With the increase in time, few numbers of cells in untreated control samples showcased budding yeast morphology (a thin ﬁlamentous tube extending from yeast), and the true germ-tube germination was observed in the control sample at the time interval of 6 h. However, the cells treated with QSSM 1112 and farnesol (at HIC) portrayed complete spread of yeast cells, lacking the classic characteristics of hyphal elongation morphologies, viz., budding yeast followed by badminton shaped germ-tubes. This inevitably reinforces the data of in vitro liquid and solid antihyphal assays, which in turn signiﬁes that QSSM QSSM 1112 and farnesol-treated cells have portrayed smooth colony morphology with abridged hyphal formation. In addition, after 7 days of incubation, the agar plates were documented using a gel documentation system (Figure 7C). The results signiﬁed the remarkable reduction of hyphal protrusion manifested with QSSM 1112 compared with the untreated control. The hyphal inhibitory effect of farnesol was better than that of QSSM 1112, as it exhibited a substantial impact even at a very low concentration, i.e., even 2 µg/ml with 25% of hyphal inhibition. In contrast, QSSM 1112 at 2 µg/ml displayed a compactly elongated hyphal formation similar to that of control. 3.8 QSSM 1112 (at Hyphal Inhibitory Concentration) Averts Candida albicans Germ-Tube Formation The dimorphic growth of C. albicans is closely linked with the thin ﬁlamentous outgrowth extending from the blastospore known as “germ-tube.” The extended germ-tube later develops A B FIGURE 8 \| (A) The inﬂuence of QSSM 1112 (at HIC) and farnesol on Candida albicans germ-tube formation at various time intervals (0–6 h). The QSSM 1112 manifested into the FBS along with the C. albicans cells. The micrograph evidently demonstrates the blockage of germ-tube formation in C. albicans cells treated with QSSM 1112 compared with that of untreated control cells (which encompasses classic badminton structures, i.e., true germ-tubes of C. albicans). (B) The impact of QSSM 1112 (at HIC) and farnesol on preformed germ-tube formation. There is no signiﬁcant difference between untreated control and QSSM 1112- and farnesol-treated cells. FBS, fetal bovine serum; HIC, hyphal inhibitory concentration. B B FIGURE 8 \| (A) The inﬂuence of QSSM 1112 (at HIC) and farnesol on Candida albicans germ-tube formation at various time intervals (0–6 h). The QSSM 1112 manifested into the FBS along with the C. albicans cells. The micrograph evidently demonstrates the blockage of germ-tube formation in C. albicans cells treated with QSSM 1112 compared with that of untreated control cells (which encompasses classic badminton structures, i.e., true germ-tubes of C. albicans). (B) The impact of QSSM 1112 (at HIC) and farnesol on preformed germ-tube formation. There is no signiﬁcant difference between untreated control and QSSM 1112- and farnesol-treated cells. FBS, fetal bovine serum; HIC, hyphal inhibitory concentration. Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org December 2021 \| Volume 11 \| Article 781790 Jothi et al. Bacterial QS Molecules as Anti-Hyphal Inhibitors 1112 followed the same mode of hyphal inhibitory action as that of C. albicans QS signaling molecule farnesol. In line with the present study, a similar kind of bacterial QSMs mediated germ- tube inhibition was observed by Zhang et al. (2011), wherein the two DSFs, i.e., cis- and trans-2-dodecenoic acid, have arrested the C. albicans cells from undergoing germ-tube induction (Zhang et al., 2011). formation. In this way, QSSM also could be used in synergistic therapy to enhance the efﬁcacy of a single dose. formation. In this way, QSSM also could be used in synergistic therapy to enhance the efﬁcacy of a single dose. 3.9 Fluorescence Microscopic Analysis Reinforces the QSSM 1112-Mediated Hyphae and Germ-Tube Inhibition In order to further reinforce the data obtained from a light microscopic analysis, ﬂuorescence microscopic analysis was carried out. As seen in Figures 9A, B, QSSM 1112 is sufﬁcient enough to inhibit the germ-tube and hyphal formation in C. albicans. Through in vitro validation of in silico results, the present investigation stands as a “proof of concept” for the reliability of in silico-based drug prediction in the drug development process. In most of the cases, the antihyphal inhibitors not only halt the germ-tube germination but also have the capability to revert the preformed germ-tube into yeast form. In order to know whether the QSSM 1112 exposure has any effect on preformed germ-tube, QSSM 1112 was added to wells that had preexisting C. albicans germ-tube. After 5-h time exposure, the preformed germ-tube cells were visualized under a light microscope. As can be seen from Figure 8B, no considerable change was observed in the germ-tube formation of both QSSM 1112-treated and untreated control cells. It was also observed that the positive control farnesol also had no effect; it neither reverted nor destroyed the germ-tube cells. This further conﬁrms that QSSM 1112 and farnesol are ably averting C. albicans dimorphism before germination, whereas they could not have any effect on C. albicans cells that had germinated already. This result is in agreement with the observation of Mosel et al. where the farnesol appears to be ineffective on preexisting hyphae even at a very high concentration (Mosel et al., 2005). Although both farnesol and QSSM 1112 did not effectively inhibit preformed germ-tube formation, it has been reported that exposure with farnesol could sensitize the drug-resistant C. albicans to antifungal treatment. With the use of this same principle, several studies also demonstrated the synergistic combinatorial effect offarnesol over the conventional antifungals (amphotericin B, ﬂuconazole, and micafungin) against C. albicans bioﬁlm REFERENCES Charro, N., and Mota, L. J. (2015). Approaches Targeting the Type III Secretion System to Treat or Prevent Bacterial Infections. Expert Opin. Drug Discov. 10 (4), 373–387. doi: 10.1517/17460441.2015.1019860 Abdulazeez, S. (2019). Molecular Simulation Studies on B-Cell Lymphoma/ Leukaemia 11A (BCL11A). Am. J. Transl. Res. 11 (6), 3689–3697. Chen, S., Xia, J., Li, C., Zuo, L., and Wei, X. (2018). The Possible Molecular Mechanisms of Farnesol on the Antifungal Resistance of C. Albicans Bioﬁlms: The Regulation of CYR1 and PDE2. BMC Microbiol. 18 (1), 203. doi: 10.1186/ s12866-018-1344-z Baell, J. B., and Nissink, J. W. M. (2018). Seven Year Itch: Pan-Assay Interference Compounds (PAINS) in 2017-Utility and Limitations. ACS Chem. 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A Novel DSF-Like Signal From Burkholderia Cenocepacia Interferes With Candida Albicans Morphological Transition. ISME J. 2 (1), 27–36. DATA AVAILABILITY STATEMENT The authors also acknowledge the DST-FIST [Grant No. SR/ FST/LSI-639/2015(C)], UGC-SAP [Grant No. F.5-1/2018/DRS- II (SAP-II)], DST-PURSE [Grant No. SR/PURSE Phase 2/38 (G)], and RUSA-Phase 2.0, Government of India [F. 24-51/2014- U, Policy (TN Multi-Gen)] for providing instrumentation facilities. RJ and NH sincerely thank Rashtriya Uchchatar Shiksha Abhiyan (RUSA), MHRD, Government of India [F. 24-51/2014-U, Policy (TN Multi-Gen), Dept. of Edn, GOI] for providing RUSA-2.0 Ph.D. Fellowship. The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors. AUTHOR CONTRIBUTIONS RJ designed the research idea, analyzed the data, carried out in vitro analysis and result interpretation, and wrote the original draft. NH carried out virtual screening, molecular docking and simulation analysis, and in silico result interpretation and prepared the ﬁgures. SG designed the research idea; performed the formal analysis, funding acquisition, investigation, administration, and validation; and critically revised the original draft. SP contributed to the conceptualization, supervision, funding acquisition, and critical revision of the original draft. All authors contributed to the article and approved the submitted version. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2021.781790/ full#supplementary-material Supplementary Table 1 \| - Physicochemical parameters, lipophilicity and solubility of QSSM 1157 & QSSM 1112. Supplementary Table 1 \| - Physicochemical parameters, lipophilicity and solubility of QSSM 1157 & QSSM 1112. FUNDING hyphal inhibitor. On the whole, through in silico and in vitro analyses, the present study opens up new avenues for considering the bacterial QSMs as potential inhibitors against fungi especially C. albicans. This study also highlights that the antagonistic interaction between bacteria and fungi can be utilized as a tool to overcome certain fungal infections. SG gratefully acknowledges DST-SERB-EEQ Project Grant (File No.:EEQ/2020/000288). The authors thankfully acknowledge the ﬁnancial support rendered by RUSA 2.0 [F.24-51/2014-U, Policy (TN Multi-Gen), Department of Education, Government of India]. SG gratefully acknowledges DST-SERB-EEQ Project Grant (File No.:EEQ/2020/000288). The authors thankfully acknowledge the ﬁnancial support rendered by RUSA 2.0 [F.24-51/2014-U, Policy (TN Multi-Gen), Department of Education, Government of India]. CONCLUSION The current study investigates the antihyphal efﬁcacy of bacterial QSMs through in silico and in vitro analyses against C. albicans dimorphic switching. Results of initial virtual screening analysis revealed hyphal inhibitory potential of the two DKPs, viz., QSSM 1112 and QSSM 1157, against CYCc and RAS1, respectively. Further, 50-ns MD simulation demonstrated the stable binding of ligands to target proteins with less conformational ﬂuctuation and greater afﬁnity. In addition, the pharmacokinetics and toxicity prediction revealed that the identiﬁed QSM hyphal inhibitors are safe for biological use. These QSMs displayed better lipophilicity and water solubility scores, lesser toxicity, and no BBB permeation. Further, in vitro investigations such as liquid and solid antihyphal assays and germ-tube inhibition assays unequivocally established QSSM 1112 as a promising A B FIGURE 9 \| Fluorescence microscopic observation showcases the inhibitory potential of QSSM 1112 on (A) yeast-to-hyphal transition (B) germ-tube formation of Candida albicans. 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Pharmacol. 5, 174. doi: 10.3389/fphar.2014.00174 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. Vyas, V. K., Ukawala, R. D., Ghate, M., and Chintha, C. (2012). Homology Modeling a Fast Tool for Drug Discovery: Current Perspectives. Indian J. Pharm. Sci. 74 (1), 1–17. doi: 10.4103/0250-474x.102537 Wang, Y. (2013). Fungal Adenylyl Cyclase Acts As a Signal Sensor and Integrator and Plays a Central Role in Interaction With Bacteria. PloS Pathog. 9 (10), e1003612. doi: 10.1371/journal.ppat.1003612 Copyright © 2021 Jothi, Hari Prasath, Gowrishankar and Pandian. 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. Yan, P. S., Song, Y., Sakuno, E., Nakajima, H., Nakagawa, H., and Yabe, K. (2004). Cyclo(L-Leucyl-L-Prolyl) Produced by Achromobacter Xylosoxidans Inhibits Aﬂatoxin Production by Aspergillus Parasiticus. Appl. Environ. Microbiol. 70 (12), 7466–7473. doi: 10.1128/AEM.70.12.7466-7473.2004 Yin, M., Yan, Y., Lohman, J. R., Huang, S. X., Ma, M., Zhao, G. R., et al. (2014). Cycloheximide and Actiphenol Production in Streptomyces Sp. YIM56141 December 2021 \| Volume 11 \| Article 781790 Frontiers in Cellular and Infection Microbiology \| www.frontiersin.org 22 22
https://openalex.org/W4214562787	https://iopscience.iop.org/article/10.1088/1748-0221/17/02/C02025/pdf	English	null	Design of the cross-polarization scattering diagnostic on the HL-2A tokamak	Journal of instrumentation	2,022	cc-by	4,395	PAPER • OPEN ACCESS PAPER • OPEN ACCESS You may also like You may also like Upgrade of an integrated Langmuir probe system on the closed divertor target plates in the HL-2A tokamak Zhihui HUANG, , Jun CHENG et al. - Energetic Particle Physics on the HL-2A Tokamak: A Review Pei-Wan Shi, , Wei Chen et al. - The ITB dynamics controlled by internal kink modes on HL-2A tokamak X X He, L W Yan, D L Yu et al. - You may also like Upgrade of an integrated Langmuir probe system on the closed divertor target plates in the HL-2A tokamak Zhihui HUANG, , Jun CHENG et al. - Energetic Particle Physics on the HL-2A Tokamak: A Review Pei-Wan Shi, , Wei Chen et al. - The ITB dynamics controlled by internal kink modes on HL-2A tokamak X X He, L W Yan, D L Yu et al. - You may also like Upgrade of an integrated Langmuir probe system on the closed divertor target plates in the HL-2A tokamak Zhihui HUANG, , Jun CHENG et al. - Energetic Particle Physics on the HL-2A Tokamak: A Review Pei-Wan Shi, , Wei Chen et al. - The ITB dynamics controlled by internal kink modes on HL-2A tokamak X X He, L W Yan, D L Yu et al. - Journal of Instrumentation To cite this article: R.H. Tong et al 2022 JINST 17 C02025 To cite this article: R.H. Tong et al 2022 JINST 17 C02025 View the article online for updates and enhancements. View the article online for updates and enhancements. This content was downloaded from IP address 195.242.1.98 on 24/10/2024 at 06:16 This content was downloaded from IP address 195.242.1.98 on 24/10/2024 at 06:16 This content was downloaded from IP address 195.242.1.98 on 24/10/2024 at 06:16 This content was downloaded from IP address 195.242.1.98 on 24/10/2024 at 06:16 Published by IOP Publishing for Sissa Medialab Published by IOP Publishing for Sissa Medialab Received: September 3, 2021 Revised: November 9, 2021 Accepted: January 10, 2022 Published: February 25, 2022 Received: September 3, 2021 Revised: November 9, 2021 Accepted: January 10, 2022 Published: February 25, 2022 4th European Conference on Plasma Diagnostics (ECPD2021) 4th European Conference on Plasma Diagnostics (ECPD2021) 7–11 June, 2021 Online 2022 JINST 17 C020 ∗Corresponding author. © 2022 The Author(s). Published by IOP Publishing Ltd on behalf of Sissa Medialab. 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. https://doi.org/10.1088/1748-0221/17/02/C02025 ∗Corresponding author. Design of the cross-polarization scattering diagnostic on the HL-2A tokamak R.H. Tong,𝑎W.L. Zhong,𝑎,∗J. Wen,𝑎Z.B. Shi,𝑎X.L. Zou,𝑏A.S. Liang,𝑎Z.C. Yang,𝑎 K.Y. Fang,𝑎M. Jiang,𝑎P.W. Shi,𝑎X. Yu,𝑎Y. Zhou,𝑎M. Xu𝑎and the HL-2A team𝑎 𝑎Southwestern Institute of Physics, P.O. Box 432, Chengdu 610041, People’s Republic of China 𝑏CEA, IRFM, 13108 Saint-Paul-lez-Durance, France E il h l@ i JINST 17 C02025 R.H. Tong,𝑎W.L. Zhong,𝑎,∗J. Wen,𝑎Z.B. Shi,𝑎X.L. Zou,𝑏A.S. Liang,𝑎Z.C. Yang,𝑎 K.Y. Fang,𝑎M. Jiang,𝑎P.W. Shi,𝑎X. Yu,𝑎Y. Zhou,𝑎M. Xu𝑎and the HL-2A team𝑎 𝑎Southwestern Institute of Physics, P.O. Box 432, Chengdu 610041, People’s Republic of China 𝑏CEA, IRFM, 13108 Saint-Paul-lez-Durance, France R.H. Tong,𝑎W.L. Zhong,𝑎,∗J. Wen,𝑎Z.B. Shi,𝑎X.L. Zou,𝑏A.S. Liang,𝑎Z.C. Yang,𝑎 K.Y. Fang,𝑎M. Jiang,𝑎P.W. Shi,𝑎X. Yu,𝑎Y. Zhou,𝑎M. Xu𝑎and the HL-2A team𝑎 𝑎Southwestern Institute of Physics, P.O. Box 432, Chengdu 610041, People’s Republic of China 𝑏CEA, IRFM, 13108 Saint-Paul-lez-Durance, France E-mail: zhongwl@swip.ac.cn https://doi.org/10.1088/1748-0221/17/02/C02025 E-mail: zhongwl@swip.ac.cn Abstract: A new cross-polarization scattering (CPS) diagnostic has been developed on HL-2A, which aims to measure the local magnetic ﬂuctuation inside the plasma. It is based on the scattering of an incident microwave beam into the perpendicular polarization by magnetic ﬂuctuations. The CPS diagnostic has been designed in the Q-band (33–50 GHz), which consists of the electronic system, quasi-optical, and polarization rejector. The ray-tracing code is used to simulate the propagation of the probe and scattered rays. To test the performance of the quasi-optical system, a 3D test platform is built and detailed test results are shown. Two methods are developed for polarization rejector on HL-2A: wire grid polarizer and dual-polarized horn antenna (DPHA). The laboratory test result shows that the polarization rejection of both methods is better than 30 dB, which meets the needs for magnetic ﬂuctuation detection. In the future, the CPS diagnosis will be used to study the electromagnetic turbulence behavior in the high-performance plasma of the HL-2A tokamak. Keywords: Plasma diagnostics — interferometry, spectroscopy and imaging; Nuclear instruments and methods for hot plasma diagnostics ∗Corresponding author. © 2022 The Author(s). Published by IOP Publishing Ltd on behalf of Sissa Medialab. 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. https://doi.org/10.1088/1748-0221/17/02/C02025 https://doi.org/10.1088/1748-0221/17/02/C02025 Contents 1 Introduction 2 CPS diagnostic on HL-2A 3 Quasi-optical system 4 Polarization rejector 5 Summary Contents 1 Introduction 1 2 CPS diagnostic on HL-2A 2 3 Quasi-optical system 3 4 Polarization rejector 4 5 Summary 6 2022 JINST 17 C02 JINST 17 C02025 1 Introduction The improved physical model of conﬁnement in plasma, especially the understanding of the high- conﬁnement operation, is particularly important in a tokamak. The gyrokinetic theories predict that as the plasma β (the ratio of volume-averaged plasma pressure ⟨p⟩to magnetic pressure B2/2μ0) increased, the electromagnetic turbulence can be dominant and drive signiﬁcant anomalous transport [1]. It is found that magnetic turbulence can inﬂuence plasma conﬁnement [2], pedestal dynamics [3], and edge-localized modes evolution [4] in experiments. In addition, the nonlinear gyro-kinetic simulation indicates that the electromagnetic turbulence, i.e. the micro-tearing modes (MTMs), is uniquely able to drive the abnormal electron transport in DIII-D high bootstrap current scenario [5, 6], which is a candidate scenario for future tokamak reactors. The magnetic ﬂuctuation ˜B measurement can provide experimental veriﬁcation of the theory and model of magnetic turbulence [7], especially in high beta plasma. In tokamak plasma, the magnetic ﬂuctuation is typically measured by magnetic coils [3], motional stark eﬀect (MSE) [8], polarimetry [4, 9], etc. . But they are generally limited to low wavenumbers of turbulence measured. Or it can be estimated indirectly by analyzing runaway electron transport [10–12]. Cross polarization scattering (CPS) is an internal magnetic ﬂuctuation diagnostic based on the microwave technique. It is a local, internal measurement due to the swelling eﬀect near the cut-oﬀlayer [13]. The advantages of CPS include ﬂexible spatial and wavenumber resolution, and the non-perturbing nature. The basic idea of the CPS is the incident microwave will be scattered into the orthogonal polarization to that of the incident polarization by local magnetic ﬂuctuation. In a tokamak, two eigenmodes exist when the wave propagation is perpendicular to B: the ordinary mode (O) with polarization parallel to B, and the extraordinary mode (X) with polarization perpendicular to B. So the CPS processes can be described as: Xi + ˜B →Os or Oi + ˜B →Xs, where the subscript indices i and s refer to the incident and scattered waves respectively. It is opposed to scattering to the same polarization by electron density ﬂuctuations ˜n: Xi + ˜n →Xs or Oi + ˜n →Os, which is typically used to measure density ﬂuctuations and the propagation velocity of turbulence, i.e. Doppler backscattering (DBS) diagnostic. For DBS, a tilt angle of the microwave beam is launched relative – 1 – to the normalofthe cutoﬀlayerand backscattered radiation is detected [14,15]. 1 Introduction The CPS diagnostic was originally demonstrated on Tore Supra tokamak [16], where the eﬀect of magnetic ﬂuctuations on electron heat transport was studied. After that, the CPS was developed on GAMMA-10 tandem mirror [17], MAST spherical tokamak [18] and DIII-D tokamak [19–21]. HL-2A is a medium-sized conventional tokamak (major radius R0 = 1.65 m and minor radius a = 0.40 m), which performs with plasma current Ip ∼100–450 kA, centre line-average electron density ne ∼(0.2–5.5) × 1019 m−3 and toroidal magnetic ﬁeld Bt ∼1.0–2.7 T. The electron and ion temperatures are up to Te = 5 keV and Ti = 3.5 keV, respectively. Recently, a high beta, high conﬁnement discharge has been achieved on HL-2A [22]. Besides, a broadband (frequency of 50–150 kHz) electromagnetic turbulence has been observed by magnetic coils in the HL-2A H-mode plasmas [3, 23]. To further study the behaviors of magnetic turbulence, which may play an important role in the achievement of high β plasma and the evolution of the pedestal, a CPS system is developed on HL-2A recently. In the following section, we ﬁrst introduced the detailed setup of the CPS diagnostic on HL-2A (section 2) followed by the design and test results for the quasi-optical (section 3) and polarization rejection system (section 4). Finally, a summary is presented in section 5. 2022 JINST 17 C0 JINST 17 C02025 2 CPS diagnostic on HL-2A To measure the magnetic turbulence in the pedestal region for typical HL-2A discharge. The CPS is designed based upon Q-band (33–50 GHz) with X mode launched wave. The electronic system is as same as the eight-channel Q-band DBS as described in ref. [15]. It is based on the Multiplexer- based Frequency Array Source (MFAS) technique. The output power for 8 frequencies is >15 dBm with ﬂatness <5 dB. A 40.5 GHz local oscillation is used, which results in the intermediate frequency varies from 0.5 to 7.5 GHz. Figure 1 shows the conﬁguration of the CPS diagnostic developed on HL-2A. It mainly consists of 3 parts: (1) quasi-optical system; (2) launch/receive system with polarization rejector; (3) electronic system for launching/receiving the microwave signals. The X-mode polarization probe beam was selected by the polarization rejector and launched from the low ﬁeld side midplane through the quasi-optical system. Then the O-mode beam will be scattered by the magnetic ﬂuctuations near the cut-oﬀlayer. Figure 1. CPS diagram on HL-2A. Figure 1. CPS diagram on HL-2A. – 2 – As the X- and O-mode waves possess diﬀerent wavenumbers along the incident ray path, the local incident and scattered wavenumbers will be diﬀerent for the CPS scattering process. Hence, the probe angle, probe frequency and plasma parameters will determine the probed wavenumber of B. Figure 2 shows ray tracing calculation for an X-mode probe beam and O-mode CPS receive beam with frequency 48 GHz. The plasma used is a 1.3 T, lower single null, HL-2A H-mode. To be received by the CPS diagnostic, the wavevector (ks = ki + k ˜B) and frequency (ωs = ωi + ω ˜B) matching conditions should be satisﬁed, where 𝒌i and 𝒌s are the incident and scattered wavevector, respectively, and k ˜B is the wavevector of the magnetic ﬂuctuation (similarly for the frequencies ωs, ωi and ω ˜B). It should be noted that although the electric swelling eﬀect can cause the most contribution of scatting near the cut-oﬀlayer [13], the multiple scattering centres may exist at any position along the entire ray path as reported in the reference [24]. 2022 JINST 17 C020 Figure 2. Ray tracing result with X-mode launch and O-mode received at 48 GHz on HL-2A H-mode plasma. JINST 17 C02025 Figure 2. Ray tracing result with X-mode launch and O-mode received at 48 GHz on HL-2A H-mode plasma. 3 Quasi-optical system The quasi-optical system consists of a spherical lens, a parabolic mirror and a plane mirror. The mechanical and optical designs are shown in ﬁgures 3(a) and (b). The design goal was to form a high-quality Gaussianbeam near the edgeof HL-2A plasmasfor the range of frequencies applicable to Q-band operation. To probe a range of wavenumber values with the CPS system, an adjustable launch angle is necessary. With the help of feedthrough, the launched angle of the beam can be changed from −10◦to +10◦in the vertical direction. A positive angle indicates the wave is launched upward. A higher mirror angular range is limited by the size of the vacuum window. To test the performance of the quasi-optical system, a 3D test platform has been developed as shown in ﬁgure 3(c). The microwave is launched by the horn antenna and passes through the quasi-optical system. The power distribution in the X-Y-Z direction can be measured through the 3D displacement platform. The high-quality Gaussian beams can be observed for diﬀerent frequencies in the Y-Z plane as shown in ﬁgure 3(c). – 3 – Figure 3. (a) The mechanical design. (1) Spherical lens; (2) parabolic mirror; (3) plane mirror. (4) Feedthrough for mirror angle control; (5) vacuum chamber. (b) Gaussian optical simulation results of the quasi-optical system. (c) The 3D test platform for the quasi-optical system: (1) CPS microwave source; (2) launch horn antenna; (3) quasi-optical system; (4) receive horn; (5) 3D displacement platform; (6) position controller; (7) spectrum analyzer; (8) control computer. The X, Y and Z direction corresponds to the radial, toroidal and vertical direction of plasma, respectively. The typical Gaussian beam measured in the Y-Z direction at f = 36 GHz is shown in (c). 2022 JINST 17 C0 Figure 3. (a) The mechanical design. (1) Spherical lens; (2) parabolic mirror; (3) plane mirror. (4) Feedthrough for mirror angle control; (5) vacuum chamber. (b) Gaussian optical simulation results of the quasi-optical system. (c) The 3D test platform for the quasi-optical system: (1) CPS microwave source; (2) launch horn antenna; (3) quasi-optical system; (4) receive horn; (5) 3D displacement platform; (6) position controller; (7) spectrum analyzer; (8) control computer. The X, Y and Z direction corresponds to the radial, toroidal and vertical direction of plasma, respectively. The typical Gaussian beam measured in the Y-Z direction at f = 36 GHz is shown in (c). 4 Polarization rejector It should be mentioned that the power level from B is much lower than ˜n for typical toka- mak parameters. The relative magnetic ﬂuctuation level ˜B/B is from about 10−4 to 10−6 measured with typical tokamak parameter [4, 16]. So the expected relative ﬂuctuation level ˜B/B 2/\| ˜n/n\|2 ∼10−2–10−5 [4, 16, 18, 25]. As mentioned in reference [13, 16], the scattered power from magnetic ﬂuctuation PCPS ∝( ˜B/B)2, and the scattered power from electron density ﬂuctuation PDBS ∝( ˜n/n)2. So one of the key points of CPS diagnostic is the signal scattered by ˜n must be rejected, i.e. the polarization of the launched and scattered microwave is the same. The isolation must be larger than 20 dB at least, which corresponds to ˜B/B 2/\| ˜n/n\|2 = 10−2. Considering the robustness in practical application, at least 30 dB should be guaranteed. To meet this requirement. The so-called Polarizing mirror eﬀect with a cut-oﬀlayer to reject the signal from ˜n is applied on Tore Supra [16], and the wire grid polarizers are applied in DIII-D [21]. Two methods are developed for polarization rejector on HL-2A: wire grid polarizer and dual-polarized horn antenna (DPHA). Another key point is the contamination by polarization mode conversion or the mismatched launch/receive polarization with the plasma magnetic ﬁeld pitch angle [16, 18, 21]. Since the power of the unwanted scattering component is proportional to sin2(δ), where δ is the mismatch angle. A polarization rejection of ∼30 dB can still satisfy with δ = 2◦mismatch angle in theory. To minimize the mismatch angle, a motorized rotation stage is used to control the angle of the polarizer or DPHA, and match the polarization of the beam with magnetic ﬁeld pitch angle with an angle resolution of 0.1 degree. The laboratory test results show that the polarization rejection of both methods is better than 30 dB, as shown below. 2022 JINST 17 C020 JINST 17 C02025 The ﬁrst method is the wire grid polarizer. It is made of 10 micron diameter tungsten wire and the wire spacing is 25 microns. The wire grid polarizer will reﬂect incoming waves where the electric ﬁeld is polarized parallel to the wires, and transmit radiation where the electric ﬁeld is polarized orthogonal to the wires. The polarizer can be used up to 3 THz with a normalized transmission close to 1. 3 Quasi-optical system JINST 17 C02025 Figure 4 shows the relationship between the launch angle and the mirror position. Here the mirror position is deﬁned as the stroke depth of the feedthrough, which is used to control the angle of the ﬂat mirror. When the launch angle is out of range (±10◦), the Gaussian beam will be destroyed due to the reﬂection of the vacuum chamber (not shown). The Gaussian beam radii distributions along the beam propagation direction are shown in ﬁgure 4(b) for 8 frequencies. By changingthe distancebetween the launch antenna(WR-22 standard gain waveguidehorn antenna in the test) and the spherical lens (L), the Gaussian beam distribution will be changed. At L = 225 mm, the beam radius is ∼40 mm at the plasma edge, and the divergence half-angle of the Gaussian beam is about 4.4 degrees. A near-parallel incident beam near the cut-oﬀlayer is considered to be better for signal detection. Figure 4. (a) The relationship between the launch angle and mirror position. A positive angle indicates the wave is launched upward. (b) The Gaussian beam radius along with the beam propagation (x) direction. L is the distance between the launch antenna and the spherical lens. The shading indicates the range of 8 frequencies and the solid line is the mean value. Locations of various plasma radii are indicated by vertical red lines. The data are laboratory test results. Figure 4. (a) The relationship between the launch angle and mirror position. A positive angle indicates the wave is launched upward. (b) The Gaussian beam radius along with the beam propagation (x) direction. L is the distance between the launch antenna and the spherical lens. The shading indicates the range of 8 frequencies and the solid line is the mean value. Locations of various plasma radii are indicated by vertical red lines. The data are laboratory test results. – 4 – 4 Polarization rejector The relationship between the power received by the rectangular horn antenna and the polarizer angle is shown in ﬁgure 5. When the polarizer angle is 0◦, the launched wave (horizontal polarization) will be reﬂected by the polarizer, resulting in the min power being received by R1 (about −50 dBm) and the max power being received by R2 (about −10 dBm). When the polarizer angle is 90◦, the wave will transmit, so the max power is received by R1 (−10 dBm). The test results show that the rejection of the polarizer is >40 dB. It should be men- tioned that the actual rejection can be better because the test result is limited by the detector noise ﬂoor. The second method is to use the dual-polarized horn antenna (DPHA), which is a three- port antenna, which can separate a circular or elliptical polarized wave into two linear, cross polarized waves. Compared with the wire grid polarizer, DPHA has the advantage of long working life, compact structure and less space occupation. To test the performance of DPHA, a linear polarization wave is launched towards DPHA with a rectangular horn antenna. And the power received by the two ports of DPHA with diﬀerent angles is shown in ﬁgure 6. The test results show that the orthogonal polarization rejection of DPHA is >30 dB, which meets the needs of CPS diagnostic. – 5 – Figure 5. The power received by antenna (a) R1 and (b) R2 with diﬀerent polarizer angles. The polarization direction for launch (L1) and receive (R1 & R2) rectangular horn antenna are all in horizontal directions. 0 degree of polarizer corresponds to the wave with horizontal polarization will be reﬂected. The data are laboratory test results. 2022 JINST 17 C020 Figure 5. The power received by antenna (a) R1 and (b) R2 with diﬀerent polarizer angles. The polarization direction for launch (L1) and receive (R1 & R2) rectangular horn antenna are all in horizontal directions. 0 degree of polarizer corresponds to the wave with horizontal polarization will be reﬂected. The data are laboratory test results. JINST 17 C02025 Figure 6. The power received by (a) port1 and (b) port2 of DPHA with diﬀerent DPHA angles. 0 degree corresponds to the polarization directions of the launch rectangular horn antenna and the port2 of DPHA are the same. The data are laboratory test results. Figure 6. 4 Polarization rejector The power received by (a) port1 and (b) port2 of DPHA with diﬀerent DPHA angles. 0 degree corresponds to the polarization directions of the launch rectangular horn antenna and the port2 of DPHA are the same. The data are laboratory test results. 5 Summary In summary, a Q-band multi-channel cross-polarization scattering diagnostic has been developed on HL-2A. The 8 working frequencies are from 34 to 48 GHz, with a frequency interval of 2 GHz. The test and calibration of the quasi-optical system have been done with a 3D test platform. It is shown that the launch angle can be controlled from −10◦to 10◦, with the beam radius ∼40 mm at the plasma edge. For polarization rejector, the cross isolation >40 dB has been achieved with wire grid polarizer and >30 dB with Dual-polarized horn antenna (DPHA) in laboratory tests. The CPS diagnostic has already been installed on HL-2A, and an experimental investigation of – 6 – electromagnetic turbulence will be carried out in the next HL-2A campaign. 3D ray-tracing and full-wave simulations will be the next steps for a better understanding of the CPS data. In addition, the toroidal angle launching capability will be evaluated in the future system upgrade to optimize high-k measurement [26] and improve signal quality. Acknowledgments The author R.H. Tong would like to gratefully acknowledge the HL-2A team for the support of the study and Doctor Y.L. Zhu for useful discussions. This work was supported by the National Key R & D Program of China under Grant No. 2017YFE0301106 and the National Natural Science Foundation of China under Grant No. 11922503. 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https://openalex.org/W2943242069	https://www.iiste.org/Journals/index.php/RJFA/article/download/47571/49124	English	null	The Effect of Corporate Governance on Profitability, Capital Structure and Corporate Value	null	2,019	cc-by	9,953	The Effect of Corporate Governance on Profitability, Capital Structure and Corporate Value Rolens E.H. Riwu Manu Student of Doctoral Program of Business Administration, Faculty of Administrative Science, Brawijaya University, Jalan M.T. Haryono No. 163 Malang, East Java - Indonesia, Postal Code 65145 Taher Alhabsji Sri Mangesti Rahayu Nila Firdausi Nuzula Department of Business Administration, Faculty of Administrative Science, Brawijaya University, Jalan M.T. Haryono No. 163 Malang, East Java - Indonesia, Postal Code 65145 Abstract This study is motivated by theoretical inconsistencies that underlie agency relations, within the corporate governance framework to increase profitability, optimal capital structure, and corporate value. The results of previous researchers' research are conflicting about the effect between variables. The originality of this study lies in providing empirical evidence of the effect of corporate governance, profitability, capital structure, on corporate value. This research is considered important to be carried out to study corporate governance within the framework of agency theory capable of directing management to make managerial decisions that minimize agency problems and improve the welfare of shareholders.This study focuses on empirical testing of the theory through testing the effect between the variables of Corporate Governance, Profitability, Capital Structure, and Corporate Value. This research was conducted in Indonesia with observation objects covering all Basic Industry Manufacture companies listed on the Indonesia Stock Exchange, the observation period from 2012 to 2017. The population was 84 companies and a sample of 25 companies. The sampling method is purposive sampling. The data analysis technique uses statistical procedures to test hypotheses with the Generalized Structured Component Analysis (GSCA) software. The findings of this study are corporate governance has a positive and significant effect on profitability, capital structure and corporate value. Profitability has a positive effect on the capital structure and corporate value. Capital structure has a positive and significant effect on corporate value. This study concludes that good corporate governance will maintain an optimal capital structure that is able to increase profitability and Corporate value. Corporate management can use these variables to predict the Corporate's future. Keywords: Corporate Governance, Profitability, Capital Structure, Corporate Value DOI 10 7176/RJFA/10 8 20 Keywords: Corporate Governance, Profitability, Capital Structure, Corporate Value DOI: 10.7176/RJFA/10-8-20 Publication date: April 30th 2019 www.iiste.org www.iiste.org Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) DOI: 10.7176/RJFA Vol.10, No.8, 2019 www.iiste.org Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) DOI: 10.7176/RJFA Vol.10, No.8, 2019 www.iiste.org ww.iiste.org have rational limitations and do not like risk. (2). In organizations there are conflicts between members and there is information asymmetry between members. (3). Information is a commodity that has economic value so it is traded. Based on these assumptions, the manager has the opportunity to make deviations. have rational limitations and do not like risk. (2). In organizations there are conflicts between members and there is information asymmetry between members. (3). Information is a commodity that has economic value so it is traded. Based on these assumptions, the manager has the opportunity to make deviations. The implementation of Corporate Governance is beneficial for the sustainable growth of the Corporate in order to increase the value of the Corporate. if management is able to place the Corporate has competitiveness in seizing the market share of the products produced so as to provide maximum benefits to the investment invested in the Corporate. Corporate governance practices (CG) direct management in carrying out the main tasks and functions of corporate financial management in decision making relating to (1) funding decisions, (2) investment decisions and (3) dividend decisions, (Hanafi, 2010: 3-4) . The three decisions are carried out simultaneously in a unity of interrelated actions (Data et al., 2017). The effect of corporate governance on profitability is explained through the principles of corporate governance to improve, competitiveness, credibility and profitability. Improve relations between stakeholders such as investors, business partners, employees, customers and others (Todorovic, 2013). Effective corporate governance over a long period of time improves Corporate performance and benefits shareholders, through increasing Corporate performance characterized by high levels of profitability followed by high dividend distribution and increasing stock value through high capital gains. The effect of corporate governance on capital structure is explained through the role of Corporate governance in preventing managerial opportunistic behavior as demonstrated by the decision of low-cost, profitable capital structure that is oriented towards positive Corporate growth. Corporate governance structure consisting of board size, board composition, internal audit committee, and external audit committee effect s the capital structure as measured by indicators of the ratio of liabilities to total assets, the ratio of total liabilities to total equity and the ratio of total long-term liabilities to total equity. The corporate governance mechanism will direct management to make capital structure decisions that have an impact on increasing the value of the Corporate. 1.1 Research Questions 1.1 Research Questions Based on the theoretical description and previous empirical research, identification of research variables and research objectives, the formulation of the problem from this study are: 1) Does corporate governance have a significant effect on profitability? 2) Does corporate governance have a significant effect on the capital structure? 3) Does corporate governance have a significant effect on Corporate value? 4) Does profitability have a significant effect on capital structure? 5) Does profitability have a significant effect on Corporate value? 6) D th it l t t h i ifi t ff t C t l ? Q Based on the theoretical description and previous empirical research, identification of research variables and research objectives, the formulation of the problem from this study are: 1) Does corporate governance have a significant effect on profitability? Based on the theoretical description and previous empirical research, identification of research variables and research objectives, the formulation of the problem from this study are: 1) Does corporate governance have a significant effect on profitability? 2) Does corporate governance have a significant effect on the capital structure 3) Does corporate governance have a significant effect on Corporate value? 4) Does profitability have a significant effect on capital structure? 5) Does profitability have a significant effect on Corporate value? ) h i l h i ifi ff l 5) Does profitability have a significant effect on Corpora 6) Does the capital structure have a significant effect on Corporate value? Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) DOI: 10.7176/RJFA Vol.10, No.8, 2019 The effect of corporate governance on corporate value is explained by Rezaee (2007) that corporate governance is a mechanism to adjust management's interests with shareholders. Especially the role of Corporate governance in reducing agency costs and creating long-term value for shareholders with a focus on the responsibility of monitoring the board of directors and senior executive management functions. The effect of profitability on capital structure is explained by Pecking order theory (Myers and Majluf, 1984) that when a Corporate needs capital, first, use internal funds, then debt, and finally issue new shares. Myers and Majluf (1984) state that companies that have high profitability do not depend on external funding to finance a Corporate's growth, because profitability has a negative correlation with the level of debt. If the Corporate issues new shares in the condition of information asymmetry, it will cause a decrease in stock prices, which will cause equity agency costs, thus issuing new shares is the last choice in certain situations. The results of Anake et al (2014) study found that profitability has a negative and significant effect on capital structure. Profitability affects the value of the Corporate, because profitability is a measure of Corporate performance as measured by the profits generated. Companies that succeed in gaining ever-increasing profits indicate that the Corporate has a good performance, thus creating positive responses from investors and encouraging a rise in the Corporate's stock price. Companies with high profitability show that the Corporate manages the Corporate's wealth effectively and efficiently. The originality of this study lies in providing empirical evidence of the effect of corporate governance variables on capital structure, profitability, and Corporate value. Motivate researchers to conduct research to reexamine arguments that state corporate governance affects, profitability, capital structure, and Corporate value. This research is considered important to do, starting from the idea that corporate governance that involves a number of board of commissioners, independent board of commissioners, representative audit committees within the framework of theoretical agencies is able to direct management to make managerial decisions that minimize agency problems. 1. Introduction The Corporate was established to achieve the main objective of maximizing the welfare of the owner, through increasing the value of the Corporate (Keown et al., 2000: 2). The management of the Corporate is entrusted by the owner to manage the Corporate in the hope of gaining profits and adding to the owner's wealth. The management of the Corporate will use its own capital and debt together in funding various Corporate operational activities. In managing the Corporate to achieve maximum value, there is often a conflict of interest between Managers and Shareholders, which is often called agency problem. Agency problems in companies affect the price of securities markets, because investors will assess the Corporate's performance through data contained in the Corporate financial report available on the stock exchange, the results of the assessment become a reference in making decisions whether the investment will be taken is right and gives benefits. Good corporate governance is needed to reduce agency problems because it is always oriented towards the goal of increasing Corporate value and aligning the interests of all parties. The purpose of implementing corporate governance is as a guide for management in controlling the Corporate to make the right decisions and in favor of the interests of the owners and all stakeholders. Good management will function as aligning management interests with shareholders and all stakeholders related to the Corporate. All available resources in the Corporate are fully utilized for the realization of the welfare of the shareholders and to increase the welfare of management to the fullest. Corporate governance is based on the agency theory concept which serves to provide confidence to investors that investment in the Corporate will provide a large return for investors. Nasution and Setiawan (2007) stated that "corporate governance is a concept proposed to mediate differences in interests between managers and Corporate owners in order to improve Corporate performance through supervision or monitoring of management performance and ensuring management accountability to stakeholders based on agreed agreements". Manager's behavior in corporate decision making is explained by agency theory (Jensen and Mecling, 1976). which explains the relationship of shareholders as principal with the manager as an agent, Eisendhart (1989) suggests there are three assumptions underlying the agency theory, namely: (1). 1. Introduction Humans have a selfish nature and 202 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) DOI: 10.7176/RJFA Vol.10, No.8, 2019 Research Journal of Finance and Accounting 2.4 The Effect of Profitability on Capital Structure Trade off theory (Modigliani and Miller, 1963) suggests that when a Corporate has high profitability, it must use more debt in order to obtain higher tax benefits for the use of debt. The research findings of Olderink (2013), Oppong-Boakye (2013), Sangeetha (2013), Kisaka (2015) found that profitability had a significant positive effect on capital structure. Pecking Order Theory (Myers and Majluf, 1984) says that when a Corporate needs capital, it first uses internal funds, then debt, and the last option is to issue new shares. Baskin (1989) suggests that using internal funds is better than relying on external capital, because internal funds are free of cost. Lots of empirical evidence such as the findings of Bevan and Danbolt (2002); Mazur (2007); Frank and Goyal, (2009); Anake et al (2014) found that profitability has a significant and negative effect on capital structure. The difference in empirical findings is a reason to re-examine the effect of profitability on capital structure, with the research hypothesis as follows: H4: Profitability has a significant effect on capital structure. 1.2 Research Objectives 1) Test and explain corporate governance effect on profitability. 203 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) DOI: 10.7176/RJFA Vol.10, No.8, 2019 2) Test and explain corporate governance effect on capital structure. 3) Test and explain corporate governance effect on Corporate value. 3) Test and explain corporate governance effect on Corporate value. ) p p g p 4) Test and explain the profitability of the effect on the capital structure. 4) Test and explain the profitability of the effect on the capital structure. 5) Test and explain the profitability of the effect on the value of the Corporate. 5) Test and explain the profitability of the effect on the value of the Corporate. 6) Test and explain the capital structure effect on Corporate value. 2.1 The Effects of Corporate Governance On Profitability 2.1 The Effects of Corporate Governance On Profitability The principles of good corporate governance can improve profitability, improve competitiveness, credibility and improve relations between key stakeholders such as investors, business partners, employees, customers, and others (Todorovic, 2013). Research by Mathur and Gill (2011), Adi et al. (2013), Yulianto (2014), Yemane (2015) found that Corporate Governance has a significant and positive effect on Corporate value, meaning that improvement (improvement) in corporate governance will increase profitability. In contrast to the findings of Coleman and Biekpe (2006), Arifin (2014) found that corporate governance has a significant and negative effect on profitability, meaning that the increase (improvement) of corporate governance will reduce profitability. The difference in empirical findings is a reason to re-examine the effect of corporate governance on profitability, with the research hypothesis as follows: H1: Corporate governance has a significant effect on profitability. 2.3 The Effect of Corporate Governance on Corporate Values 2.3 The Effect of Corporate Governance on Corporate Values Agency Theory (Jensen and Meckling, 1976) explains that corporate governance has an important role to reduce agency costs and create long-term value for shareholders with a focus on board of commissioners' monitoring responsibilities and management functions that exist in senior executives (Rezaee (2007) Managerial opportunism hypothesis (Jensen, 1986) explains that managers may hold cash in the Corporate, and provide it to be used as additional income, used for personal welfare, and invest in projects that only increase personal prestige so that it is not beneficial to shareholders. Adi et al. (2013), Ghalandari (2013), Arifin et al. (2014), Yulianto (2014), Sukmono and Yadiati (2016) found that corporate governance has a significant and positive effect on Corporate value, meaning improvement (improvement) in corporate governance will increase the value of the Corporate formulated the beriktut research hypothesis: yp H3: Corporate governance has a significant effect on Corporate value. 2.2 The Effect of Corporate Governance on Capital Structure 2.2 The Effect of Corporate Governance on Capital Structure p p Agency Theory (Jensen and Meckling, 1976) explains that corporate governance has a role in preventing managerial opportunistic behavior reflected by the Corporate's capital structure. The causality relationship between these variables is generally an effect of the structure of corporate governance, as measured by indicators of board size, board composition, internal audit committee, and external audit committee on capital structure. Hasan and Butt's (2009) study found that board size and managerial share ownership had a significant and negative effect on capital structure, meaning that increased board size and managerial share ownership reduced capital structure, but the composition of the board and CEO / Chair duality had no significant and negative effect on structure capital. Based on these explanations, the research hypothesis formulated: p yp H2: Corporate governance has a significant effect on capital structure. 2.6 The Effect of Capital Structure on Corporate Values This research, aimed at explaining the effect between variables analyzed, and drawing conclusions that are useful for the development of science and used as a method of solving actual problems that occur in the field. This study focuses on empirical testing of the theory through testing the effect between variables and measurements and analysis techniques using statistical procedures testing hypotheses. The paradigm of this research is the positivistic paradigm, where conclusions from the results of research on the sample are seen to be generally applicable to the entire study population. Figure 1. Research Concept Framework g p Source: Developed for this study (Manu, 2018) g p Source: Developed for this study (Manu, 2018) 2.5 The Effect of Profitability on Corporate Values Irrelevance theory (Modigliani and Miller, 1958) explains that investment decisions and asset use activities determine performance that impacts on Corporate value, strengthens the cash flow signaling hypothesis and permanent earnings hypothesis (Lintner, 1956). High profitability has an impact on the Corporate's financial 204 Research Journal of Finance and Accounting www.iiste.org Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) DOI: 10.7176/RJFA Vol.10, No.8, 2019 flexibility, so the Corporate is able to pay dividends to shareholders. The Corporate obtained a positive rating from the capital market, and stock prices increased. The Corporate's ability to generate profits, will determine the ability of funding to finance the Corporate's growth and dividend distribution for the owner. Ghosh and Arijit (2008) Adi et al. (2013), Asiri and Hameed (2014), Ekawati and Siswoyo (2015), Data et al. (2017) found financial performance with a high measure of profitability, had a significant and positive effect on Corporate value. The theoretical implication of the empirical study is that increasing profitability will increase the value of the Corporate. Based on these explanations, the research hypothesis formulated: H5: Profitability has a significant effect on Corporate value. 3.1 Types of Research yp This type of research is explanatory research, namely research that explains the effect between variables, using secondary data. This research, aimed at explaining the effect between variables analyzed, and drawing conclusions that are useful for the development of science and used as a method of solving actual problems that occur in the field. This study focuses on empirical testing of the theory through testing the effect between variables and measurements and analysis techniques using statistical procedures testing hypotheses. The paradigm of this research is the positivistic paradigm, where conclusions from the results of research on the sample are seen to be generally applicable to the entire study population. 2.6 The Effect of Capital Structure on Corporate Values Irrelevant Theory (Modigliani and Miller, 1958) states that the capital structure does not affect the value of the Corporate. Trade off theory (Modigliani and Miller, 1963) predicts a positive correlation between capital structure and Corporate value with the assumption that tax benefits are greater than financial costs and agency costs, then the use of debt will have a positive impact on Corporate value (Barakat, 2014). However, the Pecking order theory (Myers and Majluf, 1984) explains that if companies want to use external funding sources, they must choose debt before new equity, because debt is cheaper and less sensitive to asymmetrical information, capital markets will react positively to debt issuance and prices shares rose. If the Corporate issues new shares, costs are higher and is very sensitive to asymmetrical information, the capital market will react negatively, and stock prices fall (Ogbulu and Emeni, 2012). ) Leverage signaling theory (Ross, 1977) states that debt becomes a credible signal regarding the quality and prospects of the Corporate in the future, so that the market will react positively to the Corporate's stock price. Chowdhuri and Chowdhuri (2010) research; Ogbulu and Emeni (2012); Oluwabemiga (2013); Barakat (2014); Isaac (2014) found that the capital structure had a significant and positive effect on Corporate value, supporting trade-off theory, leverage signaling theory, and pecking order theory. The difference in empirical findings is a reason to re-examine the effect of capital structure on Corporate value, with the research hypothesis as follows: H6: Capital structure has a significant effect on Corporate value. The conceptual framework of this research was developed based on Agency Theory, capital structure theory within the corporate governance framework aims to increase profitability, optimal capital structure, and increase corporate value. Good corporate governance will affect profitability, capital structure, and Corporate value. This study will examine corporate governance within the framework of theoretical agencies capable of directing management to make managerial decisions that minimize agency problems and improve the welfare of shareholders. The conceptual framework of this research is as shown in Figure 1, as follows: Figure 1. Research Concept Framework Source: Developed for this study (Manu, 2018) Figure 1. Research Concept Framework Source: Developed for this study (Manu, 2018) 3. Research Methods 3.1 Types of Research This type of research is explanatory research, namely research that explains the effect between variables, using secondary data. Independent Board of Commissioners Total Members of the Board of Commissioners 100% Members of the Audit Committee, consisting of members of the internal audit committee and members of the external audit committee. The Internal Audit Committee (IAC) is a member of the Audit Committee that comes from within the Corporate and is formed by Corporate management in order to improve the quality of financial accountability and improve Corporate performance. Referring to the research of Yulianto et al. (2014), the Internal Audit Committee (IAC), is formulated as follows: ! "#$% &'((% = ! "#$% &'((% )' ! "#$% &'((% (+, 100% ! "#$% &'((% = ! "#$% &'((% )' ! "#$% &'((% (+, 100% The External Audit Committee (EAC) is a member of the Audit Committee originating from outside the Corporate and formed by Corporate management in order to improve the quality of financial accountability and improve Corporate performance. Referring to the research of Yulianto et al. (2014), the External Audit Committee (EAC), is formulated as follows: GPM = =',, >'5% * 4% : ; !, 100% 3.2 Operational Definition of Research Variables 3.2 Operational Definition of Research Variables p This study consists of six variables, namely Corporate Governance, Profitability, Capital Structure, and Corporate This study consists of six variables, namely Corporate 205 Research Journal of Finance and Accounting Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) DOI: 10.7176/RJFA Vol.10, No.8, 2019 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) DOI: 10.7176/RJFA Vol.10, No.8, 2019 ww.iiste.org www.iiste.org Value. The research variables are grouped into two, namely exogenous variables and endogenous va Corporate governance is a mechanism to reduce agency problems and become a means of control over the implementation of Corporate decisions by management based on managerial, investor and creditor interests. Corporate governance variables are measured by indicators of board size (BS), board composition, internal audit committee, external audit committee. Referring to the research of Yulianto et al. (2014). The Size of the Board of Commissioners (SBC) is the total of all members of the Corporate's Board of Commissioners. An independent Board of Commissioners (IBC) is a board of commissioners of companies that are not members of management, majority shareholders, officials or in other ways that directly or indirectly affect the majority shareholders of companies that oversee the management of the Corporate. Referring to the research of Yulianto et al. (2014), the Independent Board of Commissioners is formulated as follows: 3.2.2 Endogenous Profitability Variables (Y2) 3 g y V ( 2) Return on assets (ROA) is a comparison of after-tax profits with the total assets used in the Corporate's operations. ROA is an indicator that measures a Corporate's ability to generate asset returns. Referring to the study of Data et al. (2017), ROA is formulated as follows: ROA = 3 %4 5 )' ! ",, 100% ROA = 3 %4 5 )' ! ",, 100% ROA = 3 %4 5 )' ! ",, 100% Return on equity (ROE) is a comparison of post-tax profit with total equity used in the Corporate's operations. ROE is an indicator that measures a Corporate's ability to produce a return on equity. Referring to the study of Data et al. (2017), ROE is formulated as follows: ROE = 3 %4 5 )' ! 36#%7 100% ROE = 3 %4 5 )' ! 36#%7 100% Net profit margin (NPM) is a comparison of post-tax profits with total sales of the Corporate's net proceeds. NPM is an indicator that measures a Corporate's ability to generate the level of net income from each sales unit of the Corporate. Referring to the Barakat study (2014), and Data et al. (2017), ROE is formulated as follows: NPM = 3 %4 5 : ; ! , 100% Gross Profit Margin (GPM) is a comparison of the total gross profit with the total net sales of the Corporate. GPM is an indicator that measures the ability of a Corporate to generate a level of gross profit from each unit sold by the Corporate. Referring to the Barakat study (2014), and Data et al. (2017), ROE is formulated as follows: GPM = =',, >'5% * 4% : ; ! , 100% 206 Research Journal of Finance and Accounting www.iiste.org DOI: 10.7176/RJFA ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) DOI: 10.7176/RJFA Vol.10, No.8, 2019 Tobin, s Q = Market Value of Equty + Total Long Term Debt Total Asset Tobin, s Q = Market Value of Equty + Total Long Term Debt Total Asset Price to Book Value = Stock Market Price Book Value of Stock The market price reflects the expected price of the investor, if the investor's expectation of one type of stock is high, then the demand for the stock is also high so the price in the market is also relatively high, the market price can also be lower than the book value, therefore try using Tobin's approach q to measure Corporate value. Tobin's q is the market value of the equity plus the total debt of the Corporate divided by the total assets. Referring to the research of Adi et al. (2013), dan Data et al. (2017), Tobin's q is calculated by the following formula: LTDTE = Long Term Debt )' ! 36#%7 100% Corporate values are book value and stock market value. Indicators of Corporate values are: Closing Price (CP), Price to Book value (PBV), and Tobin’s Q. Closing Price is the price of shares in the capital market at the close of trading activities. Referring to the research of Yulianto et al. (2014), Data et al. (2017). Price to Book value (PBV) is the ratio of the stock market to the book value of the stock, which shows the value of the Corporate according to the capital market valuation at a certain time. Referring to the research of Adi et al. (2013) and Data et al. (2017), PBV is calculated by the following formula: Price to Book Value = Stock Market Price Book Value of Stock 3.2.3 Endogenous Variables Capital Structure (Y2) Capital structure is a combination of debt and equity in financing the assets and operations of the Corporate. Capital structure indicators are the ratio of total liabilities to total assets, ratio of total liabilities to total equity and ratio of total long-term liabilities to total equity. Debt to asset ratio is the comparison of total debt with total assets in financing the assets and operations of the Corporate. DAR indicator that measures the amount of debt in a Corporate's capital structure. Referring to Cekresi (2013), and Data et al. (2017), DAR is formulated as follows: DAR = )' ! @+ )' ! ",, 100% DAR = )' ! @+ )' ! ",, 100% Debt to equity ratio is the comparison of total debt with total equity in financing the assets and operations of the Corporate. DER indicator that measures the amount of equity in the Corporate's capital structure. Referring to Cekresi (2013), and Data et al. (2017), DER is formulated as follows: DER = )' ! @+ )' ! 36#%7 100% DER = )' ! @+ )' ! 36#%7 100% Long term debt to equity (LTDTE) is a comparison of total long-term debt with total equity in financing the assets and operations of the Corporate. LTDTE indicators that measure the amount of equity in the Corporate's capital structure. Referring to Data et al. (2017), LTDTE is formulated as follows: LTDTE = Long Term Debt )' ! 36#%7 100% 3.3 Population and Sample p p Companies are listed on the Indonesia Stock Exchange. The population in this study is an all manufacturing industry companies in Indonesia Stock Exchange (IDX), with observation period from 2012 to 2017 amounted to 84 companies. Sampling using a purposive method is the researcher determines the specific criteria or goals for the sample to be studied (Indriantoro and Supomo 1999: 146). The study is limited for the period 2012 to 2017. The unit of analysis using data from 25 corporates is multiplied by 6 years is 150 financial statements. Pooling time series and cross-section data. Companies that meet the criteria for sampling are as shown in Table 1, as follows: 207 , , Table 1. List of Corporate Name Research Samples No. Corporate Code Corporate Name 1 AMFG AsahimasFiat Glass Tbk 2 ARNA Arwana Citra Mulia Tbk 3 AKPI Argha Karya Prima Industri Tbk 4 AISA Tiga Pilar Sejahtra Food Tbk 5 DLTA Delta Jakarta Tbk 6 DVLA Darya-Varia Laboratoria Tbk 7 GGRM Gudang Garam Tbk 8 HMSP HM Sampoerna Tbk 9 INDF Indofood Sukses Makmur Tbk 10 INTP Indocement Tunggal Prakarsa Tbk 11 IPOL Indopoly Swakarsa Industri Tbk 12 KAEF Kimia Farma Tbk 13 KIAS Keramika Indonesia Assosiasi Tbk 14 KLBF Kalbe Farma Tbk 15 MERK Merk Tbk 16 MLBI Multi Bintang Indonesia Tbk 17 MYOR Mayora Indah Tbk 18 ROTI Nipon IndosariCorporindo Tbk 19 SKLT Sekar Laut Tbk 20 SMCB Hotcim Indonesia Tbk 21 TCID Mandom Indonesia Tbk 22 TKIM Pabrik Kertas Ciwi Kimia Tbk 23 TOTO Surya Toto Indonesia 24 TSPC Tempo Scan Pacific Tbk 25 UNVR Unilever Indonesia Tbk Source: Indoneisa Stock Exchange on the site http://www.idx.co.id Corporate financial report (2018) 3.4 Data Collection Method The data collection method used in this study is the documentation method. The documents that will be used in this study are audited financial statements by public accountants and annual reports, Corporate financial performance profiles, and fact book for Indonesian stock exchanges in 2012-2017. The type of data used in this study is secondary data in the form of financial report documents and Corporate annual reports, the data source used is the Indonesia Stock Exchange through the site http://www.idx.co.id. 3.5 Data Analysis Methods 3.5.1 Analysis of Descriptive Statistics Data that has been collected is analyzed by the ratio and tabulated, each calculated the minimum value, the maximum value, and the average of each research indicator. The purpose of description analysis is to describe the development trends of each research indicator, without intending to make generalizations. 3 5 2 A l i f I f i l S i i 3.5.2 Analysis of Infrential Statistics y Inferential statistical analysis of this study uses structural equation models with the Generalized Structured Component Analysis (GSCA) approach. The approach (GSCA) is varince based or component based, is a predictive model (prediction analysis), and can be used to conCorporate the theory with empirical data. The bootstrapping resampling method uses the GeSCA software which can be accessed at www.sem-gesca.org. The assumption in using GSCA analysis is linearity, meaning that the correlation between latent variables must be linear. Data to be analyzed with GSCA must be tested for linearity. Linearity test using the SPSS version 22 program, the test results are as follows: 208 Research Journal of Finance and Accounting www.iiste.org ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) DOI: 10.7176/RJFA Vol.10, No.8, 2019 Table 2. Linearity Test Results Correlation F Probabilitas Information Exogenous Variable Endogenous variable Corporate Governance (X) Profitability (Y1) 5,687 0.018 Linear Corporate Governance (X) Capital Structure (Y2) 2,178 0.143 Linear Corporate Governance (X) Corporate values (Y3) 0.964 0.328 Linear Profitability (Y1) Capital Structure (Y2) 1,180 0.280 Linear Profitability (Y1) Corporate value (Y3) 340,745 0.000 Linear Capital Structure (Y2) Corporate value (Y3) 14,880 0.000 Linear Source: Secondary Data Processed in 2018 iiste.org Table 2. Linearity Test Results Table 2. Linearity Test Results Correlation F Probabilitas Information Exogenous Variable Endogenous variable Corporate Governance (X) Profitability (Y1) 5,687 0.018 Linear Corporate Governance (X) Capital Structure (Y2) 2,178 0.143 Linear Corporate Governance (X) Corporate values (Y3) 0.964 0.328 Linear Profitability (Y1) Capital Structure (Y2) 1,180 0.280 Linear Profitability (Y1) Corporate value (Y3) 340,745 0.000 Linear Capital Structure (Y2) Corporate value (Y3) 14,880 0.000 Linear Source: Secondary Data Processed in 2018 Source: Secondary Data Processed in 2018 4. Research Findings 4. Research Findings . ese c d gs 4.1 Description of Research Variables and Indicators D i ti t ti ti l l i i t d ib d t th t h b ll t d it i ith t i t di t t k l 4.1 Description of Research Variables and Indicators 4.1 Description of Research Variables and Indicators Descriptive statistical analysis aims to describe data that has been collected as it is without intending to take general conclusions or generalizations. The data described is in the form of a minimum value, the maximum value, the average of each indicator forming latent variables, as listed in table 3, below: average of each indicator forming latent variables, as listed in table 3, below: Table 3. Description of Research Variable Value Indicators No Indikator Variabel Average Per Year Average 6 Years 2012 2013 2014 2015 2016 2017 Min Max Mean 1 Corporate Governance (X) SBC 5 5 5 5 5 5 3 8 5 IBC 2 2 2 2 2 2 1 4 2 IAC 2 2 2 2 2 2 2 3 2 EAC 1 1 1 1 1 1 1 2 1 2 Profitabilitas (Y2) ROA ( persen) 14,88 16,58 13,29 11,69 10,88 9,38 -13,58 71,51 12,78 ROE ( persen) 28,57 27,38 26,91 20,39 21,48 17,23 -24,9 143,5 23,66 NPM ( persen) 12,63 12,95 12,21 10,41 9,68 8,20 -29,23 39,44 11,01 GPM ( persen) 33,35 33,52 32,83 31,52 32,37 30,17 -6,04 73,88 32,29 3 Struktur Modal (Y2) DAR ( persen) 39,16 41,36 40,16 40 39,92 40,72 8 121 40,22 DER ( persen) 83,84 78,8 81,84 83,88 113,84 84,92 9 303 87,85 LTDTE ( persen) 25,88 26,96 31 33,76 33,04 32,72 2 179 30,56 4 Nilai Perusahaan (Y4) CP (Rp) 18.498 10.463 11.790 12.175 7.708 9.796 72 152.000 11.766 PBV (x) 7,40 6,63 7,93 6,09 6,94 7,41 0,12 82,44 7,07 Q (x) 3,98 4,77 4,53 4,70 4,24 4,33 0,21 29,79 4,43 Data source: Corporate financial report, fact book Idx situs http://www.idx.co.id data processed in 2018 4.2 Statistical Analysis with GSCA 4.2 Statistical Analysis with GSCA The overall Fit Model Test shows that the FIT value of 0.549 means that the dependent latent variable can be explained by independent latent variables in the structural model of 54.9 percent or in other words the information contained in the data can be explained by 54.9 percent by the model , while the remaining 45.1 percent is explained by other variables that have not been included in the research model and errors. Considering the variables in the model more than one independent latent will affect the dependent latent variable, it is more appropriate if the interpretation of the accuracy of the model uses a corrected FIT value (AFIT). The AFIT value of 0.547 means that the dependent latent variable can be explained by the independent latent variable in the model at 54.7 percent or in other words the information contained in the data can be explained by 54.7 percent by the model, while the remaining 45.3 percent is explained by other variables that have not been included in the research model and errors. 4.2.1 Outer Model Testing Results Outer model (Measurement Model) that describes the relationship between latent variables with indicators or manifest variables (measurement model). Based on the conceptual framework and model, the outer model is then developed which is often referred to as the outer relation that defines how each indicator block is related to its latent variables, the test results obtained with GSCA Bootstrapping calculations. Detailed test results as in table 4, as follows: 209 ste.o g Table 3. Measurement Model Test Results (Outer Model) Indicator Weight Information Estimate SE CR Measurement Model of Corporate Governance Variables (X) Size of the Board of Commissioners (SBC) 0.867 0.038 22.82* Significant Independent Board of Commissioners (IBC) 0.295 0.212 1.39 Not Significant Internal Audit Committee (IAC) 0.919 0.021 43.76* Significant External Audit Committee (EAC) -0.461 0.051 9.04* Significant Measurement Model of Profitability Variable (Y1) Return on asset (ROA) 0.154 0.097 1.59 Not Significant Return on equity (ROE) 0.428 0.077 5.56* Significant Net profit margin (NPM) 0.159 0.072 2.21* Significant Gross profit margin (GPM) 0.559 0.072 7.76* Significant Measurement Model of Capital Structure Variable (Y2) Debt to assets ratio (DAR) 0.917 0.011 83.36* Significant Debt to equity ratio (DER) 0.889 0.032 27.78* Significant Long term debt to equity (LTDTE) 0.810 0.020 40.50* Significant Measurement Model of Corporate Value Variable (Y3) Closing price (CP) 0.423 0.180 2.35* Significant Price to book value (PBV) 0.491 0.053 9.26* Significant Tobin’s q (Q) 0.357 0.060 5.95* Significant CR* = Sgnificant at 0.05 level Source: GSCA analysis results are processed in 2018. Th l f h i h d i h h h i h f h l f h i di The value of the outer weight and outer weight shows the weight of the value of each indicator as a measure of each latent variable. The indicator with the largest outer weight and outer weight indicates that the indicator is the measure of the strongest or dominant variable. The results of the outer weight and outer weight indicators of the seven latent variables measured were obtained through GSCA Bootstrap calculations which also produced the value of the critical ratio (CR) which is similar to the value of statistical t, if the weight value is above 0.4 and the CR value is greater than 1 , 96 T tables, it was decided significantly. 4.2.1 Outer Model Testing Results The results of the inner model test (structural model) explain the relationship between latent variables that describe the relationship between independent latent variables and dependent latent variables based on substantive theory that defines the relationship of each independent latent variable to the dependent latent variable. The effect path coefficient values between independent latent variables on the dependent latent variables, obtained through the calculation of Generalized Structured Compoment Analysis (GSCA) and significant tests obtained through Bootstrapping which also found the value of the critical ratio (CR) equivalent to T. Testing the inner model (structural model) essentially testing the hypothesis in the study. Hypothesis testing is done by the T test on each connecting path that effects between latent variables. Based on the results of the GSCA analysis, the hypothesis model was tested, as in table 4, as follows: Table 4. Structural Model test results (Inner Model) Hypothesis Path Path Coeficients Estimate SE CR (T) Information H1 X →Y1 0.721 0.059 12.22* Significant H1 Accepted H2 X →Y2 0.496 0.090 5.51* Significant H2 Accepted H3 X →Y3 0.810 0.052 15.58* Significant H3 Accepted H4 Y1 →Y2 0.694 0.080 8.68* Significant H4 Accepted H5 Y1 →Y3 0.905 0.030 30.17* Significant H5 Accepted H6 Y2 →Y3 0.554 0.070 7.91* Significant H6 Accepted CR* = Significant at 0,05 level Source: GSCA analysis results are processed in 2018 Table 4. Structural Model test results (Inner Model) Source: GSCA analysis results are processed in 2018. 5.2 The Effects of Corporate Governance on Capital Structure p p Corporate governance (X) has a significant effect on capital structure (Y2). Path coefficient value 0.496; CR value 5.51* greater than 1.96; at the level of P = 0.05 (table 4). The path coefficient value is 0.496, meaning that the capital structure is determined by corporate governance by 49.6%, while the remaining 50.4% is determined by other variables and errors. The test results support to accept the hypothesis 2. It means that the improvement or improvement of corporate governance will improve the capital structure. The effect of corporate governance on the capital structure is that corporate governance improvements will increase the use of debt to the optimum limit in the capital structure. The results of this study support the research of Yulianto (2014), agreeing with Donaldson 1961; Myers 1984; in Myers and Majluf, 1984. Not supporting the research findings of Hasan and Butt (2009), Kargar et al. (2014). 5. Discusion 5.1 The Effects of Corporate Governance on Profitability 5.1 The Effects of Corporate Governance on Profitability Corporate governance (X) has a significant effect on profitability (Y1). Positive path coefficient value 0.721, CR value 12.22 * is greater than 1.96; significant at the level of P = 0.05. The path coefficient value is 0.721, meaning that profitability is determined by corporate governance of 72.1%, while the remaining 27.9% is determined by other variables and errors. The test results accepting hypothesis 1 means that the effect of corporate governance 210 Research Journal of Finance and Accounting www.iiste.org Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) DOI: 10.7176/RJFA Vol.10, No.8, 2019 ww.iiste.org on profitability is that corporate governance improvements can increase profitability. The findings of this study support the results of the research by Mathur and Gill (2011), Adi et al. (2013), Danoshana and Ravivathani (2013), Yulianto (2014) and Yemane (2015) who found corporate governance had a positive and significant effect on profitability. does not support the findings of Coleman's research and Biekpe (2006) and Arifin (2014) who found corporate governance had a negative and significant effect on profitability. 5.5 The Effect of Profitability on Corporate Values Profitability (Y1) has a significant positive effect on Corporate value (Y3). Path coefficient value 0.905; the value of CR 30.17* is greater than 1.96; at the level of P = 0.05 (table 4). Path coefficient value 0.905, meaning that the value of the Corporate is determined by profitability of 90.5%, while the remaining 9.5% is determined by other variables and errors. The test results support to accept the hypothesis 5. Means that the increase in profitability will increase the value of the Corporate. The findings of this study support the research findings of Ghosh and Alijit (2008), Adi et al. (2013), Yulianto (2014), Asiri and Hameed (2014), Ekawati and Siswoyo (2015), Data et al. (2017). Supporting signaling theory (Ross, 1977), Cash flow signaling hypothesis and permanent earnings hypothesis (Lintner, 1956); (Marsh and Merton, 1987), Irrelevance theory (Modigliani and Miller, 1958), reinforcing the cash flow signaling hypothesis and permanent earnings hypothesis (Lintner, 1956). 5.4 The Effect of Profitability on Capital Structure Profitability (Y1) has a significant positive effect on capital structure (Y2). The path coefficient value is 0.694, the value of CR is 8.68 * greater than 1.96; at the level of P = 0.05 (table 4). The path coefficient value is 0.694, meaning that the capital structure is determined by profitability of 69.4%, while the remaining 30.6% is determined by other variables and errors. The test results support the acceptance of hypothesis 4. Means that the increase in profitability will improve the capital structure. The findings of this study support the findings of Oolderink (2013), Oppong-Boakye (2013), Sangeetha (2013), Kisaka (2015), consistent with trade off theory (Modigliani and Miller, 1963). The findings of this study do not support Pecking Order Theory ( Myers and Majluf, 1984) and also do not support the research findings of Frank and Goyal (2009), Anake et al. (2014) who found profitability had a significant negative effect on Capital Structure. 5.3 The Effect of Corporate Governance on Corporate Values p p Corporate Governance (X) has a significant effect on Corporate value (Y3). Path coefficient value 0.810; CR value 15.58 * greater than 1.96; at level p = 0.05 (table 4). The path coefficient value is 0.810, meaning that the value of the Corporate is determined by corporate governance at 81.0%, while the remaining 19.0% is determined by other variables and errors. The test results support to accept hypothesis 3. It means that improvements in corporate governance will increase the value of the Corporate. Supporting the research of Adi et al. (2013), Ghalandari (2013), Arifin et al. (2014), Yulianto (2014), Sukmono and Yadiati (2016) who found that corporate governance had a significant and positive effect on Corporate value. 7.1 Recommendations for Advanced Research 1) Future research can add other variables that have not been included in this research model, to obtain a more comprehensive research model. 1) Future research can add other variables that have not been included in this research model, to obtain a more comprehensive research model. 2) Using financial report data and other relevant information to overcome the limitations of historical data in financial statements. 2) Using financial report data and other relevant information to overcome the limitations of historical data in financial statements. 3) Further research can be done elsewhere in a longer time, or replace other research objects. 3) Further research can be done elsewhere in a longer time, or replace other research objects. 6. Conclusion The effect of corporate governance on profitability is the improvement of corporate governance will increase profitability. Proof with empirical data statistically shows a positive and significant path coefficient. Theoretical implications of increasing or improving corporate governance will increase profitability. The effect of corporate governance on capital structure is that improvements in corporate governance will result in an increase in capital structure. Proof with empirical data statistically shows a positive and significant path coefficient. Theoretical implications of improving or improving corporate governance will improve the capital structure. The effect of corporate governance on Corporate value can be interpreted that improvements in corporate governance will result in an increase in the value of the Corporate. Proof with empirical data statistically shows a positive and significant path coefficient. The theoretical implication is that increasing or improving corporate governance will increase the value of the Corporate. The effect of profitability on capital structure is an increase in profitability will increase the capital structure. Proof with empirical data statistically shows a positive and significant path coefficient. The theoretical implication is that increasing the ability to generate profits will increase the capital structure. The effect of profitability on the value of the Corporate is an increase in profitability will increase the value of the Corporate. Proof with empirical data statistically shows a positive and significant path coefficient. The theoretical implication is that increasing the ability to generate profits will increase the value of the Corporate. The effect of the capital structure on the value of the Corporate is an increase in the capital structure until the optimal level will increase the value of the Corporate. Proof with empirical data statistically shows a positive and significant path coefficient. The theoretical implication is that increasing dividend policy will increase the value of the Corporate. 7.2 Recommendations for Companies 1) Corporate governance improvements should be directed at a balance of increasing Corporate growth and increasing value for shareholders. Improving Corporate Governance that will consistently improve profitability, capital structure, and corporate value will lead to increased prosperity of all parties related to the Corporate. 2) U i d bt t l t l l t t b d th C t ith hi h i t t t d th i k f f il 1) Corporate governance improvements should be directed at a balance of increasing Corporate growth and increasing value for shareholders. Improving Corporate Governance that will consistently improve profitability, capital structure, and corporate value will lead to increased prosperity of all parties related to the Corporate. 2) Using debt at a low cost level so as not to burden the Corporate with high interest costs and the risk of failure. 2) Using debt at a low cost level so as not to burden the Corporate with high interest costs and the risk of failure. 3) Maintaining and increasing profitability is a good decision, because profitability is a very strong variable in determining the value of the Corporate. Investors will appreciate the shares of companies that have high financial performance at the highest prices. ) g p g 3) Maintaining and increasing profitability is a good decision, because profitability is a very strong variable in determining the value of the Corporate. Investors will appreciate the shares of companies that have high financial performance at the highest prices. p g p 4) Must use financial data along with other relevant information for the purpose of analysis to produce more precise predictions. Using other economic information to overcome the limitations of financial statements, because financial report data is historical data or past data. If there are errors in disclosures in the financial statements, the prediction results will be less precise. p g p 4) Must use financial data along with other relevant information for the purpose of analysis to produce more precise predictions. Using other economic information to overcome the limitations of financial statements, because financial report data is historical data or past data. If there are errors in disclosures in the financial statements, the prediction results will be less precise. 5.7 Limitations of Research 1) The limitations of this study occur because there are other variables that have not been included in the research model, so that not all information can be disclosed by data collected and analyzed. y y 2) This study only uses financial report data which is historical data or past data (secondary data), if there is a disclosure error in the financial statements, it also effects the results of the analysis and conclusions. 2) This study only uses financial report data which is historical data or past data (secondary data), if there is a disclosure error in the financial statements, it also effects the results of the analysis and conclusions. 3) The assumption in this study is that all Basic Industry Manufacture companies are considered to have the same characteristics because they use the same accounting standards in preparing financial statements, so that ignoring differences in one type of industry with another can cause bias from the data used in this study effectd the results of the analysis. 3) The assumption in this study is that all Basic Industry Manufacture companies are considered to have the same characteristics because they use the same accounting standards in preparing financial statements, so that ignoring differences in one type of industry with another can cause bias from the data used in this study effectd the results of the analysis. 5.6 The Effect of Capital Structure on Corporate Values Capital structure (Y2) has a significant positive effect on Corporate value (Y3), path coefficient value 0.554; the value of CR 7.91* is greater than 1.96; at the level of P = 0.05 (table 4). The path coefficient value is 0.554, meaning that the value of the Corporate is determined by the capital structure of 55.4%, while the remaining 44.6% is determined by other variables and errors. The test results accept hypothesis 6. It means that the increase in debt in the capital structure to a certain extent will increase the value of the Corporate. The findings of this study support trade off theory (Modigliani and Miller, 1963), leverage signaling theory (Ross (1977), pecking order theory (Myers and Majluf, 1984), and research findings by Chowdhuri and Chowdhuri (2010), Ogbulu and Emeni (2012 ), Ogbulu and Emeni (2012); Oluwabemiga (2013); Barakat (2014); Isaac (2014); Data et al. (2017). The findings of this study do not support the research of Ghalandari (2013), Adi et al. (2013) who find the capital structure has a significant negative effect on Corporate value, which means an increase in capital structure will reduce the value 211 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) DOI: 10.7176/RJFA Vol.10, No.8, 2019 Research Journal of Finance and Accounting ISSN 2222-1697 (Paper) ISSN 2222-2847 (Online) DOI: 10.7176/RJFA Vol.10, No.8, 2019 www.iiste.org of the Corporate. 7. Recommendations 7.1 Recommendations for Advanced Research References References Adi, Tri Wahyu. Suhadak. Siti Ragil Handayani, Sri Mangesti Rahayu (2013). The Influence of corporate governance and capital structure on risk, financial performance and Corporate value: a study on the mining Adi, Tri Wahyu. Suhadak. Siti Ragil Handayani, Sri Mangesti Rahayu (2013). 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W3112985859.txt	https://dialnet.unirioja.es/descarga/articulo/7854141.pdf	pt		Leitura no cérebro: processos no nível da palavra e da sentença	Cadernos de Tradução	2,020	cc-by	11,188	https://doi.org/10.5007/2175-7968.2020v40nesp2p149 LEITURA NO CÉREBRO: PROCESSOS NO NÍVEL DA PALAVRA E DA SENTENÇA Cyntia Bailer1 1 Universidade Regional de Blumenau, Blumenau, Santa Catarina, Brasil Lêda Maria Braga Tomitch2 2 Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brasil Resumo: Este artigo apresenta uma revisão de literatura sobre leitura no cérebro com foco na compreensão de palavras e frases. Trata-se de um recorte da tese de Bailer (b), que objetivou investigar cérebros de monolíngues do português-brasileiro e bilíngues do par linguístico português-brasileiro e inglês e sua resposta neuroanatômica ao processamento de frases escritas. Neste artigo, estudos empíricos comportamentais e de neuroimagem sobre os processos de leitura no nível da palavra e no nível da sentença são apresentados, bem como os modelos de leitura de Kintsch e van Dijk, de van Dijk e Kintsch, e de Gagné, Yekovich e Yekovich que retratam o processo de leitura no todo, das palavras ao discurso. Também, discutimos o papel das diferenças individuais, especialmente da capacidade de memória de trabalho na compreensão de leitura. O objetivo é descrever o que acontece na mente do leitor à medida que ele entende palavras, sentenças, parágrafos e constrói representações significativas de textos e também localizar o leitor com relação aos componentes que o estudo de Bailer (b) lidou. Palavras-chave: Leitura; Cérebro; Modelos de Leitura; Capacidade de Memória de Trabalho READING IN THE BRAIN: PROCESSES AT THE WORD AND SENTENCE LEVELS Abstract: This article presents a literature review on reading in the brain with a focus on the comprehension of words and sentences. It is part of Esta obra utiliza uma licença Creative Commons CC BY: https://creativecommons.org/lice Cyntia Bailer & Lêda Maria Braga Tomitch Bailer’s thesis (b), which aimed at investigating the brains of Brazilian Portuguese monolinguals and Brazilian-Portuguese and English bilinguals and their neuroanatomical response to the processing of written sentences. In this article, empirical behavioral and neuroimaging studies on reading processes at the word and sentence levels are presented, as well as the reading models by Kintsch and van Dijk, van Dijk and Kintsch, and Gagné, Yekovich and Yekovich that describe the whole reading process, from words to discourse. In addition, we discuss the role of individual differences, especially working memory capacity in reading comprehension. The objective is to describe what happens in the reader’s mind as s/he understands words, sentences, paragraphs and builds significant representations of texts and also to locate the reader in relation to the components that the Bailer’s study (b) dealt with. Keywords: Reading; Brain, Reading Models; Working Memory Capacity 1. Introdução A habilidade de entender e falar uma língua tem sido considerada como uma condição sine qua non da cognição humana e tem fascinado diversas comunidades de pesquisa: Linguística, Psicologia, Ciência da Computação e subáreas como Psicolinguística e Psicologia Cognitiva. Assim, inúmeros aspectos relacionados à produção e compreensão da linguagem têm sido investigados. Nesse contexto, é senso comum que as línguas humanas são sistemas simbólicos que estão evoluindo há milhares de anos e que servem a várias funções na comunicação. Evolutivamente, falar parece ser tão antigo quanto a humanidade, enquanto a leitura e a escrita podem ser consideradas uma invenção cultural (Dehaene (a)). A compreensão da linguagem depende, em primeiro lugar, da compreensão do significado das palavras individuais, de modo que somos capazes de entender a relação entre as palavras em uma frase, em um parágrafo, no discurso como um todo. Neste exato momento, enquanto você está lendo estas páginas, seu cérebro, com suas incontáveis conexões, está realizando um feito fascinante. Seus olhos escaneiam a página em movimentos curtos de contração. Seu olhar se move constantemente pela página. Quatro ou Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 150 Leitura no cérebro: processos no nível da palavra e da sentença cinco vezes por segundo, seu olhar pousa em uma palavra ou duas (Dehaene (a)). A fóvea, o centro da retina em seus olhos, com sua excelente resolução, permite o reconhecimento de informações escritas (Just & Carpenter (a)). Seu cérebro recebe a informação capturada por seus olhos e inicia uma surpreendente orquestração paralela de processos que visa construir uma representação mental significativa do que você está lendo. Embora os leitores possam não estar conscientes de todos os processos envolvidos no reconhecimento e compreensão do input escrito, isso acontece, a todo o momento, nas diversas situações de leitura a que somos expostos na sociedade em que vivemos. O cérebro do leitor consiste em um conjunto complexo de mecanismos perfeitamente ajustados à leitura. Com o advento de novas técnicas de investigação, os pesquisadores começaram a desvendar os princípios subjacentes aos circuitos cognitivos do cérebro. Técnicas de neuroimagem são capazes de revelar, em uma velocidade nunca antes imaginada, as áreas do cérebro que se iluminam quando lemos. Tais áreas, em conjunto com modelos teóricos e evidências comportamentais, nos ajudam a entender o que é a leitura. Além da leitura, essas técnicas podem elucidar o que acontece no cérebro quando aprendemos ou quando estamos processando informações escritas em uma nova língua. Para aprender uma nova língua, o cérebro se adapta dinamicamente. Não importa em qual idade, os indivíduos têm a capacidade de aprender, decodificar input e se comunicar em um novo idioma. Tal habilidade depende da cognição, “do ato ou processo de conhecer caracteristicamente dinâmico, engenhoso e humano” (Buchweitz 1, nossa tradução). Naturalmente, avanços envolvem esforços e parcerias interdisciplinares. O presente artigo é parte integrante de uma tese de doutorado (Bailer (b), de um estudo resultante de tais esforços e parcerias. A tese foi desenvolvida no Núcleo de Estudos em Leitura na Universidade Federal de Santa Catarina em parceria com o Center for Cognitive Brain Imaging na Carnegie Mellon University (Pittsburgh, PA, EUA). O estudo, de natureza transversal, quantitativa e exploratória, objetivou investigar cérebros de monolíngues do Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 151 Cyntia Bailer & Lêda Maria Braga Tomitch português-brasileiro e bilíngues do par linguístico português-brasileiro e inglês e sua resposta neuroanatômica ao processamento de frases escritas. Para os fins deste artigo, delimitou-se apresentar a revisão de literatura sobre leitura com foco na compreensão de palavras e frases. Na próxima seção, apresentaremos, muito brevemente, estudos empíricos comportamentais e de neuroimagem sobre os processos de leitura no nível da palavra, com a visão de Dehaene (a), seguidos dos resultados acerca dos processos de leitura no nível da sentença. Na seção intitulada “Modelos de leitura: o processo todo, das palavras ao discurso”, apresentamos os modelos de leitura de Kintsch e van Dijk, de van Dijk e Kintsch, e de Gagné, Yekovich e Yekovich. O objetivo é descrever o que acontece na mente do leitor à medida que ele entende palavras, sentenças, parágrafos e constrói representações significativas de textos e também com o objetivo de localizar o leitor com relação aos componentes que o estudo de Bailer (b) lidou. Também discutimos o papel das diferenças individuais, como o papel que a capacidade de memória de trabalho desempenha na compreensão de leitura. Por fim, apresentamos as considerações finais. 2. Processos de leitura no nível da palavra De acordo com Dehaene (a), a leitura é uma habilidade complexa que deve ser aprendida para que vivamos em sociedade. Ele acredita que o cérebro do leitor exibe um conjunto de mecanismos que foram adaptados, por meio da evolução, à leitura. Por vários séculos, tal dádiva permaneceu um mistério. Com o advento da tecnologia, “a caixa preta do cérebro” estava aberta à investigação (Dehaene (a) 1, nossa tradução). Avanços em muitos campos, como Psicologia, Lingüística e Neurociência, instigaram os pesquisadores a desvendar os mistérios envolvidos em nossa capacidade de ler. Uma variedade de estudos tem fornecido insights para a área: estudos comportamentais, de rastreamento ocular, de neuroimagem Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 152 Leitura no cérebro: processos no nível da palavra e da sentença (principalmente PET, tomografia por emissão de pósitrons, e fMRI, ressonância magnética funcional) e neurofisiológicos (ERPs, potenciais evocados). Dehaene fez uso de métodos de imageamento cerebral para entender como o cérebro decifra palavras escritas. Ele explica que nossos cérebros não foram projetados para a leitura; fomos ensinados a ler. Nosso circuito cerebral foi reciclado, adaptado para leitura. Para Dehaene e sua hipótese da reciclagem, existe uma área na região occipito-temporal chamada de área da forma visual das palavras (visual word form area, VWFA1), cujos neurônios são originalmente dedicados à atividade de responder a estímulos visuais. Esses neurônios se engajam na nova tarefa de reconhecer letras e palavras. Glezer e colaboradores testaram empiricamente essa hipótese, treinando os participantes implicitamente sobre novas palavras e concluíram que os neurônios da VWFA são seletivos para palavras inteiras, permitindo, assim, o rápido reconhecimento de palavras familiares. Assim, a capacidade de ler é o resultado de um sofisticado processo evolutivo concedido pela plasticidade do cérebro humano e o instinto humano de aprender e ensinar. Just e Carpenter (a) conduziram um estudo de rastreamento ocular para investigar como a leitura ocorre desde a fixação do olho até a compreensão. Eles assumiram que é possível aprender sobre os processos envolvidos na compreensão, examinando onde os leitores pausam, fixando seus olhos. Eles explicam que um leitor fluente é capaz de ler cerca de 200 palavras por minuto em textos científicos. Além disso, os leitores fixam cada palavra e há uma grande variação na duração das fixações individuais, bem como na duração total da mirada em palavras distintas. Os pesquisadores teorizam que as De acordo com Dehaene (a), a área VWFA está localizada no giro fusiforme, entre o giro temporal inferior e o giro para-hipocampal. As coordenadas MNI (Montreal Neurological Institute) são [-44, -58, -15]. Para Bolger, Perfetti e Schneider, eles são [-42, -57,15]. Para Glezer e colaboradores, as coordenadas devem ser [-46 +/- 7, -57 +/- 7, -15 +/- 6]. Pesquisa recente conduzida por Vogel, Petersen e Schlaggar sugere a VWFA como uma região de uso geral para tarefas que exigem propriedades de processamento, “tornando-a particularmente útil para leitura” (8). 1 Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 153 Cyntia Bailer & Lêda Maria Braga Tomitch durações das miradas refletem o tempo necessário para realizar processos de compreensão. Quando os leitores estão acessando palavras não frequentes, integrando informações e fazendo inferências, pausas mais longas ocorrem devido a maiores cargas de processamento. Dehaene (a) complementa essa visão explicando que a maioria das palavras de conteúdo, como substantivos e verbos, deve ser fixada pelo menos uma vez, enquanto palavras de função e marcadores gramaticais, por exemplo, podem ser às vezes ignorados. O reconhecimento de palavras apresentadas na modalidade visual é baseado em uma correspondência entre sequências de caracteres impressos e representações lexicais. Quando nos deparamos com uma palavra, dificilmente é possível evitar a compreensão do seu significado (Pulvermüller). Duas rotas paralelas de processamento podem ser usadas para levar ao significado: (1) a rota fonológica e (2) a rota lexical. A primeira converte letras em sons de fala, enquanto a segunda, dá acesso direto a um dicionário mental de significados de palavras (Dehaene (a); Frost, Katz & Bentin). Jobard e colegas denominam as rotas como rota grafo-fonológica e rota léxico-semântica. Apesar de receber nomes diferentes, seus significados são comparáveis. A primeira via (fonológica/grafo-fonológica) implica ligar as unidades ortográficas aos seus equivalentes fonológicos; consequentemente, o acesso às palavras acontece por meio de sua pronúncia. A segunda rota (lexical/léxico-semântica) pareia formas ortográficas de palavras inteiras e suas representações semânticas. Nas línguas alfabéticas como o inglês, os iniciantes na arte da leitura usam a rota fonológica como padrão até que sua familiaridade com a ortografia lhes permita associar formas de palavras a significados. Petersen e colegas (1988), no primeiro estudo PET sobre o processamento de palavras no cérebro com leitores habilidosos, já sinalizaram que as palavras podem ser processadas diretamente a partir de sistemas visuais sem passar por uma recodificação fonológica. De acordo com Dehaene (a), “todos os sistemas de escrita oscilam entre uma representação precisa do som e a transmissão rápida do significado” (38, nossa tradução). Ambas as rotas (fonológica e lexical) são ativadas automaticamente durante o reconhecimento de Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 154 Leitura no cérebro: processos no nível da palavra e da sentença palavras e atuam em paralelo para mediar o acesso lexical. Dehaene (a) explica que quando encontram palavras muito irregulares, raras ou novas, os leitores acessam-nas preferencialmente usando a rota fonológica: decodificando as letras, convertendo-as em pronúncia e, finalmente, acessando o significado do padrão sonoro. Por outro lado, quando os leitores são confrontados com palavras frequentes ou aquelas que têm uma pronúncia excepcional, eles usam uma rota direta que acessa o significado das palavras e, em seguida, usa a informação lexical para recuperar sua pronúncia. Geralmente, os indivíduos têm uma razão para querer pronunciar a palavra depois de ter seu significado em mãos, como interesse, necessidade ou tempo. Entre os sistemas de escrita alfabética, existem diferenças fundamentais na maneira como eles refletem a estrutura fonêmica de suas línguas faladas. Frost, Katz e Bentin, e Katz e Frost propuseram a hipótese da profundidade ortográfica que coloca as linguagens em um contínuo a partir da ortografia rasa (transparente) a profunda (opaca). Em ortografias rasas, os códigos fonêmico e ortográfico combinam, fonemas são representados por grafemas de forma direta, assim, “a ortografia segue a fonologia” (Katz & Frost 149). As ortografias profundas apresentam uma relação mais opaca entre ortografia e som: a mesma letra ou agrupamento de letras pode representar diferentes fonemas em diferentes contextos, portanto, diferentes letras podem representar o mesmo fonema. Como mencionado anteriormente, as línguas variam ao longo do continuum. Por exemplo, em um extremo, o italiano tem um sistema de ortografia transparente; com 30 fonemas, “cada letra corresponde a um único fonema” (Dehaene (a) 31, nossa tradução), facilitando a pronúncia correta das palavras. No outro extremo, o inglês exibe um sistema ortográfico opaco; dependendo do falante e do método de contagem, a língua tem de 40 a 45 fonemas, dificultando o mapeamento das letras em sons. Já o português é menos transparente que o italiano e o espanhol e menos opaco que o francês e o inglês. De acordo com Scliar-Cabral, a língua portuguesa possui 33 fonemas. Palesu e colegas realizaram estudos comportamentais e com PET com estudantes universitários italianos e ingleses. Eles con- Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 155 Cyntia Bailer & Lêda Maria Braga Tomitch cluíram que “ler em uma ortografia complexa e inconsistente tem um custo considerável” (93, nossa tradução). Os italianos leem mais rápido e mais eficientemente por causa do mapeamento consistente entre sons individuais de letras e sons de tais letras no contexto de palavras. Os leitores ingleses recorreram mais a processos semânticos/ortográficos, que pareciam ser automaticamente evocados, dado o grau de complexidade ortográfica. No que diz respeito à implementação cerebral dos processos de reconhecimento de palavras, as duas vias que permitem o acesso ao significado e aos sons parecem envolver conjuntos distintos de áreas cerebrais no hemisfério esquerdo. Regiões occipito-temporais apoiam a análise visual [-44, -58, -15]; os giros temporais superior [-53, -13, 0 e -37, -35, 12] e médio [-63, -30, 4], o giro supramarginal [-60, -41, 25] e o pars opercularis do giro frontal inferior [-50, 10, 4] estão envolvidos no processamento fonológico; e o giro temporal médio [-49, -54, 13], a região temporal basal2 [-48, -41, -16] e o pars triangularis do giro frontal inferior [-44, 23, 17] subsidiam o acesso semântico (Dehaene (a)). Palesu e colegas encontraram uma ativação mais forte em leitores italianos no planum temporale esquerdo na junção temporo -parietal, uma região do cérebro tipicamente associada ao processamento fonológico. Os leitores ingleses mostraram maior ativação na região temporal posterior inferior esquerda e na parte anterior do giro frontal inferior esquerdo. Dehaene (a), em seu livro, propõe uma alternativa ao modelo sequencial clássico de leitura no cérebro (Figura 1). Em sua perspectiva, áreas occipitais esquerdas, em particular a VWFA, recebem a entrada visual, identificando a forma visual das cadeias de letras. A partir daí, essa informação visual é distribuída para numerosas regiões em todo o hemisfério esquerdo que codificam significado, padrão de som e articulação. Embora a organização detalhada das re2 A região temporal basal é tradicionalmente conhecida como Área de Linguagem Temporal Basal. Segundo Abou-Khalil e colegas, “está localizada no giro fusiforme no lobo temporal basal; entretanto, pode se estender até o giro temporal inferior e o giro para-hipocampal” (173). Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 156 Leitura no cérebro: processos no nível da palavra e da sentença des ainda não seja totalmente conhecida, parece que a leitura implica áreas visuais e de linguagem de maneira bidirecional e simultânea. Dehaene reconhece que a conectividade cortical na leitura pode ser muito mais rica do que a descrita em seu diagrama. Figura 1 - O modelo neurológico clássico da leitura e uma perspectiva moderna de como a leitura ocorre no cérebro Fonte: Dehaene ((a) 78). Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 157 Cyntia Bailer & Lêda Maria Braga Tomitch Corroborando essa visão, Price revisou estudos sobre compreensão e produção de palavras e verificou que o acesso ao sistema semântico envolve mais regiões temporais inferiores anteriores e o córtex parietal inferior posterior no hemisfério esquerdo. Além disso, o giro supramarginal esquerdo (BA 40) está envolvido em traduzir a ortografia para a fonologia. Gitelman e colegas elaboraram um estudo para explorar a resposta neuronal a três processos fundamentais para a linguagem: ortografia, fonologia e semântica. Seus resultados revelaram uma rede comum de regiões cerebrais que apoiam o processamento da palavra: áreas ventrolaterais frontais, motoras suplementares, médio-temporais posteriores, occipito-temporais e parietais inferiores. Em sua revisão, Shaywitz e Shaywitz indicam três sistemas que são importantes para a leitura em nível de palavras: (1) o giro angular3 (região temporo-parietal), que está associado a processos fonológicos e à conversão grafemafonema; (2) a área VWFA, que responde rapidamente às palavras em cerca de 150 milisegundos após a apresentação da palavra; e (3) o giro frontal inferior esquerdo, tradicionalmente implicado na articulação, que desempenha um papel na leitura silenciosa. Bolger, Perfetti e Schneider, em sua meta-análise de estudos de leitura de palavras, encontraram uma semelhança de localização em tarefas e sistemas de escrita na área VWFA. Eles analisaram estudos com sistemas de escrita alfabéticos (inglês e francês, por exemplo), silábicos (chinês e o kana japonês) e morfo-silábicos (o kanji japonês). Eles dividiram a leitura de palavras em três processos componentes: ortográficos, fonológicos e semânticos. O primeiro processo envolve regiões occipitais bilaterais, particularmente o giro fusiforme esquerdo (onde a área VWFA está) e o O giro angular (BA39) “fica na extremidade posterior do fascículo longitudinal inferior que o liga a regiões do lobo temporal implicadas na memória semântica” (Stoeckel et al. 1092, nossa tradução). Seghier em sua revisão destacou “o papel integrador do giro angular na compreensão e raciocínio” (56, nossa tradução). A ativação do giro angular foi encontrada para a manipulação do conhecimento conceitual, processamento semântico, leitura e compreensão de palavras, recuperação de memória, atenção, e cognição espacial e social. 3 Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 158 Leitura no cérebro: processos no nível da palavra e da sentença giro temporal inferior esquerdo (BAs 18, 37, 19 e 37). O segundo processo envolve regiões lateralizadas à esquerda: o sulco temporal superior, o lobo parietal inferior, as regiões frontal inferior, ínsula e pré-motoras (BAs 22, 40, 39, 45, 6 e 9). E o terceiro processo envolve áreas lateralizadas à esquerda: o fusiforme anterior, o giro temporal inferior e médio e o giro frontal inferior (BAs 37, 21 e 44). Apesar da variação nas características visuais e em como essas línguas mapeiam a ortografia para a fonologia e a semântica, os autores concluem que diferentes sistemas de escrita utilizam uma rede comum de regiões, mas a localização dentro dessas regiões indica que existem diferenças entre os sistemas de escrita. Um estudo recente realizado por Zaccarella e Friederici investigou, com fMRI, o processamento de texto no córtex insular, “uma porção inteiramente escondida do córtex cerebral, localizada na profundidade do sulco lateral, abaixo da fissura de Sylvian” (1, nossa tradução). Essa região é normalmente associada a funções autônomas como batimentos cardíacos e respiratórios, como também a funções cognitivas como a consciência. Alguns estudos relataram ativações na ínsula durante tarefas de linguagem, como no processamento auditivo e de fala no nível da palavra e da frase. Os pesquisadores apresentaram a 22 participantes alemães palavras escritas (e pseudopalavras) e expressões. Eles usaram uma ferramenta de parcelamento multimodal para conseguir localizações mais precisas da atividade cerebral. Como achados, palavras provocaram ativação insular bilateral significativa. Os agrupamentos de voxels tiveram pico em [-33, 23, -2] (55 voxels no hemisfério esquerdo) e em [33, 23, -2] (27 voxels no hemisfério direito). Alguns estudos investigaram o efeito do comprimento da palavra na leitura. Os estudiosos acreditam que a resposta cerebral é suscetível a quantas letras são apresentadas por vez, à velocidade de apresentação das palavras e por quanto tempo permanecem no campo visual. Mechelli e colegas, por exemplo, em seu estudo PET com seis participantes examinaram como o tamanho da palavra e o contraste visual afetam a leitura. Eles usaram palavras com três, seis e nove letras e variaram o contraste de como apresentavam as Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 159 Cyntia Bailer & Lêda Maria Braga Tomitch palavras de baixo a alto. Os participantes foram solicitados a articular as palavras silenciosamente enquanto estavam no PET scan. Os resultados revelaram que, em comparação com palavras curtas, as palavras longas provocaram uma maior ativação nos giros lingual medial bilateral e fusiforme bilateral, no giro lingual superior direito, no cuneus medial e na região motora esquerda. Palavras curtas, quando comparadas com as longas, não provocaram ativação significativa. Os pesquisadores consideraram o envolvimento da área motora um efeito da subarticulação silenciosa. Em relação ao contraste, o giro lingual foi mais ativado por palavras de baixo contraste e o giro fusiforme por palavras de alto contraste. Tais resultados sugerem que as demandas no processamento de características locais e no processamento de formas globais aumentam com o aumento do comprimento das palavras. Além disso, Wehbe e colaboradores, em seu estudo sobre as regiões cerebrais envolvidas na leitura de um capítulo do livro de Harry Potter, encontraram efeitos no comprimento da palavra no córtex occipital, propagando-se pelas áreas fusiformes esquerdas (VWFA). Com todas essas informações em mente, vamos revisar alguns estudos sobre como a compreensão de sentenças é implementada no cérebro. É importante ressaltar que a maioria dos estudos revisados nesta seção foram todos conduzidos na língua materna dos participantes, a L1. 3. Processos de leitura no nível da sentença A compreensão da sentença não requer apenas o processamento e a compreensão de palavras individuais, mas exige a combinação de informações de uma sequência de palavras e frases. No nível cognitivo, envolve computar relações sintáticas e temáticas, usando o conhecimento do mundo para construir uma representação do significado da sentença. Além disso, tais processos requerem recursos mentais para compreensão, bem como para manter-se ativo durante o processamento das representações de significados de palavras e Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 160 Leitura no cérebro: processos no nível da palavra e da sentença proposições. As proposições são o resultado do processamento e podem ser definidas como unidades estruturadas coerentes no nível da frase ou unidades básicas de significado. Segundo Tomitch (a), as proposições são ideias completas. Para Gagné e colaboradores, as proposições “expressam ou ‘propõem’ relações entre conceitos” (61, nossa tradução). Paradis argumenta que quando as palavras são apresentadas fora de contexto, “elas perdem a maioria de suas propriedades específicas de linguagem” (176, nossa tradução). Corroborando essa perspectiva, Perfetti e Bolger afirmam que a leitura de sentenças envolve processos de integração de significado, referência, sintaxe e de texto, assim como requer o suporte da memória de trabalho. Esses processos não estão tão envolvidos na identificação de palavras. Just e colegas foram os primeiros a estudar a compreensão de sentenças com fMRI4. No estudo, 15 estudantes destros universitários leram frases e responderam perguntas de compreensão enquanto estavam no aparelho fMRI. Os resultados revelaram que uma rede de quatro áreas foi recrutada para processar sentenças: as áreas clássicas de linguagem do hemisfério esquerdo (BA 44 e 45, área de Broca: giro frontal inferior e BA 22, área de Wernicke: giro temporal superior) e seus homólogos no hemisfério direito. Embora o hemisfério esquerdo tenha sido mais fortemente ativado, áreas do hemisfério direito foram recrutadas quando as sentenças eram estruturalmente mais complexas (orações relativas de sujeito e de objeto). Os autores inferem que o cérebro recruta mais tecido neural de uma rede quando as demandas de compreensão são maiores. Além disso, eles concluem que não apenas uma área, mas várias contribuem para a compreensão de frases. Mazoyer e colegas foram os primeiros a publicar um estudo de neuroimagem (PET) sobre as bases neurais da fala (não apenas de palavras individuais). Eles concluíram que o processamento da fala recruta uma rede de áreas, cada uma das quais pode ser especializada em um aspecto, mas requer apoio dos outros para alcançar a compreensão. Eles sugeriram a área de Broca como crucial para o processamento lexical e conceitual. Como o foco deste artigo é a leitura, optamos por não revisar este estudo. 4 Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 161 Cyntia Bailer & Lêda Maria Braga Tomitch Hashimoto e Sakai investigaram se existem sistemas neurais especializados para compreensão de sentenças. Dezesseis falantes nativos de japonês foram testados com fMRI em uma série de decisões sintáticas e tarefas de memória de curto prazo. Na literatura, o giro frontal inferior esquerdo é tradicionalmente implicado em processos de compreensão. Acredita-se que a parte anterior (BAs 45 & 47) participe do processamento semântico e a parte posterior (BA 44 & 45) contribua para o processamento fonológico e lexical, embora outros estudos tenham implicado a parte posterior no processamento sintático. Como resultado, os autores encontraram evidências de áreas pré-frontais esquerdas especializadas na compreensão de sentenças: o córtex pré-frontal dorsal esquerdo para memória de curto prazo e o giro frontal inferior esquerdo para análise de estruturas sintáticas. Gabrieli, Poldrack e Desmond concluíram, em seu artigo de revisão de literatura, que o córtex pré-frontal esquerdo desempenha um papel crucial na linguagem e na memória, como nas análises semânticas e na memória implícita. Eles explicaram que as ativações pré-frontais do hemisfério esquerdo “parecem refletir processos que são importantes para melhorar a memória de materiais encontrados em episódios específicos” (907, nossa tradução). Além disso, podem refletir processos de seleção semântica, pois “as ativações do hemisfério esquerdo e do cerebelo direito aumentaram ou diminuíram em tandem entre as condições” (908). Como as informações sintáticas e semânticas estão tão intimamente interligadas, Newman e colegas exploraram a contribuição de duas sub-regiões da área de Broca: pars opercularis (BA 44) e pars triangularis (BA 45) no processamento de sentenças seguido por uma tarefa de julgamento de gramaticalidade. Treze participantes foram examinados por fMRI enquanto liam sentenças na voz ativa e sentenças relativas de objeto com dois tipos de agramaticalidades: concordância substantivo-verbo e adição de verbo extra. Conforme esperado, as orações relativas de objeto provocaram mais ativação cerebral devido à sua complexidade. Os dados sugerem que o pars triangularis está envolvido diferencialmente em aspectos semânti- Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 162 Leitura no cérebro: processos no nível da palavra e da sentença cos e temáticos da compreensão; e que o pars opercularis está mais envolvido na construção e na manipulação da estrutura sintática de sentenças. Além disso, os pesquisadores encontraram ativação no sulco intraparietal esquerdo, geralmente implicado no processamento visuoespacial. Eles interpretaram essa ativação como a geração de imagens visuais das ações descritas nas sentenças. Um estudo mais recente conduzido por Frankland e Greene examinou com fMRI como o cérebro codifica “quem fez o quê a quem” (1, nossa tradução) em frases visualmente apresentadas em inglês, como por exemplo, O caminhão bateu na bola e A bola bateu no caminhão. Eles descobriram que os agentes (quem fez?) são processados pela margem superior do sulco temporal superior esquerdo [-46, -18, 1] e pacientes (a quem foi feito?), pela margem lateral do giro temporal superior esquerdo [-57, -10, 2]. Eles também encontraram ativação na amígdala esquerda [-28, -7, -18], que interpretaram como processamento afetivo. Os resultados reforçam a possibilidade de “que a representação explícita de variáveis semânticas abstratas em circuitos neurais distintos desempenhe um papel crítico em permitir que cérebros humanos componham ideias complexas a partir de ideias mais simples” (6, nossa tradução). Até aqui, o hemisfério esquerdo parece ser o responsável pela compreensão da linguagem no nível da palavra e da sentença, uma vez que os estudos relatados nesta revisão não sugerem papéis específicos para áreas no hemisfério direito (apenas Just e colegas reportaram ativação em áreas do hemisfério direito quando as demandas da tarefas eram altas). No entanto, Purves e colegas explicam que o verdadeiro significado da lateralização para a linguagem “reside na subdivisão eficiente de funções complexas entre os hemisférios, em vez de residir em qualquer superioridade de um hemisfério sobre o outro” (688-689, nossa tradução). Nesta linha, Jung-Beeman sugere que, quando a compreensão da linguagem natural está em jogo, o hemisfério direito desempenha um papel importante. Ele revisou estudos de lesão cerebral, de neuroimagem e neuroanatômicos e sugeriu “pelo menos três componentes distintos, mas altamente interativos, de processamento Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 163 Cyntia Bailer & Lêda Maria Braga Tomitch semântico” (513, nossa tradução). Os três componentes recrutam áreas bilaterais. O primeiro, ativação semântica, requer a participação dos giros temporais superiores médios (área de Wernicke). A integração semântica, o segundo componente, “apoia a interpretação no nível da mensagem ao computar o grau de sobreposição semântica entre múltiplos campos semânticos” (515, nossa tradução) e recruta os giros temporais superiores anteriores, bem como os polos temporais. O último componente, seleção semântica, seleciona o conceito relevante entre os anteriormente ativados e implica o giro frontal inferior. Jung-Beeman teoriza que tais processos ocorrem bilateralmente, mas com algumas diferenças. O hemisfério esquerdo processaria “o significado dominante, literal e contextualmente relevante, ao mesmo tempo em que inibe as características relacionadas aos significados subordinados ou contextualmente irrelevantes” (514, nossa tradução); apresentando, portanto, uma boa codificação semântica que seleciona rapidamente um pequeno número de significados relevantes. O hemisfério direito manteria uma ativação semântica mais difusa, com características semânticas distantes e incomuns, trazendo significados secundários. Assim, apresenta uma codificação semântica grosseira na qual um amplo espectro de significados é fracamente ativado. Para Jung-Beeman, essas diferenças permitem que os pesquisadores investiguem a linguagem levando em consideração as conexões imediatas e estreitas no hemisfério esquerdo, e a manutenção da ativação mais ampla do significado no hemisfério direito. A hipótese da codificação grosseira de Jung-Beeman foi investigada em um estudo de fMRI que Mason e Just (b) realizaram para entender como ambiguidades lexicais são resolvidas na compreensão de sentenças em inglês. Seus estímulos eram sentenças com dois tipos de ambiguidades: (1) palavra ambígua tendenciosa, quando um sentido é dominante e o outro, subordinado, por exemplo, a palavra ball em This time the ball was moved because it was always so well attended; (2) palavra ambígua equilibrada, quando uma palavra tem dois significados igualmente possíveis, como a palavra pitcher em Of course the pitcher was often forgotten because it was kept on Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 164 Leitura no cérebro: processos no nível da palavra e da sentença the back of a high shelf. Eles também incluíram frases controle correspondentes. Como resultado, encontraram ativação extra bilateral no giro frontal inferior, bem como no caudado dos gânglios da base5 para palavras ambíguas. Eles também encontraram ativação bilateral média e superior frontal para sentenças com palavras ambíguas tendenciosas. Os autores acreditam que tal ativação seja indicador de um processo de monitoramento da coerência. Eles relacionaram a ativação do caudado com o processo de seleção e a ativação dos giros frontais inferiores com o processo de reanálise semântica. Mason e Just concluíram que “a ambiguidade lexical evoca o processamento extra que poderia ser atribuído à geração, manutenção e seleção de múltiplos significados” ((b) 118, nossa tradução). Mais recentemente, Buchweitz e colaboradores compararam padrões de ativação cerebral associados à leitura e à compreensão auditiva de sentenças em português. Como resultados, eles descobriram que a compreensão de leitura recrutava mais regiões lateralizadas à esquerda, especialmente o córtex frontal inferior e o giro fusiforme, enquanto a compreensão auditiva evocava a ativação cerebral bilateral. Eles concluíram que a área VWFA provavelmente desempenha um papel maior em ortografias mais profundas (como o inglês) que em português. Prat e Just também investigaram diferenças individuais na compreensão de sentenças com 27 estudantes de graduação. Os pesquisadores apresentaram sentenças em vários graus de complexidade. Deste estudo, três conceitos são cruciais: adaptabilidade neural, sincronização neural e eficiência neural. O primeiro, adaptabiliOs gânglios da base “consistem em vários núcleos subcorticais interconectados com grandes projeções para o córtex cerebral, tálamo e certos núcleos do tronco encefálico” (Kandel et al. 854, nossa tradução). São componentes dos gânglios da base: estriado dorsal (núcleo do caudado e putâmen), estriado ventral (núcleo accumbens e tubérculo olfativo), globo pálido, pálido ventral, substância negra e núcleo subtalâmico. Relevante para o processamento de linguagem, Bohsali e colegas encontraram conectividade estrutural entre a área de Broca e o tálamo, sugerindo que eles estão envolvidos em funções linguísticas similares àquelas servidas pelas áreas de Brodmann 44 e 45. 5 Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 165 Cyntia Bailer & Lêda Maria Braga Tomitch dade neural, refere-se a uma rede cortical que está envolvida na execução de uma tarefa, sendo capaz de se adaptar às mudanças nas demandas de processamento de informações. O segundo, sincronização neural, relaciona-se com “a medida em que os níveis de ativação de duas regiões sobem e descem em tandem” (Prat & Just 1749, nossa tradução). E o terceiro, eficiência neural, refere-se à quantidade de recursos mentais necessários para executar uma tarefa. Prat, Mason e Just explicam que a eficiência neural está relacionada a “fazer mais com menos” (1, nossa tradução). Além desses três conceitos, a hipótese do transbordamento dinâmico também é essencial para entender resultados de estudos com neuroimagem. Refere-se à ideia de que “processos lateralizados” transbordam “em hemisférios contralaterais” em tarefas “com maior dificuldade” (Prat, Mason & Just 3, nossa tradução). Isso é exatamente o que Just e colegas encontraram em seu estudo quando a complexidade sintática das frases aumentou. Xu e colegas foram os primeiros a examinar, com fMRI, os sistemas neurais envolvidos no processamento da linguagem em três níveis: palavras isoladas, em uma estrutura sintática (frases individuais) e em uma narrativa (fábulas) em um único experimento. Como resultado, a leitura ativou, em todos os níveis, as áreas da linguagem central do hemisfério esquerdo. No nível das sentenças, os pesquisadores observaram uma ativação mais abrangente em tais áreas, particularmente em regiões dentro do opérculo frontal. No nível narrativo, eles observaram demandas adicionais refletidas em ativações robustas no córtex pré-frontal medial e precúneo. À medida que os níveis aumentaram, eles encontraram a contribuição crescente do hemisfério direito. Como hipotetizado, eles descobriram que ambos os hemisférios estavam ativos na compreensão da narrativa, mas a ativação do hemisfério direito aumentou no final do segmento narrativo, à medida que os detalhes foram sintetizados e entrelaçados em um todo coerente. Tais diferenças se relacionavam “às características combinatórias e semânticas distintivas das condições lexicais, sentenciais e discursivas” (1014, nossa tradução). Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 166 Leitura no cérebro: processos no nível da palavra e da sentença Mason e Just (a) propõem redes neurais especulativas para compreensão do discurso escrito com base em achados de neuroimagem. Em sua proposta, o primeiro passo no processamento do discurso é o recrutamento de uma rede básica para compreensão de frases no hemisfério esquerdo. Tais processos básicos incluem processamento visual, fonológico, léxico-semântico e sintático. Eles destacam que o processamento do discurso “ocorre em um nível palavra por palavra, momento a momento, em paralelo com os níveis mais baixos do processamento da linguagem” (788, nossa tradução). Enquanto cada palavra está sendo processada, uma interpretação está sendo construída dentro do contexto da(s) sentença(s). Essa rede prossegue com uma rede grosseira de processamento semântico no hemisfério direito; uma rede de monitoramento de coerência préfrontal dorsolateral; uma rede de integração de texto frontal-temporal esquerda; uma rede de interpretação do protagonista/agente frontal medial; e uma rede espacial no sulco intraparietal. Como esta revisão serviu para embasar um estudo sobre o processamento de frases isoladas, tais redes não são revisadas aqui. 4. Modelos de leitura: o processo todo, das palavras ao discurso Nesta seção, escolhemos revisar alguns modelos de leitura relacionados ao estudo de Bailer (2016), como o modelo textbase de Kintsch e van Dijk, o modelo situacional de van Dijk e Kintsch e o modelo componencial de Gagné e colegas. Inicialmente, é necessário estabelecer exatamente o significado do termo modelo. Davies define-o como uma teoria formal, com suposições e previsões sobre um processo oculto, que pode ser testado através de pesquisas experimentais. No caso da leitura, a maioria dos modelos representa visualmente “o que se passa nos olhos e na mente enquanto leitores estão compreendendo (ou não compreendendo) o texto” (Davies 57, nossa tradução). Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 167 Cyntia Bailer & Lêda Maria Braga Tomitch O modelo textbase de Kintsch e van Dijk está relacionado às estruturas semânticas, que são o resultado do processamento. Estas estruturas semânticas são caracterizadas em dois níveis, o nível da microestrutura e o da macroestrutura. O nível da microestrutura envolve o nível local do discurso e a estrutura das proposições individuais e suas relações, que os autores da linguística textual chamam de coesão (Koch). Nesta linha de pensamento, a textbase é entendida como o conjunto hierarquicamente organizado de proposições da superfície do texto, incluindo as conexões entre elas. Já a macroestrutura se refere ao nível global do discurso, o discurso como um todo significativo, e retrata relações que os linguistas textuais chamam de coerência (Koch & Travaglia). Ambos os níveis se referem a regras de mapeamento semântico, as macroregras, já que se espera que o discurso seja coerente e que as proposições sejam conectadas e globalmente organizadas no nível da macroestrutura para que uma representação mental significativa do texto seja construída. A primeira macroregra proposta por Kintsch e van Dijk envolve a exclusão de informações detalhadas e redundantes. A segunda requer generalização usando termos ou categorizações superordenadas, uma vez que uma sequência de proposições pode ser substituída por uma proposição mais geral. Finalmente, a terceira macroregra envolve a construção de uma sentença tópico quando não é fornecida no texto, como os autores postulam que “cada sequência de proposições pode ser substituída por uma proposição denotando um fato global” (Kintsch & Van Dijk 366, nossa tradução). Van Dijk e Kintsch posteriormente desenvolveram o modelo de representação semântica no modelo situacional. É o resultado da interação entre a construção de uma textbase e a construção de uma compreensão geral do texto baseada no conhecimento prévio. A construção de um modelo situacional coerente requer que o leitor perceba o texto como um todo coerente ao mesmo tempo em que constrói uma textbase. O modelo situacional é o resultado do processamento, da representação cognitiva de eventos, ações, pessoas e a situação que o texto trata. Van Dijk acrescenta que os leitores Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 168 Leitura no cérebro: processos no nível da palavra e da sentença constroem uma representação mental das propriedades da situação que são atualmente relevantes para eles. Para ele, essa representação mental é “a interpretação subjetiva do contexto” (124, nossa tradução) que restringe a maneira como os leitores entendem o discurso. Nesse campo, leitores bem-sucedidos são aqueles capazes de construir uma representação mental coerente do texto, construindo uma textbase e integrando-a ao seu conhecimento prévio, construindo, assim, um modelo situacional do texto. Em consonância com esses dois modelos, Gagné e colegas entendem a compreensão leitora como a construção de um modelo mental adequado do texto, contando com a interação entre conhecimento declarativo e procedimental. O conhecimento declarativo, também considerado como entendimento conceitual, envolve todo o conhecimento que os leitores possuem sobre letras, fonemas, morfemas, palavras, ideias, esquemas e tópicos; desta forma, o conhecimento semântico. Já o conhecimento procedimental, também conhecido como habilidades e estratégias, inclui os quatro componentes da leitura: decodificação, compreensão literal, compreensão inferencial e monitoramento da compreensão. Em seu modelo, esses processos acontecem em paralelo, simultaneamente, pelo menos na leitura proficiente. Os processos de nível mais baixo envolvem a decodificação de informações impressas e a compreensão literal. A decodificação refere-se ao ato de quebrar o código impresso para torná-lo significativo, e tal componente é subdividido em emparelhamento e recodificação. O primeiro se refere a acessar o significado na memória de longo prazo e o segundo, ao pronunciar a palavra silenciosamente para ter acesso ao significado armazenado. Esse componente é enfatizado na visão de leitura de Dehaene (a) apresentada anteriormente. Pode-se relacionar emparelhamento à rota lexical e recodificação à rota fonológica. O segundo componente, compreensão literal, refere-se à compreensão literal do texto e é subdividido em acesso lexical e parseamento sintático. O primeiro se refere ao acesso à melhor interpretação da palavra, no contexto da frase, dentre todas as opções ativadas em nossos léxicos Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 169 Cyntia Bailer & Lêda Maria Braga Tomitch mentais. Enquanto o segundo envolve o uso das regras sintáticas e linguísticas para juntar palavras e formar idéias ou proposições significativas, ou seja, as “unidades de conhecimento declarativo que representam o significado do texto” (Gagné et al. 273) a que Kintsch e Van Dijk se referem. Os processos de nível mais alto envolvem a compreensão inferencial e o monitoramento da compreensão. O primeiro permite ao leitor ir além da informação literalmente declarada no texto, dando ao leitor uma compreensão mais ampla das ideias do texto. A compreensão inferencial é subdividida em integração, sumarização e elaboração. O primeiro subcomponente é responsável por conectar proposições e ocorre dentro das sentenças, entre sentenças e entre parágrafos. Nesse nível, o leitor faz as inferências necessárias para entender o texto, no nível microestrutural (Tomitch, comunicação pessoal). O segundo subcomponente, sumarização, visa produzir, na mente do leitor, uma macroestrutura que expresse as principais ideias do texto. Os processos de integração e de sumarização envolvem a produção de inferências necessárias para que o leitor seja capaz de extrair a essência do texto para produzir uma representação mental coerente do conteúdo do texto (Tomitch (b)). E o terceiro subcomponente, elaboração, permite ao leitor usar seu conhecimento prévio para complementar as novas ideias do texto, referindo-se ao que Van Dijk e Kintsch chamam de modelo situacional. Finalmente, o monitoramento da compreensão é o processo de mais alto nível, embora isso não signifique que aconteça por último. Leitores proficientes monitoram suas leituras durante todo o evento de leitura. No início, estabelecem um objetivo e selecionam as estratégias apropriadas para atingir tal objetivo. Na sequência, eles verificam se o objetivo está sendo alcançado durante a leitura e remediam, em outras palavras, mudam as estratégias quando a leitura não está atingindo o objetivo previamente definido. Nesse âmbito, é possível concluir que os modelos contribuem para entender como as sentenças são processadas. No estudo de Bailer (b), frases como A família foi feliz e O casal visitou a embaixada foram apresentadas aos participantes na modalidade escrita Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 170 Leitura no cérebro: processos no nível da palavra e da sentença e eles foram convidados a pensar sobre o significado de tais sentenças. Ao ler e pensar sobre cada palavra, cada expressão, cada frase, os participantes ativaram áreas do cérebro relacionadas aos subprocessos. Assim, nos termos de Kintsch e Van Dijk, o estudo de Bailer (b) se situou no nível da microestrutura, da textbase. No modelo de Gagné e colegas, nos processos de nível mais baixo: componentes de decodificação e compreensão literal, já que os participantes acessaram significado em suas memórias de longo prazo ou pronunciando silenciosamente as palavras (emparelhamento e recodificação) tão logo eles encontraram cada palavra na tela. Além disso, eles acessaram a melhor interpretação de tais palavras no contexto de sentenças (acesso lexical) e formaram uma representação significativa vívida de tais sentenças. Bailer (b) reconhece o fato de que trabalhou com processos componentes de baixo nível, que podem ser menos relevantes para algumas linhas de pesquisa; no entanto, não diminui a importância de se investigar a representação de frases no cérebro. De acordo com Mason e Just (b), “um dos elementos constitutivos da compreensão da linguagem é a capacidade de acessar o significado das palavras à medida que elas são encontradas e desenvolver uma interpretação que seja consistente com o contexto” (115, nossa tradução). Além disso, nós acrescentaríamos que, ao compreendermos como as sentenças são processadas em monolíngues e bilíngues, nos informamos melhor sobre como a compreensão do discurso ocorre no cérebro. Além disso, devemos ter em mente que existem diferenças individuais na compreensão de leitura, o tópico da seção a seguir. 5. Diferenças individuais na compreensão leitora: foco na memória de trabalho A linguagem é um processo de criação livre; suas leis e princípios são fixos, mas a maneira pela qual os princípios de geração são usados é livre e infinitamente variada. Até mesmo a interpretação e o uso de palavras envolve um processo de criação livre. (Noam Chomsky 152, nossa tradução) Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 171 Cyntia Bailer & Lêda Maria Braga Tomitch Em nossa perspectiva, a compreensão leitora é vista como um processo cognitivo complexo e como a interação entre texto e leitor. Varia de indivíduo para indivíduo devido a uma ampla gama de fatores tais como motivação, aptidão, capacidade de memória de trabalho (CMT), conhecimento prévio, entre outros. Esta seção apresenta uma breve revisão6 sobre a literatura relevante acerca da memória de trabalho (MT). A MT é essencial para muitas tarefas cognitivas e depende da capacidade de manter representações ativas estáveis por curtos períodos de tempo (Bledowski, Rahm & Rowe). Baddeley e colegas (Baddeley & Hitch ((a); (b)); Baddeley ((a); (b); (c)) propuseram um modelo multicomponente de MT, um sistema de controle de capacidade atencional limitada, composto por um executivo central, que é auxiliado por três sistemas subsidiários: alça fonológica, o bloco de notas visuoespaciais e o buffer episódico. Resumidamente, o executivo central desempenha um papel essencial nas funções executivas tais como foco, divisão e troca de atenção, relacionando o conteúdo da MT à memória de longo prazo. A alça fonológica armazena temporariamente e repete informações de cunho verbal até que as mesmas sejam integradas ao que está sendo processado. O bloco de notas visuoespaciais é o local onde se mantêm e se manipula informações visuais e espaciais. Finalmente, assume-se que o buffer episódico representa um sistema de armazenamento usando um código multimodal. Há concordância entre os pesquisadores de que a MT desempenha um papel importante em todos os tipos de atividades cognitivas humanas (Tomitch (a)), pois é o sistema responsável pelo armazenamento, manutenção e processamento simultâneos de informações no curto prazo. É conhecida como uma arena de computação, onde o armazenamento e o processamento competem por capacidade no sistema (Daneman & Carpenter; Just & Carpenter (b); Tomitch (a)). É o lugar onde a atividade mental acontece. Sua limitação refere-se a quanto trabalho pode ser feito por vez, quanta CMT está Para uma revisão mais aprofundada sobre o construto e modelos, sugerimos ao leitor a dissertação de Bailer (a). 6 Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 172 Leitura no cérebro: processos no nível da palavra e da sentença disponível para ser compartilhada entre os processos simultâneos. Tal capacidade limitada difere entre os indivíduos e essas diferenças são bons preditores de desempenho em tarefas cognitivas: indivíduos com CMT maior apresentam um desempenho melhor em tarefas que indivíduos com menor capacidade. Autores explicam que quem tem uma CMT maior é capaz de manter e manipular na MT mais informações relevantes para concluir tarefas complexas e, como resultado, apresentar melhor desempenho. A pesquisa sobre diferenças individuais na CMT tem sido, mais extensivamente, realizada na L1 e, menos extensivamente, na L2. Correlações positivas têm sido encontradas em uma ampla variedade de tarefas cognitivas de ordem superior, como leitura e compreensão auditiva em geral, mas também em subprocessos como construção de ideia principal, resolução de ambiguidades, compreensão inferencial, implementação de estratégias e estrutura de texto, para mencionar alguns (Bailer (a)). Em comparação aos monolíngues, os bilíngues são mais capazes de direcionar sua atenção para informações relevantes para a tarefa e manter sua atenção, apesar da interferência adversa (Yang et al.). Para medir a CMT, Daneman e Carpenter criaram o Teste de Capacidade em Leitura (Reading Span Test). Esse teste envolve a compreensão de frases além da rememoração das últimas palavras de um grupo de frases apresentadas. A capacidade em leitura de um indivíduo é o número máximo de palavras finais lembradas na ordem em que foram apresentadas. Como o teste apresenta requisitos laboriosos de processamento, a suposição subjacente (Daneman & Carpenter) é que esses requisitos podem diminuir a quantidade de informações adicionais que podem ser mantidas. Os resultados são “usados para prever o desempenho em outras habilidades cognitivas, como leitura, compreensão e raciocínio” (Tomitch (a) 33, nossa tradução). Daneman e Carpenter enfatizam que a capacidade de um indivíduo varia de acordo com a eficiência em relação aos processos correlacionados com uma tarefa específica. Seguindo esta linha, o teste é considerado um bom preditor de compreensão, pois captura muitos dos requisitos de processamento Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 173 Cyntia Bailer & Lêda Maria Braga Tomitch da compreensão de sentenças (Daneman & Merikle). Como Cantor e Engle (1102, nossa tradução) destacam, “ao ler, bons leitores têm processos de leitura rápidos e eficientes que requerem menos CMT que os processos de leitores pobres. Assim, bons leitores têm funcionalmente mais capacidade em tarefas relacionadas à leitura”. Para explicar como a CMT restringe a compreensão, Just e Carpenter (b) propuseram um modelo computacional denominado Modelo de Compreensão com Restrição de Capacidade. Os autores afirmam que “tanto o processamento quanto o armazenamento são mediados pela ativação e que a quantidade total de ativação disponível na memória de trabalho varia entre os indivíduos” (122, nossa tradução). Quando as demandas de recursos da tarefa excedem o suprimento disponível, o processamento diminui, produtos parciais são gerados e o desempenho é afetado. Os leitores com capacidade maior exibem mais capacidade residual para armazenar as palavras a serem lembradas na tarefa, porque são mais eficientes na recuperação de informações da memória de longo prazo e na alocação de recursos para atender às demandas da tarefa. Neste contexto, os estudos de neuroimagem têm fornecido informações sobre como a MT é implementada no cérebro humano. Uma grande variedade de técnicas e tarefas tem revelado o córtex pré-frontal, a área frontal do cérebro, como subjacente à MT (D’Esposito et al.; Alloway & Alloway). Essa área tem sido repetidamente relatada em estudos de MT. Perani explica que o córtex pré-frontal dorsolateral esquerdo está envolvido em muitas tarefas de linguagem, como geração de palavras, monitoramento semântico e sintático, bem como na geração e monitoramento de sequências, associações de aprendizagem entre estímulos e na MT. Nas palavras de Perani, o córtex pré-frontal “não é homogêneo, abrange muitas áreas citoarquitetônicas diferentes, cada uma exibindo um padrão único de conexões com outras áreas corticais e subcorticais” (211, nossa tradução), como com a área de Broca, o sulco intraparietal, o hipocampo e a amígdala. Cabeza e Nyberg revisaram 275 estudos com PET e fMRI e revelaram que as regiões pré-frontal, frontal e parietal do cérebro estão Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 174 Leitura no cérebro: processos no nível da palavra e da sentença associadas à MT. Eles explicam que as áreas são recrutadas de acordo com a natureza da tarefa e a dificuldade. Investigações que aplicam tarefas verbais geralmente relatam ativações na BA 44 (área de Broca) no hemisfério esquerdo. As áreas frontais normalmente implicadas na MT geral são BAs 6 (área motora suplementar, SMA), 9 e 46 (córtex pré-frontal dorsolateral). Interpreta-se a ativação de tais áreas como uma reflexão de um processo de ensaio que “atualiza o conteúdo” da MT (Cabeza & Nyberg 19). As regiões parietais, particularmente as BAs 7 e 40, estão tipicamente relacionadas a operações linguísticas, como recuperação de palavras da memória de longo prazo e o acesso ao armazenamento fonológico. Em suma, acredita-se que a MT envolve uma rede de áreas dedicadas à realização de tarefas diárias cognitivas de alta ordem, como leitura e fala. A MT medida pelo Teste de Capacidade em Leitura (Daneman & Carpenter) também é considerada uma estimativa da capacidade de leitura. Segundo Daneman e Merikle, correlaciona-se bem com testes verbais globais, como o SAT norteamericano (Teste de Aptidão Escolar Verbal), e com testes específicos que avaliam a compreensão de frases escritas e parágrafos. Para ilustrar, o estudo de Jobard e colegas considera o resultado do teste como um índice de capacidade em leitura. Eles investigaram a leitura de palavras e a habilidade leitora com 33 leitores de francês com fMRI. Eles exigiam que os participantes executassem o Teste de Capacidade em Leitura e, dentro do scanner, lessem as palavras em suas mentes (leitura oculta), uma vez que o movimento da boca na leitura em voz alta produziria ruído nos dados e ativaria áreas da linguagem relacionadas à fala. Eles usaram palavras muito frequentes como estímulos, uma vez que “se pensa que eles se beneficiam de uma ligação direta entre as representações ortográficas armazenadas e a semântica” (126, nossa tradução). Os resultados de Jobard e colegas estão de acordo com a literatura sobre as duas rotas para acessar palavras. Em termos de áreas cerebrais, eles encontraram ativação inesperada no giro pré-central esquerdo, o que pode estar refletindo o acesso aos procedimentos motores necessários para proferir palavras no contexto da leitura Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 175 Cyntia Bailer & Lêda Maria Braga Tomitch silenciosa. Análises revelaram que leitores com baixa capacidade em leitura ativam mais áreas envolvidas no processamento visual, fonológico e semântico: a VWFA, o giro pré-central, a parte média do sulco temporal, o planum temporale próximo ao giro supramarginal, a parte posterior do giro temporal médio e o pars orbitalis do giro frontal inferior. Leitores com baixa capacidade em leitura dependem mais de regiões visuais dorsais, o que pode indicar uma mudança do processamento paralelo de dados visuais - os participantes podem estar decompondo as palavras para acessá-las através de uma reconstrução grafo-fonológica e não através da rota ortográfico-semântica. Leitores com maior capacidade em leitura dependem menos das regiões acima mencionadas; eles geralmente ativam apenas as regiões implicadas no acesso direto ao significado a partir da análise visual das palavras escritas. Nas próprias palavras dos autores, nossos resultados indicam que a reconstrução grafofonológica das palavras pode ser alcançada através do recrutamento de regiões cerebrais adicionais àquelas que permitem a rota lexical quando não há ligação direta entre ortografia e semântica (Jobard et al. 127, nossa tradução). Por fim, suas descobertas mostram que a proficiência em leitura do participante (CMT, medida pelo Teste de Capacidade em Leitura) é um fator que influencia a rota que os participantes utilizam durante a leitura. Prat e Just investigaram a conectividade funcional associada às demandas de processamento em uma tarefa de leitura. Os participantes foram divididos em dois grupos: indivíduos com maior e menor CMT. Os resultados mostraram que os indivíduos com maior CMT exibiram maior eficiência, maior adaptabilidade e melhor sincronização das redes neurais de linguagem que os participantes com menor CMT na compreensão de sentenças. Cad. Trad., Florianópolis, v. 40, nº esp. 2, p. 149-184, set-dez, 2020. 176 Leitura no cérebro: processos no nível da palavra e da sentença 6. Considerações finais Conforme já mencionado, a compreensão da linguagem depende, em primeiro lugar, da compreensão do significado das palavras individuais, de modo que somos capazes de entender a relação entre as palavras em uma frase, em um parágrafo, bem como no discurso como um todo. Ao ler estas páginas, seu cérebro tem realizado um feito fascinante, com muitas conexões, áreas e processos envolvidos, os quais muitas vezes, nos passam despercebidos. O cérebro do leitor, evolutivamente, se ajustou à leitura, uma invenção cultural. Os modelos comportamentais têm tentado explicar o que acontece enquanto lemos, mas graças às técnicas de neuroimagem funcional, os pesquisadores puderam começar a desvendar como a leitura ocorre no cérebro em tempo real. Este artigo teve por objetivo apresentar a revisão de literatura sobre leitura com foco na compreensão de palavras e frases, da tese de doutorado de Bailer (b), originalmente escrita em língua inglesa e para os fins deste artigo, traduzida e adaptada para o português. É importante deixar claro que tal revisão não é exaustiva, já que existem muitos modelos para explicar a leitura e novos estudos são publicados continuamente. Agradecimento As autoras aproveitam a oportunidade para agradecer à CAPES pela bolsa de pesquisa concedida à Cyntia Bailer durante seu doutorado na UFSC (2012-2016) e pelo processo CAPES BEX 14636-13-1 da bolsa de doutorado sanduíche no exterior para que a pesquisadora pudesse conduzir seu estudo na Carnegie Mellon University (CMU) no período 2014-2015, com Dr. Marcel Just, no Center for Cognitive Brain Imaging. O artigo aqui apresentado é fruto das leituras e discussões na UFSC e na CMU. Cad. 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https://openalex.org/W2887590359	https://birmingham.elsevierpure.com/files/57408587/ATLAS_Search_for_long_lived_particles_in_final_states_Physical_Review_D_2019.pdf	English	null	Search for long-lived particles in final states with displaced dimuon vertices in <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:mi>p</mml:mi><mml:mi>p</mml:mi></mml:math> collisions at <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:msqrt><mml:mi>s</mml:mi></mml:msqrt><mml:mo>=</mml:mo><mml:mn>13</mml:mn><mml:mtext> </mml:mtext><mml:mtext> </mml:mtext><mml:mi>TeV</mml:mi></mml:math> with the ATLAS detector	Physical review. D/Physical review. 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Link to publication on Research at Birmingham portal Link to publication on Research at Birmingham portal Publisher Rights Statement: Checked for eligibility 31/01/2019 Search for long-lived particles in final states with displaced dimuon vertices in pp collisions at √s=13 TeV with the ATLAS detector ATLAS Collaboration; Newman, Paul DOI: 10.1103/PhysRevD.99.012001 License: Creative Commons: Attribution (CC BY) Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): ATLAS Collaboration & Newman, P 2019, 'Search for long-lived particles in final states with displaced dimuon vertices in pp collisions at √s=13 TeV with the ATLAS detector', Physical Review D, vol. 99, no. 1, 012001. https://doi.org/10.1103/PhysRevD.99.012001 Citation for published version (Harvard): ATLAS Collaboration & Newman, P 2019, 'Search for long-lived particles in final states with displaced dimuon vertices in pp collisions at √s=13 TeV with the ATLAS detector', Physical Review D, vol. 99, no. 1, 012001. https://doi.org/10.1103/PhysRevD.99.012001 Citation for published version (Harvard): ATLAS Collaboration & Newman, P 2019, 'Search for long-lived particles in final states with dis vertices in pp collisions at √s=13 TeV with the ATLAS detector', Physical Review D, vol. 99, no https://doi.org/10.1103/PhysRevD.99.012001 Citation for published version (Harvard): ATLAS Collaboration & Newman, P 2019, 'Search for long-lived particles in final states with displaced dimuon vertices in pp collisions at √s=13 TeV with the ATLAS detector', Physical Review D, vol. 99, no. 1, 012001. https://doi.org/10.1103/PhysRevD.99.012001 I. INTRODUCTION decay to the scale of the neutron lifetime. There are a number of BSM models where long-lived particles (LLPs) arise naturally [2,3]. Supersymmetry (SUSY) [4–9] with R-parity violation [10,11], general gauge-mediated (GGM) supersymmetry breaking [12–14], and split supersymmetry [15,16] are examples where small couplings, mass scales associated with the BSM physics, or heavy mediator particles, respectively, lead to high-mass (greater than a few hundred GeV) LLPs. Scenarios with low-mass LLPs include hidden-valley models [17], stealth supersymmetry [18], and dark-sector gauge bosons [3,19]. The ATLAS and CMS experiments at the Large Hadron Collider (LHC) were conceived to address a variety of questions not fully explained within the Standard Model (SM) of particle physics. The data collected by the LHC experiments have not yet revealed evidence of physics beyond the Standard Model (BSM). As a result, there is an increased emphasis on the exploration of unusual final-state signatures that would elude the searches based on exper- imental methods aimed at prompt signatures. In many models of BSM physics there are free parameters that influence the lifetimes of the new particle states, with no strong motivation for assuming that all the particles decay promptly1 and thus give final states investigated with standard analysis techniques. Nor are there any strong demands that these are stable on the detector scale and only weakly interacting, leading to missing transverse momen- tum signatures. Particle lifetimes in the SM, for instance, span roughly 28 orders of magnitude [1], from the strong g g Events with long-lived particles may feature vertices that are significantly displaced from the proton-proton (pp) interaction point (IP). This article presents the results of a search for displaced vertices (DVs) formed by a pair of muons of opposite-sign electric charge, denoted “OS” muons. The search is designed to be sensitive to decays of LLPs with masses between 20 and 1100 GeVand DVs at distances ranging from a few centimeters to a few meters from the IP. The data sample consists of pp collisions at ﬃﬃﬃs p ¼ 13 TeV and an integrated luminosity of 32.9 fb−1 collected with the ATLAS detector at the LHC. Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Funded by SCOAP3. Take down policy 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. 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. Download date: 24. Oct. 2024 PHYSICAL REVIEW D 99, 012001 (2019) (Received 10 August 2018; published 3 January 2019) (Received 10 August 2018; published 3 January 2019) A search is performed for a long-lived particle decaying into a final state that includes a pair of muons of opposite-sign electric charge, using proton-proton collision data collected at ﬃﬃﬃs p ¼ 13 TeV by the ATLAS detector at the Large Hadron Collider corresponding to an integrated luminosity of 32.9 fb−1. No significant excess over the Standard Model expectation is observed. Limits at 95% confidence level on the lifetime of the long-lived particle are presented in models of new phenomena including gauge-mediated supersymmetry or decay of the Higgs boson, H, to a pair of dark photons, ZD. Lifetimes in the range cτ ¼ 1–2400 cm are excluded, depending on the parameters of the model. In the supersymmetric model, the lightest neutralino is the next-to-lightest supersymmetric particle, with a relatively long lifetime due to its weak coupling to the gravitino, the lightest supersymmetric particle. The lifetime limits are determined for very light gravitino mass and various assumptions for the neutralino mass in the range 300–1000 GeV. In the dark photon model, the lifetime limits are interpreted as exclusion contours in the plane of the coupling between the ZD and the Standard Model Z boson versus the ZD mass (in the range 20–60 GeV), for various assumptions for the H →ZDZD branching fraction. DOI: 10.1103/PhysRevD.99.012001 DOI: 10.1103/PhysRevD.99.012001 Full author list given at the end of the article. 1For the purposes of this analysis, a promptly decaying particle is one with a lifetime no larger than a few tens of picoseconds. Search for long-lived particles in final states with displaced dimuon vertices in pp collisions at ﬃﬃs p = 13 TeV with the ATLAS detector M. Aaboud et al. (ATLAS Collaboration) *Full author list given at the end of the article. 1 © 2019 CERN, for the ATLAS Collaboration 1For the purposes of this analysis, a promptly decaying particle is one with a lifetime no larger than a few tens of picoseconds. g Published by the American Physical Society under the terms of 1For the purposes of this analysis, a promptly decaying particle is one with a lifetime no larger than a few tens of picoseconds. 2ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point in the center of the detector and the z axis along the beam pipe. The x axis points from the IP to the center of the LHC ring, and the y axis points upwards. Cylindrical coordinates ðr; ϕÞ are used in the transverse plane, ϕ being the azimuthal angle around the z axis. The pseudorapidity is defined in terms of the polar angle θ as η ¼ −ln tan ðθ=2Þ. Angular distances are measured in units of ΔR ≡ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ðΔηÞ2 þ ðΔϕÞ2 p . II. ATLAS DETECTOR j j Resistive-plate chambers in the barrel and thin-gap chambers in the end cap regions provide triggering capability to the detector as well as ðη; ϕÞ position measurements with a typical spatial resolution of 5– 10 mm. A precise momentum measurement is provided by three layers of monitored drift-tube chambers (MDT), with each chamber providing six to eight η measurements along the muon trajectory. For jηj > 2, the inner layer is instrumented with a quadruplet of cathode-strip chambers (CSC) instead of MDTs. The single-hit resolution in the bending plane for the MDT and the CSC is about 80 and 60 μm, respectively. The muon chambers are aligned with a precision between 30 and 60 μm. The material between the IP and the MS ranges from approximately 100 to 190 radi- ation lengths, depending on η, and consists mostly of the calorimeters. The ATLAS detector [51,52] at the LHC covers nearly the entire solid angle around the collision point.2 It consists of an inner tracking detector surrounded by a thin superconducting solenoid, electromagnetic and hadronic calorimeters, and a muon spectrometer incorporating super- conducting toroidal magnets. The inner detector (ID) is immersed in a 2 T axial magnetic field and provides charged-particle tracking in the range jηj < 2.5. A high-granularity silicon pixel detector covers the vertex region and typically provides four measurements per track, the first hit being normally in the innermost layer. It is followed by a silicon microstrip tracker, which usually provides four two-dimensional measurement points per track. These silicon detectors are complemented by a transition radiation tracker, which enables radially extended track reconstruction up to jηj ¼ 2.0. The transition radiation tracker also provides electron identification information based on the fraction of hits (typically 30 in total) above a higher energy-deposit threshold corresponding to transition radiation. Online event selection is performed with a two-level trigger system [53]. A hardware-based level-1 trigger which uses information from the MS trigger chambers and the calorimeters is followed by a software-based trigger. The calorimeter system covers the pseudorapidity range jηj < 4.9. Within the region jηj < 3.2, electromagnetic calorimetry is provided by barrel and end cap high- granularity lead/liquid-argon (LAr) sampling calorimeters, with an additional thin LAr presampler covering jηj < 1.8 to correct for energy loss in material upstream of the calorimeters. I. INTRODUCTION Although SM decay products typically consist primarily of hadrons, due to the relatively large number of color degrees of freedom for quarks, there are notable advantages to searching for DVs using only tracks of identified muons: the design of the ATLAS muon spectrometer allows detection of dimuon DVs within an unusually large decay volume, free from backgrounds associated with 012001-1 © 2019 CERN, for the ATLAS Collaboration 2470-0010=2019=99(1)=012001(32) PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. calorimeter modules optimized for electromagnetic and hadronic measurements respectively. vertices produced in interactions of hadrons with detector material [20,21]. The muon spectrometer (MS) comprises separate trigger and high-precision tracking chambers measuring the deflection of muons in a magnetic field generated by three superconducting air-core toroidal magnets, each with eight coils. The field integral of the toroids ranges between 2.0 and 6.0 T m across most of the detector. The MS is designed to detect muons in the region jηj < 2.7 and to provide momentum measurements with a relative resolu- tion better than 3% over a wide transverse momentum (pT) range and up to 10% at pT ∼1 TeV. It consists of a barrel (jηj < 1.05), with an inner radius of about 500 cm, and two end cap sections (1.05 < jηj < 2.7). Previous searches by the ATLAS Collaboration for high- mass LLPs that decay within the inner detector to give displaced dilepton vertices excluded LLP lifetimes of cτ ¼ 0.1–100 cm [22]. ATLAS has also searched for very low mass LLPs (< 10 GeV) by considering pairs of highly collimated leptons [23], with sensitivity to LLP lifetimes of cτ ¼ 0.1–20 cm. Several other LLP searches targeting a wide range of lifetimes and signatures have been conducted by the ATLAS [24–33], CMS [34–40], LHCb [41–44], CDF [45], D0 [46,47], BABAR [48], Belle [49], and ALEPH [50] collaborations. II. ATLAS DETECTOR Hadronic calorimetry is provided by a steel/ scintillator-tile calorimeter, segmented into three barrel structures within jηj < 1.7, and two copper/LAr hadronic end cap calorimeters. The solid-angle coverage is com- pleted with forward copper/LAr and tungsten/LAr A. BSM signal samples fraction for the decay ˜χ0 1 →Z ˜G can be Oð1Þ. The lifetime of the ˜χ0 1 is determined by F0 [or, alternatively, by m ˜G, according to Eq. (1)] and its mass m˜χ0 1, Monte Carlo simulated samples from two different BSM physics models are used to tune selection criteria and to evaluate signal efficiencies for use in converting signal yields into cross sections. The chosen models, a general gauge-mediated supersymmetry and dark-sector gauge boson model, represent a variety of BSM physics possibil- ities, as well as final-state topologies and kinematics, to which the analysis may be sensitive. The two processes are illustrated in Fig. 1. Samples for both models were generated with MADGRAPH5_AMC@NLO [59] using the NNPDF23LO PDF set [60] and PYTHIA8 for parton showering and hadronization. The matrix elements were calculated to next-to-leading order in the strong coupling constant. The EVTGEN generator [61] was used for weak decays of heavy-flavor hadrons. The hadronization and underlying-event parameters were set according to the A14 tune [57]. cτ˜χ0 1 ∝16πF2 0 m5 ˜χ0 1 ≈ 100 GeV m˜χ0 1 5 ﬃﬃﬃﬃﬃﬃ F0 p 300 TeV 4 × 1 cm; and hence ˜χ0 1 is long-lived (i.e., nonprompt) for ﬃﬃﬃﬃﬃﬃ F0 p ¼ 103 TeV to 104 TeV. In the GGM model, a pp interaction creates a pair of gluinos, followed by a cascade of decays leading to ˜χ0 1 →Z ˜G. A simplified model is used whereby the cascade of decays of SUSY particles is reduced to a single vertex: ˜g →qq˜χ0 1, where q represents any of the quarks lighter than the top quark, with equal probability for each. Six signal samples were generated with m˜g ¼ 1.1 TeV and ˜χ0 1 masses and lifetimes given in Table I. The value of 1.1 TeV for the gluino mass was chosen to be consistent with the value used in Ref. [22], the previous search for DVs with a GGM interpretation. The signal cross sections are calculated to next-to-leading order in the strong coupling constant, adding the resummation of soft gluon emission at next- to-leading-logarithm accuracy (NLO þ NLL) [63–67]. The nominal cross sections and their uncertainties are taken In R-parity-conserving (RPC) SUSY models where gauge interactions mediate the breaking of the supersym- metry, the gravitino ˜G acquires its mass from a “super- Higgs” mechanism and may be very light: m ˜G ¼ OðkeVÞ. III. DATA AND SIMULATED SAMPLES Proton-proton collision data, collected at the LHC during 2016, with a center-of-mass energy ﬃﬃﬃs p ¼ 13 TeV, are analyzed. After application of detector and data-quality requirements, the integrated luminosity of the data sample is 32.9 fb−1. Samples of Monte Carlo (MC) simulated events are used for studies of both the LLP signal and background processes. The detector response was simulated with GEANT4 [54,55], and the events were processed with the same reconstruction software as used for the data. The distribution of the number of additional pp collisions in the same or neighboring bunch crossings (“pileup”) is accounted for by overlaying minimum-bias events simu- lated with PYTHIA8 [56] using the A2 set of tuned parameters (tune) [57] and MSTW2008LO parton distri- bution function (PDF) set [58]. The pileup profile in the MC samples is reweighted to match the distribution observed in the data. 012001-2 PHYS. REV. D 99, 012001 (2019) SEARCH FOR LONG-LIVED PARTICLES IN FINAL … (a) (b) FIG. 1. Diagrams representing BSM processes considered signals in this article: (a) long-lived neutralino ˜χ0 1 decay in a GGM scenario, and (b) long-lived dark photons ZD produced from Higgs boson decay. The quarks, q, may have different flavors (excluding the top quark). The symbol f represents fermions lighter than half the mass of the Z boson. (a) (b) (b) (a) FIG. 1. Diagrams representing BSM processes considered signals in this article: (a) long-lived neutralino ˜χ0 1 decay in a GGM scenario, and (b) long-lived dark photons ZD produced from Higgs boson decay. The quarks, q, may have different flavors (excluding the top quark). The symbol f represents fermions lighter than half the mass of the Z boson. A. BSM signal samples mZD [GeV] cτZD [cm] BðZD →μþμ−Þ 20 50 0.1475 40 50 0.1370 40 500 0.1370 60 50 0.1066 60 500 0.1066 number of DVs across the full fiducial decay volume of the search: approximately 0 < rvtx < 400 cm. To obtain dis- tributions corresponding to a different lifetime, cτnew, each event is given a weight. The weight wi assigned to each LLP i is computed as The BSM terms in the Lagrangian density include both a hypercharge portal and a Higgs portal, providing kinetic Z-ZD mixing [i.e., mixing between Uð1ÞY and Uð1ÞD] and H-HD mixing, regulated by the small coupling parame- ters ϵ and ζ, respectively. There are two vector-boson mass eigenstates, one that is mostly ZD and another that is mostly SM Z, as well as two scalar mass eigenstates, one that is mostly HD and another that is mostly H. For simplicity, the physical (mass) states are denoted by H, HD, Z, and ZD. wiðtiÞ ¼ τgen e−ti=τgen · e−ti=τnew τnew ; where the first factor reweights the exponential decay to a constant distribution and the second factor reweights to the desired lifetime. The quantity ti is the proper decay time of the LLP and cτgen is the lifetime assumed in generating the sample. The event-level weight is the product of the weights for the two LLPs in each event. The event-level signal efficiency is then the sum of weights for all events for which at least one reconstructed dimuon vertex satisfies the selection criteria, divided by the total number of events generated. This scheme ensures that any dependence of the efficiency on the decay time of both LLPs in the event, and not just the one decaying to a dimuon final state, is properly taken into account for each choice of cτnew. In the scenario where the singlet scalar HD is heavier than the SM H boson, which means that the process H → HDHD is kinematically forbidden, and ZD is lighter than half the H mass, events with a displaced dimuon vertex signature would be observable in experiments at the LHC. The ZD bosons are produced on-shell in Higgs boson decays and decay to SM fermions due to their induced couplings to the electroweak current. A small value of ϵ (≲10−5) results in a long-lived ZD state: cτZD ∝1=ϵ2. A. BSM signal samples The mass is given by m ˜G ¼ F0 ﬃﬃﬃ 3 p MPl ¼ ﬃﬃﬃﬃﬃﬃ F0 p 100 TeV 2 × 2.4 eV; ð1Þ ð1Þ TABLE I. MC signal samples for the GGM SUSYinterpretation. For a given m˜χ0 1, the gravitino mass is chosen to give the desired lifetime. For all samples, m˜g ¼ 1100 GeV, σðpp →˜g ˜gÞ ¼ 163.5 fb, Bð˜χ0 1 →Z ˜GÞ ¼ 1.0, and BðZ →μþμ−Þ ¼ 0.03366. where ﬃﬃﬃﬃﬃﬃ F0 p is the fundamental scale of supersymmetry breaking, typically ≳100 TeV, and MPl is the Planck scale. Hence, the gravitino is the lightest supersymmetric particle (LSP). All heavier supersymmetric particles decay promptly through cascades leading to the next-to-lightest supersymmetric particle (NLSP), which then decays into the LSP gravitino via an interaction with a 1=F0 suppres- sion. The NLSP, depending on model choices, is either the lightest slepton or lightest neutralino, ˜χ0 1. For the latter case, chosen for this search and described in Ref. [62], if ˜χ0 1 has a significant wino or higgsino component the branching m˜χ0 1 [GeV] cτ˜χ0 1 [cm] 300 100 300 500 700 100 700 500 1000 100 1000 500 012001-3 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. from an envelope of cross-section predictions using differ- ent PDF sets and factorization and renormalization scales, as described in Ref. [68]. TABLE II. MC signal samples for the dark-sector interpreta- tion. For all samples, mH ¼ 125 GeV, mHD ¼ 300 GeV, σðpp →HÞ ¼ 44.1 pb (via the gluon-gluon fusion production process) and BðH →ZDZDÞ ¼ 0.10. A number of BSM theories feature a “hidden” or “dark” sector of matter that does not interact directly with SM particles but may nevertheless interact weakly with SM matter via coupling to the Higgs field. These are “Higgs portal” models that address the dark-matter problem and electroweak baryogenesis. The model considered for this search is one in which there exists a Uð1ÞD symmetry in the dark sector, and the dark vector gauge boson ZD, often called a “dark photon,” is given mass via a singlet scalar field HD that breaks the symmetry and is analogous to the Higgs field H in the visible SM sector [3,69]. A. BSM signal samples The branching fraction for H →ZDZD is determined by the value of ζ and the masses of the scalar singlets: The lifetime reweighting technique is validated by using a signal sample of a given cτgen to predict the efficiency for a different lifetime and comparing with the value directly obtained from a sample generated with that lifetime. BðH →ZDZDÞ ∝ζ m2 H jm2 HD −m2 Hj ; where values as large as 25% have not yet been ruled out by constraints from Higgs coupling fits [70,71]. For ϵ ≪1, the ZD branching fraction to muons, BðZD →μþμ−Þ, is inde- pendent of ϵ but varies with mZD [69]: from a value of 0.1475 for mZD ¼ 20 GeV to a value of 0.1066 for mZD ¼ 60 GeV. Five signal samples were generated with ZD masses and lifetimes given in Table II. The Higgs boson is produced via the gluon-gluon fusion process, assuming a cross section of 44.1 pb, calculated at next-to-next-to- leading order in the strong coupling constant, adding the resummation of soft gluon emission at next-to-next-to- leading-logarithmic accuracy [72]. The inclusion of other production processes was found to have a negligible impact on the analysis. IV. EVENT SELECTION, SIGNAL EFFICIENCIES, AND BACKGROUND ESTIMATE model). All three of the muon triggers use only measure- ments in the MS to identify muons. The thresholds for the Emiss T and collimated-dimuon triggers changed during the course of 2016 data taking. To account for these changes, the highest available thresh- old for each trigger is used and offline requirements are imposed corresponding to the thresholds listed in the table. Moreover, additional stricter requirements are imposed on the corresponding offline quantity in order to ensure that the trigger efficiency falls on the efficiency plateau. Candidate signal events are selected by identifying μþμ− pairs consistent with having been produced in a vertex displaced at least several centimeters from the IP.3 The selection criteria are designed to strongly suppress back- ground from SM processes that produce muons near the IP while efficiently accepting signal events over a wide range of LLP masses, lifetimes and velocities. To retain the greatest possible model independence, minimal require- ments are placed on other aspects of the event. For signal events with displaced dimuon vertices, the single-muon trigger efficiency falls off approximately linearly with jd0j, from a maximum of about 70% at 0 cm to approximately 10% at the fiducial limit of 400 cm, due to requirements that favor muon candidates that originate close to the IP. The calorimeter-based Emiss T trigger is employed to recover some signal efficiency. As muons leave little energy in the calorimeter and the Emiss T at the trigger level is computed only using the calorimeter signals, the Emiss T trigger is an effective muon trigger. The initial event selection is performed with a combi- nation of triggers that require either the presence of a muon candidate or large missing transverse momentum, whose magnitude is denoted Emiss T . Next, offline selection criteria are used to first identify suitable muon candidates, and then pairs of muons of opposite charge consistent with a DV. The backgrounds from all SM beam-collision and non- beam-collision processes (cosmic-ray muons or beam-halo particles) are estimated directly from the data. Finally, the number of vertices expected from background processes is compared with the observed number of vertices in data in two signal regions, distinguished by the dimuon invari- ant mass. T The collimated-dimuon trigger is based on reconstruction of muon tracks with low pT thresholds. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … SEARCH FOR LONG-LIVED PARTICLES IN FINAL … PHYS. REV. D 99, 012001 (2019) TABLE III. The MC generators, hadronization, and showering software package, underlying-event simulation and PDF sets used for the simulated background events. The mass range of the low-mass Drell-Yan sample is restricted to 6 < mμμ < 60 GeV. TABLE III. The MC generators, hadronization, and showering software package, underlying-event simulation and PDF sets used for the simulated background events. The mass range of the low-mass Drell-Yan sample is restricted to 6 < mμμ < 60 GeV. TABLE III. The MC generators, hadronization, and showering software package, underlying-event simulation and PDF sets used for the simulated background events. The mass range of the low-mass Drell-Yan sample is restricted to 6 < mμμ < 60 GeV. the simulated background events. The mass range of the low-mass Drell-Yan sample is restricted to 6 < mμμ < 60 GeV. Sample MC generator Hard-process PDF Hadronization and showering Nonperturbative tune and parton-shower PDF Z þ jets POWHEG [78,79] CT10 [80] PYTHIA8 [56]+EVTGEN [61] AZNLO+CTEQ6L1 [81] Low-mass Drell-Yan POWHEG PYTHIA8+EVTGEN AZNLO+CTEQ6L1 t¯t POWHEG CT10 PYTHIA6 [82]+EVTGEN P2012 [83]+CTEQ6L1 W þ jets POWHEG CT10 PYTHIA8 AZNLO+CTEQ6L1 ZZ POWHEG-BOX v2 [84] CT10nlo PYTHIA8 AZNLO+CTEQ6L1 WW POWHEG-BOX v2 CT10nlo PYTHIA8 AZNLO+CTEQ6L1 WZ POWHEG-BOX v2 CT10nlo PYTHIA8 AZNLO+CTEQ6L1 Single top POWHEG [85,86] CT10 PYTHIA6 P2012+CTEQ6L1 3The RMS spread of the z distribution of the pp interaction vertices is 47.7 mm and the spreads in the x and y directions are less than 0.01 mm. A. Trigger requirements Events must satisfy the requirements of at least one of four different triggers in order to achieve the best possible efficiency for a wide variety of signal topologies and kinematics. The triggers used and their descriptions are given in Table IV. The first two triggers are highly efficient for signals with high-mass states that feature muons with large transverse momentum and large transverse impact parameters, d0, such as the GGM model, while the final two allow efficient selection of signals featuring low-mass states, and therefore lower-pT muons (e.g., the dark-sector IV. EVENT SELECTION, SIGNAL EFFICIENCIES, AND BACKGROUND ESTIMATE The large rates associated with the low pT thresholds are offset by requiring two muons in the MS that are within a cone of size ΔR ¼ 0.5. The efficiency of this trigger for a given signal model is strongly dependent on the magnitude of the boost of the particle decaying to the dimuon final state, as this determines the likelihood of the two muons being found within a cone of size ΔR ¼ 0.5. The trimuon trigger increases the efficiency for selecting events with particles that have a relatively large branching fraction to muons, as is the case of the ZD in the signal model explored in this article. B. SM background samples The MC simulations of background processes are used only as a guide for some of the selection criteria and for categorization of the types of background, while the background yield itself is predicted from techniques that use solely the data. The MC generators, hadronization, and showering software packages, underlying-event sim- ulation and choice of parton distribution functions are summarized in Table III. Further details about the gen- erator settings used for these processes can also be found in Refs. [73–77]. Each of the simulated background samples is scaled to correspond to an integrated luminosity of 32.9 fb−1, the size of the data sample. The signal samples were generated with values of the LLP lifetime that were chosen to provide sufficiently large 012001-4 B. Offline reconstruction and preselection All MS track parameters are expressed at the point of closest approach to the IP and their uncertainties reflect the effects of multiple Coulomb scattering in the detector material. Although the highest track reconstruction efficiency is obtained for muons originating near the IP, appreciable efficiency is obtained for muons with transverse impact parameters as large as 200 cm. In order to optimize the resolution of the track parameters, the following criteria are imposed. The MS tracks are required to have transverse momentum greater than 10 GeV, jηj < 2.5, measurements in each of the three layers of both the precision and trigger chambers, an uncertainty in the d0 measurement less than 20 cm and to not traverse regions of the MS that are poorly aligned. Muon candidates that trigger in a small set of resistive- plate chambers that can have timing jitter are rejected. This amounts to no more than 0.3% of selected muon candidates, which has a negligible effect on the signal acceptance. Interactions between beam protons and beam collimators upstream of the IP are a source of high-momentum muons, denoted beam-induced-background (BIB) muons, that can enter the ATLAS detector nearly parallel to the beam axis. Most MS tracks generated by this process are identified and rejected with the method described in Ref. [91] and results in a negligible reduction in signal efficiency. g g g p To distinguish between muon candidates that originate from prompt and nonprompt decays, the following classi- fication of MS tracks is used. Those for which a successful ID-MS combination has been made, defined by the require- ment that the angular distance between the MS track and nearest combined-muon track is less than 0.1, are referred to as “MScomb” muon candidates and the rest are referred to as “MSonly” muon candidates, as summarized in Table V. The large majority of MS tracks are MScomb, which reflects the fact that most muons are produced very close to the IP. Track reconstruction is performed independently in the ID, and an attempt is made to match each MS track with an ID track. The two matched tracks are then used as input to a combined fit that takes into account all of the ID and MS measurements, the energy loss in the calorimeter and multiple-scattering effects. During the fit, additional MS measurements may be added to or removed from the track to improve the fit quality. B. Offline reconstruction and preselection Interaction vertices from the pp collisions are recon- structed from at least two tracks with pT larger than 400 MeV that are consistent with originating from the beam-collision region in the x-y plane. Selected events are required to have at least one reconstructed interaction vertex. 3The RMS spread of the z distribution of the pp interaction vertices is 47.7 mm and the spreads in the x and y directions are less than 0.01 mm. 012001-5 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. TABLE IV. Description of triggers used to select events. The quantity ΔRμμ is the angular distance between the two muons in the collimated-dimuon trigger. Signal type Trigger Description Thresholds High mass Emiss T Missing transverse momentum Emiss T > 110 GeV Single muon Single muon restricted to the barrel region Muon jηj < 1.05 and pT > 60 GeV Low mass Collimated dimuon Two muons with small angular separation pT of muons > 15 and 20 GeV and ΔRμμ < 0.5 Trimuon Three muons pT > 6 GeV for all three muons Jet candidates are reconstructed from topological clusters [87], built from energy deposits in the calorimeters calibrated to the electromagnetic scale, using the anti-kt algorithm [88] with radius parameter R ¼ 0.4. The reconstructed jets are calibrated to the hadronic energy scale by scaling their four- momenta to the particle level [89]. The jets are required to have pT > 20 GeV and jηj < 4.4. If a jet in an event fails the “loose” jet-quality requirements of Ref. [90], the event is vetoed in order to suppress detector noise and noncollision backgrounds [90,91]. To reduce the contamination due to jets originating from pileup interactions, an additional requirement on the jet vertex tagger [92] output is made for jets with pT < 60 GeV and jηj < 2.4. momentum greater than 400 MeV, to have a minimum number of hits in each ID subsystem and to have jd0j < 1 cm. Hence, these combined-muon candidates correspond to muons produced within ∼1 cm of the x-y position of the IP. To suppress background from misidentified jets as well as from hadron decays to muons inside jets, all muon candidates are required to have at least a minimum angular separation from all jets (muon-jet overlap removal) and to satisfy track-based isolation criteria. B. Offline reconstruction and preselection The ID track is required to be within the ID acceptance, jηj < 2.5, to have transverse B. Offline reconstruction and preselection Muon-jet overlap removal is accomplished by requiring that ΔRμ−jet > min ð0.4; 0.04 þ 10 GeV=pμ TÞ for all jets in the event, where ΔRμ−jet is the angular separation between the muon candidate and the jet in consideration. The track-based isolation quantity IID ΔR¼0.4 is defined as the ratio of the scalar sum of pT of all ID tracks matched to the primary vertex, and with pT > 0.5 GeV within a cone of size ΔR ¼ 0.4 around the muon candidate, to the muon pT. To remove the contribution of the ID track forming the muon candidate (if it exists), the ID track that is nearest to and within ΔR ¼ 0.1 of the muon candidate and has a pT within 10% of the MS-track pT is not used in the sum. Muon candidates are required to have IID ΔR¼0.4 < 0.05. The muon-jet overlap and isolation requirements are removed in defining control regions used to study backgrounds described in Sec. IV F. The muon reconstruction algorithm [93] starts by finding tracks in the MS, denoted MS tracks, and extrapolating their trajectories towards the IP. All MS track parameters are expressed at the point of closest approach to the IP and their uncertainties reflect the effects of multiple Coulomb scattering in the detector material. Although the highest track reconstruction efficiency is obtained for muons originating near the IP, appreciable efficiency is obtained for muons with transverse impact parameters as large as 200 cm. In order to optimize the resolution of the track parameters, the following criteria are imposed. The MS tracks are required to have transverse momentum greater than 10 GeV, jηj < 2.5, measurements in each of the three layers of both the precision and trigger chambers, an uncertainty in the d0 measurement less than 20 cm and to not traverse regions of the MS that are poorly aligned. Interactions between beam protons and beam collimators upstream of the IP are a source of high-momentum muons, denoted beam-induced-background (BIB) muons, that can enter the ATLAS detector nearly parallel to the beam axis. Most MS tracks generated by this process are identified and rejected with the method described in Ref. [91] and results in a negligible reduction in signal efficiency. The muon reconstruction algorithm [93] starts by finding tracks in the MS, denoted MS tracks, and extrapolating their trajectories towards the IP. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … TABLE V. Definition of categories of muon candidates. Tracks in the ID are reconstructed with maximum jd0j of 1 cm. Muon candidate Definition MScomb Successful ID-MS combination MSonly Standalone MS (no match with ID track) the dimuon invariant mass, mμμ, be larger than 15 GeV. The ability to determine the spatial location of the vertex varies with the pT of the muons in the vertex and the opening angle between them. The average resolutions of rvtx and zvtx are in the range of 2–3 cm. Cosmic-ray muons that pass through the detector in time with a pp collision are sometimes reconstructed as two separate MS tracks that have an opening angle of π: Δϕ ¼ π and Ση ¼ 0, where Δϕ is the difference in ϕ between the two MS tracks and Ση is the sum of their η values. Vertices formed by such MS tracks are effectively eliminated by requiring ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ðΣηÞ2 þ ðπ −ΔϕÞ2 p > 0.1. additional criteria are applied to form signal regions (SRs) in which data are compared to background estimates, and control regions (CRs) which are used to provide those background estimates. g Within each event, all possible pairs of muon candidates, both MScomb and MSonly, are formed. For each pair, the muon candidate with the largest pT is designated the “leading” muon, while the other is designated the “sub- leading” muon. An algorithm which assumes a straight-line extrapolation of the muon trajectory from the MS inner surface towards the IP is used to determine whether or not the two muons are consistent with originating from a common vertex. The midpoint between the points of closest approach along the trajectories of the two muon candidates is taken to be the three-dimensional location of the vertex. This simple approach is sufficient for the purposes of this analysis, as the location of the putative dimuon vertex is only used in defining the geometrical acceptance of the analysis. The decay length Lvtx and projections onto the x-y plane and z axis, rvtx and zvtx respectively, are measured relative to the IP. It is convenient to sign the vertex radius rvtx according to the following definition. C. Selection of dimuon vertices The selection criteria described below are used to define a sample of dimuon vertices (preselection) to which 012001-6 PHYS. REV. D 99, 012001 (2019) SEARCH FOR LONG-LIVED PARTICLES IN FINAL … SEARCH FOR LONG-LIVED PARTICLES IN FINAL … If the angle between the projections in the x-y plane of the vertex momentum vector (the dimuon momentum vector) and the “flight direction” (the vector connecting the IP with the displaced dimuon vertex) is less than π=2 then it is assigned a positive value, otherwise it is assigned a negative value. When the LLP decays exclusively into a pair of muons or there is a small mass difference between the LLP and the dimuon state, the two vectors are typically closely aligned and the signed rvtx more often has a positive value. Examples are the dark-sector model and the GGM model for cases where there is a relatively small mass difference between the ˜χ0 1 and the Z boson. In all cases, LLPs are distinguished by relatively large values of the magnitude of signed rvtx. Backgrounds that contribute to the preselection sample include SM proton-proton collision processes as well as events with muons that are not associated with the pp collision (e.g., cosmic-ray muons). The dominant contri- butions to the former are low-mass Drell-Yan and Z þ jets processes, collectively referred to simply as DY. At small values of mμμ, dimuon vertices from multijet processes are also substantial. Dimuon vertices reconstructed in t¯t and single-top events make small contributions, while W þ jets and diboson processes are found to be negligibly small backgrounds. Distributions of mμμ and signed rvtx for opposite-charge and same-charge (SS) dimuon vertices satisfying the preselection criteria are shown in Fig. 2. Also shown are the expected contributions from the background processes discussed above. Due to the limited number of simulated multijet events, this source of background is not included in the MC distributions. Its relative contribution is expected to be dominant for SS pairs and most pronounced for OS ones at small values of mμμ, as determined from studies of events where the muon-jet overlap and muon isolation require- ments are inverted, and this is the dominant source of difference between the data and MC distributions in those regions of Fig. 2. The fraction of events in the data with multiple dimuon vertices passing the preselection criteria is 0.065%. When the LLP decays exclusively into a pair of muons or there is a small mass difference between the LLP and the dimuon state, the two vectors are typically closely aligned and the signed rvtx more often has a positive value. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Examples are the dark-sector model and the GGM model for cases where there is a relatively small mass difference between the ˜χ0 1 and the Z boson. In all cases, LLPs are distinguished by relatively large values of the magnitude of signed rvtx. The preselected dimuon vertices are divided into two regions to be used in searches for low- and high-mass signal models, which are summarized in Table VI. To further suppress the DY background in the high-mass region, where Z þ jets production dominates, and improve the search sensitivity, the transverse boost of the dimuon pair, defined as the ratio of the transverse momentum of the dimuon system to its invariant mass, is required to be larger than 2. This reduces the DY background by approximately a factor of 20, with a small reduction in the signal efficiencies, where the decay of a heavy BSM particle produces the dimuon state (a Z boson in the GGM model) with a relatively large boost. Vertices are selected as follows. To reduce combinatorial background from random track crossings, the distance of closest approach between the two straight-line extrapola- tions is required to be less than 20 cm. As the vertex position is poorly measured for tracks that are nearly parallel to each other, vertices for which the opening angle of the muon pair is less than 0.1 are rejected. Vertices are required to be within the cylindrical fiducial volume jrvtxj < 400 cm and jzvtxj < 600 cm. Background from muons with relatively low momentum in multijet events, as well as ϒ decays to dimuons, is reduced by requiring that The next sections describe the SR and CR selection criteria based on the designation of muon candidates as MScomb or MSonly. 012001-7 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … 20 40 60 80 100 120 140 160 180 200 [GeV] μ μ m 10 2 10 3 10 4 10 5 10 6 10 7 10 Vertices / 2 GeV ATLAS = 13 TeV s -1 32.9 fb − μ + μ Drell-Yan tt Single top quark Data (a) 400 − 300 − 200 − 100 − 0 100 200 300 400 [cm] vtx Signed r 1 10 2 10 3 10 4 10 5 10 6 10 7 10 Vertices / 10 cm ATLAS = 13 TeV s -1 32.9 fb − μ + μ Drell-Yan tt Single top quark Data (b) 20 40 60 80 100 120 140 160 180 200 [GeV] μ μ m 1 10 2 10 3 10 Vertices / 2 GeV ATLAS = 13 TeV s -1 32.9 fb - μ - μ , + μ + μ Drell-Yan tt Single top quark Data (c) 400 − 300 − 200 − 100 − 0 100 200 300 400 [cm] vtx Signed r 1 10 2 10 3 10 4 10 Vertices / 10 cm ATLAS = 13 TeV s -1 32.9 fb - μ - μ , + μ + μ Drell-Yan tt Single top quark Data (d) FIG. 2. Distributions of (a) dimuon invariant mass mμμ and (b) signed vertex radius rvtx for opposite-charge dimuon vertices satisfying the preselection requirements described in the text; (c) mμμ and (d) signed rvtx for same-charge dimuon vertices satisfying the preselection requirements described in the text. The stacked histograms represent the expected contributions from various SM background processes and are derived from MC simulations scaled to an integrated luminosity of 32.9 fb−1. Multijet processes are not included in the background due to the limited number of simulated events. The contributions from these processes are most substantial at small values of mμμ. The shaded bands represent the statistical uncertainties in the simulated background. The observed distributions for data are given by the points with error bars. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … 20 40 60 80 100 120 140 160 180 200 [GeV] μ μ m 10 2 10 3 10 4 10 5 10 6 10 7 10 Vertices / 2 GeV ATLAS = 13 TeV s -1 32.9 fb − μ + μ Drell-Yan tt Single top quark Data (a) 400 − 300 − 200 − 100 − 0 100 200 300 400 [cm] vtx Signed r 1 10 2 10 3 10 4 10 5 10 6 10 7 10 Vertices / 10 cm ATLAS = 13 TeV s -1 32.9 fb − μ + μ Drell-Yan tt Single top quark Data (b) Vertices / 10 cm (b) (a) 20 40 60 80 100 120 140 160 180 200 [GeV] μ μ m 1 10 2 10 3 10 Vertices / 2 GeV ATLAS = 13 TeV s -1 32.9 fb - μ - μ , + μ + μ Drell-Yan tt Single top quark Data (c) 400 − 300 − 200 − 100 − 0 100 200 300 400 [cm] vtx Signed r 1 10 2 10 3 10 4 10 Vertices / 10 cm ATLAS = 13 TeV s -1 32.9 fb - μ - μ , + μ + μ Drell-Yan tt Single top quark Data (d) Vertices / 10 cm (d) (c) FIG. 2. Distributions of (a) dimuon invariant mass mμμ and (b) signed vertex radius rvtx for opposite-charge dimuon vertices satisfying the preselection requirements described in the text; (c) mμμ and (d) signed rvtx for same-charge dimuon vertices satisfying the preselection requirements described in the text. The stacked histograms represent the expected contributions from various SM background processes and are derived from MC simulations scaled to an integrated luminosity of 32.9 fb−1. Multijet processes are not included in the background due to the limited number of simulated events. The contributions from these processes are most substantial at small values of mμμ. The shaded bands represent the statistical uncertainties in the simulated background. The observed distributions for data are given by the points with error bars. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … 0 100 200 300 400 500 600 [cm] vtx L 0 0.05 0.1 0.15 Vertex efficiency = 300 GeV 1 0 χ∼ m = 700 GeV 1 0 χ∼ m = 1000 GeV 1 0 χ∼ m ATLAS Simulation = 13 TeV s high SR (a) 0 50 100 150 200 250 [cm] 0 Leading muon d 0 0.05 0.1 0.15 Vertex efficiency = 300 GeV 1 0 χ∼ m = 700 GeV 1 0 χ∼ m = 1000 GeV 1 0 χ∼ m ATLAS Simulation = 13 TeV s high SR (b) 0 100 200 300 400 500 600 [cm] vtx L 0 0.05 0.1 Vertex efficiency = 20 GeV D Z m = 40 GeV D Z m = 60 GeV D Z m ATLAS Simulation = 13 TeV s low SR (c) 0 50 100 150 200 250 0 Leading muon [cm] d 0 0.05 0.1 Vertex efficiency = 20 GeV D Z m = 40 GeV D Z m = 60 GeV D Z m ATLAS Simulation = 13 TeV s low SR (d) FIG. 3. The efficiency for selecting a displaced dimuon vertex that satisfies the requirements of SRhigh and SRlow as function of (a) and (c) generated decay length Lvtx, and (b) and (d) generated transverse impact parameter d0 of the leading muon. These efficiencies are calculated relative to all generated signal vertices and include geometrical acceptance and reconstruction effects. The distributions in (a) and (b) are derived from signal events with a long-lived neutralino, ˜χ0 1, decaying to a Z boson (with Z →μþμ−) and a gravitino. The distributions in (c) and (d) are derived from signal events with a long-lived dark photon, ZD, that decays to μþμ−. The shaded bands represent the statistical uncertainties in the efficiencies. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … 0 50 100 150 200 250 [cm] 0 Leading muon d 0 0.05 0.1 0.15 Vertex efficiency = 300 GeV 1 0 χ∼ m = 700 GeV 1 0 χ∼ m = 1000 GeV 1 0 χ∼ m ATLAS Simulation = 13 TeV s high SR (b) 0 100 200 300 400 500 600 [cm] vtx L 0 0.05 0.1 0.15 Vertex efficiency = 300 GeV 1 0 χ∼ m = 700 GeV 1 0 χ∼ m = 1000 GeV 1 0 χ∼ m ATLAS Simulation = 13 TeV s high SR (a) Vertex efficiency (a) (b) 0 100 200 300 400 500 600 [cm] vtx L 0 0.05 0.1 Vertex efficiency = 20 GeV D Z m = 40 GeV D Z m = 60 GeV D Z m ATLAS Simulation = 13 TeV s low SR (c) 0 50 100 150 200 250 0 Leading muon [cm] d 0 0.05 0.1 Vertex efficiency = 20 GeV D Z m = 40 GeV D Z m = 60 GeV D Z m ATLAS Simulation = 13 TeV s low SR (d) Vertex efficiency Vertex efficiency Vertex efficiency (c) (d) FIG. 3. The efficiency for selecting a displaced dimuon vertex that satisfies the requirements of SRhigh and SRlow as function of (a) and (c) generated decay length Lvtx, and (b) and (d) generated transverse impact parameter d0 of the leading muon. These efficiencies are calculated relative to all generated signal vertices and include geometrical acceptance and reconstruction effects. The distributions in (a) and (b) are derived from signal events with a long-lived neutralino, ˜χ0 1, decaying to a Z boson (with Z →μþμ−) and a gravitino. The distributions in (c) and (d) are derived from signal events with a long-lived dark photon, ZD, that decays to μþμ−. The shaded bands represent the statistical uncertainties in the efficiencies. parameters of the reconstructed MS tracks and the distri- butions are normalized to the expected yields in the signal regions. muons, while the loss at large values reflects the lower MS reconstruction efficiency for tracks with trajectories that do not extrapolate back to a region close to the IP. The value of Lvtx where maximum efficiency is achieved is different for each choice of ZD mass due to the large differences in boost. D. Signal regions and signal efficiency Signal is characterized by vertices where both muon candidates are MSonly. This requirement unavoidably leads to a reduction in efficiency for decays close to the IP. Displaced-vertex analyses that make use of ID tracks [22] effectively recover such signal events. Two orthogonal signal regions are used to increase the sensitivity to low- and high-mass signal models, SRlow and SRhigh, respec- tively. The two regions are summarized in Table VI. For both SRs, the muons are required to have opposite charge. TABLE VI. Selection criteria for low- and high-mass regions, in addition to the preselection requirements described in the text. The definitions of the low- and high-mass signal regions are also given. given. Selection Low mass High mass pμ T [GeV] > 10 > 20 mμμ [GeV] 15–60 > 60 Dimuon transverse boost > 2 SRlow SRhigh Muon candidates Both MSonly Both MSonly Muon candidate charge Opposite charge Opposite charge The product of acceptance and reconstruction efficiency determined from simulated signal events is shown in Fig. 3 as a function of generated Lvtx and leading muon d0, for the GGM model and for the dark-sector model. The lower efficiency observed for small Lvtx or small jd0j (more apparent in the ZD models) is due to the veto on MScomb 012001-8 SEARCH FOR LONG-LIVED PARTICLES IN FINAL … PHYS. REV. D 99, 012001 (2019) E. Control regions and background estimation 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 0 0.05 0.1 Total efficiency = 300 GeV 1 0 χ∼ m = 700 GeV 1 0 χ∼ m = 1000 GeV 1 0 χ∼ m ATLAS Simulation = 13 TeV s high SR (a) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 0 0.05 Total efficiency = 20 GeV D Z m = 40 GeV D Z m = 60 GeV D Z m ATLAS Simulation = 13 TeV s low SR (b) Total efficiency (b) (a) FIG. 4. Overall event-level efficiencies after the signal-region selections (combining trigger and offline selection), as a function of the lifetime of the long-lived BSM particle, for (a) the GGM model and (b) the dark-sector model. The shaded bands represent the statistical uncertainties only. E. Control regions and background estimation 20 40 60 80 100 120 140 160 180 200 [GeV] μ μ m 0 1 2 3 4 5 6 7 Vertices / 2 GeV ATLAS Simulation = 13 TeV s -1 32.9 fb high SR = 1 m τ = 300 GeV, c 1 0 χ∼ m = 1 m τ = 700 GeV, c 1 0 χ∼ m = 1 m τ = 1000 GeV, c 1 0 χ∼ m (a) 400 − 300 − 200 − 100 − 0 100 200 300 400 [cm] vtx Signed r 1 − 10 1 10 2 10 Vertices / 10 cm ATLAS Simulation = 13 TeV s -1 32.9 fb high SR = 1 m τ = 300 GeV, c 1 0 χ∼ m = 1 m τ = 700 GeV, c 1 0 χ∼ m = 1 m τ = 1000 GeV, c 1 0 χ∼ m (b) 20 30 40 50 60 70 80 [GeV] μ μ m 0 200 400 600 800 1000 1200 1400 Vertices / 2 GeV ATLAS Simulation = 13 TeV s -1 32.9 fb low SR = 0.5 m τ = 20 GeV, c D Z m = 0.5 m τ = 40 GeV, c D Z m = 0.5 m τ = 60 GeV, c D Z m (c) 400 − 300 − 200 − 100 − 0 100 200 300 400 [cm] vtx Signed r 1 10 2 10 3 10 4 10 Vertices / 10 cm ATLAS Simulation = 13 TeV s -1 32.9 fb low SR = 0.5 m τ = 20 GeV, c D Z m = 0.5 m τ = 40 GeV, c D Z m = 0.5 m τ = 60 GeV, c D Z m (d) FIG. 5. Distributions derived from MC simulations of (a) dimuon invariant mass mμμ and (b) vertex radius rvtx for signal vertices in SRhigh with a long-lived neutralino, ˜χ0 1 (m˜χ0 1 ¼ 300, 700, and 1000 GeV and cτ˜χ0 1 ¼ 100 cm) decaying to a Z boson (with Z →μþμ−) and a gravitino; (c) mμμ and (d) rvtx for signal vertices in SRlow with a long-lived dark photon, ZD (mZD ¼ 20, 40, and 60 GeV; and cτZD ¼ 50 cm), that decays to μþμ−. The shaded bands represent the statistical uncertainties. E. Control regions and background estimation The distributions are normalized to the expected yields in the signal regions for m˜g ¼ 1100 GeV, σðpp →˜g ˜gÞ ¼ 0.1635 pb, Bð˜χ0 1 →Z ˜GÞ ¼ 1.0, and BðZ →μþμ−Þ ¼ 0.03366; and mH ¼ 125 GeV, mHD ¼ 300 GeV, σðpp →HÞ ¼ 44.1 pb, BðH →ZDZDÞ ¼ 100%, and the value of BðZD →μþμ−Þ varying between 0.1475 and 0.1066 for the range mZD ¼ 20–60 GeV. 400 − 300 − 200 − 100 − 0 100 200 300 400 [cm] vtx Signed r 1 − 10 1 10 2 10 Vertices / 10 cm ATLAS Simulation = 13 TeV s -1 32.9 fb high SR = 1 m τ = 300 GeV, c 1 0 χ∼ m = 1 m τ = 700 GeV, c 1 0 χ∼ m = 1 m τ = 1000 GeV, c 1 0 χ∼ m (b) 20 40 60 80 100 120 140 160 180 200 [GeV] μ μ m 0 1 2 3 4 5 6 7 Vertices / 2 GeV ATLAS Simulation = 13 TeV s -1 32.9 fb high SR = 1 m τ = 300 GeV, c 1 0 χ∼ m = 1 m τ = 700 GeV, c 1 0 χ∼ m = 1 m τ = 1000 GeV, c 1 0 χ∼ m (a) (b) (a) 20 30 40 50 60 70 80 [GeV] μ μ m 0 200 400 600 800 1000 1200 1400 Vertices / 2 GeV ATLAS Simulation = 13 TeV s -1 32.9 fb low SR = 0.5 m τ = 20 GeV, c D Z m = 0.5 m τ = 40 GeV, c D Z m = 0.5 m τ = 60 GeV, c D Z m (c) 400 − 300 − 200 − 100 − 0 100 200 300 400 [cm] vtx Signed r 1 10 2 10 3 10 4 10 Vertices / 10 cm ATLAS Simulation = 13 TeV s -1 32.9 fb low SR = 0.5 m τ = 20 GeV, c D Z m = 0.5 m τ = 40 GeV, c D Z m = 0.5 m τ = 60 GeV, c D Z m (d) Vertices / 10 cm (c) (d) FIG. 5. E. Control regions and background estimation Dimuon vertices are categorized as described in Table VII. The observed yields of same-charge dimuon vertices in all four regions A, B, C, and D are used to estimate the background yields in the SRs due to muons produced more than about a centimeter from the IP, referred to as nonprompt muons. The observed yields in the opposite-charge B, C, and D CRs are used to predict the background yield from SM processes that produce prompt muons (those produced within about a centimeter of the IP) in the SRs (opposite- charge dimuon vertices in region A). Muons from decays of hadrons containing b and c quarks are, within the context of this analysis, considered to be prompt muons. The total event-level efficiencies, including trigger and offline selection criteria, as functions of the lifetime of the LLP, are shown in Fig. 4 and maximum values are in the cτ region 20–50 cm. The reweighted samples, as described in Sec. III, are used to estimate the efficiencies for values of the lifetime which were not used in generating the simulated samples. This event-level efficiency is defined as the fraction of generated events that are selected and have at least one dimuon DV. Distributions of mμμ and signed rvtx for signal vertices in simulated events, for both SRhigh and SRlow, are displayed in Fig. 5. The vertex properties are computed using the 012001-9 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 0 0.05 0.1 Total efficiency = 300 GeV 1 0 χ∼ m = 700 GeV 1 0 χ∼ m = 1000 GeV 1 0 χ∼ m ATLAS Simulation = 13 TeV s high SR (a) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 0 0.05 Total efficiency = 20 GeV D Z m = 40 GeV D Z m = 60 GeV D Z m ATLAS Simulation = 13 TeV s low SR (b) FIG. 4. Overall event-level efficiencies after the signal-region selections (combining trigger and offline selection), as a function of the lifetime of the long-lived BSM particle, for (a) the GGM model and (b) the dark-sector model. The shaded bands represent the statistical uncertainties only. F. Nonprompt muon vertices Nonprompt muons are those for which no matching ID track is expected. Examples of such sources of background nonprompt muons are cosmic-ray muons, BIB muons, fake MS tracks generated from random hit combinations, and those arising from pion or kaon decay. depending on the region (A, B, C, or D). As no statistical difference in Rπ=K q is observed for the low- and high-mass regions, a single value is used for both regions. For dimuon vertices composed of BIB or cosmic-ray muons, the muon charges are assumed to be entirely uncorrelated: Rcos =BIB q ¼ 1.0. Cosmic-ray and BIB muons will usually not produce ID tracks, as they rarely pass close enough to the IP to produce an ID track that satisfies the track reconstruction criteria, in particular the jd0j < 1 cm requirement. As a result, they produce mostly MSonly muon candidates. As described in Sec. IV B, dimuon vertices reconstructed from a single cosmic-ray or BIB muon that generates two MS tracks are effectively eliminated in the preselection by taking advan- tage of the fact that the angle between the two tracks will be nearly 180°. On the other hand, vertices formed when a single MS track from a cosmic-ray or BIB muon is paired with an unrelated muon candidate produced from the pp collision will satisfy these selection criteria and more readily contribute to the background. Since the relative composition of the nonprompt dimuon background is unknown, the average of Rπ=K q and Rcos =BIB q is assumed as the nominal value, Rq, with an uncertainty that is half the difference between the two values; this is shown in Table VIII (Rq ¼ 1.24 0.24 for the SRs). The numbers of OS nonprompt vertices in the regions A, B, C, and D are predicted using the number of SS vertices in each region and the appropriate Rq, as described above: Nnonprompt i ¼ Rq;iNSS i , where NSS i is the number of SS vertices observed in region i (i ¼ A, B, C, or D) and Rq;i is the charge ratio for region i. q The predicted yields of nonprompt dimuon vertices for both SRs are given in Table VIII, where the uncertainty in Rq is treated as a systematic uncertainty added in quad- rature to the statistical uncertainties. F. Nonprompt muon vertices Pions and kaons have relatively large lifetimes and feature large branching fractions to final states with one muon. Such decays often result in either no ID track being reconstructed, due to the requirement of at least a minimum number of ID hits, or the ID track of the pion/kaon failing to be matched to the muon MS track. In both of these two cases, a MSonly muon candidate will be produced. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … TABLE VIII. Observed number of SS vertices NSS, charge ratio Rq;SR and predicted yields of nonprompt dimuon vertices Nnonprompt SR in each signal region. The uncertainty in Nnonprompt SR combines in quadrature the statistical uncertainty, derived from the observed number of SS vertices, and the uncertainty in the charge ratio. The statistical uncertainty on NSS for the case where the central value is measured to be zero is taken to be the 68% one-sided Poisson confidence-level interval: þ1.14. TABLE VIII. Observed number of SS vertices NSS, charge ratio Rq;SR and predicted yields of nonprompt dimuon vertices Nnonprompt SR in each signal region. The uncertainty in Nnonprompt SR combines in quadrature the statistical uncertainty, derived from the observed number of SS vertices, and the uncertainty in the charge ratio. The statistical uncertainty on NSS for the case where the central value is measured to be zero is taken to be the 68% one-sided Poisson confidence-level interval: þ1 14 TABLE VII. Description of four regions used in estimating background yields. The ordering of the muon candidates in the description of the dimuon vertex is leading muon first, then subleading muon. Region name Muon candidates in vertex A MSonly-MSonly B MSonly-MScomb C MScomb-MSonly D MScomb-MScomb Region NSS Rq;SR Nnonprompt SR SRlow 11 1.24 0.24 13.6 4.9 SRhigh 0 1.24 0.24 0.0þ1.4 −0.0 E. Control regions and background estimation Distributions derived from MC simulations of (a) dimuon invariant mass mμμ and (b) vertex radius rvtx for signal vertices in SRhigh with a long-lived neutralino, ˜χ0 1 (m˜χ0 1 ¼ 300, 700, and 1000 GeV and cτ˜χ0 1 ¼ 100 cm) decaying to a Z boson (with Z →μþμ−) and a gravitino; (c) mμμ and (d) rvtx for signal vertices in SRlow with a long-lived dark photon, ZD (mZD ¼ 20, 40, and 60 GeV; and cτZD ¼ 50 cm), that decays to μþμ−. The shaded bands represent the statistical uncertainties. The distributions are normalized to the expected yields in the signal regions for m˜g ¼ 1100 GeV, σðpp →˜g ˜gÞ ¼ 0.1635 pb, Bð˜χ0 1 →Z ˜GÞ ¼ 1.0, and BðZ →μþμ−Þ ¼ 0.03366; and mH ¼ 125 GeV, mHD ¼ 300 GeV, σðpp →HÞ ¼ 44.1 pb, BðH →ZDZDÞ ¼ 100%, and the value of BðZD →μþμ−Þ varying between 0.1475 and 0.1066 for the range mZD ¼ 20–60 GeV. 012001-10 012001-10 PHYS. REV. D 99, 012001 (2019) G. Prompt muon vertices The number of dimuon vertices in each of the SRs arising from prompt muon processes is estimated from the observed yields in the OS low-mass and high-mass B, C, and D control regions. Sources of such background in the SRs include SM processes that produce prompt muons that are reconstructed as MSonly due to detector or reconstruction effects, such as ID inefficiencies, or poorly reconstructed combined muons, collectively described as failed combined muon reconstruction. Vertices that contain one or more nonprompt muons are referred to as “nonprompt vertices." If the vertex contains a cosmic-ray or BIB muon paired with an unrelated muon candidate, the charges of the two MS tracks will be largely uncorrelated. However, some charge correlation is expected in vertices containing muons from pion/kaon decay, because the pion/kaon is produced from the same pp collision that produces the other muon in the vertex. For the latter, the charge correlation is studied by measuring the ratio of OS to SS dimuon vertices, Rπ=K q , in the data. As muons from pion/kaon decay are not expected to be isolated from jets, the quantity Rπ=K q is measured in a sample of dimuon vertices where the selection criteria are those of the preselection, except that the jet-muon overlap and isolation requirements are removed for both muons. The value ranges from 1.39 0.09 to 1.55 0.03, To avoid double-counting of dimuons from nonprompt processes, the estimated number of nonprompt OS vertices in each region is subtracted: Nprompt i ¼ NOS i −Nnonprompt i ; i ¼ B; C; D; where NOS i is the number of OS vertices in region i, Nnonprompt i is the number of OS nonprompt vertices in region where NOS i is the number of OS vertices in region i, Nnonprompt i is the number of OS nonprompt vertices in region 012001-11 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. i (described in Sec. IV F) and Nprompt i is the estimated number of opposite-charge vertices from prompt processes in region i. The quantity Nprompt B (Nprompt C ) is the estimated number of OS vertices from prompt processes with leading (subleading) muons that fail the combined reconstruction and are identified as MSonly, while the other muon candidate is identified as being MScomb. H. Total background The predicted number of nonprompt muon vertices is summed with the predicted number of prompt muon vertices from SM background processes to give the predicted total number of background vertices in each of the SRs: 13.8 4.9 and 0.50þ1.41 −0.07 for SRlow and SRhigh, respectively, where the uncertainties include the statistical components and the systematic uncertainty in Rq. fL ¼ Nprompt B =Nprompt D ; fS ¼ Nprompt C =Nprompt D : fL ¼ Nprompt B =Nprompt D ; fS ¼ Nprompt C =Nprompt D : q The reliability of the background estimation method is validated by applying it to both the sum of the simulated background samples and to a high-mass validation region in the data. The predicted number of dimuon vertices in the simulated sample agrees with the number of observed vertices, to within the statistical precision, in both the low- and high-mass signal regions. As the simulated samples do not include multijet processes or cosmic muon back- grounds, this is primarily a validation of the technique to estimate the background from prompt dimuon vertices. The validation region in data comprises dimuon vertices that satisfy all of the selection criteria of the high-mass region, with the exception that the requirement on the transverse boost of the dimuon system is inverted: it is required to have a value less than two, which ensures that there is negligible contribution from signal processes. The results are given in Table X. These two studies validate the method within the statistical precision. The leading transfer factor multiplied by Nprompt C , or, alternatively, the subleading transfer factor multiplied by Nprompt B , thus gives for prompt muon processes the predicted number of OS vertices in region A, the SRs in this case: Nprompt A ¼ fL · Nprompt C ¼ fS · Nprompt B : The yields in the various regions are summarized in Table IX. The vertices in all CRs are used to verify that the designation of one of the MS tracks in the vertex as MScomb or MSonly is independent of the designation of the other as MScomb or MSonly (the measured correlation is negligibly small, < 0.0015). The predicted number of vertices from prompt background processes in the low- and high-mass SRs are 0.14 0.22 and 0.504 0.070, respec- tively, where the uncertainties are statistical only. G. Prompt muon vertices The quantity Nprompt D is the estimated number of OS vertices from prompt processes with muon candidates that pass the combined reconstruction and are both identified as being MScomb. With these definitions, the leading and subleading “transfer factors” are defined as follows: i (described in Sec. IV F) and Nprompt i is the estimated number of opposite-charge vertices from prompt processes in region i. The quantity Nprompt B (Nprompt C ) is the estimated number of OS vertices from prompt processes with leading (subleading) muons that fail the combined reconstruction and are identified as MSonly, while the other muon candidate is identified as being MScomb. The quantity Nprompt D is the estimated number of OS vertices from prompt processes with muon candidates that pass the combined reconstruction and are both identified as being MScomb. With these definitions, the leading and subleading “transfer factors” are defined as follows: and the transfer factors and predictions of Nprompt in the SRs are recomputed. For both the low-mass and high-mass selection, the sum over the predicted prompt background yields in each bin is consistent with the nominal value. VI. RESULTS The predicted number of nonprompt muon vertices is summed with the predicted number of prompt muon vertices from SM background processes to give the predicted total number of background vertices, Nbkgd, in each SR. The predicted background yields, along with the number of observed vertices in the data, are summarized in Table XI. The distributions of mμμ and rvtx are shown in Fig. 6 for the observed vertices in the two signal regions. Each dimuon vertex is in a separate event, and therefore the number of events observed is equivalent to the number of vertices. The dimuon vertex with the highest mass has mμμ ¼ 381 GeV, rvtx ¼ −220 cm, and zvtx ¼ 99 cm. Close inspection of the event reveals characteristics of being cosmic in origin. The observation of one such dimuon vertex in SRhigh is consistent, within the uncer- tainties, with the nonprompt background estimate of Nnonprompt ¼ 0.0þ1.4 −0.0. The other vertex in SRhigh has a mass compatible with the decay of the SM Z. The dimuon vertex with the largest value of rvtx is in SRlow and has mμμ ¼ 46 GeV, rvtx ¼ 223 cm, and zvtx ¼ 56 cm. The vertex is formed by an MS track passing through the top of the detector combined with another MS track passing through the bottom of the detector, with an angle of nearly 180° between them. This vertex is likely a cosmic- ray muon that narrowly survived the cosmic-ray veto criteria described in Sec. IV C. The systematic uncertainty from pileup effects is deter- mined by varying the pileup reweighting of simulated signal events in a manner that spans the expected uncer- tainty. This results in a systematic uncertainty of 0.2% in the signal efficiency. As no significant excess of vertices over the SM back- ground expectation is observed, 95% confidence-level (C.L.) upper limits on the signal event yields and produc- tion cross sections are calculated for various values of the proper decay distance cτ of the long-lived particle in each of the two BSM scenarios considered.4 The limits are calculated using the CLS prescription [96] with a Poisson likelihood used as the test statistic. Uncertainties in the signal efficiency and background expectation are included g y The methods used to estimate the background are entirely data-driven, with statistical uncertainties arising from the numbers of events in the CRs. 4For events that are selected exclusively by the trimuon trigger the observed signal yield will have a quadratic dependence on BðZD →μþμ−Þ. The collimated-dimuon trigger efficiency domi- nates over the trimuon trigger efficiency for the values of mZD considered in this paper. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … TABLE XI. Predicted nonprompt Nnonprompt, prompt Nprompt, and total Nbkgd background yields and number of observed vertices Nobs in data in SRlow and SRhigh. The uncertainties in the predicted background yields are statistical uncertainties and systematic uncertainties added in quadrature. The uncertainty in the 2016 integrated luminosity is 2.2%. It is derived, following a methodology similar to that detailed in Ref. [94], and using the LUCID-2 detector for the baseline luminosity measurements [95], from calibra- tion of the luminosity scale using x-y beam-separation scans. Yield SRlow SRhigh Nnonprompt 13.6 4.9 0.0þ1.4 −0.0 Nprompt 0.1þ0.2 −0.1 0.50 0.07 Nbkgd 13.8 4.9 0.50þ1.42 −0.07 Nobs 15 2 Sources of systematic uncertainties in the signal effi- ciencies include possible mismodeling of the trigger and MS efficiencies and pileup effects in the MC simulation. For the high-mass SR, the uncertainty associated with trigger and MS track reconstruction efficiency is deter- mined by comparing the observed yields in the data with MC simulation of Z þ jets events, using the selection criteria of the OS B, C, and D control regions and the additional requirement 70 < mμμ < 110 GeV. The differ- ence between the yields in data and the simulated back- ground samples is used to assign a systematic uncertainty of 1% to the combined trigger and MS track-reconstruction efficiency. For the low-mass SR, the efficiency of the trigger and MS track reconstruction is compared between MC simulation and data for J=ψ →μμ events, using a tag- and-probe technique. The efficiency is measured as a function of the angular separation between the two muons, and a maximum deviation of 6% is observed. This differ- ence is taken as an uncertainty in the signal efficiency. The agreement between data and MC simulation for the reconstruction efficiency for MS tracks with large impact parameters was studied by comparing a cosmic-ray muon simulation to cosmic-ray muon candidates in data [22]. Comparing the ratio of the muon candidate d0 distributions in the two samples yields a d0-dependent efficiency correction that is between 1% and 2.5%, with an average value of 1.5%. The systematic uncertainty on MS track reconstruction associated with this procedure is taken from the statistical uncertainty, and is 2% per track in the vertices. V. SYSTEMATIC UNCERTAINTIES As a cross-check, the B, C, and D control regions are subdivided into bins of either muon pT or muon η and ϕ, The systematic uncertainties are described in detail below. They include those in the integrated luminosity, used in converting signal yields to cross sections; the background estimate, derived entirely from the data; and the signal efficiency, determined from MC simulations. All systematic uncertainties are treated as uncorrelated. TABLE IX. The number of opposite-charge vertices, NOS, the number of same-charge vertices, NSS, the estimated ratio of opposite-charge to same-charge nonprompt dimuon vertices, Rq and the estimated number of prompt dimuon vertices Nprompt in each of the control regions B, C, and D. The values of Nprompt are obtained by subtracting the product of NSS and Rq from NOS. The quoted uncertainties in Nprompt include the statistical component and the systematic uncertainty in R TABLE X. Predicted nonprompt Nnonprompt VR , prompt Nprompt VR , and total Nbkgd VR background yields and number of observed vertices Nobs VR in data in the high-mass validation region. The uncertainty in Nprompt VR includes the statistical component and the systematic uncertainty in Rq. Yield High-mass validation region Nnonprompt VR 2.5þ2.3 −1.6 Nprompt VR 7.20 0.25 Nbkgd VR 9.7þ2.3 −1.6 Nobs VR 13 TABLE X. Predicted nonprompt Nnonprompt VR , prompt Nprompt VR , and total Nbkgd VR background yields and number of observed vertices Nobs VR in data in the high-mass validation region. The uncertainty in Nprompt VR includes the statistical component and the systematic uncertainty in Rq. and the systematic uncertainty in Rq. Region NOS NSS Rq Nprompt Low-mass region B 124 63 1.28 0.28 43 23 C 451 335 1.20 0.20 49þ74 −49 D 19599 3220 1.20 0.20 15700 660 High-mass region B 120 0 1.28 0.28 120 11 C 92 0 1.20 0.20 92 10 D 21940 24 1.20 0.20 21900 150 012001-12 PHYS. REV. D 99, 012001 (2019) VI. RESULTS 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 4 − 10 3 − 10 2 − 10 1 − 10 1 10 2 10 [pb] B x σ 95% CL upper limit (1100 GeV) = 0.163 pb g~ σ ) = 1 G~ Z → 1 0χ∼ ( B = 700 GeV 1 0 χ∼ m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 4 − 10 3 − 10 2 − 10 1 − 10 1 10 2 10 [pb] B x σ 95% CL upper limit (1100 GeV) = 0.163 pb g~ σ ) = 1 G~ Z → 1 0χ∼ ( B = 300 GeV 1 0 χ∼ m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 4 − 10 3 − 10 2 − 10 1 − 10 1 10 2 10 [pb] B x σ 95% CL upper limit (1100 GeV) = 0.163 pb g~ σ ) = 1 G~ Z → 1 0χ∼ ( B = 700 GeV 1 0 χ∼ m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 4 − 10 3 − 10 2 − 10 1 − 10 1 10 2 10 [pb] B x σ 95% CL upper limit (1100 GeV) = 0.163 pb g~ σ ) = 1 G~ Z → 1 0χ∼ ( B = 300 GeV 1 0 χ∼ m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (a) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 4 − 10 3 − 10 2 − 10 1 − 10 1 10 2 10 [pb] B x σ 95% CL upper limit (1100 GeV) = 0.163 pb g~ σ ) = 1 G~ Z → 1 0χ∼ ( B = 1000 GeV 1 0 χ∼ m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (c) (b) (a) (c) FIG. VI. RESULTS The nonprompt- vertex background estimate for both signal regions has a systematic uncertainty of 19% associated with knowledge of the charge correlation Rq, as described in Sec. IV F. Systematic uncertainties in the estimate of the prompt background are determined by varying the quantity that distinguishes MScomb from MSonly muons, the angular distance between the MS track and nearest combined-muon track, by 50% and repeating the ABCD technique described in Sec. IV G. A 9% difference in the prompt background estimate is observed, and this is taken as a systematic uncertainty. 012001-13 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. 50 100 150 200 250 300 350 400 [GeV] μ μ m 0 1 2 3 4 5 6 7 Vertices / 5 GeV ATLAS -1 32.9 fb = 13 TeV s low data, SR high data, SR (a) 300 − 200 − 100 − 0 100 200 300 [cm] vtx Signed r 0 1 2 3 4 5 6 7 8 Vertices / 10 cm ATLAS -1 32.9 fb = 13 TeV s low data, SR high data, SR (b) FIG. 6. Distributions of (a) dimuon invariant mass mμμ and (b) vertex radius rvtx for displaced dimuon vertices in the low-mass (black circles) and high-mass (red squares) signal regions. 300 − 200 − 100 − 0 100 200 300 [cm] vtx Signed r 0 1 2 3 4 5 6 7 8 Vertices / 10 cm ATLAS -1 32.9 fb = 13 TeV s low data, SR high data, SR (b) 50 100 150 200 250 300 350 400 [GeV] μ μ m 0 1 2 3 4 5 6 7 Vertices / 5 GeV ATLAS -1 32.9 fb = 13 TeV s low data, SR high data, SR (a) (a) (b) FIG. 6. Distributions of (a) dimuon invariant mass mμμ and (b) vertex radius rvtx for displaced dimuon vertices in the low-mass (black circles) and high-mass (red squares) signal regions. VI. RESULTS 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 4 − 10 3 − 10 2 − 10 1 − 10 1 10 2 10 [pb] B x σ 95% CL upper limit (1100 GeV) = 0.163 pb g~ σ ) = 1 G~ Z → 1 0χ∼ ( B = 300 GeV 1 0 χ∼ m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (a) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 4 − 10 3 − 10 2 − 10 1 − 10 1 10 2 10 [pb] B x σ 95% CL upper limit (1100 GeV) = 0.163 pb g~ σ ) = 1 G~ Z → 1 0χ∼ ( B = 700 GeV 1 0 χ∼ m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (b) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 4 − 10 3 − 10 2 − 10 1 − 10 1 10 2 10 [pb] B x σ 95% CL upper limit (1100 GeV) = 0.163 pb g~ σ ) = 1 G~ Z → 1 0χ∼ ( B = 1000 GeV 1 0 χ∼ m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (c) FIG. 7. The observed and expected 95% C.L. upper limits on the product of cross section and branching ratios for pair production of gluinos, leading to a final state of μþμ−þ X, in the GGM model, as a function of the ˜χ0 1 lifetime, for m˜g ¼ 1100 GeV and three different choices of m˜χ0 1: (a) 300 GeV, (b) 700 GeV, and (c) 1000 GeV. The shaded green (yellow) bands represent the 1σ (2σ) uncertainties in the expected limits. The dashed horizontal line represents the value of the cross section times branching fractions predicted from simulation, with m˜g ¼ 1100 GeV, σðpp →˜g ˜gÞ ¼ 0.1635 pb, Bð˜χ0 1 →Z ˜GÞ ¼ 1.0, and BðZ →μþμ−Þ ¼ 0.03366. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … 4 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 3 − 10 2 − 10 1 − 10 1 10 2 10 3 10 [pb] B x σ 95% CL upper limit ) = 0.1 D Z D Z → (H B ) = 0.01 D Z D Z → (H B ) = 0.148 μ μ → D (Z B = 20 GeV D Z m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 3 − 10 2 − 10 1 − 10 1 10 2 10 3 10 [pb] B x σ 95% CL upper limit ) = 0.1 D Z D Z → (H B ) = 0.01 D Z D Z → (H B ) = 0.148 μ μ → D (Z B = 20 GeV D Z m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 3 − 10 2 − 10 1 − 10 1 10 2 10 3 10 [pb] B x σ 95% CL upper limit ) = 0.1 D Z D Z → (H B ) = 0.01 D Z D Z → (H B ) = 0.137 μ μ → D (Z B = 40 GeV D Z m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 3 − 10 2 − 10 1 − 10 1 10 2 10 3 10 [pb] B x σ 95% CL upper limit ) = 0.1 D Z D Z → (H B ) = 0.01 D Z D Z → (H B ) = 0.137 μ μ → D (Z B = 40 GeV D Z m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (a) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 3 − 10 2 − 10 1 − 10 1 10 2 10 3 10 [pb] B x σ 95% CL upper limit ) = 0.1 D Z D Z → (H B ) = 0.01 D Z D Z → (H B ) = 0.107 μ μ → D (Z B = 60 GeV D Z m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (c) (a) (b) (c) FIG. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … PHYS. REV. D 99, 012001 (2019) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 3 − 10 2 − 10 1 − 10 1 10 2 10 3 10 [pb] B x σ 95% CL upper limit ) = 0.1 D Z D Z → (H B ) = 0.01 D Z D Z → (H B ) = 0.148 μ μ → D (Z B = 20 GeV D Z m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (a) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 3 − 10 2 − 10 1 − 10 1 10 2 10 3 10 [pb] B x σ 95% CL upper limit ) = 0.1 D Z D Z → (H B ) = 0.01 D Z D Z → (H B ) = 0.137 μ μ → D (Z B = 40 GeV D Z m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (b) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 3 − 10 2 − 10 1 − 10 1 10 2 10 3 10 [pb] B x σ 95% CL upper limit ) = 0.1 D Z D Z → (H B ) = 0.01 D Z D Z → (H B ) = 0.107 μ μ → D (Z B = 60 GeV D Z m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (c) FIG. 8. The observed and expected 95% C.L. upper limits on the product of cross section and branching ratios, σ × B ¼ σðpp →HÞ × BðH →ZDZDÞ × BðZD →μþμ−Þ, in the dark-sector model, as a function of the ZD lifetime, for three different choices of mZD: (a) 20 GeV, (b) 40 GeV, and (c) 60 GeV. The shaded green (yellow) bands represent the 1σ (2σ) uncertainties in the expected limits. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … 8. The observed and expected 95% C.L. upper limits on the product of cross section and branching ratios, σ × B ¼ σðpp →HÞ × BðH →ZDZDÞ × BðZD →μþμ−Þ, in the dark-sector model, as a function of the ZD lifetime, for three different choices of mZD: (a) 20 GeV, (b) 40 GeV, and (c) 60 GeV. The shaded green (yellow) bands represent the 1σ (2σ) uncertainties in the expected limits. The dashed horizontal lines represent the values of the cross section times branching fractions predicted by simulation, with mH ¼ 125 GeV, mHD ¼ 300 GeV, σðpp →HÞ ¼ 44.1 pb and assuming BðH →ZDZDÞ ¼ 10% or 1%. The value of BðZD →μþμ−Þ varies between 0.1475 and 0.1066 for the range mZD ¼ 20–60 GeV. 20 25 30 35 40 45 50 55 60 [GeV] D Z m 11 − 10 10 − 10 9 − 10 8 − 10 7 − 10 6 − 10 5 − 10 4 − 10 ∈ ) = 10% D Z D Z → (H B Excluded at 95% CL, ) = 1% D Z D Z → (H B Excluded at 95% CL, ATLAS = 13 TeV s -1 32.9 fb FIG. 9. The observed 95% C.L. excluded regions in the plane of ZD-Z kinetic mixing parameter, ϵ, versus ZD mass, for values of BðH →ZDZDÞ ¼ 1% or 10%, and mHD ¼ 300 GeV. The value of BðZD →μþμ−Þ varies between 0.1475 and 0.1066 for the range mZD ¼ 20–60 GeV. 20 25 30 35 40 45 50 55 60 [GeV] D Z m 11 − 10 10 − 10 9 − 10 8 − 10 7 − 10 6 − 10 5 − 10 4 − 10 ∈ ) = 10% D Z D Z → (H B Excluded at 95% CL, ) = 1% D Z D Z → (H B Excluded at 95% CL, ATLAS = 13 TeV s -1 32.9 fb as nuisance parameters, and the CLS values are calculated by generating ensembles of pseudoexperiments corre- sponding to the background-only and signal-plus-back- ground hypotheses. Both the expected and observed limits are shown in Fig. 7 for the GGM model, and in Fig. 8 for the dark-sector model, where SRhigh is used for the GGM model and SRlow is used for the dark-sector model. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … The dashed horizontal lines represent the values of the cross section times branching fractions predicted by simulation, with mH ¼ 125 GeV, mHD ¼ 300 GeV, σðpp →HÞ ¼ 44.1 pb and assuming BðH →ZDZDÞ ¼ 10% or 1%. The value of BðZD →μþμ−Þ varies between 0.1475 and 0.1066 for the range mZD ¼ 20–60 GeV. VI. RESULTS 7. The observed and expected 95% C.L. upper limits on the product of cross section and branching ratios for pair production of gluinos, leading to a final state of μþμ−þ X, in the GGM model, as a function of the ˜χ0 1 lifetime, for m˜g ¼ 1100 GeV and three different choices of m˜χ0 1: (a) 300 GeV, (b) 700 GeV, and (c) 1000 GeV. The shaded green (yellow) bands represent the 1σ (2σ) uncertainties in the expected limits. The dashed horizontal line represents the value of the cross section times branching fractions predicted from simulation, with m˜g ¼ 1100 GeV, σðpp →˜g ˜gÞ ¼ 0.1635 pb, Bð˜χ0 1 →Z ˜GÞ ¼ 1.0, and BðZ →μþμ−Þ ¼ 0.03366. 012001-14 012001-14 SEARCH FOR LONG-LIVED PARTICLES IN FINAL … SEARCH FOR LONG-LIVED PARTICLES IN FINAL … In the GGM model with a gluino mass of 1100 GeV and ˜χ0 1 masses of 300, 700, and 1000 GeV, cτ˜χ0 1 values are excluded in the ranges 3.1–1000 cm, 2.6–1500 cm, and 2.9–1800 cm, respectively. The observed limits are about 1.5σ weaker than the expected limits because of the small excess of events observed in SRhigh. In the dark-sector model with a dark-Higgs-boson mass of 300 GeV, BðH →ZDZDÞ ¼ 10% and ZD masses of 20, 40, and 60 GeV, cτZD values are excluded in the ranges 0.3– 2000 cm, 0.9–2400 cm, and 2.1–1100 cm, respectively. These limits are translated into 95% exclusion contours in the plane of the ZD-Z kinetic mixing parameter, ϵ, and the FIG. 9. The observed 95% C.L. excluded regions in the plane of ZD-Z kinetic mixing parameter, ϵ, versus ZD mass, for values of BðH →ZDZDÞ ¼ 1% or 10%, and mHD ¼ 300 GeV. The value of BðZD →μþμ−Þ varies between 0.1475 and 0.1066 for the range mZD ¼ 20–60 GeV. 012001-15 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. Colombia; MSMT CR, MPO CR and VSC CR, Czech Republic; DNRF and DNSRC, Denmark; IN2P3-CNRS, CEA-DRF/IRFU, France; SRNSFG, Georgia; BMBF, HGF, and MPG, Germany; GSRT, Greece; RGC, Hong Kong SAR, China; ISF, I-CORE and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; NWO, Netherlands; RCN, Norway; MNiSW and NCN, Poland; FCT, Portugal; MNE/IFA, Romania; MES of Russia and NRC KI, Russian Federation; JINR; MESTD, Serbia; MSSR, Slovakia; ARRS and MIZŠ, Slovenia; DST/NRF, South Africa; MINECO, Spain; SRC and Wallenberg Foundation, Sweden; SERI, SNSF and Cantons of Bern and Geneva, Switzerland; MOST, Taiwan; TAEK, Turkey; STFC, United Kingdom; DOE and NSF, United States of America. In addition, individual groups and members have received support from BCKDF, the Canada Council, CANARIE, CRC, Compute Canada, FQRNT, and the Ontario Innovation Trust, Canada; EPLANET, ERC, ERDF, FP7, Horizon 2020 and Marie Skłodowska-Curie Actions, European Union; Investissements d’Avenir Labex and Idex, ANR, R´egion Auvergne and Fondation Partager le Savoir, France; DFG and AvH Foundation, Germany; Herakleitos, Thales and Aristeia programmes co-financed by EU-ESF and the Greek NSRF; BSF, GIF and Minerva, Israel; BRF, Norway; CERCA Programme Generalitat de Catalunya, Generalitat Valenciana, Spain; the Royal Society and Leverhulme Trust, United Kingdom. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … The crucial com- puting support from all WLCG partners is acknowledged gratefully, in particular from CERN, the ATLAS Tier-1 facilities at TRIUMF (Canada), NDGF (Denmark, Norway, Sweden), CC-IN2P3 (France), KIT/GridKA (Germany), INFN-CNAF (Italy), NL-T1 (Netherlands), PIC (Spain), ASGC (Taiwan), RAL (UK) and BNL (USA), the Tier-2 facilities worldwide and large non-WLCG resource provid- ers. Major contributors of computing resources are listed in Ref. [97]. ZD mass, and are shown in Fig. 9. Values of ϵ of the order 10−8 are excluded for 20 < mZD < 60 GeV. ZD mass, and are shown in Fig. 9. Values of ϵ of the order 10−8 are excluded for 20 < mZD < 60 GeV. VII. CONCLUSION This article reports on a search for BSM long-lived particles decaying into two muons of opposite-sign electric charge in a sample of pp collisions recorded by the ATLAS detector at the LHC with a center-of-mass energy of ﬃﬃﬃs p ¼ 13 TeV and an integrated luminosity of 32.9 fb−1. The search is performed by identifying dimuon vertices with displacements from the pp interaction point in the range of 1–400 cm and having invariant mass mμμ within one of two signal regions: 20–60 GeV or > 60 GeV. In neither signal region is a significant excess observed in the number of vertices relative to the predicted background. Hence upper limits at 95% confidence level on the product of cross section and branching fraction are calculated, as a function of lifetime, for production of long-lived particles in either a dark-sector model with dark-photon masses in the range 20–60 GeV, produced from decays of the Higgs boson, or in a general gauge-mediated supersymmetric model with a gluino mass of 1100 GeV and neutralino masses in the range 300–1000 GeV. For the models considered, the lower and upper lifetime limits are set from 1 to 2400 cm in cτ, respectively, depending on the targeted model’s parameters. ACKNOWLEDGMENTS [22] ATLAS Collaboration, Search for massive, long-lived par- ticles using multitrack displaced vertices or displaced lepton pairs in pp collisions at ﬃﬃﬃs p ¼ 8 TeV with the ATLAS detector, Phys. Rev. D 92, 072004 (2015). [38] CMS Collaboration, Search for long-lived neutral particles decaying to quark-antiquark pairs in proton-proton colli- sions at ﬃﬃﬃs p ¼ 8 TeV, Phys. Rev. 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Alstaty,99 B. Alvarez Gonzalez,35 D. Álvarez Piqueras,171 M. G. Alviggi,67a,67b B. T. Amadio,18 Y. Amaral Coutinho,78b L. Ambroz,131 C. Amelung,26 D. Amidei,103 S. P. Amor Dos Santos,136a,136c S. Amoroso,35 C. S. Amrouche,52 C. Anastopoulos,146 L. S. Ancu,52 N. Andari,21 T. Andeen,11 C. F. Anders,59b J. K. Anders,20 K. J. Anderson,36 A. Andreazza,66a,66b V. Andrei,59a C. R. Anelli,173 S. Angelidakis,37 I. Angelozzi,118 A. Angerami,38 A. V. Anisenkov,120b,120a A. Annovi,69a C. Antel,59a M. T. Anthony,146 M. Antonelli,49 D. J. A. Antrim,168 F. ACKNOWLEDGMENTS Anulli,70a M. Aoki,79 L. Aperio Bella,35 G. Arabidze,104 Y. Arai,79 J. P. Araque,136a V. Araujo Ferraz,78b R. Araujo Pereira,78b A. T. H. Arce,47 R. E. Ardell,91 F. A. Arduh,86 J-F. Arguin,107 S. Argyropoulos,75 A. J. Armbruster,35 L. J. Armitage,90 A Armstrong,168 O. Arnaez,164 H. Arnold,118 M. Arratia,31 O. Arslan,24 A. Artamonov,109,a G. Artoni,131 S. Artz,97 S. Asai,160 N. Asbah,44 A. Ashkenazi,158 E. M. Asimakopoulou,169 L. Asquith,153 K. Assamagan,29 R. Astalos,28a R. J. Atkin,32a M. Atkinson,170 N. B. Atlay,148 K. Augsten,138 G. Avolio,35 R. Avramidou,58a B. Axen,18 M. K. Ayoub,15a G. Azuelos,107,e A. E. Baas,59a M. J. Baca,21 H. Bachacou,142 K. Bachas,65a,65b M. Backes,131 P. Bagnaia,70a,70b M. Bahmani,82 H. Bahrasemani,149 A. J. Bailey,171 J. T. Baines,141 M. Bajic,39 C. Bakalis,10 O. K. Baker,180 P. J. Bakker,118 D. Bakshi Gupta,93 E. M. Baldin,120b,120a P. Balek,177 F. Balli,142 W. K. Balunas,133 J. Balz,97 E. Banas,82 A. Bandyopadhyay,24 S. Banerjee,178,f A. A. E. Bannoura,179 L. Barak,158 W. M. Barbe,37 E. L. Barberio,102 D. Barberis,53b,53a M. Barbero,99 T. Barillari,113 M-S. Barisits,35 J. Barkeloo,127 T. Barklow,150 N. Barlow,31 R. Barnea,157 S. L. Barnes,58c B. M. Barnett,141 R. M. Barnett,18 Z. Barnovska-Blenessy,58a A. Baroncelli,72a G. Barone,26 A. J. Barr,131 L. Barranco Navarro,171 F. Barreiro,96 J. Barreiro Guimarães da Costa,15a R. Bartoldus,150 A. E. Barton,87 P. Bartos,28a A. Basalaev,134 A. Bassalat,128 R. L. Bates,55 S. J. Batista,164 S. Batlamous,34e J. R. Batley,31 M. Battaglia,143 M. Bauce,70a,70b F. Bauer,142 K. T. Bauer,168 H. S. Bawa,150,g J. B. Beacham,122 M. D. Beattie,87 T. Beau,132 P. H. Beauchemin,167 P. Bechtle,24 H. C. Beck,51 H. P. Beck,20,h K. Becker,50 M. Becker,97 C. Becot,44 A. Beddall,12d A. J. Beddall,12a V. A. Bednyakov,77 M. Bedognetti,118 C. P. Bee,152 T. A. Beermann,35 M. Begalli,78b M. Begel,29 A. Behera,152 J. K. Behr,44 A. S. Bell,92 G. Bella,158 L. Bellagamba,23b A. Bellerive,33 M. Bellomo,157 P. Bellos,9 K. Belotskiy,110 N. L. Belyaev,110 O. Benary,158,a D. Benchekroun,34a M. Bender,112 N. Benekos,10 Y. Benhammou,158 E. Benhar Noccioli,180 J. Benitez,75 D. P. Benjamin,47 O. S. AbouZeid,143 N. L. Abraham,153 H. Abramowicz,158 H. Abreu,157 Y. Abulaiti,6 B. S. Acharya,64a,64b,b S. Adachi,160 L. Adamczyk,81a J. Adelman,119 M. Adersberger,112 A. Adiguzel,12c,c T. Adye,141 A. A. Affolder,143 Y. Afik,157 C. Agheorghiesei,27c J. A. Aguilar-Saavedra,136f,136a F. Ahmadov,77,d G. Aielli,71a,71b S. Akatsuka,83 T. P. A. Åkesson,94 E. Akilli,52 A. V. Akimov,108 G. L. Alberghi,23b,23a J. Albert,173 P. Albicocco,49 M. J. Alconada Verzini,86 S. Alderweireldt,117 M. Aleksa,35 I. N. Aleksandrov,77 C. Alexa,27b T. Alexopoulos,10 M. Alhroob,124 B. ACKNOWLEDGMENTS Cao,158 Y. Cao,170 M. D. M. Capeans Garrido,35 I. Caprini,27b M. Caprini,27b M. Capua,40b,40a R. M. Carbone,38 R. Cardarelli,71a F. C. Cardillo,50 I. Carli,139 T. Carli,35 G. Carlino,67a B. T. Carlson,135 L. Carminati,66a,66b R. M. D. Carney,43a,43b S. Caron,117 E. Carquin,144b S. Carrá,66a,66b V. Canale,67a,67b M. Cano Bret,58c J. Cantero,125 T. Cao,158 Y. Cao,170 M. D. M. Capeans Garrido,35 I. Caprini,27b M. Caprini,27b M. Capua,40b,40a R. M. Carbone,38 R. Cardarelli,71a F. C. Cardillo,50 I. Carli,139 T. Carli,35 G. Carlino,67a B. T. Carlson,135 L. Carminati,66a,66b R. M. D. Carney,43a,43b S. Caron,117 E. Carquin,144b S. Carrá,66a,66b p n,135 L. Carminati,66a,66b R. M. D. Carney,43a,43b S. Caron,117 E. Carquin,144b S. Carrá,66a,66b 35 32b 14 j 164 14 168 G. D. Carrillo-Montoya,35 D. Casadei,32b M. P. Casado,14,j A. F. Casha,164 M. Casolino,14 D. W G. D. Carrillo-Montoya,35 D. Casadei,32b M. P. Casado,14,j A. F. Casha,164 M. Casolino,14 D. W. Casper,168 R. Castelijn,118 F L C till 171 V C till Gi 171 N F C t 136a 136e A C ti i 35 J R C t 130 A C tt i 35 J C d 24 F. L. Castillo,171 V. Castillo Gimenez,171 N. F. Castro,136a,136e A. Catinaccio,35 J. R. Catmore,13 F. L. Castillo,171 V. Castillo Gimenez,171 N. F. Castro,136a,136e A. Catinaccio,35 J. R. Catmore,130 A. Cattai,35 J. Caudron,24 V. Cavaliere,29 E. Cavallaro,14 D. Cavalli,66a M. Cavalli-Sforza,14 V. Cavasinni,69a,69b E. Celebi,12b F. Ceradini,72a,72b L. Cerda Alberich,171 A. S. Cerqueira,78a A. Cerri,153 L. Cerrito,71a,71b F. Cerutti,18 A. Cervelli,23b,23a S. A. Cetin,12b V. Cavaliere,29 E. Cavallaro,14 D. Cavalli,66a M. Cavalli-Sforza,14 V. Cavasinni,69a,69b E. Celebi,12b F. Ceradini,72a,72b L. Cerda Alberich,171 A. S. Cerqueira,78a A. Cerri,153 L. Cerrito,71a,71b F. Cerutti,18 A. Cervelli,23b,23a S. A. Cetin,12b 34 119 57 118 61 170 31 21 V. Cavaliere,29 E. Cavallaro,14 D. Cavalli,66a M. Cavalli-Sforza,14 V. Cavasinni,69a,69b E. Celebi,12b F. Ceradini,72a,72b L. Cerda Alberich,171 A. S. Cerqueira,78a A. Cerri,153 L. Cerrito,71a,71b F. Cerutti,18 A. Cervelli,23b,23a S. A. Cetin,12b A. Chafaq,34a D Chakraborty,119 S. K. Chan,57 W. S. Chan,118 Y. L. Chan,61a P. Chang,170 J. D. Chapman,31 D. G. Charlton,21 A. Chafaq,34a D Chakraborty,119 S. K. Chan,57 W. S. Chan,118 Y. L. Chan,61a P. Chang,170 J. D. Chapman,31 D. G. Charlton,21 C. C. Chau, C. A. Chavez Barajas, S. Che, A. Chegwidden, S. Chekanov, S. V. Chekulaev, G. A. Chelkov, M. A. Chelstowska,35 C. Chen,58a C. H. Chen,76 H. Chen,29 J. Chen,58a J. Chen,38 S. Chen,133 S. J. Chen,15c X. ACKNOWLEDGMENTS Ali,138 G. Alimonti,66a J. Alison,36 S. P. Alkire,145 C. Allaire,128 B. M. M. Allbrooke,153 B. W. Allen,127 P. P. Allport,21 A. Aloisio,67a,67b A. Alonso,39 F. Alonso,86 C. Alpigiani,145 A. A. Alshehri,55 M. I. Alstaty,99 B. Alvarez Gonzalez,35 012001-19 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. dyrev,111 A. E. Bolz,59b M. Bomben,132 M. Bona,90 J. S. Bonilla,127 M. Boonekamp,142 A. Bor A. S. Boldyrev,111 A. E. Bolz,59b M. Bomben,132 M. Bona,90 J. S. Bonilla,127 M. Boonekamp,142 A. Borisov,140 G. Borissov,87 J. Bortfeldt,35 D. Bortoletto,131 V. Bortolotto,71a,61b,61c,71b D. Boscherini,23b M. Bosman,14 J. D. Bossio Sola,30 A. S. Boldyrev,111 A. E. Bolz,59b M. Bomben,132 M. Bona,90 J. S. Bonilla,127 M. Boonekamp,142 A. Borisov,140 G. Borissov,87 J. Bortfeldt,35 D. Bortoletto,131 V. Bortolotto,71a,61b,61c,71b D. Boscherini,23b M. Bosman,14 J. D. Bossio Sola,30 y p G. Borissov,87 J. Bortfeldt,35 D. Bortoletto,131 V. Bortolotto,71a,61b,61c,71b D. Boscherini,23b M. Bosman,14 J. D. Bossio Sola,30 ,34a J. Boudreau,135 E. V. Bouhova-Thacker,87 D. Boumediene,37 C. Bourdarios,128 S. K. Bout K. Bouaouda,34a J. Boudreau,135 E. V. Bouhova-Thacker,87 D. Boumediene,37 C. Bourdarios,128 S. K. Boutle,55 A. Boveia,122 J. Boyd,35 I. R. Boyko,77 A. J. Bozson,91 J. Bracinik,21 N. Brahimi,99 A. Brandt,8 G. Brandt,179 O. Brandt,59a A. Boveia,122 J. Boyd,35 I. R. Boyko,77 A. J. Bozson,91 J. Bracinik,21 N. Brahimi,99 A. Brandt,8 G. Brandt,179 O. Brandt,59a F. Braren,44 U. Bratzler,161 B. Brau,100 J. E. Brau,127 W. D. Breaden Madden,55 K. Brendlinge 44 169 177 97 21 55 L. Brenner,44 R. Brenner,169 S. Bressler,177 B. Brickwedde,97 D. L. Briglin,21 D. Britton,55 D. Britzger,59b I. Brock,24 R. Brock,104 G. Brooijmans,38 T. Brooks,91 W. K. Brooks,144b E. Brost,119 J. H. Broughton,21 P. A. Bruckman de Renstrom,82 L. Brenner,44 R. Brenner,169 S. Bressler,177 B. Brickwedde,97 D. L. Briglin,21 D. Britton,55 D. Britzger,59b I. Brock,24 R B k 104 G B ij 38 T B k 91 W K B k 144b E B 119 J H B h 21 P A B k d R 82 4 G. Brooijmans,38 T. Brooks,91 W. K. Brooks,144b E. Brost,119 J. H. Broughton,21 P. A. Bruckman D. Bruncko,28b A. Bruni,23b G. Bruni,23b L. S. Bruni,118 S. Bruno,71a,71b B. H. Brunt,31 M. Bruschi,23b N. Bruscino,135 P. Bryant,36 L. Bryngemark,44 T. Buanes,17 Q. Buat,35 P. Buchholz,148 A. G. Buckley,55 I. A 130 110 8 119 88 118 M. K. Bugge,130 O. Bulekov,110 D. Bullock,8 T. J. Burch,119 S. Burdin,88 C. D. Burgard,118 A. M. Burger,5 B. ACKNOWLEDGMENTS Burghgrave,119 82 141 45 131 50 97 51 55 M. K. Bugge,130 O. Bulekov,110 D. Bullock,8 T. J. Burch,119 S. Burdin,88 C. D. Burgard,118 A. M. Burger,5 B. Burghgrave,119 K. Burka,82 S. Burke,141 I. Burmeister,45 J. T. P. Burr,131 D. Büscher,50 V. Büscher,97 E. Buschmann,51 P. Bussey,55 M. K. Bugge,130 O. Bulekov,110 D. Bullock,8 T. J. Burch,119 S. Burdin,88 C. D. Burgard,118 A. M. Burger,5 B. Burghgrave,119 K. Burka,82 S. Burke,141 I. Burmeister,45 J. T. P. Burr,131 D. Büscher,50 V. Büscher,97 E. Buschmann,51 P. Bussey,55 K. Burka, S. Burke, I. Burmeister, J. T. P. Burr, D. Büscher, V. Büscher, E. Buschmann, P. Bussey, J. M. Butler,25 C. M. Buttar,55 J. M. Butterworth,92 P. Butti,35 W. Buttinger,35 A. Buzatu,155 A. R. Buzykaev,120b,120a G Cabras 23b,23a S Cabrera Urbán 171 D Caforio 138 H Cai 170 V M M Cairo 2 O Cakir 4a N Calace 52 P Calafiura 18 y J. M. Butler,25 C. M. Buttar,55 J. M. Butterworth,92 P. Butti,35 W. Buttinger,35 A. Buzatu,155 A. R. Buzykaev,120b,120a G. Cabras,23b,23a S. Cabrera Urbán,171 D. Caforio,138 H. Cai,170 V. M. M. Cairo,2 O. Cakir,4a N. Calace,52 P. Calafiura,18 J. M. Butler,25 C. M. Buttar,55 J. M. Butterworth,92 P. Butti,35 W. Buttinger,35 A. Buzatu,155 A. R. Buzykaev,120b,120a G. Cabras,23b,23a S. Cabrera Urbán,171 D. Caforio,138 H. Cai,170 V. M. M. Cairo,2 O. Cakir,4a N. Calace,52 P. Calafiura,18 99 132 63 40b 40 78b 96 37 37 A. Calandri,99 G. Calderini,132 P. Calfayan,63 G. Callea,40b,40a L. P. Caloba,78b S. Calvente Lopez,96 D. Calvet,37 S. Calvet,37 T P Calvet 152 M Calvetti 69a,69b R Camacho Toro 132 S Camarda 35 P Camarri 71a,71b D Cameron 130 A. Calandri,99 G. Calderini,132 P. Calfayan,63 G. Callea,40b,40a L. P. Caloba,78b S. Calvente Lopez,96 D. Calvet,37 S. Calvet,37 T. P. Calvet,152 M. Calvetti,69a,69b R. Camacho Toro,132 S. Camarda,35 P. Camarri,71a,71b D. Cameron,130 R. Caminal Armadans,100 C. Camincher,35 S. Campana,35 M. Campanelli,92 A. Camplani,39 A. Campoverde,148 V. Canale,67a,67b M. Cano Bret,58c J. Cantero,125 T. Cao,158 Y. Cao,170 M. D. M. Capeans Garrido,35 I. Caprini,27b M. Caprini,27b M. Capua,40b,40a R. M. Carbone,38 R. Cardarelli,71a F. C. Cardillo,50 I. Carli,139 T. Carli,35 G. Carlino,67a B T Carlson 135 L Carminati 66a,66b R M D Carney 43a,43b S Caron 117 E Carquin 144b S Carrá 66a,66b , , , , , , R. Caminal Armadans,100 C. Camincher,35 S. Campana,35 M. Campanelli,92 A. Camplani,39 A. Campoverde,148 V. Canale,67a,67b M. Cano Bret,58c J. Cantero,125 T. ACKNOWLEDGMENTS Chen,15b,l 80 44 103 15d 77 140 34 5 C. Chen,58a C. H. Chen,76 H. Chen,29 J. Chen,58a J. Chen,38 S. Chen,133 S. J. Chen,15c X. Che . Chen,44 H. C. Cheng,103 H. J. Cheng,15d A. Cheplakov,77 E. Cheremushkina,140 R. Cherkaoui El Y. Chen,80 Y-H. Chen,44 H. C. Cheng,103 H. J. Cheng,15d A. Cheplakov,77 E. Cheremushkina,140 E. Cheu,7 K. Cheung,62 L. Chevalier,142 V. Chiarella,49 G. Chiarelli,69a G. Chiodini,65a A. S. Chisholm,35 A. Chitan,27b I. Chiu,160 Y. H. Chiu,173 M. V. Chizhov,77 K. Choi,63 A. R. Chomont,128 S. Chouridou,159 Y. S. Chow,118 V. Christodoulou,92 M. C. Chu,61a J. Chudoba,137 A. J. Chuinard,101 J. J. Chwastowski,82 L. Chytka,126 D. Cinca,45 E. Cheu, K. Cheung, L. Chevalier, V. Chiarella, G. Chiarelli, G. Chiodini, A. S. Chisholm, A. Chitan, I. Chiu,160 Y. H. Chiu,173 M. V. Chizhov,77 K. Choi,63 A. R. Chomont,128 S. Chouridou,159 Y. S. Chow,118 V Ch i t d l 92 M C Ch 61a J Ch d b 137 A J Ch i d 101 J J Ch t ki 82 L Ch tk 126 D Ci 45 ou,92 M. C. Chu,61a J. Chudoba,137 A. J. Chuinard,101 J. J. Chwastowski,82 L. Chytka,126 D. Ci V. Cindro,89 I. A. Cioară,24 A. Ciocio,18 F. Cirotto,67a,67b Z. H. Citron,177 M. Citterio,66a A. Clark,52 M. R. Clark,38 8 C. Clement,43a,43b Y. Coadou,99 M. Cobal,64a,64c A. Coccaro,53b,53a J. Cochran,76 A. E. C. Co 5 5 P. J. Clark,48 C. Clement,43a,43b Y. Coadou,99 M. Cobal,64a,64c A. Coccaro,53b,53a J. Cochran P. J. Clark, C. Clement, Y. Coadou, M. Cobal, A. Coccaro, J. Cochran, A. E. C. Coimbra, L. Colasurdo,117 B. Cole,38 A. P. Colijn,118 J. Collot,56 P. Conde Muiño,136a,136b E. Coniavitis,50 S. H. Connell,32b L. Colasurdo,117 B. Cole,38 A. P. Colijn,118 J. Collot,56 P. Conde Muiño,136a,136b E. Coniavitis,50 S. H. Connell,32b 98 27b 67 131 172 164 L. Colasurdo,117 B. Cole,38 A. P. Colijn,118 J. Collot,56 P. Conde Muiño,136a,136b E. Coniav 17 B. Cole,38 A. P. Colijn,118 J. Collot,56 P. Conde Muiño,136a,136b E. Coniavitis,50 S. H. Conn I. A. Connelly,98 S. Constantinescu,27b F. Conventi,67a,m A. M. Cooper-Sarkar,131 F. Cormier,172 K. J. R. Cormier,164 M. Corradi,70a,70b E. E. Corrigan,94 F. Corriveau,101,n A. Cortes-Gonzalez,35 M. J. Costa,171 D. Costanzo,146 G. Cottin,31 I. A. Connelly, S. Constantinescu, F. Conventi, A. M. Cooper Sarkar, F. Cormier, K. J. R. Cormier, M. Corradi,70a,70b E. E. Corrigan,94 F. Corriveau,101,n A. Cortes-Gonzalez,35 M. J. Costa,171 D. Costanzo,146 G. Cottin,31 g B. E. Cox,98 J. M. Benoit,52 J. R. Bensinger,26 S. Bentvelsen,118 L. Beresford,131 M. Beretta,49 D. Berge,44 E. Bergeaas Kuutmann,169 N. Berger,5 L. J. Bergsten,26 J. Beringer,18 S. Berlendis,7 N. R. Bernard,100 G. Bernardi,132 C. Bernius,150 F. U. Bernlochner,24 T. Berry,91 P. Berta,97 C. Bertella,15a G. Bertoli,43a,43b I. A. Bertram,87 G. J. Besjes,39 ACKNOWLEDGMENTS Crane,98 K. Cranmer,121 S. J. Crawley,55 R. A. Creager,133 G. Cree,33 S. Cr´ep´e-R G. Cowan,91 B. E. Cox,98 J. Crane,98 K. Cranmer,121 S. J. Crawley,55 R. A. Creager,133 G. Cree,33 S. Cr´ep´e-Renaudin,56 F. Crescioli,132 M. Cristinziani,24 V. Croft,121 G. Crosetti,40b,40a A. Cueto,96 T. Cuhadar Donszelmann,146 F. Crescioli,132 M. Cristinziani,24 V. Croft,121 G. Crosetti,40b,40a A. Cueto,96 T. Cuhadar Donszelmann,146 150 M. Curatolo,49 J. Cúth,97 S. Czekierda,82 P. Czodrowski,35 M. J. Da Cunha Sargedas De Sou A. R. Cukierman,150 M. Curatolo,49 J. Cúth,97 S. Czekierda,82 P. Czodrowski,35 M. J. Da Cunha Sargedas De Sousa,58b,136b C. Da Via,98 W. Dabrowski,81a T. Dado,28a,i S. Dahbi,34e T. Dai,103 F. Dallaire,107 C. Dallapiccola,100 M. Dam,39 G. D’amen,23b,23a J. Damp,97 J. R. Dandoy,133 M. F. Daneri,30 N. P. Dang,178,f N.D Dann,98 M. Danninger,172 V. Dao,35 , , , , , g , C. Da Via,98 W. Dabrowski,81a T. Dado,28a,i S. Dahbi,34e T. Dai,103 F. Dallaire,107 C. Dallapiccola,100 M. Dam,39 G D’amen 23b,23a J Damp 97 J R Dandoy 133 M F Daneri 30 N P Dang 178,f N D Dann 98 M Danninger 172 V Dao 35 , , , , , g , C. Da Via,98 W. Dabrowski,81a T. Dado,28a,i S. Dahbi,34e T. Dai,103 F. Dallaire,107 C. Dallapiccola,100 M. Dam,39 012001-20 PHYS. REV. D 99, 012001 (2019) SEARCH FOR LONG-LIVED PARTICLES IN FINAL … SEARCH FOR LONG-LIVED PARTICLES IN FINAL … G. Darbo,53b S. Darmora,8 O. Dartsi,5 A. Dattagupta,127 T. Daubney,44 S. D’Auria,55 W. Davey,24 C. David,44 T. Davidek,139 D. R. Davis,47 E. Dawe,102 I. Dawson,146 K. De,8 R. De Asmundis,67a A. De Benedetti,124 S. De Castro,23b,23a G. Darbo,53b S. Darmora,8 O. Dartsi,5 A. Dattagupta,127 T. Daubney,44 S. D’Auria,55 W. Davey,24 C. David,44 T. Davidek,139 D. R. Davis,47 E. Dawe,102 I. Dawson,146 K. De,8 R. De Asmundis,67a A. De Benedetti,124 S. De Castro,23b,23a S. De Cecco,70a,70b N. De Groot,117 P. de Jong,118 H. De la Torre,104 F. De Lorenzi,76 A. De Maria,51,o D. De Pedis,70a A. De Salvo,70a U. De Sanctis,71a,71b A. De Santo,153 K. De Vasconcelos Corga,99 J. B. De Vivie De Regie,128 S. De Cecco,70a,70b N. De Groot,117 P. de Jong,118 H. De la Torre,104 F. De Lorenzi,76 A. De Maria,51,o D. De Pedis,70a A. De Salvo,70a U. De Sanctis,71a,71b A. De Santo,153 K. De Vasconcelos Corga,99 J. B. De Vivie De Regie,128 g g C. M. Delitzsch,7 M. Della Pietra,67a,67b D. Della Volpe,52 A. Dell’Acqua,35 L. Dell’Asta,25 M. Delmastro,5 C. Delporte,128 P A D l 56 D A D M 164 S D 180 M D i h 77 S P D i 140 D D i k 118 L D’E 132 A. Di Simone,50 R. Di Sipio,164 D. Di Valentino,33 C. Diaconu,99 M. Diamond,164 F. A. Dias,39 T. Dias Do Vale,136a 5 M. A. Diaz,144a J. Dickinson,18 E. B. Diehl,103 J. Dietrich,19 S. Díez Cornell,44 A. Dimitrievska,18 J. Dingfelder,24 F. Dittus,35 F. Djama,99 T. Djobava,156b J. I. Djuvsland,59a M. A. B. Do Vale,78c M. Dobre,27b D. Dodsworth,26 C. Doglioni,94 j j j J. Dolejsi,139 Z. Dolezal,139 M. Donadelli,78d J. Donini,37 A. D’onofrio,90 M. D’Onofrio,88 J. D 86 55 51 149 51 10 58b M. T. Dova,86 A. T. Doyle,55 E. Drechsler,51 E. Dreyer,149 T. Dreyer,51 M. Dris,10 Y. Du,58b J. Duarte-Campderros,158 F Dubinin 108 M Dubovsky 28a A Dubreuil 52 E Duchovni 177 G Duckeck 112 A Ducourthial 132 O A Ducu 107,p D. Duda,113 A. Dudarev,35 A. C. Dudder,97 E. M. Duffield,18 L. Duflot,128 M. Dührssen,35 C. Dülsen,179 M. Dumancic,177 A. E. Dumitriu,27b,q A. K. Duncan,55 M. Dunford,59a A. Duperrin,99 H. Duran Yildiz,4a M. Düren,54 A. Durglishvili,156b D. Duschinger,46 B. Dutta,44 D. Duvnjak,1 M. Dyndal,44 S. Dysch,98 B. S. Dziedzic,82 C. Eckardt,44 K. M. Ecker,113 C. García,171 J. E. García Navarro,171 J. A. García Pascual,15a M. Garcia-Sciveres,18 R. W. Gardner,36 N. Garelli,150 V. Garonne,130 K. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Ha 112 14 48 142 20 104 J. D. Hansen, M. C. Hansen, P. H. Hansen, K. Hara, A. S. Hard, T. Harenberg, S. Harkusha, P. F. Harrison, N. M. Hartmann,112 Y. Hasegawa,147 A. Hasib,48 S. Hassani,142 S. Haug,20 R. Hauser,104 L. Hauswald,46 L. B. Havener,38 138 21 35 104 152 131 90 N. M. Hartmann,112 Y. Hasegawa,147 A. Hasib,48 S. Hassani,142 S. Haug,20 R. Hauser,104 L. Hauswald,46 L. B. Havener,38 M. Havranek,138 C. M. Hawkes,21 R. J. Hawkings,35 D. Hayden,104 C. Hayes,152 C. P. Hays,131 J. M. Hays,90 H. S. Hayward,88 S. J. Haywood,141 M. P. Heath,48 V. Hedberg,94 L. Heelan,8 S. Heer,24 K. K. Heidegger,50 J. Heilman,33 S. Heim,44 T. Heim,18 B. Heinemann,44,aa J. J. Heinrich,112 L. Heinrich,121 C. Heinz,54 J. Hejbal,137 L. Helary,35 A. Held,172 S. Hellesund,130 S. Hellman,43a,43b C. Helsens,35 R. C. W. Henderson,87 Y. Heng,178 S. Henkelmann,172 5 5 S. Hellesund,130 S. Hellman,43a,43b C. Helsens,35 R. C. W. Henderson,87 Y. Heng,178 S. Henkelmann,172 A. M. Henriques Correia,35 G. H. Herbert,19 H. Herde,26 V. Herget,174 Y. Hernández Jim´enez 112 35 133 92 165 A. M. Henriques Correia, G. H. Herbert, H. Herde, V. Herget, Y. Hernández Jimenez, H. Herr, G. Herten, R. Hertenberger,112 L. Hervas,35 T. C. Herwig,133 G. G. Hesketh,92 N. P. Hessey,165a J. W. Hetherly,41 S. Higashino,79 q g R. Hertenberger,112 L. Hervas,35 T. C. Herwig,133 G. G. Hesketh,92 N. P. Hessey,165a J. W. Hetherly,41 S. Higashino,79 g g y E. Higón-Rodriguez,171 K. Hildebrand,36 E. Hill,173 J. C. Hill,31 K. K. Hill,29 K. H. Hiller,44 E. Higón-Rodriguez,171 K. Hildebrand,36 E. Hill,173 J. C. Hill,31 K. K. Hill,29 K. H. Hiller,44 S. J. Hillier,21 M. Hils,46 I. Hinchliffe,18 M. Hirose,129 D. Hirschbuehl,179 B. Hiti,89 O. Hladik,137 D. R. Hlaluku,32c X. Hoad,48 J. Hobbs,152 N. Hod,165a M. C. Hodgkinson,146 A. Hoecker,35 M. R. Hoeferkamp,116 F. Hoenig,112 D. Hohn,24 D. Hohov,128 I. Hinchliffe,18 M. Hirose,129 D. Hirschbuehl,179 B. Hiti,89 O. Hladik,137 D. R. Hlaluku,32c X. Hoad,48 J. Hobbs,152 N. Hod,165a M. C. Hodgkinson,146 A. Hoecker,35 M. R. Hoeferkamp,116 F. Hoenig,112 D. Hohn,24 D. Hohov,128 T. R. Holmes,36 M. Holzbock,112 M. Homann,45 S. Honda,166 T. Honda,79 T. M. Hong,135 A. Hönle,113 B. H. Hooberman,170 W. H. Hopkins,127 Y. Horii,115 P. Horn,46 A. J. Horton,149 L. A. Horyn,36 J-Y. Hostachy,56 A. Hostiuc,145 S. Hou,155 A. Hoummada,34a J. Howarth,98 J. Hoya,86 M. Hrabovsky,126 J. Hrdinka,35 I. Hristova,19 J. Hrivnac,128 A. Hrynevich,106 T. Hryn’ova,5 P. J. Hsu,62 S.-C. Hsu,145 Q. D. Goujdami,34c A. G. Goussiou,145 N. Govender,32b,u C. Goy,5 E. Gozani,157 I. Grabowska-Bold,81a P. O. J. Gradin,169 E. C. Graham,88 J. Gramling,168 E. Gramstad,130 S. Grancagnolo,19 V. Gratchev,134 P. M. Gravila,27f C. Gray,55 H. M. Gray,18 Z. D. Greenwood,93,v C. Grefe,24 K. Gregersen,92 I. M. Gregor,44 P. Grenier,150 K. Grevtsov,44 J. Griffiths,8 A. A. Grillo,143 K. Grimm,150 S. Grinstein,14,w Ph. Gris,37 J.-F. Grivaz,128 S. Groh,97 E. Gross,177 J. Grosse-Knetter,51 G. C. Grossi,93 Z. J. Grout,92 C. Grud,103 A. Grummer,116 L. Guan,103 W. Guan,178 J. Guenther,35 A. Guerguichon,128 F. Guescini,165a D. Guest,168 R. Gugel,50 B. Gui,122 T. Guillemin,5 S. Guindon,35 U. Gul,55 C. Gumpert,35 J. Guo,58c W. Guo,103 Y. Guo,58a,x Z. Guo,99 R. Gupta,41 S. Gurbuz,12c G. Gustavino,124 B. J. Gutelman,157 P. Gutierrez,124 C. Gutschow,92 C. Guyot,142 M. P. Guzik,81a C. Gwenlan,131 C. B. Gwilliam,88 A. Haas,121 C. Haber,18 H. K. Hadavand,8 N. Haddad,34e A. Hadef,58a SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Gasnikova,44 A. Gaudiello,53b,53a G. Gaudio,68a I. L. Gavrilenko,108 A. Gavrilyuk,109 C. Gay,172 G. Gaycken,24 E. N. Gazis,10 C. N. P. Gee,141 J. Geisen,51 M. Geisen,97 M. P. Geisler,59a K. Gellerstedt,43a,43b C. Gemme,53b M. H. Genest,56 C. Geng,103 S. Gentile,70a,70b C. Gentsos,159 S. George,91 D. Gerbaudo,14 G. Gessner,45 S. Ghasemi,148 M. Ghasemi Bostanabad,173 M. Ghneimat,24 B. Giacobbe,23b S. Giagu,70a,70b N. Giangiacomi,23b,23a P. Giannetti,69a S. M. Gibson,91 M. Gignac,143 D. Gillberg,33 G. Gilles,179 D. M. Gingrich,3,e M. P. Giordani,64a,64c F. M. Giorgi,23b P. F. Giraud,142 P. Giromini,57 G. Giugliarelli,64a,64c D. Giugni,66a F. Giuli,131 M. Giulini,59b S. Gkaitatzis,159 I. Gkialas,9,t E. L. Gkougkousis,14 P. Gkountoumis,10 L. K. Gladilin,111 C. Glasman,96 J. Glatzer,14 P. C. F. Glaysher,44 A. Glazov,44 M. Goblirsch-Kolb,26 J. Godlewski,82 S. Goldfarb,102 T. Golling,52 D. Golubkov,140 A. Gomes,136a,136b,136d R. Goncalves Gama,78a R. Gonçalo,136a G. Gonella,50 L. Gonella,21 A. Gongadze,77 F. Gonnella,21 J. L. Gonski,57 E. L. Gkougkousis, P. Gkountoumis, L. K. Gladilin, C. Glasman, J. Glatzer, P. C. F. Glaysher, A. Glazov, M. Goblirsch-Kolb,26 J. Godlewski,82 S. Goldfarb,102 T. Golling,52 D. Golubkov,140 A. Gomes,136a,136b,136d R G l G 78a R G l 136a G G ll 50 L G ll 21 A G d 77 F G ll 21 J L G ki 57 ç g S. González de la Hoz,171 S. Gonzalez-Sevilla,52 L. Goossens,35 P. A. Gorbounov,109 H. A. Gordon,29 B. Gorini,35 E. Gorini,65a,65b A. Gorišek,89 A. T. Goshaw,47 C. Gössling,45 M. I. Gostkin,77 C. A. Gottardo,24 C. R. Goudet,128 S. González de la Hoz,171 S. Gonzalez-Sevilla,52 L. Goossens,35 P. A. Gorbounov,109 H. A. Gordon,29 B. Gorini,35 E G i i 65a,65b A G iš k 89 A T G h 47 C Gö li 45 M I G tki 77 C A G tt d 24 C R G d t 128 012001-21 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. , , , , , , , , S. Hageböck,24 M. Hagihara,166 H. Hakobyan,181,a M. Haleem,174 J. Haley,125 G. Halladjian,104 G. D. Hallewell,99 K. Hamacher,179 P. Hamal,126 K. Hamano,173 A. Hamilton,32a G. N. Hamity,146 K. Han,58a,y L. Han,58a S. Han,15d K. Hamacher,179 P. Hamal,126 K. Hamano,173 A. Hamilton,32a G. N. Hamity,146 K. Han,58a,y L y K. Hanagaki,79,z M. Hance,143 D. M. Handl,112 B. Haney,133 R. Hankache,132 P. Hanke,59a E. Hansen,94 J. B. Hansen,39 J. D. Hansen,39 M. C. Hansen,24 P. H. Hansen,39 K. Hara,166 A. S. Hard,178 T. Harenberg,179 S. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Jakobs,50 S. Jakobsen,74 T. Jakoubek,137 D. O. Jamin,125 D. K. Jana,93 R. Jansky,52 J. Janssen,24 M. Janus,51 P. A. Janus,81a G. Jarlskog,94 N. Javadov,77,d T. Javůrek,50 M. Javurkova,50 F. Jeanneau,142 L. Jeanty,18 J. Jejelava,156a,dd A. Jelinskas,175 P. Jenni,50,ee J. Jeong,44 C. Jeske,175 S J´ ´ l 5 H Ji 178 J Ji 152 H Ji 76 Y Ji 58a Z Ji 150 ff S Ji i 50 F A Ji M l 37 J Ji P 171 S. Jin,15c A. Jinaru,27b O. Jinnouchi,162 H. Jivan,32c P. Johansson,146 K. A. Johns,7 C. A. Johnson,63 W. J. Johnson,145 K. Jon-And,43a,43b R. W. L. Jones,87 S. D. Jones,153 S. Jones,7 T. J. Jones,88 J. Jongmanns,59a P. M. Jorge,136a,136b J. Jovicevic,165a X. Ju,178 J. J. Junggeburth,113 A. Juste Rozas,14,w A. Kaczmarska,82 M. Kado,128 H. Kagan,122 M. Kagan,150 T. Kaji,176 E. Kajomovitz,157 C. W. Kalderon,94 A. Kaluza,97 S. Kama,41 A. Kamenshchikov,140 L. Kanjir,89 Y. Kano,160 110 79 121 178 32 165b 10 77 T. Kaji,176 E. Kajomovitz,157 C. W. Kalderon,94 A. Kaluza,97 S. Kama,41 A. Kamenshchikov,140 L. Kanjir,89 Y. Kano,160 V. A. Kantserov,110 J. Kanzaki,79 B. Kaplan,121 L. S. Kaplan,178 D. Kar,32c M. J. Kareem,165b E. Karentzos,10 S. N. Karpov,77 Z. M. Karpova,77 V. Kartvelishvili,87 A. N. Karyukhin,140 K. Kasahara,166 L. Kashif,178 R. D. Kass,122 A. Kastanas,151 Y. Kataoka,160 C. Kato,160 J. Katzy,44 K. Kawade,80 K. Kawagoe,85 T. Kawamoto,160 G. Kawamura,51 E. F. Kay,88 V. F. Kazanin,120b,120a R. Keeler,173 R. Kehoe,41 J. S. Keller,33 E. Kellermann,94 J. J. Kempster,21 J. Kendrick,21 O. Kepka,137 S Kersten 179 B P Kerševan 89 R A Keyes 101 M Khader 170 F Khalil Zada 13 A Khanov 125 A G Kharlamov 120b,120a V. A. Kantserov,110 J. Kanzaki,79 B. Kaplan,121 L. S. Kaplan,178 D. Kar,32c M. J. Kareem,165b E. K Z. M. Karpova,77 V. Kartvelishvili,87 A. N. Karyukhin,140 K. Kasahara,166 L. Kashif,178 R. D. Kass,122 A. Kastanas,151 Y. Kataoka,160 C. Kato,160 J. Katzy,44 K. Kawade,80 K. Kawagoe,85 T. Kawamoto,160 G. Kawamura,51 E. F. Kay,88 V. F. Kazanin,120b,120a R. Keeler,173 R. Kehoe,41 J. S. Keller,33 E. Kellermann,94 J. J. Kempster,21 J. Kendrick,21 O. Kepka,137 S. Kersten,179 B. P. Kerševan,89 R. A. Keyes,101 M. Khader,170 F. Khalil-Zada,13 A. Khanov,125 A. G. Kharlamov,120b,120a T. Kharlamova,120b,120a A. Khodinov,163 T. J. Khoo,52 E. Khramov,77 J. Khubua,156b S. Kido,80 M. Kiehn,52 C. R. Kilby,91 S. H. Kim,166 Y. K. Kim,36 N. Kimura,64a,64c O. M. Kind,19 B. T. King,88 D. Kirchmeier,46 J. Kirk,141 A. E. Kiryunin,113 T. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Hu,29 S. Hu,58c Y. Huang,15a Z. Hubacek,138 F. Hubaut,99 M. Huebner,24 F. Huegging,24 T. B. Huffman,131 E. W. Hughes,38 M. Huhtinen,35 R. F. H. Hunter,33 P. Huo,152 A. M. Hupe,33 N. Huseynov,77,d J. Huston,104 J. Huth,57 R. Hyneman,103 G. Iacobucci,52 G. Iakovidis,29 I. Ibragimov,148 L. Iconomidou-Fayard,128 Z. Idrissi,34e P. Iengo,35 R. Ignazzi,39 O. Igonkina,118,bb R. Iguchi,160 T. Iizawa,52 Y. Ikegami,79 M. Ikeno,79 D. Iliadis,159 N. Ilic,150 F. Iltzsche,46 G. Introzzi,68a,68b M. Iodice,72a K. Iordanidou,38 V. Ippolito,70a,70b M. F. Isacson,169 N. Ishijima,129 M. Ishino,160 M. Ishitsuka,162 W. Islam,125 C. Issever,131 S. Istin,12c,cc F. Ito,166 , j , , , , , , , J. M. Iturbe Ponce,61a R. Iuppa,73a,73b A. Ivina,177 H. Iwasaki,79 J. M. Izen,42 V. Izzo,67a S. Jabbar,3 P. Jacka,137 P. Jackson,1 R. M. Jacobs,24 V. Jain,2 G. Jäkel,179 K. B. Jakobi,97 K. Jakobs,50 S. Jakobsen,74 T. Jakoubek,137 D. O. Jamin,125 D. K. Jana,93 R. Jansky,52 J. Janssen,24 M. Janus,51 P. A. Janus,81a G. Jarlskog,94 N. Javadov,77,d T. Javůrek,50 M. Javurkova,50 F. Jeanneau,142 L. Jeanty,18 J. Jejelava,156a,dd A. Jelinskas,175 P. Jenni,50,ee J. Jeong,44 C. Jeske,175 S. J´ez´equel,5 H. Ji,178 J. Jia,152 H. Jiang,76 Y. Jiang,58a Z. Jiang,150,ff S. Jiggins,50 F. A. Jimenez Morales,37 J. Jimenez Pena,171 S Ji 15c A Ji 27b O Ji hi 162 H Ji 32c P J h 146 K A J h 7 C A J h 63 W J J h 145 J. M. Iturbe Ponce,61a R. Iuppa,73a,73b A. Ivina,177 H. Iwasaki,79 J. M. Izen,42 V. Izzo,67a S. Jabbar,3 P. Jacka,137 P. Jackson,1 R. M. Jacobs,24 V. Jain,2 G. Jäkel,179 K. B. Jakobi,97 K. Jakobs,50 S. Jakobsen,74 T. Jakoubek,137 D. O. Jamin,125 D. K. Jana,93 R. Jansky,52 J. Janssen,24 M. Janus,51 P. A. Janus,81a G. Jarlskog,94 N. Javadov,77,d T. Javůrek,50 M. Javurkova,50 F. Jeanneau,142 L. Jeanty,18 J. Jejelava,156a,dd A. Jelinskas,175 P. Jenni,50,ee J. Jeong,44 C. Jeske,175 S. J´ez´equel,5 H. Ji,178 J. Jia,152 H. Jiang,76 Y. Jiang,58a Z. Jiang,150,ff S. Jiggins,50 F. A. Jimenez Morales,37 J. Jimenez Pena,171 S. Jin,15c A. Jinaru,27b O. Jinnouchi,162 H. Jivan,32c P. Johansson,146 K. A. Johns,7 C. A. Johnson,63 W. J. Johnson,145 K. Jon-And,43a,43b R. W. L. Jones,87 S. D. Jones,153 S. Jones,7 T. J. Jones,88 J. Jongmanns,59a P. M. Jorge,136a,136b J. Jovicevic,165a X. Ju,178 J. J. Junggeburth,113 A. Juste Rozas,14,w A. Kaczmarska,82 M. Kado,128 H. Kagan,122 M. Kagan,150 176 157 94 97 41 140 89 160 R. M. Jacobs,24 V. Jain,2 G. Jäkel,179 K. B. Jakobi,97 K. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Kishimoto,160 D. Kisielewska,81a V. Kitali,44 O. Kivernyk,5 E. Kladiva,28b T. Klapdor-Kleingrothaus,50 M. H. Klein,103 012001-22 012001-22 PHYS. REV. D 99, 012001 (2019) SEARCH FOR LONG-LIVED PARTICLES IN FINAL … SEARCH FOR LONG-LIVED PARTICLES IN FINAL … C. Leggett,18 N. Lehmann,179 G. Lehmann Miotto,35 W. A. Leight,44 A. Leisos,159,hh M. A. L. Leite,78d R. Leitner,139 177 51 92 24 35 7 69 48 G. Lerner,153 C. Leroy,107 R. Les,164 A. A. J. Lesage,142 C. G. Lester,31 M. Levchenko,134 J. Levêque,5 D. Levin,103 L. J. Levinson,177 D. Lewis,90 B. Li,103 C-Q. Li,58a H. Li,58b L. Li,58c Q. Li,15d Q. Y. Li,58a S. Li,58d,58c X. Li,58c Y. Li,148 Z. Liang,15a B. Liberti,71a A. Liblong,164 K. Lie,61c S. Liem,118 A. Limosani,154 C. Y. Lin,31 K. Lin,104 T. H. Lin,97 R. A. Linck,63 B. E. Lindquist,152 A. L. Lionti,52 E. Lipeles,133 A. Lipniacka,17 M. Lisovyi,59b T. M. Liss,170,ii A. Lister,172 A. M. Litke,143 J. D. Little,8 B. Liu,76 B.L Liu,6 H. B. Liu,29 H. Liu,103 J. B. Liu,58a J. K. K. Liu,131 K. Liu,132 M. Liu,58a A. M. Litke,143 J. D. Little,8 B. Liu,76 B.L Liu,6 H. B. Liu,29 H. Liu,103 J. B. Liu,58a J. K. K. Liu,131 K. Liu,132 M. Liu,58a . , . , . . , . W. , . v , . , J. , S. . y , C. . , F. Lo Sterzo,41 E. M. Lobodzinska,44 P. Loch,7 F. K. Loebinger,98 A. Loesle,50 K. M. Loew,26 T. Lohse,19 K. Lohwasser,146 M L k ji k 137 B A L 25 J D L 170 R E L 87 L L 65a 65b K A L 122 J A L 144b I L P 14 F. Lo Sterzo,41 E. M. Lobodzinska,44 P. Loch,7 F. K. Loebinger,98 A. Loesle,50 K. M. Loew,26 T. Lohse,19 K. Lohwasser,146 137 25 170 87 65a 65b 122 144b 14 j , g, g, g, g , p , p , p , A. Lopez Solis,146 J. Lorenz,112 N. Lorenzo Martinez,5 M. Losada,22 P. J. Lösel,112 X. Lou,44 X. Lou,15a A. Lounis,128 J Love 6 P A Love 87 J J Lozano Bahilo 171 H Lu 61a M Lu 58a N Lu 103 Y J Lu 62 H J Lubatti 145 C Luci 70a,70b A. Lopez Solis,146 J. Lorenz,112 N. Lorenzo Martinez,5 M. Losada,22 P. J. Lösel,112 X. Lou,44 X. Lou,15a A. Lounis,128 A. Lopez Solis,146 J. Lorenz,112 N. Lorenzo Martinez,5 M. Losada,22 P. J. Lösel,112 X. Lou,44 X. Lou,15a A. Lounis,128 6 87 171 61 58 103 62 145 70 70b A. Lucotte,56 C. Luedtke,50 F. Luehring,63 I. Luise,132 W. Lukas,74 L. Luminari,70a B. T. Klioutchnikova,35 F. F. Klitzner,112 P. Kluit,118 S. Kluth,113 E. Kneringer,74 E. B. F. G. Knoops,99 A. Knue,50 A. Kobayashi,160 D. Kobayashi,85 T. Kobayashi,160 M. Kobel,46 M. Kocian,150 P. Kodys,139 T. Koffas,33 E. Koffeman,118 N. M. Köhler,113 T. Koi,150 M. Kolb,59b I. Koletsou,5 T. Kondo,79 N. Kondrashova,58c K. Köneke,50 A. C. König,117 T. Kono,79 R. Konoplich,121,gg V. Konstantinides,92 N. Konstantinidis,92 B. Konya,94 R. Kopeliansky,63 S. Koperny,81a K. Korcyl,82 K. Kordas,159 A. Korn,92 I. Korolkov,14 E. V. Korolkova,146 O. Kortner,113 S. Kortner,113 T. Kosek,139 V. V. Kostyukhin,24 A. Kotwal,47 A. Koulouris,10 A. Kourkoumeli-Charalampidi,68a,68b C. Kourkoumelis,9 E. Kourlitis,146 V. Kouskoura,29 A. B. Kowalewska,82 R. Kowalewski,173 T. Z. Kowalski,81a C. Kozakai,160 W. Kozanecki,142 A. S. Kozhin,140 V. A. Kramarenko,111 G. Kramberger,89 D. Krasnopevtsev,110 M. W. Krasny,132 A. Krasznahorkay,35 D. Krauss,113 J. A. Kremer,81a J. Kretzschmar,88 P. Krieger,164 K. Krizka,18 K. Kroeninger,45 H. Kroha,113 J. Kroll,137 J. Kroll,133 J. Krstic,16 U. Kruchonak,77 H. Krüger,24 N. Krumnack,76 M. C. Kruse,47 T. Kubota,102 S. Kuday,4b J. T. Kuechler,179 S. Kuehn,35 A. Kugel,59a F. Kuger,174 T. Kuhl,44 V. Kukhtin,77 R. Kukla,99 Y. Kulchitsky,105 S. Kuleshov,144b Y. P. Kulinich,170 M. Kuna,56 T. Kunigo,83 A. Kupco,137 T. Kupfer,45 O. Kuprash,158 H. Kurashige,80 L. L. Kurchaninov,165a Y. A. Kurochkin,105 M. G. Kurth,15d E. S. Kuwertz,173 M. Kuze,162 J. Kvita,126 T. Kwan,101 A. La Rosa,113 J. L. La Rosa Navarro,78d L. La Rotonda,40b,40a F. La Ruffa,40b,40a C. Lacasta,171 F. Lacava,70a,70b J. Lacey,44 D. P. J. Lack,98 H. Lacker,19 D. Lacour,132 E. Ladygin,77 R. Lafaye,5 B. Laforge,132 T. Lagouri,32c S. Lai,51 S. Lammers,63 W. Lampl,7 E. Lançon,29 U. Landgraf,50 M. P. J. Landon,90 M. C. Lanfermann,52 V. S. Lang,44 J. C. Lange,14 R. J. Langenberg,35 A. J. Lankford,168 F. Lanni,29 K. Lantzsch,24 A. Lanza,68a A. Lapertosa,53b,53a S. Laplace,132 J. F. Laporte,142 T. Lari,66a F. Lasagni Manghi,23b,23a M. Lassnig,35 T. S. Lau,61a A. Laudrain,128 A. T. Law,143 P. Laycock,88 M. Lazzaroni,66a,66b B. Le,102 O. Le Dortz,132 E. Le Guirriec,99 E. P. Le Quilleuc,142 M. LeBlanc,7 T. LeCompte,6 F. Ledroit-Guillon,56 C. A. Lee,29 G. R. Lee,144a L. Lee,57 S. C. Lee,155 B. Lefebvre,101 M. Lefebvre,173 F. Legger,112 C. Leggett,18 N. Lehmann,179 G. Lehmann Miotto,35 W. A. Leight,44 A. Leisos,159,hh M. A. L. Leite,78d R. Leitner,139 D. Lellouch,177 B. Lemmer,51 K. J. C. Leney,92 T. Lenz,24 B. Lenzi,35 R. Leone,7 S. Leone,69a C. Leonidopoulos,48 G. Lerner,153 C. Leroy,107 R. Les,164 A. A. J. Lesage,142 C. G. Lester,31 M. Levchenko,134 J. Levêque,5 D. Levin,103 L. J. Levinson,177 D. Lewis,90 B. Li,103 C-Q. Li,58a H. Li,58b L. Li,58c Q. Li,15d Q. Y. Li,58a S. Li,58d,58c X. Li,58c Y. Li,148 Z. Liang,15a B. Liberti,71a A. Liblong,164 K. Lie,61c S. Liem,118 A. Limosani,154 C. Y. Lin,31 K. Lin,104 T. H. Lin,97 R. A. Linck,63 B. E. Lindquist,152 A. L. Lionti,52 E. Lipeles,133 A. Lipniacka,17 M. Lisovyi,59b T. M. Liss,170,ii A. Lister,172 A. M. Litke,143 J. D. Little,8 B. Liu,76 B.L Liu,6 H. B. Liu,29 H. Liu,103 J. B. Liu,58a J. K. K. Liu,131 K. Liu,132 M. Liu,58a P. Liu,18 Y. Liu,15a Y. L. Liu,58a Y. W. Liu,58a M. Livan,68a,68b A. Lleres,56 J. Llorente Merino,15a S. L. Lloyd,90 C. Y. Lo,61b F. Lo Sterzo,41 E. M. Lobodzinska,44 P. Loch,7 F. K. Loebinger,98 A. Loesle,50 K. M. Loew,26 T. Lohse,19 K. Lohwasser,146 M Lokajicek 137 B A Long 25 J D Long 170 R E Long 87 L Longo 65a,65b K A Looper 122 J A Lopez 144b I Lopez Paz 14 SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Lund-Jensen,151 M. S. Lutz,100 132 29 137 94 15 77 29 58b 58b A. Lucotte,56 C. Luedtke,50 F. Luehring,63 I. Luise,132 W. Lukas,74 L. Luminari,70a B. Lund-Jensen,151 M. S. Lutz,100 P M Luzi 132 D Lynn 29 R Lysak 137 E Lytken 94 F Lyu 15a V Lyubushkin 77 H Ma 29 L L Ma 58b Y Ma 58b ynn,29 R. Lysak,137 E. Lytken,94 F. Lyu,15a V. Lyubushkin,77 H. Ma,29 L. L. Ma,58b Y. Ma,58b Macchiolo,113 C. M. Macdonald,146 J. Machado Miguens,133,136b D. Madaffari,171 R. Madar,37 er,46 A. Madsen,44 N. Madysa,46 J. Maeda,80 K. Maekawa,160 S. Maeland,17 T. Maeno,29 A. S W. F. Mader,46 A. Madsen,44 N. Madysa,46 J. Maeda,80 K. Maekawa,160 S. Maeland,17 T. Maeno,29 A. S. Maevskiy,111 V. Magerl,50 C. Maidantchik,78b T. Maier,112 A. Maio,136a,136b,136d O. Majersky,28a S. Majewski,127 Y. Makida,79 N. Makovec,128 B. Malaescu,132 Pa. Malecki,82 V. P. Maleev,134 F. Malek,56 U. Mallik,75 D. Malon,6 C. Malone,31 S Maltezos 10 S Malyukov 35 J Mamuzic 171 G Mancini 49 I Mandić 89 J Maneira 136a L Manhaes de Andrade Filho 78a J. Manjarres Ramos,46 K. H. Mankinen,94 A. Mann,112 A. Manousos,74 B. Mansoulie,142 J. D. Mansour,15a M. Mantoani,51 S Manzoni 66a,66b G Marceca 30 L March 52 L Marchese 131 G Marchiori 132 M Marcisovsky 137 C A Marin Tobon 35 J. Manjarres Ramos,46 K. H. Mankinen,94 A. Mann,112 A. Manousos,74 B. Mansoulie,142 J. D. Mansour,15a M. Mantoani,51 66 66b 30 52 131 132 137 35 S. Manzoni,66a,66b G. Marceca,30 L. March,52 L. Marchese,131 G. Marchiori,132 M. Marcisovsky,137 C. A. Marin Tobon,35 M. Marjanovic,37 D. E. Marley,103 F. Marroquim,78b Z. Marshall,18 M. U. F. Martensson,169 S. Marti-Garcia,171 tin,175 V. J. Martin,48 B. Martin dit Latour,17 M. Martinez,14,w V. I. Martinez Outschoorn,100 S. Martin-Haugh,141 V. S. Martoiu,27b A. C. Martyniuk,92 A. Marzin,35 L. Masetti,97 T. Mashimo,160 R. Mashinistov,108 J. Masik,98 A. L. Maslennikov,120b,120a L. H. Mason,102 L. Massa,71a,71b P. Mastrandrea,5 A. Mastroberardino,40b,40a T. Masubuchi,160 P. Mättig,179 J. Maurer,27b B. Maček,89 S. J. Maxfield,88 D. A. Maximov,120b,120a R. Mazini,155 I. Maznas,159 S. M. Mazza,143 N. C. Mc Fadden,116 G. Mc Goldrick,164 S. P. Mc Kee,103 A. McCarn,103 T. G. McCarthy,113 T. Masubuchi,160 P. Mättig,179 J. Maurer,27b B. Maček,89 S. J. Maxfield,88 D. A. Maximov,120b,120a R. Mazini,155 I. Maznas,159 S. M. Mazza,143 N. C. Mc Fadden,116 G. Mc Goldrick,164 S. P. Mc Kee,103 A. McCarn,103 T. G. McCarthy,113 L. I. McClymont,92 E. F. McDonald,102 J. A. Mcfayden,35 G. Mchedlidze,51 M. S. Meehan,145 T. M. Megy,50 S. Mehlhase,112 A. Mehta,88 T. Meideck,56 B. Meirose,42 D. Melini,171,jj B. R. Mellado Garcia,32c J. D. Mellenthin,51 M. Melo,28a F. Meloni,20 A. Melzer,24 S. B. Menary,98 E. D. Mendes Gouveia,136a L. Meng,88 X. T. Meng,103 A. Mengarelli,23b,23a S. Menke,113 E. Meoni,40b,40a S. Mergelmeyer,19 C. Merlassino,20 P. Mermod,52 L. Merola,67a,67b C. Meroni,66a F. S. Merritt,36 A. Messina,70a,70b J. Metcalfe,6 A. S. Mete,168 C. Meyer,133 J. Meyer,157 J-P. Meyer,142 H. Meyer Zu Theenhausen,59a F. Miano,153 R. P. Middleton,141 L. Mijović,48 G. Mikenberg,177 M. Mikestikova,137 M. Mikuž,89 M. Milesi,102 A. Milic,164 D. A. Millar,90 D. W. Miller,36 A. Milov,177 D. A. Milstead,43a,43b A. A. Minaenko,140 M. Miñano Moya,171 I. A. Minashvili,156b A. I. Mincer,121 B. Mindur,81a M. Mineev,77 Y. Minegishi,160 Y. Ming,178 L. M. Mir,14 A. Mirto,65a,65b K. P. Mistry,133 T. Mitani,176 J. Mitrevski,112 V. A. Mitsou,171 A. Miucci,20 P. S. Miyagawa,146 A. Mizukami,79 J. U. Mjörnmark,94 T. Mkrtchyan,181 M. Mlynarikova,139 T. Moa,43a,43b K. Mochizuki,107 P. Mogg,50 S. Mohapatra,38 S. Molander,43a,43b R. Moles-Valls,24 M. C. Mondragon,104 K. Mönig,44 J. Monk,39 E. Monnier,99 A. Montalbano,149 J. Montejo Berlingen,35 F. Monticelli,86 S. Monzani,66a R. W. Moore,3 N. Morange,128 D. Moreno,22 M. Moreno Llácer,35 P. Morettini,53b M. Morgenstern,118 S. Morgenstern,35 D. Mori,149 T. Mori,160 M. Morii,57 M. Morinaga,176 V. Morisbak,130 A. K. Morley,35 G. Mornacchi,35 A. P. Morris,92 J. D. Morris,90 L. Morvaj,152 P. Moschovakos,10 M. Mosidze,156b H. J. Moss,146 J. Moss,150,kk K. Motohashi,162 R. Mount,150 E. Mountricha,35 E. J. W. Moyse,100 S. Muanza,99 F. Mueller,113 J. Mueller,135 R. S. P. Mueller,112 D. Muenstermann,87 P. Mullen,55 G. A. Mullier,20 F. J. Munoz Sanchez,98 P. Murin,28b W. J. Murray,175,141 A. Murrone,66a,66b M. Muškinja,89 C. Mwewa,32a A. G. Myagkov,140,ll J. Myers,127 M. Myska,138 B. P. Nachman,18 O. Nackenhorst,45 K. Nagai,131 K. Nagano,79 Y. Nagasaka,60 K. Nagata,166 M. Nagel,50 E. Nagy,99 A. M. Nairz,35 Y. Nakahama,115 K. Nakamura,79 T. Nakamura,160 I. Nakano,123 H. Nanjo,129 F. Napolitano,59a R. F. Naranjo Garcia,44 R. Narayan,11 D. I. Narrias Villar,59a I. Naryshkin,134 T. Naumann,44 G. Navarro,22 R. Nayyar,7 H. A. Neal,103 P. Y. Nechaeva,108 T. J. Neep,142 A. Negri,68a,68b M. Negrini,23b S. Nektarijevic,117 C. Nellist,51 M. E. Nelson,131 S. Nemecek,137 P. Nemethy,121 M. Nessi,35,mm M. S. Neubauer,170 M. Neumann,179 P. R. Newman,21 T. Y. Ng,61c Y. S. Ng,19 H. D. N. Nguyen,99 T. Nguyen Manh,107 E. Nibigira,37 R. B. Nickerson,131 R. Nicolaidou,142 J. Nielsen,143 N. Nikiforou,11 V. Nikolaenko,140,ll I. Nikolic-Audit,132 K. Nikolopoulos,21 P. Nilsson,29 Y. Ninomiya,79 A. Nisati,70a N. Nishu,58c R. Nisius,113 I. Nitsche,45 T. Nitta,176 T. Nobe,160 Y. Noguchi,83 M. Nomachi,129 I. Nomidis,132 M. A. Nomura,29 T. Nooney,90 M. Nordberg,35 N. Norjoharuddeen,131 T. Novak,89 O. Novgorodova,46 R. Novotny,138 M. Nozaki,79 L. Nozka,126 K. Ntekas,168 E. Nurse,92 F. Nuti,102 F. G. Oakham,33,e H. Oberlack,113 T. Obermann,24 J. Ocariz,132 A. Ochi,80 I. Ochoa,38 J. P. Ochoa-Ricoux,144a K. O’Connor,26 S. Oda,85 S. Odaka,79 A. Oh,98 S. H. Oh,47 C. C. Ohm,151 H. Oide,53b,53a H. Okawa,166 Y. Okazaki,83 Y. Okumura,160 T. Okuyama,79 A. Olariu,27b L. F. Oleiro Seabra,136a S. A. Olivares Pino,144a D. Oliveira Damazio,29 SEARCH FOR LONG-LIVED PARTICLES IN FINAL … A. McKay,41 K. D. McLean,173 S. J. McMahon,141 P. C. McNamara,102 C. J. McNicol,175 R. A. McPherson,173,n J. E. Mdhluli,32c Z. A. Meadows,100 , , , , , y, L. I. McClymont,92 E. F. McDonald,102 J. A. Mcfayden,35 G. Mchedlidze,51 M. A. McKay,41 K. D. McLean,173 012001-23 012001-23 M. AABOUD et al. PHYS. REV. D 99, 012001 (2019) , , gg, p , , , g , K. Mönig,44 J. Monk,39 E. Monnier,99 A. Montalbano,149 J. Montejo Berlingen,35 F. Monticelli,86 S. Monzani,66a R. W. Moore,3 N. Morange,128 D. Moreno,22 M. Moreno Llácer,35 P. Morettini,53b M. Morgen D. Mori,149 T. Mori,160 M. Morii,57 M. Morinaga,176 V. Morisbak,130 A. K. Morley,35 G. Mornacchi,35 A. P. Morris,92 E. Mountricha,35 E. J. W. Moyse,100 S. Muanza,99 F. Mueller,113 J. Mueller,135 R. S. P. Mueller,112 D. Muenstermann,87 55 20 98 28b 175 141 66 66b 89 P. Mullen,55 G. A. Mullier,20 F. J. Munoz Sanchez,98 P. Murin,28b W. J. Murray,175,141 A. Murrone,66a,66b M. Muškinja,89 C. Mwewa,32a A. G. Myagkov,140,ll J. Myers,127 M. Myska,138 B. P. Nachman,18 O. Nackenhorst,45 K. Nagai,131 P. Mullen, G. A. Mullier, F. J. Munoz Sanchez, P. Murin, W. J. Murray, A. Murrone, M. Muškinja, C. Mwewa,32a A. G. Myagkov,140,ll J. Myers,127 M. Myska,138 B. P. Nachman,18 O. Nackenhorst,45 K. Nagai,131 K. Nagano,79 Y. Nagasaka,60 K. Nagata,166 M. Nagel,50 E. Nagy,99 A. M. Nairz,35 Y. Naka T. Nakamura,160 I. Nakano,123 H. Nanjo,129 F. Napolitano,59a R. F. Naranjo Garcia,44 R. Narayan,11 D. I. Narrias Villar,59a T. Nakamura,160 I. Nakano,123 H. Nanjo,129 F. Napolitano,59a R. F. Naranjo Garcia,44 R. Na I. Naryshkin,134 T. Naumann,44 G. Navarro,22 R. Nayyar,7 H. A. Neal,103 P. Y. Nechaeva,108 T. J. Neep,142 A. Negri,68a,68b M. Negrini,23b S. Nektarijevic,117 C. Nellist,51 M. E. Nelson,131 S. Nemecek,137 P. Nemethy,121 M. Nessi,35,mm y yy p g M. Negrini,23b S. Nektarijevic,117 C. Nellist,51 M. E. Nelson,131 S. Nemecek,137 P. Nemethy,121 M. Nessi,35,mm E. Nibigira,37 R. B. Nickerson,131 R. Nicolaidou,142 J. Nielsen,143 N. Nikiforou,11 V. Nikolaenko,140,ll I. Nikolic-Audit,132 K. Nikolopoulos,21 P. Nilsson,29 Y. Ninomiya,79 A. Nisati,70a N. Nishu,58c R. Nisius,113 I. Nitsch p y Y. Noguchi,83 M. Nomachi,129 I. Nomidis,132 M. A. Nomura,29 T. Nooney,90 M. Nordberg,35 N. Norjoharuddeen,131 T. Novak,89 O. Novgorodova,46 R. Novotny,138 M. Nozaki,79 L. Nozka,126 K. Ntekas,168 E. Nurse,92 F. Nuti,102 F. G. Oakham,33,e H. Oberlack,113 T. Obermann,24 J. Ocariz,132 A. Ochi,80 I. Ochoa,38 J. P. Ochoa-Ricoux,144a Y. Noguchi,83 M. Nomachi,129 I. Nomidis,132 M. A. Nomura,29 T. Nooney,90 M. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Nordberg, K. O’Connor,26 S. Oda,85 S. Odaka,79 A. Oh,98 S. H. Oh,47 C. C. Ohm,151 H. Oide,53b,53a H Y. Okumura,160 T. Okuyama,79 A. Olariu,27b L. F. Oleiro Seabra,136a S. A. Olivares Pino,144a D. Oliveira Damazio,29 J. L. Oliver,1 M. J. R. Olsson,36 A. Olszewski,82 J. Olszowska,82 D. C. O’Neil,149 A. Onofre,136a,136e K. Onogi,115 P. U. E. Onyisi,11 H. Oppen,130 M. J. Oreglia,36 Y. Oren,158 D. Orestano,72a,72b E. C. Orgill,98 N. Orlando,61b 44 164 53b 53 55 58 30 85 A. A. O’Rourke,44 R. S. Orr,164 B. Osculati,53b,53a,a V. O’Shea,55 R. Ospanov,58a G. Otero M. Ouchrif,34d F. Ould-Saada,130 A. Ouraou,142 Q. Ouyang,15a M. Owen,55 R. E. Owen,21 V. E. Ozcan,12c N. Ozturk,8 J. Pacalt,126 H. A. Pacey,31 K. Pachal,149 A. Pacheco Pages,14 L. Pacheco Rodriguez,142 C. Padilla Aranda,14 M. Ouchrif,34d F. Ould-Saada,130 A. Ouraou,142 Q. Ouyang,15a M. Owen,55 R. E. Owen,21 V J. Pacalt,126 H. A. Pacey,31 K. Pachal,149 A. Pacheco Pages,14 L. Pacheco Rodriguez,142 S. Pagan Griso,18 M. Paganini,180 G. Palacino,63 S. Palazzo,40b,40a S. Palestini,35 M. Palka,81b D. Pallin,37 I. Panagoulias,10 C. E. Pandini,35 J. G. Panduro Vazquez,91 P. Pani,35 G. Panizzo,64a,64c L. Paolozzi,52 T. D. Papadopoulou,10 K. Papageorgiou,9,t A. Paramonov,6 D. Paredes Hernandez,61b S. R. Paredes Saenz,131 B. Parida,58c A. J. Parker,87 K. A. Parker,44 M. A. Parker,31 F. Parodi,53b,53a J. A. Parsons,38 U. Parzefall,50 V. R. Pascuzzi,164 J. M. P. Pasner,143 E. Pasqualucci,70a S. Passaggio,53b F. Pastore,91 P. Pasuwan,43a,43b S. Pataraia,97 J. R. Pater,98 A. Pathak,178,f T. Pauly,35 B. Pearson,113 M. Pedersen,130 L. Pedraza Diaz,117 S. Pedraza Lopez,171 R. Pedro,136a,136b S. V. Peleganchuk,120b,120a O. Penc,137 C. Peng,15d H. Peng,58a B. S. Peralva,78a M. M. Perego,142 A. P. Pereira Peixoto,136a D. V. Perepelitsa,29 F. Peri,19 L. Perini,66a,66b H. Pernegger,35 S. Perrella,67a,67b V. D. Peshekhonov,77,a K. Peters,44 R. F. Y. Peters,98 B. A. Petersen,35 T. C. Petersen,39 E. Petit,56 A. Petridis,1 C. Petridou,159 P. Petroff,128 E. Petrolo,70a M. Petrov,131 F. Petrucci,72a,72b M. Pettee,180 N. E. Pettersson,100 A. Peyaud,142 R. Pezoa,144b T. Pham,102 F. H. Phillips,104 P. W. Phillips,141 G. Piacquadio,152 E. Pianori,18 A. Picazio,100 M. A. Pickering,131 R. Piegaia,30 J. E. Pilcher,36 A. D. Pilkington,98 M. Pinamonti,71a,71b J. L. Pinfold,3 M. Pitt,177 M-A. Pleier,29 V. Pleskot,139 E. Plotnikova,77 D. Pluth,76 P. Podberezko,120b,120a R. Poettgen,94 R. Poggi,52 L. Poggioli,128 I. Pogrebnyak,104 D. Pohl,24 I. Pokharel,51 G. Polesello,68a A. Poley,44 A. Policicchio,40b,40a R. Polifka,35 A. Polini,23b C. S. Pollard,44 V. Polychronakos,29 D. Ponomarenko,110 L. Pontecorvo,70a G. A. Popeneciu,27d D. M. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Portillo Quintero,132 S. Pospisil,138 K. Potamianos,44 I. N. Potrap,77 C. J. Potter,31 H. Potti,11 S. Pagan Griso,18 M. Paganini,180 G. Palacino,63 S. Palazzo,40b,40a S. Palestini,35 M. Palka,81b D. Pallin,37 I. Panagoulias,10 C. E. Pandini,35 J. G. Panduro Vazquez,91 P. Pani,35 G. Panizzo,64a,64c L. Paolozzi,52 T. D. Papadopoulou,10 K. Papageorgiou,9,t A. Paramonov,6 D. Paredes Hernandez,61b S. R. Paredes Saenz,131 B. Parida,58c A. J. Parker,87 K. A. Parker,44 M. A. Parker,31 F. Parodi,53b,53a J. A. Parsons,38 U. Parzefall,50 V. R. Pascuzzi,164 J. M. P. Pasner,143 E. Pasqualucci,70a S. Passaggio,53b F. Pastore,91 P. Pasuwan,43a,43b S. Pataraia,97 J. R. Pater,98 A. Pathak,178,f T. Pauly,35 B. Pearson,113 M. Pedersen,130 L. Pedraza Diaz,117 S. Pedraza Lopez,171 R. Pedro,136a,136b S. V. Peleganchuk,120b,120a O. Penc,137 C. Peng,15d H. Peng,58a B. S. Peralva,78a M. M. Perego,142 A. P. Pereira Peixoto,136a D. V. Perepelitsa,29 F. Peri,19 L. Perini,66a,66b H. Pernegger,35 S. Perrella,67a,67b V. D. Peshekhonov,77,a K. Peters,44 R. F. Y. Peters,98 B. A. Petersen,35 T. C. Petersen,39 E. Petit,56 A. Petridis,1 C. Petridou,159 P. Petroff,128 E. Petrolo,70a M. Petrov,131 F. Petrucci,72a,72b M. Pettee,180 N. E. Pettersson,100 A. Peyaud,142 R. Pezoa,144b T. Pham,102 F. H. Phillips,104 P. W. Phillips,141 G. Piacquadio,152 E. Pianori,18 A. Picazio,100 M. A. Pickering,131 R. Piegaia,30 J. E. Pilcher,36 A. D. Pilkington,98 M. Pinamonti,71a,71b J. L. Pinfold,3 M. Pitt,177 M-A. Pleier,29 V. Pleskot,139 E. Plotnikova,77 D. Pluth,76 P. Podberezko,120b,120a R. Poettgen,94 R. Poggi,52 L. Poggioli,128 I. Pogrebnyak,104 D. Pohl,24 I. Pokharel,51 G. Polesello,68a A. Poley,44 A. Policicchio,40b,40a R. Polifka,35 A. Polini,23b C. S. Pollard,44 V. Polychronakos,29 D. Ponomarenko,110 L. Pontecorvo,70a G. A. Popeneciu,27d D. M. Portillo Quintero,132 S. Pospisil,138 K. Potamianos,44 I. N. Potrap,77 C. J. Potter,31 H. Potti,11 K. A. Parker,44 M. A. Parker,31 F. Parodi,53b,53a J. A. Parsons,38 U. Parzefall,50 V. R. Pascuzzi,164 J. M. P. Pasner,143 E. Pasqualucci,70a S. Passaggio,53b F. Pastore,91 P. Pasuwan,43a,43b S. Pataraia,97 J. R. Pater,98 A. Pathak,178,f T. Pauly,35 B. Pearson,113 M. Pedersen,130 L. Pedraza Diaz,117 S. Pedraza Lopez,171 R. Pedro,136a,136b S. V. Peleganchuk,120b,120a O. Penc,137 C. Peng,15d H. Peng,58a B. S. Peralva,78a M. M. Perego,142 A. P. Pereira Peixoto,136a D. V. Perepelitsa,29 F. Peri,19 L. Perini,66a,66b H. Pernegger,35 S. Perrella,67a,67b V. D. Peshekhonov,77,a K. Peters,44 R. F. Y. Peters,98 B. A. Petersen,35 T. C. Petersen,39 E. Petit,56 A. Petridis,1 C. Petridou,159 P. Petroff,128 E. Petrolo,70a M. Petrov,131 F. Petrucci,72a,72b M. Pettee,180 N. E. Pettersson,100 A. Peyaud,142 R. Pezoa,144b T. Pham,102 F. H. Phillips,104 P. W. Phillips,141 G. Piacquadio,152 E. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Richter,113 S. Rich g E. D. Resseguie,133 S. Rettie,172 E. Reynolds,21 O. L. Rezanova,120b,120a P. Reznicek,139 R. Ri E. Richter-Was,81b O. Ricken,24 M. Ridel,132 P. Rieck,113 C. J. Riegel,179 O. Rifki,44 M. Rijssenbeek,152 A. Rimoldi,68a,68b L. Rinaldi,23b G. Ripellino,151 B. Ristić,87 E. Ritsch,35 I. Riu,14 J. C. Rivera Vergara,144a F. Riz M. Rimoldi,20 L. Rinaldi,23b G. Ripellino,151 B. Ristić,87 E. Ritsch,35 I. Riu,14 J. C. Rivera Vergara,144a F. Rizatdinova,125 E. Rizvi,90 C. Rizzi,14 R. T. Roberts,98 S. H. Robertson,101,n A. Robichaud-Veronneau,101 D. Robinson,31 , , p , , , , g , , E. Rizvi,90 C. Rizzi,14 R. T. Roberts,98 S. H. Robertson,101,n A. Robichaud-Veronneau,101 D. Robinson,31 vi,90 C. Rizzi,14 R. T. Roberts,98 S. H. Robertson,101,n A. Robichaud-Veronneau,101 D. Robinso son,44 A. Robson,55 E. Rocco,97 C. Roda,69a,69b Y. Rodina,99 S. Rodriguez Bosca,171 A. Rodrigu J. E. M. Robinson,44 A. Robson,55 E. Rocco,97 C. Roda,69a,69b Y. Rodina,99 S. Rodriguez Bosca,171 A. Rodriguez Perez,14 D. Rodriguez Rodriguez,171 A. M. Rodríguez Vera,165b S. Roe,35 C. S. Rogan,57 O. Røhne,130 R. Röhrig,113 D. Rodriguez Rodriguez,171 A. M. Rodríguez Vera,165b S. Roe,35 C. S. Rogan,57 O. Røhne,130 R. Röhrig,113 d,63 J. Roloff,57 A. Romaniouk,110 M. Romano,23b,23a N. Rompotis,88 M. Ronzani,121 L. Roos,132 S C. P. A. Roland,63 J. Roloff,57 A. Romaniouk,110 M. Romano,23b,23a N. Rompotis,88 M. Ronzani K. Rosbach,50 P. Rose,143 N-A. Rosien,51 E. Rossi,67a,67b L. P. Rossi,53b L. Rossini,66a,66b J. H. N. Rosten,31 R. Rosten,14 J. Rothberg,145 D. Rousseau,128 D. Roy,32c A. Rozanov,99 Y. Rozen,157 X. Ruan,32c F. Rubbo,15 M. Rotaru,27b J. Rothberg,145 D. Rousseau,128 D. Roy,32c A. Rozanov,99 Y. Rozen,157 X. Ruan,32c F. Rubbo,150 F. Rühr,50 A. Ruiz-Martinez,33 Z. Rurikova,50 N. A. Rusakovich,77 H. L. Russell,101 J. P. Rutherfoord,7 N. Ruthmann,35 A. Ruiz-Martinez,33 Z. Rurikova,50 N. A. Rusakovich,77 H. L. Russell,101 J. P. Rutherfoord,7 N. Ruthmann,35 E. M. Rüttinger,44,nn Y. F. Ryabov,134 M. Rybar,170 G. Rybkin,128 S. Ryu,6 A. Ryzhov,140 G. F. Rzehorz,51 P. Sabatini,51 G. Sabato,118 S. Sacerdoti,128 H. F-W. Sadrozinski,143 R. Sadykov,77 F. Safai Tehrani,70a P. Saha,119 M. Sahinsoy,59a g y y y y y G. Sabato,118 S. Sacerdoti,128 H. F-W. Sadrozinski,143 R. Sadykov,77 F. Safai Tehrani,70a P. Saha,119 M. Sahinsoy,59a y A. Sahu,179 M. Saimpert,44 M. Saito,160 T. Saito,160 H. Sakamoto,160 A. Sakharov,121,gg D. Salam J. E. Salazar Loyola,144b D. Salek,118 P. H. Sales De Bruin,169 D. Salihagic,113 A. Salnikov,150 J. Salt,171 D. Salvatore,40b,40a F. Salvatore,153 A. Salvucci,61a,61b,61c A. Salzburger,35 D. Sammel,50 D. Sampsonidis,159 D. Sampsonidou,159 J. Sánchez,171 A. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … S g , g, g y, , , , C. Schiavi,53b,53a S. Schier,143 L. K. Schildgen,24 Z. M. Schillaci,26 E. J. Schioppa,35 M. Schioppa,40b,40a K. E. Schleicher,50 35 113 35 97 44 97 50 C. Schiavi,53b,53a S. Schier,143 L. K. Schildgen,24 Z. M. Schillaci,26 E. J. Schioppa,35 M. Schioppa,40b,40a K. E. Schleicher,50 S S hl k 35 K R S h idt S f ld 113 K S h i d 35 C S h itt 97 S S h itt 44 S S h it 97 U S h 50 Schiavi,53b,53a S. Schier,143 L. K. Schildgen,24 Z. M. Schillaci,26 E. J. Schioppa,35 M. Schioppa,4 L. Schoeffel,142 A. Schoening,59b E. Schopf,24 M. Schott,97 J. F. P. Schouwenberg,117 J. Schovancova,35 S. Schramm,52 A. Schulte,97 H-C. Schultz-Coulon,59a M. Schumacher,50 B. A. Schumm,143 Ph. Schune,142 A. Schwartzman,150 T. A. Schwarz,103 H. Schweiger,98 Ph. Schwemling,142 R. Schwienhorst,104 A. Sciandra,24 G. Sciolla,26 M. Scornajenghi,40b,40a F. Scuri,69a F. Scutti,102 L. M. Scyboz,113 J. Searcy,103 C. D. Sebastiani,70a,70b P. Seema,24 S. C. Seidel,116 A. Seiden,143 T. Seiss,36 J. M. Seixas,78b G. Sekhniaidze,67a K. Sekhon,103 S. J. Sekula,41 N. Semprini-Cesari,23b,23a S. Sen,47 S. Senkin,37 C. Serfon,130 L. Serin,128 L. Serkin,64a,64b M. esari,23b,23a S. Sen,47 S. Senkin,37 C. Serfon,130 L. Serin,128 L. Serkin,64a,64b M. Sessa,72a,72b H. p T. Šfiligoj,89 F. Sforza,167 A. Sfyrla,52 E. Shabalina,51 J. D. Shahinian,143 N. W. Shaikh,43a,43b L. Y. Shan,15a R. Shang,170 25 18 1 131 109 153 98 134 J. T. Shank,25 M. Shapiro,18 A. S. Sharma,1 A. Sharma,131 P. B. Shatalov,109 K. Shaw,153 S. M. Shaw,98 A. Shcherbakova,134 Y Sh 124 N Sh f ti 33 A D Sh 25 P Sh d 92 L Shi 155,oo S Shi i 80 C O Shi i 180 M Shi ji 114 J. T. Shank,25 M. Shapiro,18 A. S. Sharma,1 A. Sharma,131 P. B. Shatalov,109 K. Shaw,153 S. M. S 124 33 25 92 155 80 , , , , , , , j , I. P. J. Shipsey,131 S. Shirabe,85 M. Shiyakova,77 J. Shlomi,177 A. Shmeleva,108 D. Shoaleh Saadi,107 M. J. Shochet,36 S. Shojaii,102 D. R. Shope,124 S. Shrestha,122 E. Shulga,110 P. Sicho,137 A. M. Sickles,170 P. E. Sidebo,151 E. Sideras Haddad,32c O. Sidiropoulou,174 A. Sidoti,23b,23a F. Siegert,46 Dj. Sijacki,16 J. Silva,136a M. Silva Jr.,178 M. V. Silva Oliveira,78a S. B. Silverstein,43a L. Simic,77 S. Simion,128 E. Simioni,97 M. Simon,97 P. Sinervo,164 N. B. Sinev,127 M. Sioli,23b,23a G. Siragusa,174 I. Siral,103 S.Yu. Sivoklokov,111 J. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Sjölin,43a,43b M. B. Skinner,87 P. Skubic,124 M. Slater,21 T. Slavicek,138 M. Slawinska,82 K. Sliwa,167 R. Slovak,139 V. Smakhtin,177 B. H. Smart,5 J. Smiesko,28a N. Smirnov,110 S.Yu. Smirnov,110 Y. Smirnov,110 L. N. Smirnova,111 O. Smirnova,94 J. W. Smith,51 M. N. K. Smith,38 R. W. Smith,38 j I. P. J. Shipsey,131 S. Shirabe,85 M. Shiyakova,77 J. Shlomi,177 A. Shmeleva,108 D. Shoaleh Saadi,107 M. J. Shochet,36 S. Shojaii,102 D. R. Shope,124 S. Shrestha,122 E. Shulga,110 P. Sicho,137 A. M. Sickles,170 P. E. Sidebo,151 E. Sideras Haddad,32c O. Sidiropoulou,174 A. Sidoti,23b,23a F. Siegert,46 Dj. Sijacki,16 J. Silva,136a M. Silva Jr.,178 E. Sideras Haddad,32c O. Sidiropoulou,174 A. Sidoti,23b,23a F. Siegert,46 Dj. Sijacki,16 J. Silva,136a M. Silva Jr.,178 M. V. Silva Oliveira,78a S. B. Silverstein,43a L. Simic,77 S. Simion,128 E. Simioni,97 M. Simon,97 P. Sinervo,164 N. B. Sinev,127 M. Sioli,23b,23a G. Siragusa,174 I. Siral,103 S.Yu. Sivoklokov,111 J. Sjölin,43a,43b M. B. Skinner,87 P. Skubic,124 M. Slater,21 T. Slavicek,138 M. Slawinska,82 K. Sliwa,167 R. Slovak,139 V. Smakhtin,177 B. H. Smart,5 J. Smiesko,28a N. Smirnov,110 S.Yu. Smirnov,110 Y. Smirnov,110 L. N. Smirnova,111 O. Smirnova,94 J. W. Smith,51 M. N. K. Smith,38 R. W. Smith,38 M. Smizanska,87 K. Smolek,138 A. A. Snesarev,108 I. M. Snyder,127 S. Snyder,29 R. Sobie,173,n A. M. Soffa,168 A. Soffer,158 A. Søgaard,48 D. A. Soh,155 G. Sokhrannyi,89 C. A. Solans Sanchez,35 M. Solar,138 E. Yu. Soldatov,110 U. Soldevila,171 A. A. Solodkov,140 A. Soloshenko,77 O. V. Solovyanov,140 V. Solovyev,134 P. Sommer,146 H. Son,167 W. Song,141 M. V. Silva Oliveira, S. B. Silverstein, L. Simic, S. Simion, E. Simioni, M. Simon, P. Sinervo, N. B. Sinev, M. Sioli,23b,23a G. Siragusa,174 I. Siral,103 S.Yu. Sivoklokov,111 J. Sjölin,43a,43b M. B. Skinner,87 P. Skubic,124 M. Slater,21 T. Slavicek,138 M. Slawinska,82 K. Sliwa,167 R. Slovak,139 V. Smakhtin,177 B. H. Smart,5 J. Smiesko,28a N. Smirnov,110 S.Yu. Smirnov,110 Y. Smirnov,110 L. N. Smirnova,111 O. Smirnova,94 J. W. Smith,51 M. N. K. Smith,38 R. W. Smith,38 M. Sioli,23b,23a G. Siragusa,174 I. Siral,103 S.Yu. Sivoklokov,111 J. Sjölin,43a,43b M. B. Skinner,87 P. Skubic,124 M. Slater,21 T. Slavicek,138 M. Slawinska,82 K. Sliwa,167 R. Slovak,139 V. Smakhtin,177 B. H. Smart,5 J. Smiesko,28a N. Smirnov,110 S.Yu. Smirnov,110 Y. Smirnov,110 L. N. Smirnova,111 O. Smirnova,94 J. W. Smith,51 M. N. K. Smith,38 R. W. Smith,38 87 138 108 127 29 173 168 158 A. Søgaard,48 D. A. Soh,155 G. Sokhrannyi,89 C. A. Solans Sanchez,35 M. Solar,138 E. Yu. Soldatov,110 U. Soldevila,171 A. A. Solodkov,140 A. Soloshenko,77 O. V. Solovyanov,140 V. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Pianori,18 A. Picazio,100 M. A. Pickering,131 R. Piegaia,30 J. E. Pilcher,36 A. D. Pilkington,98 M Pinamonti 71a,71b J L Pinfold 3 M Pitt 177 M A Pleier 29 V Pleskot 139 E Plotnikova 77 D Pluth 76 P Podberezko 120b,120a E. Pasqualucci,70a S. Passaggio,53b F. Pastore,91 P. Pasuwan,43a,43b S. Pataraia,97 J. R. Pater,98 A. Pathak,178,f T. Pauly,35 B. Pearson,113 M. Pedersen,130 L. Pedraza Diaz,117 S. Pedraza Lopez,171 R. Pedro,136a,136b S. V. Peleganchuk,120b,120a O. Penc,137 C. Peng,15d H. Peng,58a B. S. Peralva,78a M. M. Perego,142 A. P. Pereira Peixoto,136a D. V. Perepelitsa,29 F. Peri,19 L. Perini,66a,66b H. Pernegger,35 S. Perrella,67a,67b V. D. Peshekhonov,77,a K. Peters,44 R. F. Y. Peters,98 B. A. Petersen,35 T. C. Petersen,39 E. Petit,56 A. Petridis,1 C. Petridou,159 P. Petroff,128 E. Petrolo,70a M. Petrov,131 F. Petrucci,72a,72b 180 100 142 144b 102 104 141 T. C. Petersen,39 E. Petit,56 A. Petridis,1 C. Petridou,159 P. Petroff,128 E. Petrolo,70a M. Petrov,131 F. Petrucci,72a,72b M. Pettee,180 N. E. Pettersson,100 A. Peyaud,142 R. Pezoa,144b T. Pham,102 F. H. Phillips,104 P. W. Phillips,141 . A. Popeneciu,27d D. M. Portillo Quintero,132 S. Pospisil,138 K. Potamianos,44 I. N. Potrap,77 C 012001-24 012001-24 PHYS. REV. D 99, 012001 (2019) SEARCH FOR LONG-LIVED PARTICLES IN FINAL … j g p p D. M. Rauch,44 F. Rauscher,112 S. Rave,97 B. Ravina,146 I. Ravinovich,177 J. H. Rawling,98 M. , , , , , g, y , , N. P. Readioff,56 M. Reale,65a,65b D. M. Rebuzzi,68a,68b A. Redelbach,174 G. Redlinger,29 R. Reece,143 R. G. Reed,32c K. Reeves,42 L. Rehnisch,19 J. Reichert,133 A. Reiss,97 C. Rembser,35 H. Ren,15d M. Rescigno,70a S. Resconi,66a E. D. Resseguie,133 S. Rettie,172 E. Reynolds,21 O. L. Rezanova,120b,120a P. Reznicek,139 R. Richter,113 S. Richter,92 N. P. Readioff, M. Reale, D. M. Rebuzzi, A. Redelbach, G. Redlinger, R. Reece, R. G. Reed, K. Reeves,42 L. Rehnisch,19 J. Reichert,133 A. Reiss,97 C. Rembser,35 H. Ren,15d M. Rescigno,70a S. Resconi,66a E. D. Resseguie,133 S. Rettie,172 E. Reynolds,21 O. L. Rezanova,120b,120a P. Reznicek,139 R. Richter,113 S. Richter,92 g L. Rehnisch,19 J. Reichert,133 A. Reiss,97 C. Rembser,35 H. Ren,15d M. Rescigno,70a S. Rescon K. Reeves, L. Rehnisch, J. Reichert, A. Reiss, C. Rembser, H. Ren, M. Rescigno, S. Resconi, E. D. Resseguie,133 S. Rettie,172 E. Reynolds,21 O. L. Rezanova,120b,120a P. Reznicek,139 R. Richter,113 S. Richter,92 81b 24 132 113 179 44 152 68 68b , , , , , g , e,133 S. Rettie,172 E. Reynolds,21 O. L. Rezanova,120b,120a P. Reznicek,139 R. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Sanchez Pineda,64a,64c H. Sandaker,130 C. O. Sander,44 M. Sandhoff,179 C. Sandoval,22 D. P. C. Sankey,141 M. Sannino,53b,53a Y. Sano,115 A. Sansoni,49 C. Santoni,37 H. Santos,136a I. Santoyo Castillo,153 A. Sapronov,77 J. G. Saraiva,136a,136d O. Sasaki,79 K. Sato,166 E. Sauvan,5 P. Savard,164,e N. Savic,113 R. Sawada,160 C. Sawyer,141 L. Sawyer,93,v C. Sbarra,23b A. Sbrizzi,23b,23a T. Scanlon,92 J. Schaarschmidt,145 P. Schacht,113 B. M. Schachtner,112 D. Schaefer,36 L. Schaefer,133 J. Schaeffer,97 S. Schaepe,35 U. Schäfer,97 A. C. Schaffer,128 D. Schaile,112 R. D. Schamberger,152 N. Scharmberg,98 V. A. Schegelsky,134 D. Scheirich,139 F. Schenck,19 M. Schernau,168 53b 53 143 24 26 35 40b 40 50 F. Salvatore,153 A. Salvucci,61a,61b,61c A. Salzburger,35 D. Sammel,50 D. Sampsonidis,159 D. Sampsonidou,159 J. Sánchez,171 A. Sanchez Pineda,64a,64c H. Sandaker,130 C. O. Sander,44 M. Sandhoff,179 C. Sandoval,22 D. P. C. Sankey,141 M. Sannino,53b,53a Y. Sano,115 A. Sansoni,49 C. Santoni,37 H. Santos,136a I. Santoyo Castillo,153 A. Sapronov,77 J. G. Saraiva,136a,136d O. Sasaki,79 K. Sato,166 E. Sauvan,5 P. Savard,164,e N. Savic,113 R. Sawada,160 C. Sawyer,141 L. Sawyer,93,v C. Sbarra,23b A. Sbrizzi,23b,23a T. Scanlon,92 J. Schaarschmidt,145 P. Schacht,113 B. M. Schachtner,112 F. Salvatore, A. Salvucci, A. Salzburger, D. Sammel, D. Sampsonidis, D. Sampsonidou, J. Sánchez, A. Sanchez Pineda,64a,64c H. Sandaker,130 C. O. Sander,44 M. Sandhoff,179 C. Sandoval,22 D. P. C. Sankey,141 M. Sannino,53b,53a Y. Sano,115 A. Sansoni,49 C. Santoni,37 H. Santos,136a I. Santoyo Castillo,153 A. Sapronov,77 J G Saraiva 136a,136d O Sasaki 79 K Sato 166 E Sauvan 5 P Savard 164,e N Savic 113 R Sawada 160 C Sawyer 141 , , g , , p , p , , A. Sanchez Pineda,64a,64c H. Sandaker,130 C. O. Sander,44 M. Sandhoff,179 C. Sandoval,22 D. P. C. Sankey,141 , , g , , p , p , , A. Sanchez Pineda,64a,64c H. Sandaker,130 C. O. Sander,44 M. Sandhoff,179 C. Sandoval,22 D. P. C. Sankey,141 M Sannino 53b,53a Y Sano 115 A Sansoni 49 C Santoni 37 H Santos 136a I Santoyo Castillo 153 A Sapronov 77 M. Sannino,53b,53a Y. Sano,115 A. Sansoni,49 C. Santoni,37 H. Santos,136a I. Santoyo Castillo,153 A. Sapronov,77 J. G. Saraiva,136a,136d O. Sasaki,79 K. Sato,166 E. Sauvan,5 P. Savard,164,e N. Savic,113 R. Sawada,160 C. Sawyer,141 L. Sawyer,93,v C. Sbarra,23b A. Sbrizzi,23b,23a T. Scanlon,92 J. Schaarschmidt,145 P. Schacht,113 y D. Schaefer,36 L. Schaefer,133 J. Schaeffer,97 S. Schaepe,35 U. Schäfer,97 A. C. Schaffer,128 D. Schaile,112 p D. Schamberger,152 N. Scharmberg,98 V. A. Schegelsky,134 D. Scheirich,139 F. Schenck,19 M. T. Poulsen,94 J. Poveda,35 T. D. Powell,146 M. E. Pozo Astigarraga,35 P. Pralavorio,99 S. Prell,76 D. Price,98 M. Primavera,65a S. Prince,101 N. Proklova,110 K. Prokofiev,61c F. Prokoshin,144b S. Protopopescu,29 J. Proudfoot,6 M. Przybycien,81a A. Puri,170 P. Puzo,128 J. Qian,103 Y. Qin,98 A. Quadt,51 M. Queitsch-Maitland,44 A. Qureshi,1 P. Rados,102 F. Ragusa,66a,66b G R h l 95 J A R i 98 S R j l 29 A R i M l 90 T R hid 128 S R 5 M G R tti 66a,66b SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Solovyev,134 P. Sommer,146 H. Son,167 W. Song,141 012001-25 012001-25 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. PHYS. REV. D 99, 012001 (2019) A. Strubig,48 S. A. Stucci,29 B. Stugu,17 J. Stupak,124 N. A. Styles,44 D. Su,150 J. Su,135 S. Suchek,59a Y. Sugaya,129 M. Suk,138 V. V. Sulin,108 D. M. S. Sultan,52 S. Sultansoy,4c T. Sumida,83 S. Sun,103 X. Sun,3 K. Suruliz,153 C. J. E. Suster,154 M. R. Sutton,153 S. Suzuki,79 M. Svatos,137 M. Swiatlowski,36 S. P. Swift,2 A. Sydorenko,97 I. Sykora,28a T. Sykora,139 D. Ta,97 K. Tackmann,44,qq J. Taenzer,158 A. Taffard,168 R. Tafirout,165a E. Tahirovic,90 N. Taiblum,158 H. Takai,29 R. Takashima,84 E. H. Takasugi,113 K. Takeda,80 T. Takeshita,147 Y. Takubo,79 M. Talby,99 A. A. Talyshev,120b,120a J. Tanaka,160 M. Tanaka,162 R. Tanaka,128 R. Tanioka,80 B. B. Tannenwald,122 S. Tapia Araya,144b S. Tapprogge,97 A. Tarek Abouelfadl Mohamed,132 S. Tarem,157 G. Tarna,27b,q G. F. Tartarelli,66a P. Tas,139 M. Tasevsky,137 T. Tashiro,83 40b 40 136 136b 34 116 48 102 102 E. Tassi,40b,40a A. Tavares Delgado,136a,136b Y. Tayalati,34e A. C. Taylor,116 A. J. Taylor,48 G. N. Taylor,102 P. T. E. Taylor,102 W. Taylor,165b A. S. Tee,87 P. Teixeira-Dias,91 H. Ten Kate,35 P. K. Teng,155 J. J. Teoh,129 F. Tepel,179 S. Terada,79 E. Tassi, A. Tavares Delgado, Y. Tayalati, A. C. Taylor, A. J. Taylor, G. N. Taylor, P. T. E. Taylor, W. Taylor,165b A. S. Tee,87 P. Teixeira-Dias,91 H. Ten Kate,35 P. K. Teng,155 J. J. Teoh,129 F. Tepel,179 S. Terada,79 K. Terashi,160 J. Terron,96 S. Terzo,14 M. Testa,49 R. J. Teuscher,164,n S. J. Thais,180 T. Theve J. P. Thomas,21 A. S. Thompson,55 P. D. Thompson,21 L. A. Thomsen,180 E. Thomson,133 Y. Tian,38 R. E. Ticse Torres,51 V. O. Tikhomirov,108,rr Yu.A. Tikhonov,120b,120a S. Timoshenko,110 P. Tipton,180 S. Tisserant,99 K. Todome,162 J. P. Thomas,21 A. S. Thompson,55 P. D. Thompson,21 L. A. Thomsen,180 E. Thomson,133 Y. Tian,38 R. E. Ticse Torres,51 V. O. Tikhomirov,108,rr Yu.A. Tikhonov,120b,120a S. Timoshenko,110 P. Tipton,180 S. Tisserant,99 K. Todome,162 S. Todorova-Nova,5 S. Todt,46 J. Tojo,85 S. Tokár,28a K. Tokushuku,79 E. Tolley,122 K. G. Tomiwa,32c M. Tomoto,115 L. Tompkins,150,ff K. Toms,116 B. Tong,57 P. Tornambe,50 E. Torrence,127 H. Torres,46 E. Torró Pastor,145 C. Tosciri,131 J. Toth,99,ss F. Touchard,99 D. R. Tovey,146 C. J. Treado,121 T. Trefzger,174 F. Tresoldi,153 A. Tricoli,29 I. M. Trigger,165a 132 14 164 56 128 66a 173 F. Trovato,153 L. Truong,32b M. Trzebinski,82 A. Trzupek,82 F. Tsai,44 J.C-L. Tseng,131 P. V. Tsiareshka,105 N. Tsirintanis,9 V. Tsiskaridze,152 E. G. Tskhadadze,156a I. I. Tsukerman,109 V. Tsulaia,18 S. Tsuno,79 D. Tsybychev,152 Y. Tu,61b A. Tudorache,27b V. Tudorache,27b T. T. Tulbure,27a A. N. PHYS. REV. D 99, 012001 (2019) Vercesi,68a M V d i 72a 72b C M V l I f 76 W V k k 118 A T V l 118 J C V l 118 M C V li 149 e N. Viaux Maira,144b O. Viazlo,94 I. Vichou,170,a T. Vickey,146 O. E. Vickey Boeriu,146 G. H. A. Viehhauser,131 S. Viel,18 L. Vigani,131 M. Villa,23b,23a M. Villaplana Perez,66a,66b E. Vilucchi,49 M. G. Vincter,33 V. B. Vinogradov,77 A. Vishwakarma,44 C. Vittori,23b,23a I. Vivarelli,153 S. Vlachos,10 M. Vogel,179 P. Vokac,138 G. Volpi,14 32 24 139 110 171 88 16 S. E. von Buddenbrock,32c E. Von Toerne,24 V. Vorobel,139 K. Vorobev,110 M. Vos,171 J. H. V S. E. von Buddenbrock, E. Von Toerne, V. Vorobel, K. Vorobev, M. Vos, J. H. Vossebeld, N. Vranjes, M. Vranjes Milosavljevic,16 V. Vrba,138 M. Vreeswijk,118 R. Vuillermet,35 I. Vukotic,36 P. Wagner,24 W. Wagner,179 M. Vranjes Milosavljevic,16 V. Vrba,138 M. Vreeswijk,118 R. Vuillermet,35 I. Vukotic,36 P. Wagner,24 W. Wagner,179 J. Wagner-Kuhr, H. Wahlberg, S. Wahrmund, K. Wakamiya, V. M. Walbrecht, J. Walder, R. Walker, W. Walkowiak,148 V. Wallangen,43a,43b A. M. Wang,57 C. Wang,58b,q F. Wang,178 H. Wang,18 H. Wang,3 J. Wang,154 J. Wang,59b P. Wang,41 Q. Wang,124 R.-J. Wang,132 R. Wang,58a R. Wang,6 S. M. Wang,155 W. T. Wang,58a W. Wang,155,uu W. X. Wang,58a,vv Y. Wang,58a Z. Wang,58c C. Wanotayaroj,44 A. Warburton,101 C. P. Ward,31 D. R. Wardrope,92 A. Washbrook,48 P. M. Watkins,21 A. T. Watson,21 M. F. Watson,21 G. Watts,145 S. Watts,98 B. M. Waugh,92 A. F. Webb,11 S. Webb,97 C. Weber,180 M. S. Weber,20 S. A. Weber,33 S. M. Weber,59a J. S. Webster,6 A. R. Weidberg,131 B. Weinert,63 J W i t 51 M W i i h 97 C W i 50 P S W ll 35 T W 29 T W l 35 S W i 35 N W 24 J. Wagner-Kuhr, H. Wahlberg, S. Wahrmund, K. Wakamiya, V. M. Walbrecht, J. Walder, R. Walker, W. Walkowiak,148 V. Wallangen,43a,43b A. M. Wang,57 C. Wang,58b,q F. Wang,178 H. Wang,18 H. Wang,3 J. Wang,154 J. Wang,59b P. Wang,41 Q. Wang,124 R.-J. Wang,132 R. Wang,58a R. Wang,6 S. M. Wang,155 W. T. Wang,58a W. Wang,155,uu W. X. Wang,58a,vv Y. Wang,58a Z. Wang,58c C. Wanotayaroj,44 A. Warburton,101 C. P. Ward,31 D. R. Wardrope,92 S. Webb,97 C. Weber,180 M. S. Weber,20 S. A. Weber,33 S. M. Weber,59a J. S. Webster,6 A. R. Weidberg,131 B. Weinert,63 J. Weingarten,51 M. Weirich,97 C. Weiser,50 P. S. Wells,35 T. A. Sopczak,138 F. Sopkova,28b D. Sosa,59b C. L. Sotiropoulou,69a,69b S. Sottocornola,68a,68b R. Soualah,64a,64c,pp A. M. Soukharev,120b,120a D. South,44 B. C. Sowden,91 S. Spagnolo,65a,65b M. Spalla,113 M. Spangenberg,175 F. Spanò,91 D. Sperlich,19 F. Spettel,113 T. M. Spieker,59a R. Spighi,23b G. Spigo,35 L. A. Spiller,102 D. P. Spiteri,55 M. Spousta,139 A. Stabile,66a,66b R. Stamen,59a S. Stamm,19 E. Stanecka,82 R. W. Stanek,6 C. Stanescu,72a B. Stanislaus,131 M. M. Stanitzki,44 B. Stapf,118 S. Stapnes,130 E. A. Starchenko,140 G. H. Stark,36 J. Stark,56 S.H Stark,39 P. Staroba,137 P. Starovoitov,59a S. Stärz,35 R. Staszewski,82 M. Stegler,44 P. Steinberg,29 B. Stelzer,149 H. J. Stelzer,35 O. Stelzer-Chilton,165a H. Stenzel,54 T. J. Stevenson,90 G. A. Stewart,55 M. C. Stockton,127 G. Stoicea,27b P. Stolte,51 S. Stonjek,113 A. Straessner,46 J. Strandberg,151 S. Strandberg,43a,43b M. Strauss,124 P. Strizenec,28b R. Ströhmer,174 D. M. Strom,127 R. Stroynowski,41 A. Strubig,48 S. A. Stucci,29 B. Stugu,17 J. Stupak,124 N. A. Styles,44 D. Su,150 J. Su,135 S. Suchek,59a Y. Sugaya,129 M. Suk,138 V. V. Sulin,108 D. M. S. Sultan,52 S. Sultansoy,4c T. Sumida,83 S. Sun,103 X. Sun,3 K. Suruliz,153 C. J. E. Suster,154 M. R. Sutton,153 S. Suzuki,79 M. Svatos,137 M. Swiatlowski,36 S. P. Swift,2 A. Sydorenko,97 I. Sykora,28a T. Sykora,139 D. Ta,97 K. Tackmann,44,qq J. Taenzer,158 A. Taffard,168 R. Tafirout,165a E. Tahirovic,90 N. Taiblum,158 H. Takai,29 R. Takashima,84 E. H. Takasugi,113 K. Takeda,80 T. Takeshita,147 Y. Takubo,79 M. Talby,99 A. A. Talyshev,120b,120a J. Tanaka,160 M. Tanaka,162 R. Tanaka,128 R. Tanioka,80 B. B. Tannenwald,122 S. Tapia Araya,144b S. Tapprogge,97 A. Tarek Abouelfadl Mohamed,132 S. Tarem,157 G. Tarna,27b,q G. F. Tartarelli,66a P. Tas,139 M. Tasevsky,137 T. Tashiro,83 E. Tassi,40b,40a A. Tavares Delgado,136a,136b Y. Tayalati,34e A. C. Taylor,116 A. J. Taylor,48 G. N. Taylor,102 P. T. E. Taylor,102 W. Taylor,165b A. S. Tee,87 P. Teixeira-Dias,91 H. Ten Kate,35 P. K. Teng,155 J. J. Teoh,129 F. Tepel,179 S. Terada,79 K. Terashi,160 J. Terron,96 S. Terzo,14 M. Testa,49 R. J. Teuscher,164,n S. J. Thais,180 T. Theveneaux-Pelzer,44 F. Thiele,39 J. P. Thomas,21 A. S. Thompson,55 P. D. Thompson,21 L. A. Thomsen,180 E. Thomson,133 Y. Tian,38 R. E. Ticse Torres,51 V. O. Tikhomirov,108,rr Yu.A. Tikhonov,120b,120a S. Timoshenko,110 P. Tipton,180 S. Tisserant,99 K. Todome,162 S. Todorova-Nova,5 S. Todt,46 J. Tojo,85 S. Tokár,28a K. Tokushuku,79 E. Tolley,122 K. G. Tomiwa,32c M. Tomoto,115 L. Tompkins,150,ff K. Toms,116 B. Tong,57 P. Tornambe,50 E. Torrence,127 H. Torres,46 E. Torró Pastor,145 C. Tosciri,131 J. Toth,99,ss F. Touchard,99 D. R. Tovey,146 C. J. Treado,121 T. Trefzger,174 F. Tresoldi,153 A. Tricoli,29 I. M. Trigger,165a PHYS. REV. D 99, 012001 (2019) Tuna,57 S. Turchikhin,77 D. Turgeman,177 I. Turk Cakir,4b,tt R Turra 66a P M Tuts 38 E Tzovara 97 G Ucchielli 23b,23a I Ueda 79 M Ughetto 43a,43b F Ukegawa 166 G Unal 35 A. Tudorache, V. Tudorache, T. T. Tulbure, A. N. Tuna, S. Turchikhin, D. Turgeman, I. Turk Cakir, R. Turra,66a P. M. Tuts,38 E. Tzovara,97 G. Ucchielli,23b,23a I. Ueda,79 M. Ughetto,43a,43b F. Ukegawa,166 G. Unal,35 A. Undrus,29 G. Unel,168 F. C. Ungaro,102 Y. Unno,79 K. Uno,160 J. Urban,28b P. Urquijo,102 P. Urrejola,97 G. Usai,8 J. Usui,79 L. Vacavant,99 V. Vacek,138 B. Vachon,101 K. O. H. Vadla,130 A. Vaidya,92 C. Valderanis,112 E. Valdes Santurio,43a,43b M. Valente,52 S. Valentinetti,23b,23a A. Valero,171 L. Val´ery,44 R. A. Vallance,21 A. Vallier,5 J. A. Valls Ferrer,171 T R V D l 14 W V D W ll b 118 H V d G f 118 P V G 6 J V Ni k 149 L. Vacavant,99 V. Vacek,138 B. Vachon,101 K. O. H. Vadla,130 A. Vaidya,92 C. Valderanis,112 E. Valdes Santurio,43a,43b M. Valente,52 S. Valentinetti,23b,23a A. Valero,171 L. Val´ery,44 R. A. Vallance,21 A. Vallier,5 J. A. Valls Ferrer,171 T R Van Daalen 14 W Van Den Wollenberg 118 H Van der Graaf 118 P Van Gemmeren 6 J Van Nieuwkoop 149 L. Vacavant, V. Vacek, B. Vachon, K. O. H. Vadla, A. Vaidya, C. Valderanis, E. Valdes Santurio, M. Valente,52 S. Valentinetti,23b,23a A. Valero,171 L. Val´ery,44 R. A. Vallance,21 A. Vallier,5 J. A. Valls Ferrer,171 T R V D l 14 W V D W ll b 118 H V d G f 118 P V G 6 J V Ni k 149 I. Van Vulpen,118 M. C. van Woerden,118 M. Vanadia,71a,71b W. Vandelli,35 A. Vaniachine,163 P. Vankov,118 R. Vari,70a E. W. Varnes,7 C. Varni,53b,53a T. Varol,41 D. Varouchas,128 A. Vartapetian,8 K. E. Varvell,154 G. A. Vasquez,144b J. G. Vasquez,180 F. Vazeille,37 D. Vazquez Furelos,14 T. Vazquez Schroeder,101 J. Veatch,51 V. Vecchio,72a,72b L. M. Veloce,164 F. Veloso,136a,136c S. Veneziano,70a A. Ventura,65a,65b M. Venturi,173 N. Venturi,35 V. Vercesi,68a E. W. Varnes, C. Varni, T. Varol, D. Varouchas, A. Vartapetian, K. E. Varvell, G. A. Vasquez, J. G. Vasquez,180 F. Vazeille,37 D. Vazquez Furelos,14 T. Vazquez Schroeder,101 J. Veatch,51 V. Vecchio,72a,72b L. M. Veloce,164 F. Veloso,136a,136c S. Veneziano,70a A. Ventura,65a,65b M. Venturi,173 N. Venturi,35 V. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … S. Young,35 C. Young,150 J. Yu,8 J. Yu,76 X. Yue,59a S. P. Y. Yuen,24 I. Yusuff,31,ww B. Zabinski,82 G. Zacharis,10 E. Zaffaroni,52 R. Zaidan,14 A. M. Zaitsev,140,ll N. Zakharchuk,44 J. Zalieckas,17 S. Zambito,57 D. Zanzi,35 D. R. Zaripovas,55 Y. Zhou,7 C. G. Zhu,58b H. L. Zhu,58a H. Zhu,15a J. Zhu,103 Y. Zhu,58a X. Zhuang,15a K. Z 5 A. Zibell,174 D. Zieminska,63 N. I. Zimine,77 S. Zimmermann,50 Z. Zinonos,113 M. Zinser,97 M. Ziolkowski,148 L. Živković,16 G. Zobernig,178 A. Zoccoli,23b,23a K. Zoch,51 T. G. Zorbas,146 R. Zou,36 M. Zur Nedden,19 and L. Zwalinski35 PHYS. REV. D 99, 012001 (2019) Wenaus,29 T. Wengler,35 S. Wenig,35 N. Wermes,24 012001-26 PHYS. REV. D 99, 012001 (2019) M. D. Werner,76 P. Werner,35 M. Wessels,59a T. D. Weston,20 K. Whalen,127 N. L. Whallon,145 A. M. Wharton,87 A. S. White,103 A. White,8 M. J. White,1 R. White,144b D. Whiteson,168 B. W. Whitmore,87 F. J. Wickens,141 W. Wiedenmann,178 M. Wielers,141 C. Wiglesworth,39 L. A. M. Wiik-Fuchs,50 A. Wildauer,113 F. Wilk,98 H. G. Wilkens,35 L. J. Wilkins,91 H. H. Williams,133 S. Williams,31 C. Willis,104 S. Willocq,100 J. A. Wilson,21 I. Wingerter-Seez,5 E. Winkels,153 F. Winklmeier,127 O. J. Winston,153 B. T. Winter,24 M. Wittgen,150 M. Wobisch,93 A. Wolf,97 T. M. H. Wolf,118 R. Wolff,99 M. W. Wolter,82 H. Wolters,136a,136c V. W. S. Wong,172 N. L. Woods,143 S. D. Worm,21 B. K. Wosiek,82 K. W. Woźniak,82 K. Wraight,55 M. Wu,36 S. L. Wu,178 X. Wu,52 Y. Wu,58a T. R. Wyatt,98 B. M. Wynne,48 S. Xella,39 Z. Xi,103 L. Xia,175 D. Xu,15a H. Xu,58a L. Xu,29 T. Xu,142 W. Xu,103 B. Yabsley,154 S. Yacoob,32a K. Yajima,129 D. P. Yallup,92 D. Yamaguchi,162 Y. Yamaguchi,162 A. Yamamoto,79 T. Yamanaka,160 F. Yamane,80 M. Yamatani,160 T. Yamazaki,160 Y. Yamazaki,80 Z. Yan,25 H. J. Yang,58c,58d H. T. Yang,18 S. Yang,75 Y. Yang,160 Z. Yang,17 W-M. Yao,18 Y. C. Yap,44 Y. Yasu,79 E. Yatsenko,58c,58d J. Ye,41 S. Ye,29 I. Yeletskikh,77 E. Yigitbasi,25 E. Yildirim,97 K. Yorita,176 K. Yoshihara,133 C. J. S. Young,35 C. Young,150 J. Yu,8 J. Yu,76 X. Yue,59a S. P. Y. Yuen,24 I. Yusuff,31,ww B. Zabinski,82 G. Zacharis,10 E. Zaffaroni,52 R. Zaidan,14 A. M. Zaitsev,140,ll N. Zakharchuk,44 J. Zalieckas,17 S. Zambito,57 D. Zanzi,35 D. R. Zaripovas,55 S. V. Zeißner,45 C. Zeitnitz,179 G. Zemaityte,131 J. C. Zeng,170 Q. Zeng,150 O. Zenin,140 T. Ženiš,28a D. Zerwas,128 M. Zgubič,131 D. F. Zhang,58b D. Zhang,103 F. Zhang,178 G. Zhang,58a,vv H. Zhang,15c J. Zhang,6 L. Zhang,50 L. Zhang,58a M. Zhang,170 P. Zhang,15c R. Zhang,58a,q R. Zhang,24 X. Zhang,58b Y. Zhang,15d Z. Zhang,128 P. Zhao,47 X. Zhao,41 Y. Zhao,58b,128,y Z. Zhao,58a A. Zhemchugov,77 B. Zhou,103 C. Zhou,178 L. Zhou,41 M. S. Zhou,15d M. Zhou,152 N. Zhou,58c Y. Zhou,7 C. G. Zhu,58b H. L. Zhu,58a H. Zhu,15a J. Zhu,103 Y. Zhu,58a X. Zhuang,15a K. Zhukov,108 V. Zhulanov,120b,120a A. Zibell,174 D. Zieminska,63 N. I. Zimine,77 S. Zimmermann,50 Z. Zinonos,113 M. Zinser,97 M. Ziolkowski,148 L. Živković,16 G. Zobernig,178 A. Zoccoli,23b,23a K. Zoch,51 T. G. Zorbas,146 R. Zou,36 M. Zur Nedden,19 and L. Zwalinski35 Barcelona, Spain 17Department for Physics and Technology, University of Bergen, Bergen, Norway 18Physics Division, Lawrence Berkeley National Laboratory and University of California, (ATLAS Collaboration) 1Department of Physics, University of Adelaide, Adelaide, Australia 2 2Physics Department, SUNY Albany, Albany, New York, USA 3Department of Physics, University of Alberta, Edmonton, Alberta, Canada 4 4aDepartment of Physics, Ankara University, Ankara, Turkey 4b 4bIstanbul Aydin University, Istanbul, Turkey Istanbul Aydin University, Istanbul, Turkey 4cDivision of Physics, TOBB University of Economics and Technology, Ankara, Turkey 5LAPP, Universit´e Grenoble Alpes, Universit´e Savoie Mont Blanc, CNRS/IN2P3, Annecy, France 6High Energy Physics Division, Argonne National Laboratory, Argonne, Illinois, USA 7Department of Physics, University of Arizona, Tucson, Arizona, USA 8Department of Physics, University of Texas at Arlington, Arlington, Texas, USA 9Physics Department, National and Kapodistrian University of Athens, Athens, Greece 10Physics Department, National Technical University of Athens, Zografou, Greece 11Department of Physics, University of Texas at Austin, Austin, Texas, USA 12aBahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey 12bIstanbul Bilgi University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey 12cDepartment of Physics, Bogazici University, Istanbul, Turkey 12dDepartment of Physics Engineering, Gaziantep University, Gaziantep, Turkey 13Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 14Institut de Física d’Altes Energies (IFAE), Barcelona Institute of Science and Technology, B l S i Berkeley, California, USA 19Institut für Physik, Humboldt Universität zu Berlin, Berlin, Germany 19Institut für Physik, Humboldt Universität zu Berlin, Berlin, Germany SEARCH FOR LONG-LIVED PARTICLES IN FINAL … SEARCH FOR LONG-LIVED PARTICLES IN FINAL … M. D. Werner,76 P. Werner,35 M. Wessels,59a T. D. Weston,20 K. Whalen,127 N. L. Whallon,145 A. M. Wharton,87 A. S. White,103 A. White,8 M. J. White,1 R. White,144b D. Whiteson,168 B. W. Whitmore,87 F. J. Wickens,141 W. Wiedenmann,178 M. Wielers,141 C. Wiglesworth,39 L. A. M. Wiik-Fuchs,50 A. Wildauer,113 F. Wilk,98 H. G. Wilkens,35 L. J. Wilkins,91 H. H. Williams,133 S. Williams,31 C. Willis,104 S. Willocq,100 J. A. Wilson,21 I. Wingerter-Seez,5 E. Winkels,153 F. Winklmeier,127 O. J. Winston,153 B. T. Winter,24 M. Wittgen,150 M. Wobisch,93 A. Wolf,97 T. M. H. Wolf,118 R. Wolff,99 M. W. Wolter,82 H. Wolters,136a,136c V. W. S. Wong,172 N. L. Woods,143 S. D. Worm,21 B. K. Wosiek,82 K. W. Woźniak,82 K. Wraight,55 M. Wu,36 S. L. Wu,178 X. Wu,52 Y. Wu,58a T. R. Wyatt,98 B. M. Wynne,48 S. Xella,39 Z. Xi,103 L. Xia,175 D. Xu,15a H. Xu,58a L. Xu,29 T. Xu,142 W. Xu,103 B. Yabsley,154 S. Yacoob,32a K. Yajima,129 D. P. Yallup,92 D. Yamaguchi,162 Y. Yamaguchi,162 A. Yamamoto,79 T. Yamanaka,160 F. Yamane,80 M. Yamatani,160 T. Yamazaki,160 Y. Yamazaki,80 Z. Yan,25 H. J. Yang,58c,58d H. T. Yang,18 S. Yang,75 Y. Yang,160 Z. Yang,17 W-M. Yao,18 Y. C. Yap,44 Y. Yasu,79 E. Yatsenko,58c,58d J. Ye,41 S. Ye,29 I. Yeletskikh,77 E. Yigitbasi,25 E. Yildirim,97 K. Yorita,176 K. Yoshihara,133 C. J. S. Young,35 C. Young,150 J. Yu,8 J. Yu,76 X. Yue,59a S. P. Y. Yuen,24 I. Yusuff,31,ww B. Zabinski,82 G. Zacharis,10 E. Zaffaroni,52 R. Zaidan,14 A. M. Zaitsev,140,ll N. Zakharchuk,44 J. Zalieckas,17 S. Zambito,57 D. Zanzi,35 D. R. Zaripovas,55 S. V. Zeißner,45 C. Zeitnitz,179 G. Zemaityte,131 J. C. Zeng,170 Q. Zeng,150 O. Zenin,140 T. Ženiš,28a D. Zerwas,128 M. Zgubič,131 D. F. Zhang,58b D. Zhang,103 F. Zhang,178 G. Zhang,58a,vv H. Zhang,15c J. Zhang,6 L. Zhang,50 L. Zhang,58a M. Zhang,170 P. Zhang,15c R. Zhang,58a,q R. Zhang,24 X. Zhang,58b Y. Zhang,15d Z. Zhang,128 P. Zhao,47 X. Zhao,41 Y. Zhao,58b,128,y Z. Zhao,58a A. Zhemchugov,77 B. Zhou,103 C. Zhou,178 L. Zhou,41 M. S. Zhou,15d M. Zhou,152 N. Zhou,58c Y. Zhou,7 C. G. Zhu,58b H. L. Zhu,58a H. Zhu,15a J. Zhu,103 Y. Zhu,58a X. Zhuang,15a K. Zhukov,108 V. Zhulanov,120b,120a A. Zibell,174 D. Zieminska,63 N. I. Zimine,77 S. Zimmermann,50 Z. Zinonos,113 M. Zinser,97 M. Ziolkowski,148 L. Živković,16 G. Zobernig,178 A. Zoccoli,23b,23a K. Zoch,51 T. G. Zorbas,146 R. Zou,36 M. Zur Nedden,19 and L. Zwalinski35 M. D. Werner,76 P. Werner,35 M. Wessels,59a T. D. Weston,20 K. Whalen,127 N. L. Whallon,145 A. M. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Wharton,87 A. S. White,103 A. White,8 M. J. White,1 R. White,144b D. Whiteson,168 B. W. Whitmore,87 F. J. Wickens,141 W. Wiedenmann,178 M. Wielers,141 C. Wiglesworth,39 L. A. M. Wiik-Fuchs,50 A. Wildauer,113 F. Wilk,98 H. G. Wilkens,35 L. J. Wilkins,91 H. H. Williams,133 S. Williams,31 C. Willis,104 S. Willocq,100 J. A. Wilson,21 I. Wingerter-Seez,5 E. Winkels,153 F. Winklmeier,127 O. J. Winston,153 B. T. Winter,24 M. Wittgen,150 M. Wobisch,93 A. Wolf,97 T. M. H. Wolf,118 R. Wolff,99 M. W. Wolter,82 H. Wolters,136a,136c V. W. S. Wong,172 N. L. Woods,143 S. D. Worm,21 B. K. Wosiek,82 K. W. Woźniak,82 K. Wraight,55 M. Wu,36 S. L. Wu,178 X. Wu,52 Y. Wu,58a T. R. Wyatt,98 B. M. Wynne,48 S. Xella,39 Z. Xi,103 L. Xia,175 D. Xu,15a H. Xu,58a L. Xu,29 T. Xu,142 W. Xu,103 B. Yabsley,154 S. Yacoob,32a K. Yajima,129 D. P. Yallup,92 D. Yamaguchi,162 Y. Yamaguchi,162 A. Yamamoto,79 T. Yamanaka,160 F. Yamane,80 M. Yamatani,160 T. Yamazaki,160 Y. Yamazaki,80 Z. Yan,25 H. J. Yang,58c,58d H. T. Yang,18 S. Yang,75 Y. Yang,160 Z. Yang,17 W-M. Yao,18 Y. C. Yap,44 Y. Yasu,79 E. Yatsenko,58c,58d J. Ye,41 S. Ye,29 I. Yeletskikh,77 E. Yigitbasi,25 E. Yildirim,97 K. Yorita,176 K. Yoshihara,133 C. J. S. Young,35 C. Young,150 J. Yu,8 J. Yu,76 X. Yue,59a S. P. Y. Yuen,24 I. Yusuff,31,ww B. Zabinski,82 G. Zacharis,10 E. Zaffaroni,52 R. Zaidan,14 A. M. Zaitsev,140,ll N. Zakharchuk,44 J. Zalieckas,17 S. Zambito,57 D. Zanzi,35 D. R. Zaripovas,55 S. V. Zeißner,45 C. Zeitnitz,179 G. Zemaityte,131 J. C. Zeng,170 Q. Zeng,150 O. Zenin,140 T. Ženiš,28a D. Zerwas,128 M. Zgubič,131 D. F. Zhang,58b D. Zhang,103 F. Zhang,178 G. Zhang,58a,vv H. Zhang,15c J. Zhang,6 L. Zhang,50 L. Zhang,58a M. Zhang,170 P. Zhang,15c R. Zhang,58a,q R. Zhang,24 X. Zhang,58b Y. Zhang,15d Z. Zhang,128 P. Zhao,47 X. Zhao,41 Y. Zhao,58b,128,y Z. Zhao,58a A. Zhemchugov,77 B. Zhou,103 C. Zhou,178 L. Zhou,41 M. S. Zhou,15d M. Zhou,152 N. Zhou,58c Y. Zhou,7 C. G. Zhu,58b H. L. Zhu,58a H. Zhu,15a J. Zhu,103 Y. Zhu,58a X. Zhuang,15a K. Zhukov,108 V. Zhulanov,120b,120a A. Zibell,174 D. Zieminska,63 N. I. Zimine,77 S. Zimmermann,50 Z. Zinonos,113 M. Zinser,97 M. Ziolkowski,148 L. Živković,16 G. Zobernig,178 A. Zoccoli,23b,23a K. Zoch,51 T. G. Zorbas,146 R. Zou,36 M. Zur Nedden,19 and L. Zwalinski35 Y. Yamazaki,80 Z. Yan,25 H. J. Yang,58c,58d H. T. Yang,18 S. Yang,75 Y. Yang,160 Z. Yang,17 W-M. Yao,18 Y. C. Yap,44 Y. Yasu,79 E. Yatsenko,58c,58d J. Ye,41 S. Ye,29 I. Yeletskikh,77 E. Yigitbasi,25 E. Yildirim,97 K. Yorita,176 K. Yoshihara,133 C. J. 35CERN, Geneva, Switzerland 51II. Physikalisches Institut, Georg-August-Universität Göttingen, Göttingen, Germany ´ t t d Ph i N l´ i t C l i U i it´ d G ` G ` S it II. Physikalisches Institut, Georg August Universität Göttingen, Göttingen, Germ 2D´epartement de Physique Nucl´eaire et Corpusculaire, Universit´e de Gen`eve, Gen`eve, S 53aDipartimento di Fisica, Universit`a di Genova, Genova, Italy 53b University of Bern, Bern, Switzerland 21School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 012001-27 M. AABOUD et al. PHYS. REV. University of Bern, Bern, Switzerland D 99, 012001 (2019) 22Centro de Investigaciónes, Universidad Antonio Nariño, Bogota, Colombia 23aDipartimento di Fisica e Astronomia, Universit`a di Bologna, Bologna, Italy 23bINFN Sezione di Bologna, Italy 24Physikalisches Institut, Universität Bonn, Bonn, Germany 25Department of Physics, Boston University, Boston, Massachusetts, USA 26Department of Physics, Brandeis University, Waltham, Massachusetts, USA 27aTransilvania University of Brasov, Brasov, Romania 27bHoria Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania 27cDepartment of Physics, Alexandru Ioan Cuza University of Iasi, Iasi, Romania 27dNational Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj-Napoca, Romania 27eUniversity Politehnica Bucharest, Bucharest, Romania 27fWest University in Timisoara, Timisoara, Romania 28aFaculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovak Republic 28bDepartment of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 29Physics Department, Brookhaven National Laboratory, Upton, New York, USA 30Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Argentina 31Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 32aDepartment of Physics, University of Cape Town, Cape Town, South Africa 32bDepartment of Mechanical Engineering Science, University of Johannesburg, Johannesburg, South Africa 32cSchool of Physics, University of the Witwatersrand, Johannesburg, South Africa 33Department of Physics, Carleton University, Ottawa, Ontario, Canada 34aFacult´e des Sciences Ain Chock, R´eseau Universitaire de Physique des Hautes Energies - Universit´e Hassan II, Casablanca, Morocco 34bCentre National de l’Energie des Sciences Techniques Nucleaires (CNESTEN), Rabat, Morocco 34cFacult´e des Sciences Semlalia, Universit´e Cadi Ayyad, LPHEA-Marrakech, Morocco 34dFacult´e des Sciences, Universit´e Mohamed Premier and LPTPM, Oujda, Morocco 34eFacult´e des sciences, Universit´e Mohammed V, Rabat, Morocco 35CERN, Geneva, Switzerland 36Enrico Fermi Institute, University of Chicago, Chicago, Illinois, USA 37LPC, Universit´e Clermont Auvergne, CNRS/IN2P3, Clermont-Ferrand, France 38Nevis Laboratory, Columbia University, Irvington, New York, USA 39Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 40aDipartimento di Fisica, Universit`a della Calabria, Rende, Italy 40bINFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati, Italy 41Physics Department, Southern Methodist University, Dallas, Texas, USA 42Physics Department, University of Texas at Dallas, Richardson, Texas, USA 43aDepartment of Physics, Stockholm University, Sweden 43bOskar Klein Centre, Stockholm, Sweden 44Deutsches Elektronen-Synchrotron DESY, Hamburg and Zeuthen, Germany 45Lehrstuhl für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 46Institut für Kern- und Teilchenphysik, Technische Universität Dresden, Dresden, Germany 47Department of Physics, Duke University, Durham, North Carolina, USA 48SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 49INFN e Laboratori Nazionali di Frascati, Frascati, Italy 50Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany 51II. 53bINFN Sezione di Genova, Italy 54II. Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 55 55SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingd 56LPSC U i it´ G bl Al CNRS/IN2P3 G bl INP G bl F 57Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge, Massachusetts, USA 58aDepartment of Modern Physics and State Key Laboratory of Particle Detection and Electronics, University of Science and Technology of China, Hefei, China b University of Science and Technology of China, Hefei, China 58bInstitute of Frontier and Interdisciplinary Science and Key Laboratory of Particle P Irradiation (MOE), Shandong University, Qingdao, China y f gy f f 58bInstitute of Frontier and Interdisciplinary Science and Key Laboratory of Particle Physics and Particle Irradiation (MOE), Shandong University, Qingdao, China Institute of Frontier and Interdisciplinary Science and Key Laboratory of Particle P Irradiation (MOE), Shandong University, Qingdao, China University of Bern, Bern, Switzerland Physikalisches Institut, Georg-August-Universität Göttingen, Göttingen, Germany 52D´epartement de Physique Nucl´eaire et Corpusculaire, Universit´e de Gen`eve, Gen`eve, Switzerland 53aDipartimento di Fisica, Universit`a di Genova, Genova, Italy 53bINFN Sezione di Genova, Italy 54II. Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 55SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 56LPSC, Universit´e Grenoble Alpes, CNRS/IN2P3, Grenoble INP, Grenoble, France 57Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge, Massachusetts, USA 58aDepartment of Modern Physics and State Key Laboratory of Particle Detection and Electronics, University of Science and Technology of China, Hefei, China 58bInstitute of Frontier and Interdisciplinary Science and Key Laboratory of Particle Physics and Particle I di i (MOE) Sh d U i i Qi d Chi 22Centro de Investigaciónes, Universidad Antonio Nariño, Bogota, Colombia 23 24Physikalisches Institut, Universität Bonn, Bonn, Germany 25Department of Physics, Boston University, Boston, Massachusetts, USA 6 26Department of Physics, Brandeis University, Waltham, Massachusetts, USA 27 27eUniversity Politehnica Bucharest, Bucharest, Romania 27f Krakow, Poland 81bMarian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland 82 82Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland 83 83Faculty of Science, Kyoto University, Kyoto, Japan 84 84Kyoto University of Education, Kyoto, Japan 84Kyoto University of Education, Kyoto, Japan 85 85Research Center for Advanced Particle Physics and Department of Physics, Kyushu University, 012001-28 012001-28 PHYS. REV. D 99, 012001 (2019) 73aINFN-TIFPA, Italy y 73bUniversit`a degli Studi di Trento, Trento, Italy 74Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria 75University of Iowa Iowa City Iowa USA f p y p 75University of Iowa, Iowa City, Iowa, USA 76 76Department of Physics and Astronomy, Iowa State University, Ames, Iowa, USA 77 77Joint Institute for Nuclear Research, Dubna, Russia f 78aDepartamento de Engenharia El´etrica, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora Brazil 78aDepartamento de Engenharia El´etrica, Universidade Federal de Juiz de Fora (UFJF), Fukuoka, Japan 86Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentin 87 87Physics Department, Lancaster University, Lancaster, United Kingdom 88 88Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 89Department of Experimental Particle Physics, Jožef Stefan Institute and Department of Physics, University of Ljubljana, Ljubljana, Slovenia 90 90School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom 91Department of Physics, Royal Holloway University of London, Egham, United Kingdom 92 90School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom 91Department of Physics, Royal Holloway University of London, Egham, United Kingdom 92Department of Physics and Astronomy, University College London, London, United Kingdom 92Department of Physics and Astronomy, University College London, London, United Kingdom g 64bICTP, Trieste, Italy 65bDipartimento di Matematica e Fisica, Universit`a del Salento, Lecce, Italy 66 66aINFN Sezione di Milano, Italy 66bDipartimento di Fisica, Universit`a di Milano, Milano, Italy 67 67bDipartimento di Fisica, Universit`a di Napoli, Napoli, Italy 68 67bDipartimento di Fisica, Universit`a di Napoli, Napoli, Italy 68 68aINFN Sezione di Pavia, Italy 68bDipartimento di Fisica, Universit`a di Pavia, Pavia, Italy 69 68bDipartimento di Fisica, Universit`a di Pavia, Pavia, Italy 69 69aINFN Sezione di Pisa, Italy 69bDipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy 70 69bDipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy 70 70bDipartimento di Fisica, Sapienza Universit`a di Roma, Roma, Italy 71 70bDipartimento di Fisica, Sapienza Universit`a di Roma, Roma, Italy 71 71aINFN Sezione di Roma Tor Vergata, Italy 71aINFN Sezione di Roma Tor Vergata, Italy 71bDipartimento di Fisica, Universit`a di Roma Tor Vergata, Roma, Italy 72a 71bDipartimento di Fisica, Universit`a di Roma Tor Vergata, Roma, Italy 72 72aINFN Sezione di Roma Tre, Italy 72bDipartimento di Matematica e Fisica, Universit`a Roma Tre, Roma, Italy 73aINFN-TIFPA, Italy Juiz de Fora, Brazil 78bUniversidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro, Brazil 78c d d d l d d l ( ) d l l 79KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 80 te School of Science, Kobe University, Kobe, Japan 81aAGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow Poland SEARCH FOR LONG-LIVED PARTICLES IN FINAL … SEARCH FOR LONG-LIVED PARTICLES IN FINAL … 58cSchool of Physics and Astronomy, Shanghai Jiao Tong University, KLPPAC-MoE, SKLPPC, Shanghai, China 58dTsung-Dao Lee Institute, Shanghai, China 012001-29 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. 93Louisiana Tech University, Ruston, Louisiana, USA 94Fysiska institutionen, Lunds universitet, Lund, Sweden 95Centre de Calcul de l’Institut National de Physique Nucl´eaire et de Physique des Particules (IN2P3), Villeurbanne, France 96Departamento de Física Teorica C-15 and CIAFF, Universidad Autónoma de Madrid, Madrid, Spain 97Institut für Physik, Universität Mainz, Mainz, Germany 98School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 99CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France 100Department of Physics, University of Massachusetts, Amherst, Massachusetts, USA 101Department of Physics, McGill University, Montreal, Quebec, Canada 102School of Physics, University of Melbourne, Victoria, Australia 103Department of Physics, University of Michigan, Ann Arbor, Michigan, USA 104Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan, USA 105B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Belarus 106Research Institute for Nuclear Problems of Byelorussian State University, Minsk, Belarus 107Group of Particle Physics, University of Montreal, Montreal, Quebec, Canada 108P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow, Russia 109Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia 110National Research Nuclear University MEPhI, Moscow, Russia 111D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow, Russia 112Fakultät für Physik, Ludwig-Maximilians-Universität München, München, Germany 113Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 114Nagasaki Institute of Applied Science, Nagasaki, Japan 115 Norman, Oklahoma, USA 125Department of Physics, Oklahoma State University, Stillwater, Oklahoma, USA 126Palacký University, RCPTM, Joint Laboratory of Optics, Olomouc, Czech Republic 127 125Department of Physics, Oklahoma State University, Stillwater, Oklahoma, USA 126P l ký U i i RCPTM J i L b f O i Ol C h R bli 127Center for High Energy Physics, University of Oregon, Eugene, Oregon, USA 128 128LAL, Universit´e Paris-Sud, CNRS/IN2P3, Universit´e Paris-Saclay, Orsay, France 129 129Graduate School of Science, Osaka University, Osaka, Japan 130 ment of Physics, Oxford University, Oxford, United p f y , f y, f , g 132LPNHE, Sorbonne Universit´e, Paris Diderot Sorbonne Paris Cit´e, CNRS/IN2P3, Paris, France 133Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania, USA 134Konstantinov Nuclear Physics Institute of National Research Centre “Kurchatov Institute”, PNPI St Petersburg Russia NHE, Sorbonne Universit´e, Paris Diderot Sorbonne Paris Cit´e, CNRS/IN2P3, Paris, France 133Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania, USA Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania, USA onstantinov Nuclear Physics Institute of National Research Centre “Kurchatov Institute”, PNPI, St. Petersburg, Russia 135Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, Pennsylvania, USA 136aLaboratório de Instrumentação e Física Experimental de Partículas - LIP, Portugal 6 ç p g 136bDepartamento de Física, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal 136cDepartamento de Física, Universidade de Coimbra, Coimbra, Portugal 136dCentro de Física Nuclear da Universidade de Lisboa, Lisboa, Portugal 136eDepartamento de Física, Universidade do Minho, Braga, Portugal 36fDepartamento de Física Teorica y del Cosmos, Universidad de Granada, Granada (Spain), Spain 6gDep Física and CEFITEC of Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 136fDepartamento de Física Teorica y del Cosmos, Universidad de Granada, Granada (Spain), Spain 136gDep Física and CEFITEC of Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal y Moscow, Russia 112Fakultät für Physik, Ludwig-Maximilians-Universität München, München, Germany 113 113Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 114 114Nagasaki Institute of Applied Science, Nagasaki, Japan 115Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan 116Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, USA 117Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands 116Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, USA 117Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, 116Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, USA 117 f h h d l h db d kh f Nijmegen, Netherlands hef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands 119 119Department of Physics, Northern Illinois University, DeKalb, Illinois, USA 120 20aBudker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 120b 0aBudker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 120bN ibi k St t U i it N ibi k R i 20bNovosibirsk State University Novosibirsk, Russia 121Department of Physics, New York University, New York, New York, USA 122 nt of Physics, New York University, New York, New 122The Ohio State University, Columbus, Ohio, USA 23 123Faculty of Science, Okayama University, Okayama, Japan 124Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, Oklahoma, USA 125 Caparica, Portugal 137Institute of Physics, Academy of Sciences of the Czech Republic, Prague, Czech Republic 138Czech Technical University in Prague, Prague, Czech Republic 139 139Charles University, Faculty of Mathematics and Physics, Prague, Czech Republic 139Charles University, Faculty of Mathematics and Physics, Prague, Czech Republic 93Louisiana Tech University, Ruston, Louisiana, USA 94 Tokyo, Japan 161Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 163Tomsk State University, Tomsk, Russia 164Department of Physics, University of Toronto, Toronto, Ontario, Canada 165 165aTRIUMF, Vancouver, British Columbia, Canada 165bDepartment of Physics and Astronomy, York University, Toronto, Ontario, Canada 166Di i i f Ph i d T C f h Hi f h U i 165bDepartment of Physics and Astronomy, York University, Toronto, Ontario, Canada 166 Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan 167Department of Physics and Astronomy, Tufts University, Medford, Massachusetts, USA 68Department of Physics and Astronomy, University of California Irvine, Irvine, California, USA 169 Bergische Universität Wuppertal, Wuppertal, Germany 180Department of Physics, Yale University, New Haven, Connecticut, USA 181 Yale University, New Haven, Connecticut, USA of Physics, Yale University, New Haven, Connect 181 181Yerevan Physics Institute, Yerevan, Armenia aDeceased. bAlso at Department of Physics, King’s College London, London, United Kingdom. cAlso at Istanbul University, Department of Physics, Istanbul, Turkey. dAlso at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan. eAlso at TRIUMF, Vancouver, British Columbia, Canada. fAlso at Department of Physics and Astronomy, University of Louisville, Louisville, Kentucky, US gAlso at Department of Physics, California State University, Fresno, Stanford, California, USA. hAlso at Department of Physics, University of Fribourg, Fribourg, Switzerland. iAlso at II. Physikalisches Institut, Georg-August-Universität Göttingen, Göttingen, Germany. jAlso at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain. aDeceased. b bAlso at Department of Physics, King’s College London, London, United Kingdom. bAlso at Department of Physics, King’s College London, London, United Kingdom. cAlso at Istanbul University, Department of Physics, Istanbul, Turkey. d dAlso at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan eAlso at TRIUMF, Vancouver, British Columbia, Canada. f eAlso at TRIUMF, Vancouver, British Columbia, Canada. f p y y y y gAlso at Department of Physics, California State University, Fresno, Stanford, California, USA. h p y y y y gAlso at Department of Physics, California State University, Fresno, Stanford, California, USA. h hAlso at Department of Physics, University of Fribourg, Fribourg, Switzerland. i hAlso at Department of Physics, University of Fribourg, Fribourg, Switzerland. i iAlso at II. Physikalisches Institut, Georg-August-Universität Göttingen, Göttingen, Germany. j jAlso at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain. 012001-30 PHYS. REV. D 99, 012001 (2019) SEARCH FOR LONG-LIVED PARTICLES IN FINAL … SEARCH FOR LONG-LIVED PARTICLES IN FINAL … 140State Research Center Institute for High Energy Physics, NRC KI, Protvino, Russia 141Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 142 140State Research Center Institute for High Energy Physics, NRC KI, Protvino, Russia 141Particle Physics Department Rutherford Appleton Laboratory Didcot United Kingdom y p f pp y 142IRFU, CEA, Universit´e Paris-Saclay, Gif-sur-Yvette, France 43 y f 143Santa Cruz Institute for Particle Physics, University of California Santa Cruz, 143Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, California, USA 144aDepartamento de Física, Pontificia Universidad Católica de Chile, Santiago, Chile 144b artment Physik, Universität Siegen, Siegen, German 149Department of Physics, Simon Fraser University, Burnaby, British Columbia, Canada 150 y f f 151Physics Department, Royal Institute of Technology, Stockholm, Sweden tment, Royal Institute of Technology, Stockholm, Sw y p y f gy 152Departments of Physics and Astronomy, Stony Brook University, Stony Brook, New York, USA 153 p f y y y y y 153Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 154 154School of Physics, University of Sydney, Sydney, Australia 155 156aE. Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbilisi, Georgia 156bHigh Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 157 157Department of Physics, Technion, Israel Institute of Technology, Haifa, Israel 158Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israe 159Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 012001-31 M. AABOUD et al. PHYS. REV. D 99, 012001 (2019) kAlso at Tomsk State University, Tomsk, and Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia. lAlso at The Collaborative Innovation Center of Quantum Matter (CICQM), Beijing, China. Also at Tomsk State University, Tomsk, and Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia. lAlso at The Collaborative Innovation Center of Quantum Matter (CICQM), Beijing, China. y, , y gy y, g p y, lAlso at The Collaborative Innovation Center of Quantum Matter (CICQM), Beijing, China. y, , y lAlso at The Collaborative Innovation Center of Quantum Matter (CIC mAlso at Universita di Napoli Parthenope, Napoli, Italy. mAlso at Universita di Napoli Parthenope, Napoli, Italy. nAlso at Institute of Particle Physics (IPP), Canada. nAlso at Institute of Particle Physics (IPP), Canada. y ( ) oAlso at Dipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy. oAlso at Dipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy. Also at Dipartimento di Fisica E. Fermi, Universit`a p y pAlso at Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania. pAlso at Horia Hulubei National Institute of Physics and Nuclear Engine qAlso at CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France. qAlso at CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France. qAlso at CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France. sAlso at Borough of Manhattan Community College, City University of New York, New York, USA. t sAlso at Borough of Manhattan Community College, City University of New York, New York, USA. tAlso at Department of Financial and Management Engineering, University of the Aege at Department of Financial and Management Engineering, University of the Aegean, Chios, Greece uAlso at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Tow at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Town, South Africa. g p g p vAlso at Louisiana Tech University, Ruston, Louisiana, USA. vAlso at Louisiana Tech University, Ruston, Louisiana, USA. y wAlso at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain. wAlso at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spai p xAlso at Department of Physics, University of Michigan, Ann Arbor, Michigan, USA. xAlso at Department of Physics, University of Michigan, Ann Arbor, Michigan, USA. yAlso at LAL, Universit´e Paris-Sud, CNRS/IN2P3, Universit´e Paris-Saclay, Orsay, France. yAlso at LAL, Universit´e Paris-Sud, CNRS/IN2P3, Universit´e Paris-Sacla zAlso at Graduate School of Science, Osaka University, Osaka, Japan. 012001-31 zAlso at Graduate School of Science, Osaka University, Osaka, Japan. aaAlso at Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany. bb aaAlso at Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freib bb bbAlso at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands. bbAlso at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands. st University, Nicosia, North Cyprus, Mersin, Turke ddAlso at Institute of Theoretical Physics, Ilia State University, Tbilisi, G ddAlso at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia. eeAlso at CERN, Geneva, Switzerland. ff ffAlso at Department of Physics, Stanford University, USA. ffAlso at Department of Physics, Stanford University, USA. ggAlso at Manhattan College, New York, New York, USA. hh ggAlso at Manhattan College, New York, New York, USA. hh hhAlso at Hellenic Open University, Patras, Greece. ii p y iiAlso at The City College of New York, New York, New York, USA. jj iiAlso at The City College of New York, New York jj iiAlso at The City College of New York, New York, New York, USA. jj y g Departamento de Física Teorica y del Cosmos, Universidad de Granada, Granada (Spain), Spain. jjAlso at Departamento de Física Teorica y del Cosmos, Universidad de kk kkAlso at Department of Physics, California State University, Sacramento, Stanford, California, ll llAlso at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia. mmAlso at D´epartement de Physique Nucl´eaire et Corpusculaire, Universit´e de Gen`eve, Gen`eve, nnAlso at Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom. nnAlso at Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom. l of Physics, Sun Yat-sen University, Guangzhou, C y y g ppAlso at Department of Applied Physics and Astronomy, University of Sharjah, Sharjah, United Arab Emirates. qq ppAlso at Department of Applied Physics and Astronomy, University of Sharjah, Sharjah, United Arab Emirates ppAlso at Department of Applied Physics and Astronomy, University of S qqAlso at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany. qqAlso at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany. rrAlso at National Research Nuclear University MEPhI, Moscow, Russia. rrAlso at National Research Nuclear University MEPhI, Moscow, Russia. y ssAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary. 012001-31 tt l i i i l f i i i k ssAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, H tt ssAlso at Institute for Particle and Nuclear Physics, Wigner Research Cen ttAlso at Giresun University, Faculty of Engineering, Giresun, Turkey. ttAlso at Giresun University, Faculty of Engineering, Giresun, Turkey. y y g g y uuAlso at Department of Physics, Nanjing University, Nanjing, China. uuAlso at Department of Physics, Nanjing University, Nanjing, China. vvAlso at Institute of Physics, Academia Sinica, Taipei, Taiwan. vvAlso at Institute of Physics, Academia Sinica, Taipei, Taiwan. wwAlso at Department of Physics, University of Malaya, Kuala Lumpur, Malaysia. 012001-32 012001-32
https://openalex.org/W3037164797	http://www.ochsnerjournal.org/content/ochjnl/20/2/232.full.pdf	English	null	Pulmonary Adenocarcinoma Presenting as a Pineal Gland Mass With Obstructive Hydrocephalus	The Ochsner journal	2,020	cc-by	2,529	INTRODUCTION 84 pack-year smoking history but no alcohol use. His home medications included albuterol, atorvastatin, umeclidinium- vilanterol, pantoprazole, aspirin, and clopidogrel. Vital signs were normal. Physical examination was notable for a bilat- eral sixth nerve palsy with bilateral papilledema, mild ataxia of both lower extremities accompanied by truncal ataxia, and hyperreflexia in both upper and lower extremities with upgoing plantar reflexes bilaterally. The patient was alert and oriented, could follow commands, and had no aphasia or dysarthria. His pupils were equal, round, and reactive to light bilaterally. Adenocarcinoma of the lungs accounts for nearly 40% of all lung cancers and is the most prevalent type of non– small cell carcinoma of the lung.1 These patients commonly present with cough, dyspnea, and weight loss.2 Pulmonary carcinomas are the most common primary source of brain metastasis3; however, metastasis to the pineal gland is very rare, accounting for only 0.4% of all brain metastases.1 We describe a unique presentation of pulmonary adenocarci- noma in a patient presenting with ataxia, presyncopal spells, and falls secondary to pineal metastasis discovered on brain imaging. y Head computed tomography (CT) scan performed in the ED showed a soft tissue mass in the pineal gland region with lateral and third ventricular enlargement. Brain mag- netic resonance imaging (MRI) showed a 2.1 cm × 2.2 cm pineal mass with marked hydrocephalus from compres- sion of the aqueduct and associated vasogenic brain edema (Figures 1A, 1B, and 1C). CT imaging of the chest, abdomen, and pelvis with intravenous (IV) contrast showed a 2.0 cm × 2.0 cm left hilar spiculated nodule with adjacent lymphadenopathy concerning for malignancy (Figure 1D). Pulmonary Adenocarcinoma Presenting as a Pineal Gland Mass With Obstructive Hydrocephalus Mohamed A. Abdallah, MD,1 Mahum Shahid, MD,1 Moataz Ellithi, MD,1 Ahmed Yeddi, MD,2 Arwyn Cunningham, DO,3 Ryan Askeland, MD,4 Jamal Dodin, MD1 1Department of Internal Medicine, University of South Dakota Sanford School of Medicine, Sioux Falls, SD 2Department of Internal Medicine, Wayne State University/Detroit Medical Center, Detroit, MI 3Department of Pathology, University of South Dakota Sanford School of Medicine, Sioux Falls, SD 4Sanford Health Pathology Clinic, Sioux Falls, SD Background: Adenocarcinoma is the most prevalent type of non–small cell carcinoma of the lungs. Patients with lung adenocarci- noma often present with cough, dyspnea, pain, and weight loss. They can also present with signs and symptoms of brain metastasis because the lungs are one of the most common origins of metastatic brain cancer. We describe a rare case of adenocarcinoma of the lungs presenting with pineal region metastasis. Case Report: A 61-year-old male presented to the emergency department with dizzy spells and gait disturbance. Magnetic reso- nance imaging of the brain demonstrated a solitary mass in the pineal region with marked obstructive hydrocephalus. A stereotac- tic biopsy was performed, and metastatic adenocarcinoma consistent with pulmonary origin was diagnosed. Computed tomog- raphy scan of the chest revealed a spiculated mass. The patient died shortly after the diagnosis was made. nance imaging of the brain demonstrated a solitary mass in the pineal region with marked obstructive hydrocephalus. A stereotac- tic biopsy was performed, and metastatic adenocarcinoma consistent with pulmonary origin was diagnosed. Computed tomog- raphy scan of the chest revealed a spiculated mass. The patient died shortly after the diagnosis was made. Conclusion: The pineal region is an unusual site for brain metastasis. Although such metastasis has rarely been described, it should be considered in the differential diagnosis of pineal region tumors, especially for patients with suggestive clinical or histopatho- logic features of primary malignancy elsewhere. Conclusion: The pineal region is an unusual site for brain metastasis. Although such metastasis has rarely been described, it should be considered in the differential diagnosis of pineal region tumors, especially for patients with suggestive clinical or histopatho- logic features of primary malignancy elsewhere. Keywords: Adenocarcinoma of lung, carcinoma–non small cell lung, hydrocephalus, neoplasm metastasis, pineal gland Keywords: Adenocarcinoma of lung, carcinoma–non small cell lung, hydrocephalus, neoplasm metastasis, pi Address correspondence to Ahmed Yeddi, MD, Department of Internal Medicine, Wayne State University/Detroit Medical Center, 4201 St. Antoine, Ste. 2E, Detroit, MI 48201. Tel: (312) 928-9142. Email: ayeddi@med.wayne.edu CASE REPORT CASE REPORT Ochsner Journal 20:232–235, 2020 Ochsner Journal 20:232–235, 2020 ©2020 by the author(s); Creative Commons Attribution License (CC BY) DOI: 10.31486/toj.18.0159 CASE REPORT A 61-year-old male presented to the emergency depart- ment (ED) with a 3-week history of early-morning headaches, presyncopal episodes, blurry vision, and gait disturbance. He also described nausea, dizziness, presyncope, and mul- tiple falls during the same period. The patient denied history of trauma or symptoms of palpitations, weakness, or speech difficulties. The patient’s medical history was pertinent for coronary artery disease, chronic obstructive pulmonary dis- ease, peripheral vascular disease, hypertension, and nico- tine use disorder. He was an active cigarette smoker with an The neurosurgery service evaluated the patient and rec- ommended brain biopsy. The patient requested a second Ochsner Journal Ochsner Journal 232 Abdallah, MA Figure1. (A)Sagittalsectionsofmagneticresonanceimaging(MRI)ofthebrainshowingthepinealglandmass.(B)Coronaland (C) transverse sections of brain MRI showing the same pineal tumor. (D) Computed tomography of the chest with intravenous contrast showing a lung nodule over the left hilar region in the lower right corner. Figure1. (A)Sagittalsectionsofmagneticresonanceimaging(MRI)ofthebrainshowingthepinealglandmass.(B)Coronaland (C) transverse sections of brain MRI showing the same pineal tumor. (D) Computed tomography of the chest with intravenous contrast showing a lung nodule over the left hilar region in the lower right corner. Figure1. (A)Sagittalsectionsofmagneticresonanceimaging(MRI)ofthebrainshowingthepinealglandmass.(B)Coronaland (C) transverse sections of brain MRI showing the same pineal tumor. (D) Computed tomography of the chest with intravenous contrast showing a lung nodule over the left hilar region in the lower right corner. on immunoreactivity for cytokeratins and negativity for GFAP and synaptophysin. opinion and was transferred to a referral center with neu- rosurgical expertise. At that center, the patient underwent endoscopic ventriculostomy to relieve hydrocephalus and pineal mass region biopsy. Histologic examination was sug- gestive of an epithelioid neoplasm with low proliferative activity. The immunophenotype of the tumor was positive for cytokeratin CAM 5.2, cytokeratin OSCAR, and epithelial membrane antigen (EMA), and negative for synaptophysin, glial fibrillary acidic protein (GFAP), neurofilament, S100, and OCT 3/4. Differential diagnosis was low-grade neoplasm such as papillary tumor of the pineal region (PTPR) based Postoperatively, the patient’s gait and headaches markedly improved, but he had persistent diplopia. Alter- nate eye patching was recommended. The patient was discharged with plans for repeat outpatient brain imaging to monitor the size of the tumor given the negative initial brain biopsy. Four weeks later, the patient presented to the ED after a fall. He was found to have a right hip fracture. CASE REPORT The patient reported that shortly after his brain biopsy, his neurologic 233 Volume 20, Number 2, Summer 2020 Pulmonary Adenocarcinoma Presenting as a Pineal Gland Mass Figure 2. (A) Epithelioid neoplasm (hematoxylin and eosin [H&E] stain, ×20). (B) Strong and diffuse staining for pankeratin (H&E stain, ×20). (C) Epithelioid neoplasm demonstrating marked nuclear pleomorphism with eosinophilic cytoplasm (H&E stain, ×400). (D) Strong and diffuse staining of the neoplasm for thyroid transcription factor-1, supporting the diagnosis of adenocarcinoma of pulmonary origin (H&E stain, ×40). Pulmonary Adenocarcinoma Presenting as a Pineal Gland Mass Figure 2. (A) Epithelioid neoplasm (hematoxylin and eosin [H&E] stain, ×20). (B) Strong and diffuse staining for pankeratin (H&E stain, ×20). (C) Epithelioid neoplasm demonstrating marked nuclear pleomorphism with eosinophilic cytoplasm (H&E stain, ×400). (D) Strong and diffuse staining of the neoplasm for thyroid transcription factor-1, supporting the diagnosis of adenocarcinoma of pulmonary origin (H&E stain, ×40). symptoms had worsened. He required a walker to ambu- late and fell more often because of imbalance and ataxia. He continued to have headaches, dizziness, and double vision. MRI of the brain in the ED showed an increase in mass size from 2.1 cm × 2.2 cm to 3.8 cm × 3.3 cm with vasogenic edema around the pineal mass. A decision to proceed with repeat stereotactic biopsy of the pineal tumor was made. Postoperatively, the patient failed to wake up from anesthe- sia. He had large pupils on neurologic examination. He was transferred to the neurointensive care unit for close mon- itoring and mechanical ventilation. Repeat CT scan of the head revealed hemorrhage within the mass, as well as small intraventricular hemorrhage. Serial CT head scans demon- strated progressive enlargement of the lateral ventricles. An emergent external ventricular drain was inserted; however, the patient did not show any clinical improvement. itoring and mechanical ventilation. Repeat CT scan of the head revealed hemorrhage within the mass, as well as small intraventricular hemorrhage. Serial CT head scans demon- strated progressive enlargement of the lateral ventricles. An emergent external ventricular drain was inserted; however, the patient did not show any clinical improvement. Results of the second biopsy demonstrated fragments of solid tumor intermixed with brain parenchyma and blood. The neoplastic cells had eosinophilic cytoplasm, marked nuclear pleomorphism with irregular nuclear contours, Results of the second biopsy demonstrated fragments of solid tumor intermixed with brain parenchyma and blood. DISCUSSION Pineal metastases from lung cancer are extremely rare. Patients with pineal metastases are typically asymptomatic, and the cancer is found incidentally on autopsy in most patients.1 Small cell carcinoma is the most reported lung cancer associated with pineal metastasis.1 Other his- tologic types, including squamous cell carcinoma4 and adenocarcinoma,1 have also been reported. Our case describes an atypical presentation of lung adenocarcinoma with hydrocephalus caused by mass effect of metastasis to the pineal gland. In addition, the initial pineal gland biopsy had a broad differential, including a PTPR secondary to the nonspecific staining pattern of the small tumor fragments, with immunoreactivity for cytokeratins and negativity for GFAP and synaptophysin.5 PTPR is a rare entity itself, and in 2007, the World Health Organization described PTPR as “a rare neuroepithelial tumor of the pineal region in adults, char- acterized by papillary architecture and epithelial cytology, immunopositivity for cytokeratin and ultra-structural features suggesting ependymal differentiation.”6 This article meets the Accreditation Council for Graduate Medical Education and the American Board of Medical Specialties Maintenance of Certiﬁcation competencies for Patient Care and Medical Knowledge. ©2020 by the author(s); licensee Ochsner Journal, Ochsner Clinic Foundation, New Orleans, LA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (creativecommons.org/licenses/by/4.0/legalcode) that permits unrestricted use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. CONCLUSION The pineal gland is an extremely rare site for both primary brain tumor and metastatic cancers. In patients with high suspicion for malignant pineal gland tumor, repeat pineal biopsy might be required if the first biopsy is not consistent with the overall clinical picture. As lungs are the most com- mon primary site for brain metastasis, chest imaging and immunohistochemical staining are useful tools for diagnosis of metastatic lung cancer to the pineal gland. CASE REPORT The neoplastic cells had eosinophilic cytoplasm, marked nuclear pleomorphism with irregular nuclear contours, Ochsner Journal 234 Abdallah, MA conspicuous nucleoli, and occasional atypical mitoses. The neoplastic cells stained positive for pankeratin, CK7, and thyroid transcription factor-1 (TTF-1). The neoplastic cells were negative for CK20, p40, placental alkaline phos- phatase, S100, GFAP, chromogranin A, and synaptophysin. Given the positivity of the CK7 and TTF-1 immunohisto- chemical stains, the tumor was diagnosed as metastatic adenocarcinoma consistent with pulmonary origin (Figure 2). The patient remained comatose for 48 hours after the procedure. After discussion with the family about treatment options and prognosis, the family opted for comfort care. The patient was extubated and died shortly thereafter. gland region should always prompt a detailed investigation to look for any primary malignancy, as both types of tumors have drastically different treatment modalities. Because the lungs are the most common primary site for brain metas- tasis, followed by breast cancer and malignant melanoma,1 the index of suspicion should always be high for a possible pineal metastasis, and workup should be done to look for a primary source. conspicuous nucleoli, and occasional atypical mitoses. The neoplastic cells stained positive for pankeratin, CK7, and thyroid transcription factor-1 (TTF-1). The neoplastic cells were negative for CK20, p40, placental alkaline phos- phatase, S100, GFAP, chromogranin A, and synaptophysin. Given the positivity of the CK7 and TTF-1 immunohisto- chemical stains, the tumor was diagnosed as metastatic adenocarcinoma consistent with pulmonary origin (Figure 2). The patient remained comatose for 48 hours after the procedure. After discussion with the family about treatment options and prognosis, the family opted for comfort care. The patient was extubated and died shortly thereafter. In cases of metastatic malignancy to the brain, after exam- ination of the histology on hematoxylin and eosin stained slides, immunohistochemical stains may be used to help elucidate the site of origin. In metastatic nonmucinous pulmonary adenocarcinoma, TTF-1 is typically positive in >85% of tumor cells. Also helpful in the diagnosis of pul- monary adenocarcinoma is positivity of napsin A and CK7, with negative staining of CK20.7 ACKNOWLEDGMENTS The authors have no ﬁnancial or proprietary interest in the subject matter of this article. REFERENCES 1. Committee of Brain Tumor Registry of Japan. Report of brain tumor registry of Japan (1969-1996). Neurol Med Chir (Tokyo). 2003 Sep;43 Suppl:i-vii, 1-111. Looking retrospectively, the markers in our patient might have suggested a metastatic etiology initially, considering EMA positivity and GFAP negativity.5 However, the rarity of both tumor types—primary pineal tumors and metastatic carcinoma—in this region made reaching the correct diag- nosis challenging. 2. Kocher F, Hilbe W, Seeber A, et al. Longitudinal analysis of 2293 NSCLC patients: a comprehensive study from the TYROL registry. Lung Cancer. 2015 Feb;87(2):193-200. doi: 10.1016/j.lungcan.2014.12.006. 3. Singh J, Velho V, Kharosekar H. Parinaud’s syndrome: a rare presentation of lung carcinoma with solitary pineal metastasis. Ann Indian Acad Neurol. 2014 Oct;17(4):478-480. doi: 10.4103/0972-2327.144047. Our patient had an isolated brain metastasis to the pineal region, and the unremarkable features of the first pineal biopsy might have obscured the clinical picture initially, leading to a delay in diagnosis. However, multiple biopsies from the same mass can clarify the diagnosis in cases of rare tumors, as the second biopsy in our case did yield a diagnosis. 4. Vaquero J, Martínez R, Magallón R, Ramiro J. Intracranial metastases to the pineal region. Report of three cases. J Neurosurg Sci. 1991 Jan-Mar;35(1):55-57. 5. Ballester LY, Fuller GN, Powell SZ, et al. Retrospective analysis of molecular and immunohistochemical characterization of 381 primary brain tumors. J Neuropathol Exp Neurol. 2017 Mar 1;76(3):179-188. doi: 10.1093/jnen/nlw119. Another important consideration is the aggressive nature of the adenocarcinoma in this patient, manifested by the increase in size of the pineal mass during the 4-week inter- val after the first presentation. The rapid progression of this tumor demonstrates that the time window can be very nar- row from the time of presentation to initiation of treatment with such malignancies. Because the incidence of primary pineal tumors is low in older patients, any mass in the pineal 6. Louis DN, Ohgaki H, Wiestler OD, et al. The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol. 2007 Aug;114(2):97-109. 6. Louis DN, Ohgaki H, Wiestler OD, et al. The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol. 2007 Aug;114(2):97-109. 7. Rekhtman N, Bishop JA. Quick Reference Handbook for Surgical Pathologists. Berlin, Germany: Springer; 2011. Volume 20, Number 2, Summer 2020 235
https://openalex.org/W3081901204	https://www.nature.com/articles/s41467-020-18211-3.pdf	English	null	Anharmonic quantum nuclear densities from full dimensional vibrational eigenfunctions with application to protonated glycine	Nature communications	2,020	cc-by	9,964	ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 I I nfrared and Raman vibrational spectroscopies are funda- mental tools for chemists. In particular, the mid-infrared region is traditionally interpreted by assigning the character- istic vibration of functional groups to each band. These assign- ments have been inferred from experiments1, and handbooks have been compiled with characteristic group frequencies2,3. This approach is indisputably useful for chemical analysis and very appealing to chemical intuition. However, spectral features cor- relates precisely with molecular structure only after appropriate quantum mechanical theoretical modeling. Despite the impor- tance of vibrational spectroscopy, theoretical chemistry approa- ches may perform accurate simulations of quantum vibrational spectra only for few-atom systems4–8. space. Moreover, as mentioned above4–8, the exact solution of the quantum vibrational problem is in general limited to low- dimensional systems. Speciﬁc methods have been devised to get eigenenergies for higher molecular dimensions27–36, but the eigenfunction estimates are usually accessible only for the ground state 31,37. The multiple coherent time averaging semiclassical initial value representation (MC SCIVR)38–41 can successfully simulate anharmonic vibrational spectra for large systems42–46, even in extreme anharmonic cases47–49. MC SCIVR can be rigorously derived from the stationary phase approximation of the exact Feynman’s path integral, and expresses the quantum time- evolution propagator in terms of a few classical trajectories, whose energy is close to the vibrational eigenvalues of the system (see Supplementary Methods). Moreover, MC SCIVR is also able to recover a reliable approximation of the vibrational ground and excited eigenstates by expanding the eigenfunction of each vibrational eigenvalue as a combination of harmonic eigenfunc- tions50–52. The combination coefﬁcients are calculated by Fourier transforming the semiclassical approximate quantum mechanical time correlation function of each harmonic eigenfunction at the vibrational eigenvalue obtained from a preliminary MC SCIVR power spectrum calculation (see Supplementary Methods). p y y The standard approach is normal-mode analysis. Normal modes are linearly independent atomic displacements that describe molecular vibrations in terms of harmonic forces. In other words, it is assumed that the interaction of the infrared (IR) radiation with a molecule results in a vibrational excitation conﬁned to the small- amplitude regime. More speciﬁcally, the Hessian matrix, whose elements are the mass-weighted force constants, is diagonalized at the molecule equilibrium geometry. The link of spectral peaks to molecular motions is therefore provided by the correspondence between Hessian matrix eigenvalues, which are directly related to normal-mode frequencies, and the eigenvectors, which are sets of nuclear displacements. ARTICLE The normal-mode approximation justiﬁes in part the traditional group frequency hypothesis. Indeed, it is true that for small and symmetric molecules the normal-mode analysis mostly results in motions of local groups. However, for larger non- symmetric molecular systems, the eigenvector displacements are necessarily spread all over the molecular structure, and the mode assignment in terms of functional group frequencies must be tentative1,9,10. Instead, chemical intuition is tempted to assert that a given vibrational normal mode is only weakly coupled to other modes in faraway parts of the molecule11–13, so that distance-based truncation of couplings is a common view in chemistry. Conse- quently, many methods have been proposed to localize normal modes12,14–18, to help with the interpretation of spectra in terms of chemically intuitive motions also for large systems. p p pp y In this work, we introduce the calculation of one-nucleus marginal densities by Monte Carlo integration of the anhar- monic eigenfunctions obtained with MC SCIVR simulations. Speciﬁcally, we employ the eigenfunction modulus square to weight a Monte Carlo Cartesian space sampling at each mole- cular conformation, and we obtain the ﬁnal molecular nuclear density as a direct sum of each nucleus contribution53, (see Supplementary Methods). This quantity is the nuclear analogue of electron density in Density Functional Theory for electronic structure calculations. The Cartesian coordinate space repre- sentation allows for the visualization of probability density isosurfaces in 3D, as for the nuclear ground state distributions obtained from Diffusion Monte Carlo calculations31,37,54–58, and in a similar fashion as routinely done for electron density59,60. We are therefore able to represent with nuclear density differences the nuclear motion associated with each peak in vibrational spectra in three-dimensional (3D) space, and to visually spot couplings resulting in speciﬁc non-local nuclear density patterns in a quantum mechanical framework. In addition, we compute the anharmonic quantum density dis- tributions of other observables, such as bond lengths, angles, and dihedrals using the same procedure. A further problem is how to account for anharmonicity, which is particularly relevant when molecules undergo large-amplitude motions such as internal rotations. Classical molecular dynamics (MD) offers a way to go beyond the harmonic approximation. Speciﬁcally, trajectories that run on the actual nuclear potential energy surface (PES) at different energies may span regions far from the potential minimum surroundings. ARTICLE The Fourier transform of the velocity autocorrelation function from MD simulations yields at low temperature the same frequencies as the normal-mode analysis, but in addition, anharmonic frequency shifts can be estimated from high-temperature simulations19,20. Systematic methods have been reported to decompose the whole classical nuclear dynamics into an approximate sum of effective mode contributions, which correspond to the peaks in the spec- trum 11,21–26. NATURE COMMUNICATIONS \| (2020) 11:4348 \| https://doi.org/10.1038/s41467-020-18211-3 \| www.nature.com/naturecommunicatio Anharmonic quantum nuclear densities from full dimensional vibrational eigenfunctions with application to protonated glycine Chiara Aieta 1, Marco Micciarelli1, Gianluca Bertaina 1,2 & Michele Ceotto 1✉ The interpretation of molecular vibrational spectroscopic signals in terms of atomic motion is essential to understand molecular mechanisms and for chemical characterization. The signals are usually assigned after harmonic normal mode analysis, even if molecular vibrations are known to be anharmonic. Here we obtain the quantum anharmonic vibrational eigenfunctions of the 11-atom protonated glycine molecule and we calculate the density distribution of its nuclei and its geometry parameters, for both the ground and the O-H stretch excited states, using our semiclassical method based on ab initio molecular dynamics trajectories. Our quantum mechanical results describe a molecule elongated and more ﬂexible with respect to what previously thought. More importantly, our method is able to assign each spectral peak in vibrational spectroscopy by showing quantitatively how normal modes involving different functional groups cooperate to originate that spectroscopic signal. The method will possibly allow for a better rationalization of experimental spectroscopy. 1 Dipartimento di Chimica, Università degli Studi di Milano, via C. Golgi 19, 20133 Milano, Italy. 2 Istituto Nazionale di Ricerca Metrologica, Strada delle Cacce 91, 10135 Torino, Italy. ✉email: michele.ceotto@unimi.it 1 Dipartimento di Chimica, Università degli Studi di Milano, via C. Golgi 19, 20133 Milano, Italy. 2 Istituto Nazionale di Rice 91, 10135 Torino, Italy. ✉email: michele.ceotto@unimi.it NATURE COMMUNICATIONS \| (2020) 11:4348 \| https://doi.org/10.1038/s41467-020-18211-3 \| www.nature.com/naturecommunications 1 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 Instead, the harmonic O–H stretch excited state is the same direct product as the ground state for the ﬁrst 26 DOFs, but with the eigenfunction depending on the 27th normal-mode coordinate in the ﬁrst excited state solution. For the anharmonic case, we represent the wavefunction with a combination of harmonic wavefunctions, as deﬁned in Supplementary Eq. 14. The combination coefﬁcients are obtained with the semiclassical procedure described in the Supplementary Methods51. The character of the anharmonic wavefunction is determined by considering the relative contribution of the different harmonic basis functions. In particular, the ground state wavefunction has the largest coefﬁcient on the harmonic ground state (see Supplementary Table 1), while the O–H stretch excited state has the largest coefﬁcient on the harmonic basis function with one quantum of excitation on the 27th component which depends on the 27th normal mode and whose displacement correspond to the stretching of the hydroxyl group (see Supplementary Table 2). However, in the latter case, we ﬁnd other important contributions coming from other harmonic states that are mixing with the fundamental O–H stretch one. For example, mode 26 which is the asymmetric N–H stretching at fundamental frequency equal to 3504 cm−1, mode 25, which is the symmetric N–H stretching at frequency 3445 cm−1, and modes 23 and 22 which are the C–H symmetric and asymmetric stretching with harmonic frequencies equal to 3116 and 3105 cm−1 respectively form combination states with low-frequency modes that have large coefﬁcients in the anharmonic wavefunction expansion. Also, there is a contribution from the overtone of the O–H stretching with two quanta of excitation. Protonated glycine nuclear densities. In the previous paragraph we showed the GlyH+ molecular system has a signiﬁcant amount of ZPE, which is overlooked by classical approaches. Therefore, one may wonder to what extent the molecular average geometry at the ZPE level differs from the classical geometry at the bottom of the well (i.e., the position expectation value in the harmonic approximation). To answer this question, we calculate the full quantum distribution of the geometry parameters, using the nuclear densities both in the harmonic and anharmonic case, as described in details in the Supplementary Methods. Even if the ground state vibrational eigenfunction is mainly peaked around the classical minimum, we observe a slight elongation of almost all the bond distances and angle distortions in the anharmonic case with respect to the harmonic ones. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 semiclassical power spectrum with the red continuous line in the same panel, where we have singled out the ZPE and the fundamental O–H stretch peaks. The value of the ZPE energy from the global molecular minimum is well above 20,000 wavenumbers, which is a huge amount of energy in comparison with the harmonic vibrational level spacing. This quantum quantity cannot be grasped by any classical simulation and determines the physical behavior of the system as we will show in the next section. Here, we look at the inﬂuence of the harmonic approximation on the estimate of this key quantity. Figure 1b pictorially represents the semiclassical and harmonic vibrational energy level estimates. The red and the green arrows correspond respectively to the semiclassical and harmonic O–H stretch transition frequency. These frequencies can be directly compared with the peak positions of the experimental IR spectrum (black continuous line in Fig. 1a)69. The accuracy of our on-the-ﬂy semiclassical approach outperforms the normal mode approach. The harmonic estimates of the ZPE and the O–H stretch do not provide an accurate value for the IR fundamental transition associated to the O–H stretch excitation. Actually, the harmonic transition frequency is signiﬁcantly worse than the semiclassical prediction when compared to the experimental value, as shown in the inset Table of Fig. 1b. This conﬁrms that the semiclassical treatment is remarkably able to describe precisely the actual spectral decomposition of the nuclear vibrational Hamiltonian, beyond the harmonic approximation. Moreover, from the computational point of view, GlyH+ is an ideal application to demonstrate the capabilities of MC SCIVR, since neither pre-computed PES nor exact quantum vibrational calculations have been reported with current state-of-the-art methods for this 11-atom molecule. We evolve the needed classical trajectories on-the-ﬂy, and in this case we compute the electronic potential along the dynamics at the DFT-B3LYP/aug- cc-pVDZ level of theory (see the “Methods” section below). p y ( ) In the harmonic case, the 27-dimensional vibrational Hamilto- nian becomes separable in the normal-mode coordinates. Therefore, the harmonic wavefunctions are the direct product of 27 one- dimensional eigenfunctions, each one depending on a single normal- mode coordinate. The degree of excitation of each one-dimensional wavefunction deﬁnes the state. For instance, for the harmonic ground state, all the eigenfunctions in the product are the ground state solution of each separate one-dimensional Schrödinger equation with harmonic potential. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 Moreover, if we compare the anharmonic quantum nuclear densities with the corre- sponding harmonic ones, we can appreciate how the synergy of these small effects results in speciﬁc deviations of the global vibrational behavior. The actual nuclear harmonic ground state density is displayed in Supplementary Figs. 6a1–4, while Sup- plementary Figs. 6c1–4 shows the actual anharmonic ground state density. To clearly visualize the differences between these two nuclear densities, we report in Fig. 2b the difference between the anharmonic and the harmonic nuclear densities for the ground state, along with the atomic labeling to guide the discussion of the results in Fig. 2a. Protonated glycine vibrational eigenvalues. We start from the normal-mode analysis of GlyH+ at the potential global mini- mum. Gas phase calculations have already been reported for this structure, in which the protonated amino group presumably forms an ionic intramolecular hydrogen bond with the carbonyl oxygen67,68. Our DFT optimized structure displays Cs symmetry. In a normal mode approach, we diagonalize the Hessian matrix at this geometry, and with the frequencies and harmonic zero point energy we build the stick power spectrum represented in green dashed lines in Fig. 1a. The O–H stretch is the highest frequency mode (3694 cm−1) and it belongs to the A0 irreducible representation. More importantly for our discussion, the eigen- vector displacements associated with this frequency result almost exclusively in the variation of the O–H distance, thus indicating a very localized O–H stretch motion, as pictorially highlighted in the ball-and-stick molecule reported on the inset in Fig. 1a. We detail the calculated Cartesian displacements along this normal mode coordinate in Supplementary Table 3 and Supplementary Fig. 7. To go beyond the harmonic approximation, we calculate the The most relevant differences are found in the densities of light hydrogen atoms. Speciﬁcally, the three red lobes on the H1, H2, and H3 nuclei indicate an enrichment of anharmonic density in the inner part of the protonated amino group umbrella, which offsets the density depletion represented by the blue lobes on the outer part. This picture reveals that the effect of anharmonicity is to drive the ammonium protons closer together with respect to the harmonic picture (as highlighted in magenta in Fig. 2n, and as also conﬁrmed by the angle distributions in Fig. 2h). Results Protonated glycine vibrational eigenfunctions. In this work, we calculate the 27-dimensional vibrational eigenfunctions and the one-nucleus marginal density for the protonated glycine (GlyH+) molecule. Neutral glycine is the simplest amino-acid, and it has been extensively studied with both theoretical and experimental methods43,61–63. We choose to study its proto- nated form, because it is a typical product during infrared multiple photon dissociation (IRMPD) spectroscopy, one of the most effective experimental approaches for structural investi- gation of biomolecules64,65. Actually, in the gas phase, proto- nation is the dominant ionization pathway when analyzing peptides by mass spectrometry64. Among all possible vibrational eigenfunctions, we focus on the ground state and on the O–H stretch excited vibrational state with intake of one quantum of excitation. We focus on this excitation because the O–H stretching peak is usually very intense and it is employed as the reference to scale the calculated harmonic vibrational spectra to match the experimental ones66. Nevertheless, features such as zero-point energies (ZPEs), overtones, and tunneling splittings, require a quantum mechanical treatment of vibrational spectroscopy. In the quantum framework, spectral peaks correspond to transitions between vibrational states. Exact quantum methods for spectroscopy involve a solution of the nuclear Schrödinger equation to get the vibrational levels. How- ever, peak assignment in quantum mechanics is more cumbersome than in classical mechanics. In fact, the dimensionality of internal coordinate eigenfunctions corresponding to eigenenergies for a (linear) molecule containing N atoms is Nv = 3N −6(5). Therefore, differently from the classical picture which can be represented by classical trajectories, the normal-mode displacements are not directly interpreted as single point atom displacements in Cartesian TURE COMMUNICATIONS \| (2020) 11:4348 \| https://doi.org/10.1038/s41467-020-18211-3 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 displacement of the average position of the heavier O1, C1, C2, and N nuclei, thus rendering the backbone of the molecule slightly longer (as highlighted in green in Fig. 2n). This is conﬁrmed by the bond-length distribution calculation, where the equilibrium distances C1–O1, C1–C2, and C2–N are longer for the anharmonic ground state wavefunction (see Fig. 2c, d, g). Also, the N–C2–C1–O1 anharmonic dihedral angle distribution of Fig. 2e is broader than the harmonic one. From bond-length distributions we also observe that both the N–H3 and the O2–H3 average distances are longer with anharmonicity inclusion (see Fig. 2i, l), hinting at a weaker O2–H3 hydrogen bond. However, the whole nuclear density picture of Fig. 2b shows that multiple local density deformations cooperate to the change of the H3 atom average position. Anharmonicity closes the protonated amine umbrella while opening the O2–C1–C2 angle. The combination of these two effects pushes the H3 atom farther away from the center of the molecule. O–H stretch state nuclear density. Also in this case, anharmonic effects are distributed all over the molecule. In addition, we observe a general elongation of bond lengths by comparing the bond-length distributions obtained from the harmonic and the anharmonic excited state wavefunctions, especially for the CH2 NH þ 3 part of the molecule (see Fig. 3b, e, f). Once more, looking at the complete nuclear density picture in Fig. 3a, one can deduce that these elongations balance the density distortion due to anharmonicity along the O–H stretching direction. Remarkably, an interesting lobe pattern appears along the O1–H6 stretching direction, as can be rationalized with the inspection of bond- length distributions in Fig. 3b. Here, the bond length analysis conﬁrms the global picture of anharmonicity obtained by visual inspection of density differences in Fig. 3a. The unidimensional plot reported in Fig. 3b compares the harmonic distribution (black continuous line) with the semiclassical anharmonic one, which presents the average shifted towards longer bond distances and hence it is represented with the green continuous line. In the harmonic picture, a node can be observed along the O–H stretch direction. This means that the vibrational excitation can be effectively approximated as the excitation of a harmonic one- dimensional oscillator whose potential varies along this direction. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 The nuclear density shift on Hydrogen nuclei H4 and H5 determines an elongation of the C2-H4/5 bonds, as conﬁrmed by the bond length distribution in Fig. 2f). Also, anharmonic effects lead to a 3 3 NATURE COMMUNICATIONS \| (2020) 11:4348 \| https://doi.org/10.1038/s41467-020-18211-3 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 492 20,000 20,800 21,600 22,400 3551 3694 143 3539 12 23,200 O–H stretch freq. (cm–1) Δ Experiment (EXP) Semiclassic (SC) Harmonic (Har) 24,400 O-H stretch ZPE E (cm–1) 0 1292 Intensity (Arb. units) O-H stretch coordinate 2092 (cm–1) SC Har EXP Har SC 2892 3692 a b Fig. 1 Vibrational power spectrum of the protonated glycine molecule. a GlyH+ vibrational spectrum. Red continuous line is the semiclassical power spectrum, and green dashed lines correspond to the stick spectrum in the harmonic normal mode approximation. The peak intensity of the power spectra has been scaled to match the semiclassical ZPE peak one. Black continuous line is the experimental IR absorption spectrum69. In the same panel, ball-and- stick molecular representation of GlyH+ at the equilibrium Cs geometry where the atoms involved in the O–H stretch normal mode displacements are highlighted. b Pictorial representation of the potential well along the O–H stretch elongation. Red and green lines are respectively the semiclassical and the harmonic ZPE and O–H stretch excitation vibrational levels. Red and green arrows are respectively the semiclassical and the harmonic normal mode O–H stretch fundamental excitation frequencies. These values are reported in the inset table together with their deviations from the experimental value (Δν). a Intensity (Arb. units) b O-H stretch coordinate Fig. 1 Vibrational power spectrum of the protonated glycine molecule. a GlyH+ vibrational spectrum. Red continuous line is the semiclassical power spectrum, and green dashed lines correspond to the stick spectrum in the harmonic normal mode approximation. The peak intensity of the power spectra has been scaled to match the semiclassical ZPE peak one. Black continuous line is the experimental IR absorption spectrum69. In the same panel, ball-and- stick molecular representation of GlyH+ at the equilibrium Cs geometry where the atoms involved in the O–H stretch normal mode displacements are highlighted. b Pictorial representation of the potential well along the O–H stretch elongation. Red and green lines are respectively the semiclassical and the harmonic ZPE and O–H stretch excitation vibrational levels. Red and green arrows are respectively the semiclassical and the harmonic normal mode O–H stretch fundamental excitation frequencies. These values are reported in the inset table together with their deviations from the experimental value (Δν). O–H stretch state nuclear density. Also in this case, anharmonic effects are distributed all over the molecule. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 In addition, we observe a general elongation of bond lengths by comparing the bond-length distributions obtained from the harmonic and the anharmonic excited state wavefunctions, especially for the CH2 NH þ 3 part of the molecule (see Fig. 3b, e, f). Once more, looking at the complete nuclear density picture in Fig. 3a, one can deduce that these elongations balance the density distortion due to anharmonicity along the O–H stretching direction. Remarkably, an interesting lobe pattern appears along the O1–H6 stretching direction, as can be rationalized with the inspection of bond- length distributions in Fig. 3b. Here, the bond length analysis conﬁrms the global picture of anharmonicity obtained by visual inspection of density differences in Fig. 3a. The unidimensional plot reported in Fig. 3b compares the harmonic distribution (black continuous line) with the semiclassical anharmonic one, which presents the average shifted towards longer bond distances and hence it is represented with the green continuous line. In the harmonic picture, a node can be observed along the O–H stretch direction. This means that the vibrational excitation can be effectively approximated as the excitation of a harmonic one- dimensional oscillator whose potential varies along this direction. This agrees with the standard harmonic normal-mode picture which shows the major displacement along the O–H stretch displacement of the average position of the heavier O1, C1, C2, and N nuclei, thus rendering the backbone of the molecule slightly longer (as highlighted in green in Fig. 2n). This is conﬁrmed by the bond-length distribution calculation, where the equilibrium distances C1–O1, C1–C2, and C2–N are longer for the anharmonic ground state wavefunction (see Fig. 2c, d, g). Also, the N–C2–C1–O1 anharmonic dihedral angle distribution of Fig. 2e is broader than the harmonic one. From bond-length distributions we also observe that both the N–H3 and the O2–H3 average distances are longer with anharmonicity inclusion (see Fig. 2i, l), hinting at a weaker O2–H3 hydrogen bond. However, the whole nuclear density picture of Fig. 2b shows that multiple local density deformations cooperate to the change of the H3 atom average position. Anharmonicity closes the protonated amine umbrella while opening the O2–C1–C2 angle. The combination of these two effects pushes the H3 atom farther away from the center of the molecule. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 This agrees with the standard harmonic normal-mode picture which shows the major displacement along the O–H stretch y We next turn to the excited vibrational state obtained when the O–H stretch normal mode has acquired one quantum of vibrational energy. Figure 3 shows for this eigenstate the density difference between the anharmonic and the harmonic nuclear densities. The actual excited harmonic O–H stretch nuclear density is displayed in Supplementary Figs. 6b1–4, while Supplementary Figs. 6d1–4 shows the actual excited anharmonic NATURE COMMUNICATIONS \| (2020) 11:4348 \| https://doi.org/10.1038/s41467-020-18211-3 \| www.nature.com/naturecommunications 4 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 i h 1.04 1.06 1.08 1.1 1.12 d(N-H3) (Å) Harmonic Anharmonic decrease Anharmonic increase Anharmonic broadening O2 a m b l i h g n c d e f H3 C2 C1 O1 H6 H4 H5 H1 H2 1.84 1.86 1.88 1.9 1.92 1.94 1.96 1.04 1.06 1.08 1.1 1.12 105 106 107 108 109 110 111 112 1.49 1.5 1.3 1.31 118 120 θ2(O2-C1-C2) (deg) θ2 θ1 φ θ1(H1/2-N-H3) (deg) d(O2-H3) (Å) d(N-H3) (Å) d(C2-N) (Å) d(C1-O1) (Å) 122 124 1.32 1.33 1.34 1.35 1.51 1.52 1.53 1.54 N Harmonic Anharmonic decrease Anharmonic increase Anharmonic broadening l 1.84 1.86 1.88 1.9 1.92 1.94 1.96 d(O2-H3) (Å) i 1.04 1.06 1.08 1.1 1.12 d(N-H3) (Å) O2 a H3 C2 C1 O1 H6 H4 H5 H1 H2 N a h 105 106 107 108 109 110 111 112 θ1(H1/2-N-H3) (deg) h m 118 120 θ2(O2-C1-C2) (deg) 122 124 b m g 1.49 1.5 d(C2-N) (Å) 1.51 1.52 1.53 1.54 g c 1.3 1.31 d(C1-O1) (Å) 1.32 1.33 1.34 1.35 c f 1.16 1.14 1.12 1.1 1.08 1.06 2-C1-O1) (deg) d(C2-H4/5) (Å) 1.80 1.90 200 n n e f 1.16 1.14 1.12 1.1 1.08 1.06 1.60 φ(N-C2-C1-O1) (deg) d(C2-H4/5) (Å) 1.70 1.80 1.90 200 d d 1.51 1.52 1.53 1.54 1.55 1.56 d(C1-C2) (Å) e φ(N-C2-C1-O1) (deg) d(C1-C2) (Å) d(C2-H4/5) (Å) Fig. 2 Comparison of harmonic and anharmonic nuclear densities for the vibrational ground state. a The Cs symmetry equilibrium geometry of GlyH+ at DFT-B3LYP/aug-cc-pVDZ level of theory with the atomic labeling that we adopt as reference throughout the paper. b Two isosurfaces of the difference between the anharmonic and the corresponding harmonic nuclear densities for the vibrational ground state. Red indicates positive contributions, where the density concentrates due to anharmonicity, while blue stands for the negative contributions, where the density is depleted. The density isosurfaces are respectively set to 0.15a.u. and −0.15a.u. In addition, we report the comparison of the maxima of relevant geometry parameter distributions. c, d, f, g, i, l Bond lengths distributions. h, m bond angles distributions. e Dihedral distribution. The black curves are obtained from the harmonic approximation while the colored ones are calculated from the full anharmonic wavefunction. Green curves present the maximum shifted towards longer bond lengths or wider angles (Anharmonic increase), on the contrary red indicates contraction (Anharmonic decrease). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 Blue stands for broadening of the anharmonic distribution with respect to the harmonic one, without maximum shift (Anharmonic broadening). n Pictorial representation of the overall effect of anharmonicity and couplings on the quantum nuclear density which leads to speciﬁc nuclear density redistribution (indicated by the green arrows) giving two distinctive structural effects, the backbone elongation (highlighted in green) and the closing of NHþ 3 umbrella (highlighted in magenta). These are deduced from the consideration of geometrical parameters distributions reported in c–i, m and in the Supplementary Figs. 1, 2, 3. semiclassical distribution clearly shows quantum features, since it extends beyond the classical turning points located at the extremes of the histogram, and it displays higher probability in the middle. direction in Cartesian coordinates, as reported in Supplementary Table 3. Instead, after inclusion of anharmonicity, we can barely detect a reminiscence of the node, and the density concentrates closer to the equilibrium position, as can be also seen in Fig. 3c, where the difference between the anharmonic and harmonic distributions is reported. In the anharmonic picture, the O–H stretch excitation can therefore not be regarded as the excitation of a single bond oscillation, and it is not sufﬁcient, for instance, to consider a separable Morse potential along the O–H bond to model this vibration. We stress that the anharmonic distribution of Fig. 3b, which was obtained with the semiclassical approxima- tion of the wavefunction, has a quantum nature even if the calculation is based on classical information. This is evident when we build a histogram by binning instantaneous bond lengths along the quasi-classical trajectory employed for the semiclassical calculation (i.e., the constant energy classical trajectory with excited O–H stretch kinetic energy in harmonic approximation, as described in the Supplementary Methods). The quasi-classical distribution is peaked at the turning points. On the contrary, the direction in Cartesian coordinates, as reported in Supplementary Table 3. Instead, after inclusion of anharmonicity, we can barely detect a reminiscence of the node, and the density concentrates closer to the equilibrium position, as can be also seen in Fig. 3c, where the difference between the anharmonic and harmonic distributions is reported. In the anharmonic picture, the O–H stretch excitation can therefore not be regarded as the excitation of a single bond oscillation, and it is not sufﬁcient, for instance, to consider a separable Morse potential along the O–H bond to model this vibration. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 ARTICLE O2 a m b l i h g n c d e f H3 C2 C1 O1 H6 H4 H5 H1 H2 1.84 1.86 1.88 1.9 1.92 1.94 1.96 1.04 1.06 1.08 1.1 1.12 105 106 107 108 109 110 111 112 1.49 1.5 1.16 1.14 1.12 1.1 1.08 1.06 1.60 1.51 1.52 1.53 1.54 1.55 1.56 1.3 1.31 118 120 θ2(O2-C1-C2) (deg) θ2 θ1 φ φ(N-C2-C1-O1) (deg) θ1(H1/2-N-H3) (deg) d(O2-H3) (Å) d(N-H3) (Å) d(C2-N) (Å) d(C2-H4/5) (Å) d(C1-O1) (Å) d(C1-C2) (Å) 122 124 1.32 1.33 1.34 1.35 1.70 1.80 1.90 200 1.51 1.52 1.53 1.54 N Harmonic Anharmonic decrease Anharmonic increase Anharmonic broadening Fig. 2 Comparison of harmonic and anharmonic nuclear densities for the vibrational ground state. a The Cs symmetry equilibrium geometry of GlyH+ at DFT-B3LYP/aug-cc-pVDZ level of theory with the atomic labeling that we adopt as reference throughout the paper. b Two isosurfaces of the difference between the anharmonic and the corresponding harmonic nuclear densities for the vibrational ground state. Red indicates positive contributions, where the density concentrates due to anharmonicity, while blue stands for the negative contributions, where the density is depleted. The density isosurfaces are respectively set to 0.15a.u. and −0.15a.u. In addition, we report the comparison of the maxima of relevant geometry parameter distributions. c, d, f, g, i, l Bond lengths distributions. h, m bond angles distributions. e Dihedral distribution. The black curves are obtained from the harmonic approximation while the colored ones are calculated from the full anharmonic wavefunction. Green curves present the maximum shifted towards longer bond lengths or wider angles (Anharmonic increase), on the contrary red indicates contraction (Anharmonic decrease). Blue stands for broadening of the anharmonic distribution with respect to the harmonic one, without maximum shift (Anharmonic broadening). n Pictorial representation of the overall effect of anharmonicity and couplings on the quantum nuclear density which leads to speciﬁc nuclear density redistribution (indicated by the green arrows) giving two distinctive structural effects, the backbone elongation (highlighted in green) and the closing of NHþ 3 umbrella (highlighted in magenta). These are deduced from the consideration of geometrical parameters distributions reported in c–i, m and in the Supplementary Figs. 1, 2, 3. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 We stress that the anharmonic distribution of Fig. 3b, which was obtained with the semiclassical approxima- tion of the wavefunction, has a quantum nature even if the calculation is based on classical information. This is evident when we build a histogram by binning instantaneous bond lengths along the quasi-classical trajectory employed for the semiclassical calculation (i.e., the constant energy classical trajectory with excited O–H stretch kinetic energy in harmonic approximation, as described in the Supplementary Methods). The quasi-classical distribution is peaked at the turning points. On the contrary, the Normal mode vs. quantum anharmonic O–H stretch excita- tion. Finally, in Fig. 4 we propose a 3D real-space representation of the anharmonic motion which the molecule undergoes when it is excited by IR radiation, using the observation of nuclear density depletion and accumulation, as an improvement over the classical harmonic normal-mode displacements. Speciﬁcally, we calculate the difference between the anharmonic nuclear density of the O–H stretch excited vibrational state and the ground-state one (Fig. 4b, d). We also compare it to the corresponding quantum harmonic estimate, reported in Fig. 4a, c. Considering that the excitation linked to the vibrational spectra signal at 3553 cm−1 in the experimental IR spectra is interpreted as the O–H stretch, we ﬁrst concentrate on the nuclear density deformation by changing the vibrational state from the ground to NATURE COMMUNICATIONS \| (2020) 11:4348 \| https://doi.org/10.1038/s41467-020-18211-3 \| www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 Harmonic Anharmonic distribution Anharmonic increase Anharmonic broadening 90 1.08 1.1 1.12 1.14 1.16 95 100 105 110 115 120 θ1(C2-C1-N) (deg) d(C2-N) (Å) 0.7 0.75 0 b c f g a d e 0 θ1 0.8 0.85 0.95 1.0 1.05 1.1 1.15 1.2 1.25 1.3 1.48 1.5 1.52 1.54 1.56 0.9 d(O1-H6) (Å) d(C2-H4/5) (Å) Fig. 3 Comparison of harmonic and anharmonic nuclear densities for the O-H stretch vibrational excited state. a The same as in Fig. 2b but for the O–H stretch vibrational excited state. b–f Relevant geometry parameter distributions with the same color code as in Fig. 2. The solid black curves are obtained from the harmonic approximation of the wavefunction, while the colored ones are calculated from the full anharmonic wavefunction. Green curves present the maximum shifted towards longer bond lengths (Anharmonic increase), while the blue one stands for broadening of the anharmonic distribution with respect to the harmonic one and without maximum shift (Anharmonic broadening). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 The harmonic normal mode distribution is small but not equal to zero at equilibrium distance because the variation of O–H stretch normal mode does involve, even if minimally, also other atom displacements. c Difference between anharmonic and harmonic curves of b. The statistical error bars for the distributions are smaller than the line width. d C2–C1–N angle distribution. f, e enlargement of the distribution peaks for the C2–H4/5 and C2–N bond distances. g Summary of the overall effect of anharmonicity and couplings on the quantum nuclear density which leads to speciﬁc nuclear density redistribution (indicated by the green arrows) giving effects which counterbalance each other in two separate regions of the molecule, the hydroxyl (highlighted in magenta) and the CH2NHþ 3 (highlighted in green). These are deduced from the consideration of geometrical parameters distributions reported in b, f, e, d, and in the Supplementary Figs. 4, 5. densities clearly indicates that all the nuclei are involved in the excitation, even if the anharmonic density difference exhibits (with distortions) the typical lobe pattern of the O1–H6 stretch excitation. To give a quantitative assessment of the involvement of each nucleus in the excitation in the harmonic and anharmonic case we can also refer to the density standard deviation position differences (see Supplementary Discussion) shown in Supple- mentary Tables 4, 5. These data conﬁrm what is clearly visible from the nuclear density differences in Fig. 4c, d, i.e., that many more atoms are involved in the O–H stretch excitation when the anharmonic density is employed. More importantly, the anhar- monic nuclear density picture highlights the relevant coupling of the hydroxyl group with the far protonated amino group, which is signiﬁcantly involved in the O–H stretch vibrational excitation. This is proved by the values of the harmonic basis set functions reported in Supplementary Table 1, where the contribution of each normal mode to the spectroscopic signal at 3553 cm−1 is quantiﬁed. The vibrational coupling is therefore non-local in this case, i.e., it is not conﬁned to the nearest neighbor atoms as shown in Fig. 4e for the harmonic approximation. In particular, the anharmonic vibration of the hydroxyl functional group triggers the vibration of the CH2NHþ 3 groups, as pictorially summarized by Fig. 4f. the excited one. We start with an analysis focused on the hydroxyl group of the molecule (Fig. 4a, b). The harmonic case reported in Fig. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 Speciﬁcally, b Bond-length distributions along the radial O1–H6 stretch distance. The ﬁlled area is the histogram of the classical distribution obtained from the quasi-classical trajectory used to generate the semiclassical wavefunction. The harmonic normal mode distribution is small but not equal to zero at equilibrium distance because the variation of O–H stretch normal mode does involve, even if minimally, also other atom displacements. c Difference between anharmonic and harmonic curves of b. The statistical error bars for the distributions are smaller than the line width. d C2–C1–N angle distribution. f, e enlargement of the distribution peaks for the C2–H4/5 and C2–N bond distances. g Summary of the overall effect of anharmonicity and couplings on the quantum nuclear density which leads to speciﬁc nuclear density redistribution (indicated by the green arrows) giving effects which counterbalance each other in two separate regions of the molecule, the hydroxyl (highlighted in magenta) and the CH2NHþ 3 (highlighted in green). These are deduced from the consideration of geometrical parameters distributions reported in b, f, e, d, and in the Supplementary Figs. 4, 5. Harmonic Anharmonic distribution Anharmonic increase Anharmonic broadening 90 1.08 1.1 1.12 1.14 1.16 95 100 105 110 115 120 θ1(C2-C1-N) (deg) d(C2-N) (Å) 0 b a d e θ1 90 1.08 1.1 1.12 1.14 1.16 95 100 105 110 115 120 θ1(C2-C1-N) (deg) d(C2-N) (Å) d e d a 0.7 0.75 0 b c 0 0.8 0.85 0.95 1.0 1.05 1.1 1.15 1.2 1.25 1.3 0.9 d(O1-H6) (Å) b e d(C2-N) (Å) f 1.48 1.5 1.52 1.54 1.56 d(C2-H4/5) (Å) g f g d(O1-H6) (Å) Fig. 3 Comparison of harmonic and anharmonic nuclear densities for the O-H stretch vibrational excited state. a The same as in Fig. 2b but for the O–H stretch vibrational excited state. b–f Relevant geometry parameter distributions with the same color code as in Fig. 2. The solid black curves are obtained from the harmonic approximation of the wavefunction, while the colored ones are calculated from the full anharmonic wavefunction. Green curves present the maximum shifted towards longer bond lengths (Anharmonic increase), while the blue one stands for broadening of the anharmonic distribution with respect to the harmonic one and without maximum shift (Anharmonic broadening). Speciﬁcally, b Bond-length distributions along the radial O1–H6 stretch distance. The ﬁlled area is the histogram of the classical distribution obtained from the quasi-classical trajectory used to generate the semiclassical wavefunction. Methods Computatio Computational details. The semiclassical calculation of GlyH+ eigenfunctions is performed using on-the-ﬂy classical trajectories at the DFT-B3LYP level of theory using the aug-cc-pVDZ basis set with the NWChem package70. We employ one trajectory for the ground state and another one for the O–H stretch excited state. For each one, the Hessian matrix is calculated at each time-step. For our DFT simulations the timing is: 16 h for the trajectory evolution and 552 h for the Hessians employing the NWChem code on 20 CPUs with clock frequency of 2.6 GHz. We also point out that the calculation of the Hessian along the trajectory is embarrassingly parallel, and the scaling with the number of cores employed is linear. Each GlyH+ eigenfunction has been symmetrized so that it belongs to one of the Cs irreducible representations and is given by a combination of 12,799 har- monic functions, which are obtained from considering all possible single and simultaneous excitations of two modes with maximum quantum number nα = 6. However, only the coefﬁcients greater than 10−3 are important for the eigen- function shape. The excited eigenfunction is orthonormalized via Gram-Schmidt with respect to the ground state one. The 3D histogram for the one-nucleus density is composed of cubes whose edge is 0.049 Å. We represent these histograms in the standard cube ﬁle format, which can be read by any visualization software which supports it. In particular, we used the VMD software to produce all the nuclear density pictures in the present work71. Instead, the density space resolution of the bond-length distributions in Fig. 3b is equal to 0.008 Å, the one for angle and dihedral distributions is 0.45 degrees. The evaluation of wavefunction coefﬁcients and the calculation of the densities takes few minutes on a personal computer and it does not necessarily require many cores or large memory. Fig. 4 Nuclear density difference plots for the excited O-H stretch ib ti l i t t f th Gl H+ Diff b t th h Fig. 4 Nuclear density difference plots for the excited O-H stretch vibrational eigenstate of the GlyH+. a Difference between the harmonic density of the excited eigenstate and the corresponding ground-state one on the hydroxyl group. b The same but for the anharmonic case. c, d The same differences but for the entire molecular structure. Red indicates positive contributions, while blue stands for the negative contributions. Methods Computatio All isodensities are set to 0.15a.u. and −0.15a.u. e, f Pictorial representation of different interpretations of the vibrational excitation as derived by the harmonic picture (e) and the anharmonic one (f). The green arrows indicate the most signiﬁcant shifts of the nuclear densities, while the magenta and green halos highlight the molecular regions most affected by the geometrical distortions caused by excitation respectively in the harmonic and anharmonic case. These are deduced from the consideration of geometrical parameters distributions reported in the Supplementary Figs. 1–5. Discussion l Any data generated and analyzed for this study that are not included in this Article and its Supplementary Information are available from the authors upon request. In conclusion, the present method allows for the calculation of high dimensional quantum eigenfunctions of vibrational ground and excited states for molecules of moderate size, and provides an immediately informative real space nuclear density representation of molecular vibrations, overcoming the major limitations of the normal-mode approach and going beyond the quantum harmo- nic picture53. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 ARTICLE functional groups in the molecule, and the normal mode picture that we are accustomed to. Instead, our semiclassical method provides a quantum mechanical anharmonic picture of molecular vibrations which reveals quantitatively that more than one mode can be responsible for a given fundamental excitation and how much the modes are mixing. When we consider the complete potential, which includes all the anharmonicities without any other approximation apart from the level of electronic structure theory adopted, our calculations quantitatively characterize the involvement of other functional groups, also in the case of spectral features which are usually assigned to very localized normal modes, such as the O–H stretch one of the GlyH+. Harmonic a c d e f b Anharmonic Fig. 4 Nuclear density difference plots for the excited O-H stretch ib ti l i t t f th Gl H+ Diff b t th h i d f b Anharmonic a c e b a d c c Eventually, this method allows a more accurate, physically sound assignment of fundamental and overtone vibrational absorption bands. This approach should stimulate a better rationalization of the experimental results, providing a reliable tool to gauge the extent and importance of couplings for a comprehensive understanding of anharmonic vibrational beha- vior in molecules. f Code availability The computer code and the data ﬁles used to produce all the ﬁgures presented in this study are available as Supplementary Code (zip ﬁle). The code is released under the GNU General Public Licence v3. p The analysis of the quantum nuclear densities for a biologically important and experimentally signiﬁcant molecule, GlyH+, shows that anharmonic contributions, that can be assessed thanks to our semiclassical methodology, have a signiﬁcant impact on the interpretation of relevant vibrational excitations. Overall, our anharmonic quantum simulation provides a ﬂexible molecule picture with a greater adaptability to the various binding situa- tions than the harmonic approximation. As an example, we cal- culate the eigenfunction corresponding to the free O–H stretch energy peak and ﬁnd that other modes, in addition to the O–H stretch one, contribute to this spectroscopic signal. More speci- ﬁcally, the amino group plays a signiﬁcant synergistic role in the O–H stretch excitation. Even if the mixing of normal modes is the well known consequence of the introduction of couplings, the determination of the amount of mixing as resulting from our calculations is not trivial, also given the position of the two Received: 23 January 2020; Accepted: 29 July 2020; NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-18211-3 4a provides lobes with perpendicular nodal planes with respect to the O1–H6 bond. There is a minor density deformation contribution on the O6 nucleus, while the bigger one is on the lighter H6. Turning to the anharmonic picture shown in Fig. 4b, we ﬁnd a similar pattern but with the lobes that have no longer parallel nodal planes. The lobes are in this case distributed along a curved line. This is an effect of anharmonicity and vibrational couplings which leads to a less symmetric oscillation of the O–H bond. By zooming out to the whole molecule structure in Fig. 4d, we can appreciate how anharmonicity calls into play all the other nuclei. The difference between the harmonic picture of Fig. 4c is striking. The harmonic picture in panel c is compliant with the chemical intuition and with the classical normal-mode displace- ment picture. The excitation of the O–H stretch normal mode results in a very localized density deformation, which causes a depletion and a consequent increment of density exclusively for the hydroxyl functional group, while in the other parts of the molecule the ground state and the excited state densities cancels perfectly upon subtraction. Instead, the quantum mechanical anharmonic picture provided by the semiclassical vibrational TURE COMMUNICATIONS \| (2020) 11:4348 \| https://doi.org/10.1038/s41467-020-18211-3 \| www.nature.com/naturecommunications 6 ARTICLE Micciarelli, M., Conte, R., Suarez, J. & Ceotto, M. Anharmonic vibrational eigenfunctions and infrared spectra from semiclassical molecular dynamics. J. Chem. Phys. 149, 064115 (2018). y y 25. Imoto, S. & Marx, D. 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Peer reviewer reports are available. 69. Voss, J. M., Fischer, K. C. & Garand, E. Accessing the vibrational signatures of amino acid ions embedded in water clusters. J. Phys. Chem. Lett. 9, 2246–2250 (2018). Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints Additional information 66. Wu, R. & McMahon, T. B. Infrared multiple photon dissociation spectra of proline and glycine proton-bound homodimers. Evidence for zwitterionic structure. J. Am. Chem. Soc. 129, 4864–4865 (2007). Supplementary information is available for this paper at https://doi.org/10.1038/s41467- 020-18211-3. 67. Zhang, K. & Chung-Phillips, A. Conformers of gaseous protonated glycine. J. Comput. Chem. 19, 1862–1876 (1998). © The Author(s) 2020 Acknowledgements The authors thank Prof. E. Garand for providing the experimental spectrum of proto- nated glycine. The authors thank Dr. F. Gabas for contribution in the early stages of this work, Prof. L. Lo Presti for discussion, and Prof. A. Gavezzotti for a critical reading of the manuscript. The authors acknowledge ﬁnancial support from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme [Grant Agreement No. (647107)—SEMICOMPLEX—ERC-2014-CoG] and from the Italian Ministry of Education, University, and Research (MIUR) (FARE pro- gramme R16KN7XBRB project QURE). Part of the needed CPU time was provided by CINECA (Italian Supercomputing Center) under ISCRAB project QUASP, ISCRAC project MCSCMD, and ISCRAC project heavyTUN. © The Author(s) 2020 © The Author(s) 2020 NATURE COMMUNICATIONS \| (2020) 11:4348 \| https://doi.org/10.1038/s41467-020-18211-3 \| www.nature.com/naturecommunications 9
https://openalex.org/W2170770595	https://pubs.rsc.org/en/content/articlepdf/2014/ra/c4ra90013h	English	null	Correction: White light emission from a mixed organogel of lanthanide(<scp>iii</scp>)-containing organogelators	RSC advances	2,014	cc-by	334	RSC Advances RSC Advances Department of Materials Science and Engineering, College of Engineering, Seoul National University, Seoul, 151-744, Korea. E-mail: jichang@snu.ac.kr; Fax: +82 2 885 1748; Tel: +82 2 880 7190 This journal is © The Royal Society of Chemistry 2014 CORRECTION Correction: White light emission from a mixed organogel of lanthanide(III)-containing organogelators Correction: White light emission from a mixed organogel of lanthanide(III)-containing organogelators Correction for ‘White light emission from a mixed organogel of lanthanide(III)-containing organogelators’ by Hyungwoo Kim et al., RSC Adv., 2013, 3, 1774–1780. Fig. 2a in the original manuscript was mistakenly a repeat of Fig. 3a. The correct Fig. 2 is as follows: Fig. 2a in the original manuscript was mistakenly a repeat of Fig. 3a. The correct Fig. 2 is as follows: Fig. 2 TEM images of dry gels obtained from Eu2 (a) and Tb2 (b) in n-decane (2 wt%). Access Article. Published on 15 September 2014. Downlo This article is licensed under a Creative Common Fig. 2a in the original manuscript was mistakenly a repeat of Fig. 3a. The correct Fig. 2 is as follows: Fig. 2 TEM images of dry gels obtained from Eu2 (a) and Tb2 (b) in n-decane (2 wt%). Access Article. Published on 15 September 2014. Downlo This article is licensed under a Creative Common Fig. 2 TEM images of dry gels obtained from Eu2 (a) and Tb2 (b) in n-decane (2 wt%). The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers. Royal Society of Chemistry apologises for these errors and any consequent inconvenience to autho Department of Materials Science and Engineering, College of Engineering, Seoul National University, Seoul, 151-744, Korea. E-mail: jichang@snu.ac.kr; Fax: +82 2 885 1748; Tel: 82 2 880 7190 Department of Materials Science and Engineering, College of Engineering, Seoul National University, Seoul, 151-744, Korea. E-mail: jichang@snu.ac.kr; Fax: +82 2 885 1748; Tel: +82 2 880 7190 RSC Adv., 2014, 4, 43549 \| 43549 This journal is © The Royal Society of Chemistry 2014
https://openalex.org/W1161177104	https://www.pure.ed.ac.uk/ws/files/21562567/Variable_exon_usage_of_differentially_expressed_genes_associated_with_resistance_of_sheep_to_Teladorsagia_circumcincta.pdf	English	null	Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta	Veterinary parasitology	2,015	cc-by	7,934	Edinburgh Research Explorer Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta Citation for published version: Wilkie, H, Xu, S, Gossner, A & Hopkins, J 2015, 'Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta', Veterinary Parasitology, vol. 212, no. 3- 4, pp. 206-213. https://doi.org/10.1016/j.vetpar.2015.08.023 Citation for published version: Wilkie, H, Xu, S, Gossner, A & Hopkins, J 2015, 'Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta', Veterinary Parasitology, vol. 212, no. 3- 4, pp. 206-213. https://doi.org/10.1016/j.vetpar.2015.08.023 General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and investigate your claim. 1. Introduction Stear et al., 2006) but the identiﬁcation of genetic markers has the potential for marker assisted selection (Davies et al., 2006). Both quantitative trait loci mapping and genome-wide association stud- ies have been used to identify selectable genetic markers (Davies et al., 2005; Dominik, 2005; Riggio et al., 2013). More recently high throughput microarrays (Andronicos et al., 2010; Diez-Tascon et al., 2005) and digital gene expression (Pemberton et al., 2011) have been used to identify potential candidate genes for resistance to sheep gastrointestinal nematodes including Haemonchus contortus (Rowe et al., 2008) and T. circumcincta (Gossner et al., 2013; Knight et al., 2011). The aim of these studies was to identify genes that were differentially-expressed in pre-exposed (immune) vs. naïve or resistant vs. susceptible sheep and to try and explain the molecular basis of differential polarization of the immune response associated with each phenotype. The abomasal nematode, Teladorsagia circumcincta is one of the most common parasites of sheep in cool temperate regions (Urquhart et al., 1987) and is a major drain on the economics of sheep production (Nieuwhof and Bishop, 2005). Several studies have described genetic variation for parasite susceptibility within and between sheep breeds (Dominik, 2005; Mugambi et al., 1997; Terefe et al., 2007), and that faecal egg count (FEC) is a heritable characteristic (Davies et al., 2005) and can be used as a parameter for selective breeding for parasite resistance (Davies et al., 2005; Windon, 1990; Woolaston, 1997). Sheep selected for low FEC con- trol infection by the acquisition of anti-parasite antibody (Stear et al., 1999) driven byTh2 cytokines (Craig et al., 2014), and there is a signiﬁcant genetic relationship between IgA antibody levels and the ability to control infection (Stear et al., 1997; Strain et al., 2002). In contrast, sheep selected for high FEC produce little IgA antibody but instead generate an inﬂammatory Th1/Th17 response that fails to control infection and egg production (Gossner et al., 2012). Microarray analysis of abomasal lymph nodes from infected sheep, selected on the basis of FEC, highlighted genes and physi- ological pathways related to the polarization of T cells and control of inﬂammation as being associated with resistance (Gossner et al., 2013). These data were obtained using the Affymetrix Ovine Gene 1.1 ST whole-genome array consisting of probes for all exons of each annotated transcript in the sheep Oar v2 genome assembly. jou rn al h om epage: www.elsevier.com/locate/vetpar Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta Hazel Wilkie 1, Siyang Xu 1, Anton Gossner, John Hopkins The Roslin Institute & R(D)SVS, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK a r t i c l e i n f o The resistance and susceptibility of sheep to the common abomasal nematode parasite, Teladorsagia cir- cumcincta is strongly associated with the differential polarization of the immune response. Resistant animals control larval colonization by the production of a protective antibody response regulated by Th2 T cells. Susceptible sheep respond to infection by developing an inﬂammatory Th1/Th17 response that fails to control infection. Previous microarray analysis identiﬁed genes associated with T cell polarization that were differentially expressed between the resistant and susceptible sheep. RT-qPCR conﬁrmed the microarray data for ALOX15 and IL13. Both ALOX15 exon 9 and IL13 exon 4 were signiﬁcantly increased in resistant animals and copy number RT-qPCR showed that expression levels of these exons were signif- icantly negatively correlated with quantitative phenotypic traits, including abomasal worm counts and faecal egg counts. Sequencing of the intronic regions 5′ to these genes failed to identify any potential genetic links to differential exon usage. Article history: Received 9 June 2015 Received in revised form 17 August 2015 Accepted 20 August 2015 Keywords: Sheep Teladorsagia Resistance Cytokines Gene variants Article history: Received 9 June 2015 Received in revised form 17 August 2015 Accepted 20 August 2015 © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). ARTICLE IN PRESS G Model VETPAR-7752; No. of Pages 8 G Model G Model VETPAR-7752; No. of Pages 8 Veterinary Parasitology xxx (2015) xxx–xxx ∗Corresponding author. Fax: +44 131 651 9107. E-mail address: john.hopkins@ed.ac.uk (J. Hopkins). 1 These authors contributed equally to this work. Take down policy Download date: 24. Oct. 2024 http://dx.doi.org/10.1016/j.vetpar.2015.08.023 0304-4017/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). H. Wilkie et al. / Veterinary Parasitology xxx (2015) xxx–xxx 2 II Reverse Transcription Kit (Invitrogen, UK) using oligo-dT(15) primer and RNaseOUT (Invitrogen). (or part of) an exon or intron creates multiple distinct transcripts (Blencowe, 2006). Alternative splicing can modify coding sequence, but within the 5′ and 3′ UTRs it can also alter miRNA-binding and regulatory motifs and inﬂuence transcript expression levels or translation, and therefore can have a profound effect on the identity or expression levels of a protein (Martinez and Lynch, 2013). 2.3. Cloning of ovine exons The sequences of differentially-expressed probes of the Affymetrix Ovine Gene 1.1 ST Array (http://www.affymetrix.com/ analysis/index.affx) were used to obtain full exon sequences using NCBI-BLAST against the sheep genome assembly, Oar v3.1 (http://www.livestockgenomics.csiro.au/sheep/oar3.1.php/). Exon-speciﬁc primers were selected using Primer-BLAST (http:// www.ncbi.nlm.nih.gov/tools/primer-blast/) and reanalysed using Net Primer (www.premierbiosoft.com/netprimer/). Primers used for RT-PCR and RT-qPCR are shown in Table S2A. The location of the primers and their relationship to the locations of the Affymetrix Ovine Gene 1.1 ST whole-genome array probe sets is shown in Table S3. RT-PCR used the FastStart Taq DNA Polymerase Kit (Roche) following the manufacturer’s protocol. PCR products were ana- lyzed by agarose gel electrophoresis, visualized by GelRed/UV transillumination, puriﬁed using MinElute PCR Puriﬁcation Kit (Qiagen), ligated into pGEM-T Easy vector (Promega) and trans- formed into JM109High Efﬁciency Competent Cells (Promega). A random selection of color-screened clones were sequenced (Edin- burgh Genomics; https://genomics.ed.ac.uk/) with SP6 and T7 primers using the BigDye® Terminator v3.1Cycle Sequencing Kit (Applied Biosystems, UK). The focus of this current study was to examine the differential usage of exons of selected genes in the resistant and suscepti- ble sheep, as identiﬁed by microarray analysis (Gossner et al., 2013). The eight genes were chosen on the basis that they were differentially-expressed in resistant and susceptible sheep and have a role in T cell biology and inﬂammation and included arachi- donate 15-lipoxygenase (ALOX15) and interleukin 13 (IL13), both of which were signiﬁcantly increased in resistant animals and had variable exon usage. The microarray data were ﬁrst validated by fold-change RT-qPCR for two exons of each selected gene; copy number RT-qPCR was then used to test the hypotheses that resis- tance/susceptibility to T. circumcincta is associated with differential exon usage within the selected genes and that individual exon usage correlates with selected quantitative parameters of resis- tance. 2.4. Quantitative real-time PCR analysis qPCR was performed using 1 l template cDNA or linearized plasmid DNA (for the copy number analysis), 7.5 l FastStart Universal SYBRgreen Master (Rox) 2× concentration (Roche), 0.1––0.3 l primers and nuclease-free water to 15 l ﬁnal volume. All reactions were prepared using a CAS-1200TM Precision Liquid Handling System and performed on the Rotor-Gene Q (Qiagen). Cycle conditions were: 95 ◦C for 10 min and then 40 cycles of 95 ◦C for 10 s, annealing (Table S2A) for 15 s and 72 ◦C for 30 s followed by melt curve analysis. Optimised RT-qPCR primers had an efﬁciency between 95 and 105% and R2 of 0.98 and 0.99. Relative analysis was performed on the 7 most resistant sheep (ranked 1–7) and the 7 most susceptible sheep (ranked 39–45) (Table S1). Abso- lute expression (copy number) analysis was performed on all 45 infected animals. Transcripts were quantiﬁed in cDNA from three separate RT-qPCR reactions for each biological sample; each cDNA sample was assayed in triplicate with GAPDH housekeeping and no-template controls included in all runs. Relative gene expression levels were calculated in GenEx 5.3.4.157 (MultiD Analyses AB, Sweden) using the comparative 2 − (Cq) method and normalized to the geometric mean of GAPDH and SDHA. Fold changes were calculated from Cq values using GenEx. To derive the copy number of the target sequence in all 45 sheep, a standard curve (linearized sheep ALOX15 or IL13 plasmid DNA) was used with a dynamic range that spanned at least ﬁve orders of magnitude. Copy numbers were calcu- lated from Cq values using the following formula: molecules per g = ((1 × 10−6)/(M g/mol)× [6.03 × 10−23 molecules/mol]) 2.1. Animals and tissues Details of animals, infection protocols, phenotypic parameters and population genetic analyses have been described previously (Beraldi et al., 2008). Brieﬂy, ∼3 months old Blackface female lambs originated from a ﬂock used for quantitative trait analyses (Davies et al., 2006); all animals were housed in worm-free conditions. Ten sheep were sham infected controls and 45 sheep were infected experimentally with ∼2300 L3 T. circumcincta larvae three times a week for three months and sacriﬁced two days after the last infection. At post mortem ten of the infected group had no adult worms in the abomasal contents, while the other infected ani- mals had adult worm count (AWC) within the abomasum at post mortem ranging from 80 to 11300 (Table S1). Animals were ranked (1–45) according to AWC and FEC (Beraldi et al., 2008). The relative concentration of serum IgA anti-T. circumcincta antibody was also measured for all these animals; and it was determined that both AWC and FEC were signiﬁcantly negatively correlated with anti- body levels and with body weight (BW) (Beraldi et al., 2008). The 7 most resistant (R) sheep (infection rank 1–7) had no detectable AWC or FEC, high IgA antibody levels and high body weight (BW). The 7 most susceptible (S) sheep (infection rank 39–45) were those with the highest AWC (mean 6000, maximum 11300), high FEC (mean 414, maximum 950), low IgA antibody levels and low body weight. Animal experiments were approved by University of Edin- burgh Ethical Review Committee and conducted under an Animals (Scientiﬁc Procedures) Act 1986 Project Licence. Abomasal (gas- tric) lymph nodes (ALN) were removed at post mortem and stored at −80 ◦C in RNAlater (Ambion, Huntingdon, UK). 2.2. RNA extraction and cDNA synthesis M = size of plasmid × 660 g/mol per bp. The expression levels were normalized by dividing the copy number derived from the standard curve by the calculated normalization factor for each indi- vidual sample. Total RNA was isolated from 0.02 g of tissue using the Ribop- ure Kit (Ambion, UK) according to the manufacturers’ instructions. Contaminating DNA was removed by On-column PureLink® DNase I treatment (Ambion). The quantity, quality and integrity of the RNA samples were determined using a NanoDrop ND-1000 spectropho- tometer (Labtech International Ltd.) and Agilent 2200 TapeStation system (Agilent Technologies); all had an RNA Integrity Num- ber of >7.3. cDNA synthesis from 1 g RNA was by SuperScriptTM 1. Introduction The data were originally analysed at the gene-level, with exons summarized to genes, but this analysis also revealed that individual exons for some genes were not equally expressed. FEC and antibody levels have both been used as selectable mark- ers of resistance (McRae et al., 2014; Sayers and Sweeney, 2005; ∗Corresponding author. Fax: +44 131 651 9107. E-mail address: john.hopkins@ed.ac.uk (J. Hopkins). 1 These authors contributed equally to this work. Alternative splicing is a post-transcriptional mechanism for reg- ulating gene expression; and within a gene the variable usage of Please cite this article in press as: Wilkie, H., et al., Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.023 http://dx.doi.org/10.1016/j.vetpar.2015.08.023 0304-4017/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 5.08.023 shed by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Please cite this article in press as: Wilkie, H., et al., Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.023 ARTICLE IN PRESS G Model VETPAR-7752; No. of Pages 8 2 H. Wilkie et al. / Veterinary Parasitology xxx (2015) xxx–xxx ARTICLE IN PRESS G Model VETPAR-7752; No. of Pages 8 2 H. Wilkie et al. / Veterinary Parasitology xxx (2015) xxx–xxx Please cite this article in press as: Wilkie, H., et al., Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.023 ARTICLE IN PRESS H. Wilkie et al. / Veterinary Parasitology xxx (2015) xxx–xxx G Model VETPAR-7752; No. of Pages 8 G Model VETPAR-7752; No. of Pages 8 Table 1 iff DNA Puriﬁcation System (Promega). 10–20 mg of ALN tissue was incubated in the digestion solution at 55 ◦C with 300 rpm shak- ing overnight and then processed following the manufacturer’s protocol; 200 ng gDNA per PCR reaction was used as a template for cloning and sequencing of ALOX15 and IL13 promoter regions (primers and parameters Table S2B). Comparison of the array and RT-qPCR fold change analysis for the exons of the selected genes is shown in Table 1. Signiﬁcant differential exon expression was validated, by RT-qPCR, only for ALOX15 and IL13. Exons 9 and 14 of ALOX15 were 22.02 and 7.44 fold increased respectively, in the resistant compared to susceptible sheep as assessed by array, and 5.3 (p = 0.001) and 1.5 fold (p = 0.03) by RT-qPCR. Exons 1 and 4 of IL13 were 3.6 and 6.91 fold increased by array and 3.77 (p = 0.0002) and 5.16 fold (p = 5.2E − 6) by RT- qPCR. When the data were summarized to the whole gene, ALOX15 was 5.81 fold higher, and IL13 was 3.17 fold higher in the resistant sheep (Gossner et al., 2013). Differential exon expression of the other six genes was not conﬁrmed by RT-qPCR, although all but CD109 demonstrated signiﬁcant differential expression for at least one exon in the R vs. S comparison. 3.2. Association of exon copy number and quantitative phenotype ALOX15 and IL13 were chosen for copy number measurement in the ALN in all 45 infected animals. The expression levels of ALOX15 exon 9 was signiﬁcantly higher than exon 14 (mean 11313 ± 4547 and 7729 ± 2360 copies per g total RNA respectively, p = 0.0003). Similarly IL13 exon 4 was expressed signiﬁcantly higher that exon 1 (152.377 ± 81.222 and 72.406 ± 43.858, p < 0.0001). The data were further analysed by dividing the 45 animals into three groups; the ﬁfteen most resistant sheep with mean AWC of 59 and mean FEC of 1.67; a susceptible group of ﬁfteen sheep with mean AWC of 5167 and mean FEC of 288; and the ﬁfteen intermediate sheep with mean AWC of 1508 and mean FEC of 82 (Table S1). 2.6. Statistical analysis Relative gene expression levels were analyzed statistically in GenEx using an unpaired, 2-tailed t-test to determine the differ- ence between groups. ALOX15 and IL13 copy number results were analyzed in GraphPad Prism v 5 (Graph Pad Software, USA). The data were grouped into resistant, susceptible and intermediate (n = 15 per group) and a Kruskal–Wallis test was performed to determine overall signiﬁcance, with Dunn’s multiple comparison test within the Kruskal–Wallis to determine signiﬁcance between groups. The correlations between transcript levels and quantitative phenotypes were analyzed with Spearman’s rank correlation coefﬁcient (rs). p-values less than 0.05 were considered statistically signiﬁcant. 2.5. Sequencing 5′ intronic regions 2.5. Sequencing 5′ intronic regions Genomic DNA (gDNA) was extracted from the ALN of 6 resis- tant (infection rank 1, 3, 4, 5, 6, 7) and 6 susceptible (infection rank 39, 41, 42, 43, 44, 45) sheep using the Wizard® SV Genomic Please cite this article in press as: Wilkie, H., et al., Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.023 ARTICLE IN PRESS G Model VETPAR-7752; No. of Pages 8 H. Wilkie et al. / Veterinary Parasitology xxx (2015) xxx–xxx H. Wilkie et al. / Veterinary Parasitology xxx (2015) xxx–xxx 3 3 H. Wilkie et al. / Veterinary Parasitology xxx (2015) xxx xxx 3 Table 1 Differentially-expressed genes in R vs. S comparison with differential exon usage. Gene symbol Gene name Exon FCa p value FCa p valueb Microarray RT-qPCR ALOX15 Arachidonate 15-lipoxygenase 9 22.03 0.003 5.3c 0.001 14 7.44 0.001 1.5 0.03 CD109 CD109 molecule 11 6.36 0.01 −1.0 0.09 19 1 0.05 −1.1 0.08 CD163 CD163 molecule 1 1 0.05 −2.4 0.01 9 −5.42 0.02 −1.6 0.04 CPA3 Carboxypeptidase A3 (mast cell) 3 8.0 0.004 2.0 0.02 5 2.2 0.03 1.5 0.04 EMR3 egf-like module containing, mucin-like, hormone receptor like-3 9 6.83 0.008 2.2 0.06 11 3.58 0.01 2.0 0.01 IL13 Interleukin 13 1 3.6 0.0001 3.77c 0.0002 4 6.91 0.0002 5.16 5.2E − 6 KIT v-Kit Hardy–Zuckerman 4 feline sarcoma viral oncogene homolog 16 6.16 0.006 1.1 0.08 21 2.06 0.01 1.2 0.05 MAP3K5 Mitogen-activated protein kinase kinase kinase 26 1 0.05 −1.5 0.02 28 7.73 0.01 −1.3 0.03 a Fold change R vs S comparison. b p value R vs S. c p ≤ 0.05 in the comparison of the two exons. 3.1. RT-qPCR validation of array analysis Transcriptome analysis of the ALN has been described previ- ously (Gossner et al., 2013). In brief, cDNA from the abomasal lymph node of 7 resistant, 7 susceptible and 7 uninfected sheep was hybridized to Affymetrix Ovine Gene 1.1 ST Array and ana- lysed, with exons summarized to genes, using the mean expression of all the exons of a gene. These data and protocols are available at ArrayExpress accession number E-MTAB-1580. In this current study the same dataset was used but without summarizing to genes. Of the 165740 exons interrogated, 1196 were signiﬁcantly differentially-expressed within their gene in the R vs. S comparison, with a fold change ≥2 and p-value ≤0.05 (one-way between-subject ANOVA, unpaired); 930 exons were increased and 266 exons were repressed. Eight genes were selected, based on each being signiﬁ- cantly differentially-expressed in the R vs. S comparison, their role in T cell biology and inﬂammation as well as signiﬁcant differential exon usage within the gene (Table S4). RT-qPCR was developed for two exons of each gene including one with signiﬁcant differential- expression. Quantiﬁcation of ALOX15 (Fig. 1) showed that exons 9 and 14 expression levels were signiﬁcantly different in the resistant, rank 1–15 (15.842 ± 4434 for exon 9 and 9360 ± 2382 copies per g RNA for exon 14, p < 0.0001), intermediate, rank 16–32 (10377 ± 2272 and 7381 ± 2330, p = 0.013) and susceptible groups, rank 31–45 (7719 ± 1846 and 6446 ± 1286, p = 0.04). Measurement of the IL13 exons also showed signiﬁcant differential expression of exon 1 and exon 4 in all three groups; 83418 ± 36148 for exon 1 and 205953 ± 73295 for exon 4, p < 0.0001 in the resistant group; 69660 ± 50.941 and 148318 ± 84464, p = 0.0045 in the intermedi- ate group, and 64139 ± 43.959 and 102858 ± 50067, p = 0.03 in the susceptible group. Spearman’s rank correlation analysis was used to quantify the correlation of expression levels of each exon in relation to the quan- titative phenotypes, FEC, AWC, BW and IgA antibody levels (Table S1). ALOX15 exon 9 was signiﬁcantly negatively correlated with Please cite this article in press as: Wilkie, H., et al., Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta. Vet. Parasitol. 3.1. RT-qPCR validation of array analysis Copy number per (FEC < 10). B, rank 16–30, intermediate (FEC 10–219). C, rank 31–45 susceptible (FEC > 220). Error bars are means ± SD. Fig. 1. Expression of ALOX15 exons 9 and 14, and IL13 exons 1 and 4 in the ALN of T. circumcincta infected sheep. Copy number per g total RNA. A, rank 1–15 resistant sheep (FEC < 10). B, rank 16–30, intermediate (FEC 10–219). C, rank 31–45 susceptible (FEC > 220). Error bars are means ± SD. cantly negatively correlated with FEC (rs −0.255, p = 0.046) but there were no signiﬁcant correlations between exon 1 and AWC (rs −0.15, p = 0.17), BW (rs 0.18, p = 0.12) or IgA (rs 0.001, p = 0.50). AWC (rs −0.59, p < 0.0001) and FEC (rs −0.62, p < 0.0001), and signif- icantly positively correlated with BW (rs 0.75, p < 0.0001) and IgA (rs 0.59, p < 0.0001), exon 14 was also signiﬁcantly negatively cor- related with AWC (rs −0.40, p = 0.004) and FEC (rs −0.31, p = 0.02), and signiﬁcantly positively correlated with BW (rs 0.40, p < 0.004), but not with IgA (rs 0.18, p = 0.11) (Fig. 2). 3.1. RT-qPCR validation of array analysis Susceptible, rank 31 – 45 E xo n 1 E xo n 4 0 5 0 0 0 0 1 0 0 0 0 0 1 5 0 0 0 0 2 0 0 0 0 0 p = 0.03 E xo n 1 E xo n 4 0 1 0 0 0 0 0 2 0 0 0 0 0 3 0 0 0 0 0 4 0 0 0 0 0 E xo n 1 E xo n 4 0 1 0 0 0 0 0 2 0 0 0 0 0 3 0 0 0 0 0 4 0 0 0 0 0 E xo n 9 E xo n 1 4 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 E xo n 9 E xo n 1 4 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 p = 0.013 p = 0.04 ALOX15 E xo n 9 E xo n 1 4 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 2 5 0 0 0 IL13 p <0.0001 p <0.0001 p = 0.0045 1. Expression of ALOX15 exons 9 and 14, and IL13 exons 1 and 4 in the ALN of T. circumcincta infected sheep. Copy number per g total RNA. A, rank 1–15 resistan C < 10). B, rank 16–30, intermediate (FEC 10–219). C, rank 31–45 susceptible (FEC > 220). Error bars are means ± SD. WC (rs −0.59, p < 0.0001) and FEC (rs −0.62, p < 0.0001), and signif- ntly positively correlated with BW (rs 0.75, p < 0.0001) and IgA 0.59, p < 0.0001), exon 14 was also signiﬁcantly negatively cor- ated with AWC (rs −0.40, p = 0.004) and FEC (rs −0.31, p = 0.02), d signiﬁcantly positively correlated with BW (r 0 40 p < 0 004) cantly negatively correlated with FEC (rs −0.255, p = 0.04 there were no signiﬁcant correlations between exon AWC (rs −0.15, p = 0.17), BW (rs 0.18, p = 0.12) or IgA (rs p = 0.50). 4 IL13 ALOX15 NA A. 3.1. RT-qPCR validation of array analysis Resistant, rank 1 – 15 1 2 3 4 ALOX15 E xo n 9 E xo n 1 4 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 2 5 0 0 0 p <0.0001 E xo n 1 E xo n 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 p <0.0001 E xo n 1 B. Intermediate, rank 16 – 30 Copy number per μg total R B. Intermediate, rank 16 – 30 E xo n 9 E xo n 1 4 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 p = 0.013 E xo n 1 E xo n 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 p = 0.0045 E xo n 1 E xo n 1 C. Susceptible, rank 31 – 45 E xo n 9 E xo n 1 4 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 p = 0.04 C. Susceptible, rank 31 – 45 E xo n 1 E xo n 4 0 5 0 0 0 0 1 0 0 0 0 0 1 5 0 0 0 0 2 0 0 0 0 0 p = 0.03 E xo n 9 E xo n 1 4 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 p = 0.04 Fig. 1. Expression of ALOX15 exons 9 and 14, and IL13 exons 1 and 4 in the ALN of T. circumcincta infected sheep. Copy number per g total RNA. A, rank 1–15 resistant she (FEC < 10). B, rank 16–30, intermediate (FEC 10–219). C, rank 31–45 susceptible (FEC > 220). Error bars are means ± SD. C. Susceptible, rank 31 – 45 E xo n 1 0 5 0 0 0 0 0 0 0 0 0 5 0 0 0 0 2 0 0 0 0 0 p = 0. E xo n 1 E xo n 1 4 E xo n 9 E xo n 4 Fig. 1. Expression of ALOX15 exons 9 and 14, and IL13 exons 1 and 4 in the ALN of T. circumcincta infected sheep. 3.1. RT-qPCR validation of array analysis (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.023 Please cite this article in press as: Wilkie, H., et al., Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.023 ARTICLE IN PRESS Model TPAR-7752; No. of Pages 8 ARTICLE IN PRESS VETPAR 7752; No. of Pages 8 4 H. Wilkie et al. / Veterinary Parasitology xxx (2015) xxx–xxx Copy number per μg total RNA A. Resistant, rank 1 – 15 B. Intermediate, rank 16 – 30 C. Susceptible, rank 31 – 45 E xo n 1 E xo n 4 0 5 0 0 0 0 1 0 0 0 0 0 1 5 0 0 0 0 2 0 0 0 0 0 p = 0.03 E xo n 1 E xo n 4 0 1 0 0 0 0 0 2 0 0 0 0 0 3 0 0 0 0 0 4 0 0 0 0 0 E xo n 1 E xo n 4 0 1 0 0 0 0 0 2 0 0 0 0 0 3 0 0 0 0 0 4 0 0 0 0 0 E xo n 9 E xo n 1 4 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 E xo n 9 E xo n 1 4 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 p = 0.013 p = 0.04 ALOX15 E xo n 9 E xo n 1 4 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 2 0 0 0 0 2 5 0 0 0 IL13 p <0.0001 p <0.0001 p = 0.0045 Fig. 1. Expression of ALOX15 exons 9 and 14, and IL13 exons 1 and 4 in the ALN of T. circumcincta infected sheep. Copy number per g total RNA. A, rank 1–15 resistan (FEC < 10). B, rank 16–30, intermediate (FEC 10–219). C, rank 31–45 susceptible (FEC > 220). Error bars are means ± SD. H. Wilkie et al. / Veterinary Parasitology xxx (2015) xxx–xxx Copy number per μg total RNA A. Resistant, rank 1 – 15 B. Intermediate, rank 16 – 30 C. 3.3. Sequence of 5′ intronic or UTR regions of ALOX15 and IL13 3.3. Sequence of 5′ intronic or UTR regions of ALOX15 and IL13 Similar analysis for IL13 (Fig. 3) showed that exon 4 expres- sion was signiﬁcantly negatively correlated with AWC (rs −0.49, p = 0.0003) and FEC (rs −0.474, p = 0.0005) and positively cor- related with BW (rs 0.52, p = 0.0001) but not with IgA levels (rs 0.08, p = 0.31). IL13 exon 1 expression was also signiﬁ- Polymorphisms in intronic promoter or 5′ UTR regulatory ele- ments (Black, 2003), possibly associated with differential exon usage, were examined by sequencing approximately 1000 bp 5′ to the translation start site of both ALOX15 (LN864492) and IL13 Please cite this article in press as: Wilkie, H., et al., Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.023 G Model VETPAR-7752; No. of Pages 8 ARTICLE IN PRESS G Model VETPAR-7752; No. of Pages 8 ARTICLE IN PRESS ARTICLE IN PRESS -7752; No. of Pages 8 H. Wilkie et al. / Veterinary Parasitology xxx (2015) xxx–xxx Correlation analysis of the phenotypic parameters, AWC, FEC, BW and IgA with ALOX15 exon 9 and 14 copy number per g total RNA in ALN of T. circumci s—Spearman’s rank correlation coefﬁcient. Fig. 2. Correlation analysis of the phenotypic parameters, AWC, FEC, BW and IgA with ALOX15 exon 9 and 14 copy number per g total RNA in ALN of T. circumcincta infected sheep. rs—Spearman’s rank correlation coefﬁcient. Fig. 2. Correlation analysis of the phenotypic parameters, AWC, FEC, BW and IgA with ALOX15 exon 9 and 14 copy number per g t sheep. rs—Spearman’s rank correlation coefﬁcient. Please cite this article in press as: Wilkie, H., et al., Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.023 ARTICLE IN PRESS G Model VETPAR-7752; No. of Pages 8 ARTICLE IN PRESS Model TPAR-7752; No. of Pages 8 ARTICLE IN PRESS -7752; No. of Pages 8 H. Wilkie et al. / Veterinary Parasitology xxx (2015) xxx–xxx Correlation analysis of the phenotypic parameters, AWC, FEC, BW and IgA with IL13 exon 1 and 4 copy number per g total RNA in ALN of T. circumcin s—Spearman’s rank correlation coefﬁcient. 4491) from 6 R and 6 S sheep. 4. Discussion These regions were sequenced for both ALOX15 and IL13 from 6 resistant (rank 1–6) and 6 susceptible (rank 40–45) sheep; and although variants were identiﬁed for both genes, none segregated with resistance and sus- ceptibility phenotype. In conclusion, we have validated differential exon usage of two genes that were expressed in the ALN of sheep showing differential resistance to the abomasal nematode T. circumcincta. Both ALOX15 exon 9 and IL13 exon 4 were signiﬁcantly increased in resistant animals and expression levels of these exons were negatively corre- lated with quantitative phenotypic traits, including AWC and FEC. Consequently, they represent potential markers for selection for resistance to a common and economically important gastrointesti- nal parasite. gy Fold-change RT-qPCR resulted in the validation of the microar- ray data of two of the eight selected genes, ALOX15 and IL13. IL13 plays a major role in resistance to gastrointestinal nema- todes. In T. circumcincta infected sheep it was the most signiﬁcant up-regulated gene in the R vs. S comparison by both microarray and RT-qPCR array (Gossner et al., 2013). It is also the dominant cytokine in the control of murine Nippostrongylus brasiliensis infec- tion (Mckenzie et al., 1998) and seems to be equally involved with IL4 in the development of protective immunity to Trichuris muris and Heligomosoides polygyrus (Bancroft et al., 1998). IL13 is largely a product of antigen-activated Th2 cells but is also synthesized by mast cells and eosinophils (Gessner et al., 2005). IL13 shares many biological functions with IL4, including the regulation of Th2 development and consequently antibody production and heavy chain switch; it also inhibits inﬂammatory cytokine production and promotes mast cell proliferation (Minty et al., 1993) and tis- sue remodelling after parasite-induced injury (Wynn, 2003). IL13 is encoded by four exons on the minus strand of ovine chromo- some 5 (NC 019462.1); exon 4 encodes 108 bp at the 3′ end of the coding region (19.262.871–19262764) and the 862 bp 3′ UTR (19.262.763–19261902). The consistent differential expression of both exons between the R and S groups, and the highly signiﬁcant correlation of exon 4 levels and sheep rank phenotypes (AWC and FEC) as well as BW, argues against a simple technical explanation for differential exon usage. 4. Discussion ALOX15 sequence (NM 174501.2) identiﬁes that the sheep gene is encoded by 14 exons on the plus strand of chromosome 11 (NC 019468.1). Exon 9 encodes 90 bp within the coding region (1247–1337 from the translation start site, 26326237–26326327) and exon 14 encodes the terminal 180 bp of the coding region and the complete 3′ UTR (26327916–26328935). As with IL13, it is likely that the ALOX15 transcript that includes exon 9 encodes the full length and functional enzyme, and therefore there is a highly signif- icant relationship between exon 9 expression levels and phenotypic parameters of parasite resistance. One transcript and three pre- dicted transcript variants of ALOX15 have been described in cattle, with the X2 variant (XM 005220190.2) having a deleted exon 7. However, no functional consequences of these variants have been described. The resistance of sheep to the gastrointestinal nematode para- site T. circumcincta has a major host genetic component (Murphy et al., 2010; Riggio et al., 2013) and differential susceptibility to this parasite is clearly associated with T cell activation and inﬂam- mation (Gossner et al., 2013; Gossner et al., 2012). In addition, mouse models of nematode infections show that variations of genes associated with Th1, Th2 and Th17 T cell maturation are involved in the genetics of resistance (Filbey et al., 2014; Finkelman et al., 1997). Most studies in sheep have focussed on the important role for transcription regulation and primary sequence differences of these genes (Gossner et al., 2013; Riggio et al., 2013); however this current study concentrates on differential exon usage as a source of gene variants. Alternative splicing is a signiﬁcant cause of protein variation (Martinez and Lynch, 2013) especially in genes expressed in the immune system (Lynch, 2004) including cytokines and recep- tors involved in T cell polarization (Gaudreau et al., 2012; Martinez et al., 2012); and the original selection of the eight genes was made on the basis that they were differentially-expressed in resistant and susceptible sheep (Gossner et al., 2013) as well as being involved in T cell biology and inﬂammation. Control of exon usage and differential splicing can be associated with intronic regulatory elements (Black, 2003), often in the prox- imal 1000 bp 5′ to the translation start site. ARTICLE IN PRESS G Model VETPAR-7752; No. of Pages 8 H. Wilkie et al. / Veterinary Parasitology xxx (2015) xxx–xxx G Model VETPAR-7752; No. of Pages 8 G Model VETPAR-7752; No. of Pages 8 7 4. Discussion Expression levels of exon 4 are likely to represent full length and functional IL13 transcripts, which may explain why quantitative levels of IL13 exon 4 are signiﬁcantly pos- itively correlated BW and consequently negatively correlated with AWC and FEC. The lack of correlation between IL13 exon 4 and IgA (r2 0.08, p = 0.31) may be due to the fact that the IL13 levels are transcripts at a single time point (12 weeks post-infection), but that serum IgA had accumulated over time. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vetpar.2015.08. 023. Another major function of IL13 (and IL4) is the promotion of ALOX15 expression (Nassar et al., 1994) and the consistent up- regulation of all the ALOX15 exons in resistant sheep could be explained by the high levels of these cytokines in those sheep. ALOX15 is one of a range of at least six isoenzymes that oxy- genate arachidonic acid leading to the production of the lipoxins (Samuelsson et al., 1987). These function to inhibit leukotrienes and therefore are broadly anti-inﬂammatory. In addition ALOX15 stimulates the clearance of apoptotic cells and promotes tis- sue healing (McMahon et al., 2001), which is seen in resistant sheep (Gossner et al., 2012). ALOX15 has not been annotated in the Oar v3.1 genome assembly but analysis using the Bos taurus Acknowledgements This project was funded initially by the Natural Environment Research Council (NE/D000645/1) and by the Biotechnology and Biological Sciences Research Council (BBSRC) Institute Strategic Programme Grant to The Roslin Institute. HW is a BBSRC Industrial CASE scholar, sponsored by Sheep Improved Genetics Ltd and Eblex (grant BB/J012653/1). The funders had no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. We thank Professor Josephine Pemberton (Institute of Evolutionary Biology, University of Edinburgh) and Dr Dario Beraldi (CRUK, Li Ka Shing Centre, Cambridge) for original collaboration; Barbara Craig for the original parasitological measurements and Joan Docherty for animal husbandry and collection of weight data and blood samples. Please cite this article in press as: Wilkie, H., et al., Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.023 3.3. Sequence of 5′ intronic or UTR regions of ALOX15 and IL13 Ten single base variations were ﬁed in the ALOX15 region (g.66T>C; g.227C>G; g.246C>G; T>C; g.654G>A; g.656G>C; g.697T>C; g.836T>C; g.960G>A; 6A>G) but none segregated with phenotype. An initiator has already been identiﬁed in other sheep breeds (NCB rs422045752). The only variation found with IL13 was t tion of three nucleotides (g.674 675insGAA) 222 nucleot the translation start site in one animal (lamb 110, rank H. Wilkie et al. / Veterinary Parasitology xxx (2015) xxx–xxx 6 Fig. 3. Correlation analysis of the phenotypic parameters, AWC, FEC, BW and IgA with IL13 exon 1 and 4 copy number per sheep. rs—Spearman’s rank correlation coefﬁcient. Fig. 3. Correlation analysis of the phenotypic parameters, AWC, FEC, BW and IgA with IL13 exon 1 and 4 copy number per g total RNA in ALN of T. circumcincta infected sheep. rs—Spearman’s rank correlation coefﬁcient. has already been identiﬁed in other sheep breeds (NCBI dbSNP; rs422045752). The only variation found with IL13 was the inser- tion of three nucleotides (g.674 675insGAA) 222 nucleotides 5′ to the translation start site in one animal (lamb 110, rank 7), which was heterozygous at this locus. has already been identiﬁed in other sheep breeds (NCBI dbSNP; rs422045752). The only variation found with IL13 was the inser- tion of three nucleotides (g.674 675insGAA) 222 nucleotides 5′ to the translation start site in one animal (lamb 110, rank 7), which was heterozygous at this locus. (LN864491) from 6 R and 6 S sheep. Ten single base variations were identiﬁed in the ALOX15 region (g.66T>C; g.227C>G; g.246C>G; g.606T>C; g.654G>A; g.656G>C; g.697T>C; g.836T>C; g.960G>A; g.1036A>G) but none segregated with phenotype. An initiator codon variant (A/G) was also identiﬁed in two animals, which Please cite this article in press as: Wilkie, H., et al., Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.023 H. Wilkie et al. / Veterinary Parasitology xxx (2015) xxx–xxx 8 with divergent phenotypes for nematode resistance. Vet. Parasitol. 206, 200–207. with divergent phenotypes for nematode resistance. Vet. Parasitol. 206, 200–207. Black, D.L., 2003. Mechanisms of alternative pre-messenger RNA splicing. Annu. Rev. Biochem. 72, 291–336. Blencowe, B.J., 2006. Alternative splicing: new insights from global analyses. Cell 126, 37–47. 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Alternative splicing networks regulated by signaling in human T cells. RNA 18, 1029–1040. Urquhart, G.M., Armour, J., Duncan, J.L., Dunn, A.M., Jennings, F.W., 1987. Veterinary Parasitology. Longman Scientiﬁc and Technical, Avon. Mckenzie, G.J., Bancroft, A., Grencis, R.K., McKenzie, A.N., 1998. A distinct role for interleukin-13 in Th2-cell-mediated immune responses. Curr. Biol. 8, 339. y gy g , Windon, R.G., 1990. Selective breeding for the control of nematodiasis in sheep. Rev. Sci. Tech. 9, 555–576. McMahon, B., Mitchell, S., Brady, H.R., Godson, C., 2001. Lipoxins: revelations on resolution. Trends Pharmacol. Sci 22, 391–395. Woolaston, R.R., 1997. Detecting genetic differences between groups of sheep during parasite infection. Int. J. Parasitol. 27, 839–841. McRae, K.M., Good, B., Hanrahan, J.P., Glynn, A., O’Connell, M.J., Keane, O.M., 2014. Response to Teladorsagia circumcincta infection in Scottish Blackface lambs Wynn, T.A., 2003. IL-13 effector functions. Annu. Rev. Immunol. 21, 425–456. Please cite this article in press as: Wilkie, H., et al., Variable exon usage of differentially-expressed genes associated with resistance of sheep to Teladorsagia circumcincta. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.023
https://openalex.org/W3100659773	https://www.epj-conferences.org/10.1051/epjconf/202022800011/pdf	English	null	Biases in the estimation of velocity dispersions and dynamical masses for galaxy clusters	EPJ web of conferences	2,020	cc-by	3,262	1 Introduction Several authors have used the velocity dispersion mass proxy to study and characterise scal- ing relations between SZ and dynamical mass [1–3]. In order to have sample with enough statistical power, it is necessary to estimate the velocity dispersion for hundreds of galaxy clusters (GCs). Although this goal can be achieved through spectroscopic follow-up [e.g. 4, 5], these studies are extremely expensive in terms of observational time and data reduc- tion. For these reasons, it is extremely diﬃcult to estimate radial velocities for more than few members (typically ∼20) for each cluster target. In this article we present our study of statistical and physical biases introduced in the estimation of velocity dispersion and dynamical mass. ∗e-mail: ferragamo_ext@iac.es © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1051/epjconf/202022800011 https://doi.org/10.1051/epjconf/202022800011 EPJ Web of Conferences 228, 00011 (2020) mm Universe @ NIKA2 Biases in the estimation of velocity dispersions and dy- namical masses for galaxy clusters A. Ferragamo1,2,∗, J. A. Rubiño-Martín1,2, J. Betancort-Rijo1,2, E. Munari3,4, B. Sartoris3,4, and R. Barrena1,2 A. Ferragamo1,2,∗, J. A. Rubiño-Martín1,2, J. Betancort-Rijo1,2, E. Munari3,4, B. Sartoris3,4, and R. Barrena1,2 1Instituto de Astrofísica de Canarias (IAC), C/ Vía Láctea s/n, E-38205 La Laguna, Tenerife, Spain 2Universidad de La Laguna, Departamento de Astrofísica, C/ Astrofísico Francisco Sánchez s/n, E- 38206 La Laguna, Tenerife, Spain 3Dipartimento di Fisica, Sezione di Astronomia, Università di Trieste, Via Tiepolo 11, I-34143 Trieste, Italy 3Dipartimento di Fisica, Sezione di Astronomia, Università di Trieste, Via Tiepolo 11, I-34143 Trieste, It l 4INAF/Osservatorio Astronomico di Trieste, Via Tiepolo 11, I-34143 Trieste, Italy 4INAF/Osservatorio Astronomico di Trieste, Via Tiepolo 11, I-34143 Trieste, Italy Abstract. Using a set of 73 numerically simulated galaxy clusters, we have characterised the statistical and physical biases for three velocity dispersion and mass estimators, namely biweight, gapper and standard deviation, in the small number of galaxies regime (Ngal ≤75), both for the determination of the ve- locity dispersion and the dynamical mass of the clusters via the σ–M relation. These results are used to deﬁne a new set of unbiased estimators, that are able to correct for those statistical biases. By applying these new estimators to a subset of simulated observations, we show that they can retrieve bias-corrected values for both the mean velocity dispersion and the mean mass. 2 Statistical biases in Velocity Dispersion estimation For our analysis we use a sample of 73 simulated massive clusters selected from the simula- tions described in [6]. Our selected sample contains clusters with masses M200 > 2 × 1014M⊙ and located at ﬁve redshifts between 0.12 ≤z ≤0.82. There are several method to estimate mean and scale of a distribution. We focus our attention on the estimators that, in the last decades, became standard tools in GC analyses, EPJ Web of Conferences 228, 00011 (2020) mm Universe @ NIKA2 https://doi.org/10.1051/epjconf/202022800011 Figure 1. Left panel: mean velocity dispersion S X/S std(< R200) as a function of the number of galaxies Ngal. Right panel: corrected velocity dispersion estimators S ′ X/S std(< R200) as a function of Ngal. The dispersion S X(Ngal) is calculated for the standard deviation (green line), biweight (blue line), and gapper (red line) estimators. Figure 1. Left panel: mean velocity dispersion S X/S std(< R200) as a function of the number of galaxies Ngal. Right panel: corrected velocity dispersion estimators S ′ X/S std(< R200) as a function of Ngal. The dispersion S X(Ngal) is calculated for the standard deviation (green line), biweight (blue line), and gapper (red line) estimators. Table 1. Best-ﬁt parameters to be used in the parametric function given in equation 1, describing th bias of the three estimators. See text for details. BWT GAP S TD D 0.72 ± 0.03 0 0.25 B −0.0225 ± 0.0002 −0.0080 ± 0.0002 −0.0037 ± 0.0003 β 1.28 ± 0.03 1 1 Table 1. Best-ﬁt parameters to be used in the parametric function given in equation 1, describing the bias of the three estimators. See text for details. bias of the three estimators. See text for details. BWT GAP S TD D 0.72 ± 0.03 0 0.25 B −0.0225 ± 0.0002 −0.0080 ± 0.0002 −0.0037 ± 0.0003 β 1.28 ± 0.03 1 1 BWT GAP S TD D 0.72 ± 0.03 0 0.25 B −0.0225 ± 0.0002 −0.0080 ± 0.0002 −0.0037 ± 0.0003 β 1.28 ± 0.03 1 1 namely biweight and gapper, compared with the standard deviation. A detailed description of these estimators can be found in [7]. In order to investigate bias and variance of the three scale estimators S X(Ngal), being X= “std”, “bwt” or “gap”, for each one of the three cases, we have explored the low number of galaxies regime between Ngal = 8 and Ngal = 75. 2 Statistical biases in Velocity Dispersion estimation We have generated 2500 conﬁgurations of randomly selecting galaxies within the projected circle of radius R200 and then we averaged the 73 × 2250 velocity dispersions (750 for each mayor axis). Left panel of Fig. 1 shows the average S X(Ngal) normalised with respect to S std(< R200), which represents the velocity dispersion of all the galaxies in the simulation within a circle of projected radius R200, and calculated using the standard deviation estimator. In the low-Ngal regime, all estimators are biased showing very diﬀerent behaviours. The standard deviation estimator (green line) shows a dependence with the number of elements used for the esti- mation. However, this dependence can be theoretically predicted to be 1 −1/(4(Ngal −1)). The biweight (blue line) shows a stronger drop for Ngal ≤30 underestimating the reference dispersion by up to 4 % at Ngal = 7. Finally, the gapper (red line) shows an estimate of the velocity dispersion almost constantly biased at any Ngal. In order to construct an unbiased velocity dispersion estimators, S ′ X(Ngal), we use a parametrisation of the curves in left panel of Fig. 1: S ′ X(Ngal) ≡S X(Ngal) 1 +  D (Ngal −1) β + B  . (1) (1) Table 1 shows the best-ﬁt values for the parameters D, β and B, for each one of the three estimators (biweight, gapper and standard deviation). Right panel of Fig. 1 shows that the corrected estimators S ′ X(Ngal) are actually unbiased by construction. 2 https://doi.org/10.1051/epjconf/202022800011 EPJ Web of Conferences 228, 00011 (2020) mm Universe @ NIKA2 Figure 2. Eﬀect of interlopers on S ′ X/S std(< R200), as a function of the number of galaxies. The velocity dispersion, S ′ X(Ngal), is ﬁrst computed using a pure galaxie sample (solid lines). We also evaluated the response of biweight (blue lines), standard deviation (green lines), and gapper (red lines) using samples contaminated by 0% (solid line), 5% (dashed lines), 10% (dot dashed lines), 15% (three-dot-dashed lines), 20% (two-dot-long-dashed lines) and 30% (dotted lines) of interlopers at any Ngal. Figure 2. Eﬀect of interlopers on S ′ X/S std(< R200), as a function of the number of galaxies. The velocity dispersion, S ′ X(Ngal), is ﬁrst computed using a pure galaxie sample (solid lines). 3 Biases by interlopers contamination Any spectroscopic sample of cluster members is contaminated by galaxies belonging to the large scale structure that surrounds the cluster itself. This population of ‘pseudo cluster mem- bers’, called interlopers, modiﬁes the velocity distribution and therefore aﬀects the estimation of velocity dispersion. According to the deﬁnition of interlopers given in [8], one must distin- guish between two very diﬀerent types of contaminants: i. galaxies gravitationally bound to the clusters that are outside the virial sphere (according to the deﬁnition given in [9]), but that, due to projection eﬀects appear within a smaller radius; ii. background/foreground galaxies with similar redshifts to the cluster, but belonging to the large scale structure that surrounds the cluster itself. Detailed study of these interlopers is beyond the scope of this work. Here, we illustrate the robustness of the three estimators in exam, by ﬁxing the fraction of contam- inants at any Ngal. Fig. 2 shows the results obtained for ﬁve diﬀerent fractions of interlopers: 5% (dashed lines), 10% (dot-dashed lines), 15%(three-dot-dashed lines), 20% (two-dot-long- dashed lines) and 30% (dotted lines). We note that the three estimators are similarly aﬀected by interloper contamination. The eﬀect of the interlopers contamination consists of a velocity dispersion overestimation which is as high as their relative fraction (up to 30%) representing the most damaging source of biases in the velocity estimation estimation. 2 Statistical biases in Velocity Dispersion estimation We also evaluated the response of biweight (blue lines), standard deviation (green lines), and gapper (red lines) using samples contaminated by 0% (solid line), 5% (dashed lines), 10% (dot dashed lines), 15% (three-dot-dashed lines), 20% (two-dot-long-dashed lines) and 30% (dotted lines) of interlopers at any Ngal. 4 Physical biases in Velocity Dispersion estimation Observational strategies and technical limitations generally force us to observe only massive clusters members sampled in a fraction of R200. We studied how these limitations might produce biased velocity dispersion estimates. 4.2 Effect of aperture sub-sampling There are evidence in the literature that the velocity dispersion has a radial dependence [e.g., 2, 9]. This implies that sampling galaxies in diﬀerent fractions of the cluster’s virial radius should lead to a biased velocity dispersion. In order to investigate and quantify this eﬀect, we averaged the 73 velocity dispersions calculated using all galaxies inside a cylinder of variable radius 0.2 ≤r/R200 ≤1.2. Fig. 3 (right panel) shows that the velocity dispersion is (on average) overestimated in region smaller than R200 and slightly underestimated for r > R200, in analogy with that recover by [2]. 4.1 Effects due to the selected fraction of massive galaxies For each case, we reproduce the procedure explained in Sect. 2. Fig. 3 (left panel) shows S ′ std(Ngal)/S std(< R200) as function of Ngal calculated with the corrected standard deviation estimator, and using galaxies picked up from 100 % (black line), 1/2 (blue line), 1/3 (red line) and 1/4 (green line) of the complete cluster member samples. We see that the velocity dispersion is sensitive to the fraction of massive galaxies used to estimate it, reaching a bias of almost 2% using only the most massive fraction. However, it is almost insensitive to Ngal. For this reason, we can use the curves in the left panel of Fig. 3 to correct this physical eﬀect. 4.1 Effects due to the selected fraction of massive galaxies In the ideal case we can observe any cluster member regardless of its brightness. However, the telescope aperture limits the detection magnitude and prevents us from detecting faint 3 https://doi.org/10.1051/epjconf/202022800011 EPJ Web of Conferences 228, 00011 (2020) mm Universe @ NIKA2 Figure 3. Left panel: Mean (bias) of S ′ std(Ngal)/S std(< R200) as a function of the number of galaxies Ngal, calculated by choosing galaxies within 100% (black solid line), 1/2 (blue solid line), 1/3 (red solid line), and 1/4 (green solid line) of the complete cluster member samples. Right panel: Average velocity dispersion proﬁle within a given enclosed radius r, < S ′ std(< r)/S std(< R200) >, normalised to R200. The red line represents the mean at each radius of the individual 73 simulated GC proﬁles (grey lines). The dashed blue line represents the [2] proﬁle, which is almost coincident with our derived proﬁle. Figure 3. Left panel: Mean (bias) of S ′ std(Ngal)/S std(< R200) as a function of the number of galaxies Ngal, calculated by choosing galaxies within 100% (black solid line), 1/2 (blue solid line), 1/3 (red solid line), and 1/4 (green solid line) of the complete cluster member samples. Right panel: Average velocity dispersion proﬁle within a given enclosed radius r, < S ′ std(< r)/S std(< R200) >, normalised to R200. The red line represents the mean at each radius of the individual 73 simulated GC proﬁles (grey lines). The dashed blue line represents the [2] proﬁle, which is almost coincident with our derived proﬁle. objects, for a ﬁxed integration time. Therefore, cluster samples contain only a fraction of the brightest galaxy members, which are also the most massive. In order to simulate this eﬀect, we mimicked observational conditions by selecting three percentages of all visible galaxies in the simulation, i.e. 50 %, 33 %, and 25 %, by sorting the cluster members by mass and dividing the sample in 2, 3, and 4 mass bins, starting from the most massive object. For each case, we reproduce the procedure explained in Sect. 2. In order to simulate this eﬀect, we mimicked observational conditions by selecting three percentages of all visible galaxies in the simulation, i.e. 50 %, 33 %, and 25 %, by sorting the cluster members by mass and dividing the sample in 2, 3, and 4 mass bins, starting from the most massive object. 5 Statistical bias in the estimation of M200 When we use velocity dispersion as a mass proxy we apply a scaling relation σ1D −M, it is a power law that has been previously calibrated either with simulations [6, 10, 11]. The left panel of Fig. 4 shows how applying the scaling relation σ1D −M to the unbiased velocity dispersion estimator S ′ X the resulting masses M(S ′ X)/M(S std(< R200)) are biased. 4 4 4 https://doi.org/10.1051/epjconf/202022800011 EPJ Web of Conferences 228, 00011 (2020) mm Universe @ NIKA2 Figure 4. Left panel: mean of M S ′ X(Ngal) /M S ′ std(< R200) , which represent the standard mass es- timator, described in [6], applied to normal and unbiased velocity dispersion estimators, standard de- viation (green), gapper (red), and biweight (blue). Right panel: mean of the corrected mass estimator M′ S ′ X(Ngal) /M S ′ std(< R200) . Figure 4. Left panel: mean of M S ′ X(Ngal) /M S ′ std(< R200) , which represent the standard mass es- timator, described in [6], applied to normal and unbiased velocity dispersion estimators, standard de- viation (green), gapper (red), and biweight (blue). Right panel: mean of the corrected mass estimator M′ S ′ X(Ngal) /M S ′ std(< R200) . Table 2. Best-ﬁt parameters for the function describing the bias in M(S ′ X)/M(S std(< R200)) for simulated clusters, as described in equation 2. BWT GAP S TD E′ 1.31 ± 0.03 1.50 ± 0.03 1.53 ± 0.03 F′ 1 1 1 γ′ 1.24 ± 0.03 1.17 ± 0.04 1.11 ± 0.04 We can calculate analytically the functional form of the mass estimator as a function of the number of galaxies 1−2α 4α2(Ngal−1), with A = 1177.0 km s−1 and α = 0.364 parameters of the [6] scaling relation. g As in Sect. 2, we ﬁtted a parametric form description of the bias as a function of Ngal by using three parameters (E′,F′ and γ′, listed in Table 2): M′ S ′ X(Ngal) = M S ′ X(Ngal) 1 −E′α (E′α)2(Ngal −1)γ′ + F′ −1 . (2) (2) The right panel of Fig. 4 shows the bias of M′(S ′ X). The new mass estimator is actually unbiased by construction. The right panel of Fig. 4 shows the bias of M′(S ′ X). The new mass estimator is actually unbiased by construction. 5 Statistical bias in the estimation of M200 Finally, in order to test the corrections described in previous sections, we have applied them to a set of mock cluster catalogues. To do this we have simulated a realistic observa- tional strategy based on the observations described in [4, 5]. We generated 100 mock samples out of the 73 GCs object simulated in this study. For each of these samples we calculated the mean ratio between the estimated and the reference velocity dispersion of each cluster, as well as the velocity dispersion calculated using S ′ X deﬁned in eq. 1 with the parameters in Table 1. Averaging over all the mock samples we obtained a biased mean velocity dispersion S std(Ngal, r)/S bwt(< R200) = 0.96 ± 0.02, S ′ std(Ngal, r)/S bwt(< R200) = 1.00 ± 0.02. (3) (3) The normal S std estimator shows to be biased, whereas the S ′ X lead to an unbiased esti- mation of the velocity dispersion. 5 5 EPJ Web of Conferences 228, 00011 (2020) mm Universe @ NIKA2 https://doi.org/10.1051/epjconf/202022800011 mm Universe @ NIKA2 Using the bias corrected velocity dispersions, we calculated cluster masses, M(S ′ X) and M′ S ′ std , obtaining M S ′ std(Ngal, r) /M (S bwt(< R200)) = 1.13 ± 0.07, M′ S ′ std(Ngal, r) /M′ (S bwt(< R200)) = 1.00 ± 0.06. (4) (4) As described above, these normal mass estimator is overestimated, while primed mass estimator, M′ is actually unbiased. As described above, these normal mass estimator is overestimated, while primed mass estimator, M′ is actually unbiased. 6 Conclusions We have used 73 simulated GCs from hydrodynamic simulations including AGN feedback and star formation, in order to characterise the statistical and physical biases in three velocity dispersion (and mass) estimators frequently used in the literature: the biweight, the gapper, and the standard deviation. We have focused our study in the (common) case of a low number of galaxy members (Ngal ∼< 75). g y g ∼ We showed that each of these estimators (dispersion and mass) presents a statistical bias. Therefore, we propose bias corrected velocity dispersion (S ′ X) and mass (M′(S ′ X)) estimators. We have tested the robustness of the new estimators against the contamination by inter- lopers. We found that the velocity dispersion estimators S ′ X are similarly aﬀected by the contamination for all the three cases in this low-Ngal limit. g We observed that the most likely sources of physical bias are i) the selection eﬀect the fraction of massive galaxies used to estimate the velocity dispersion; and ii) the fraction of the virial radius explored. We showed that in the ﬁrst case the bias is estimated to be around 2 % when considering only 1/4 of the most massive galaxies. Concerning the eﬀect produced by the sampling aperture, we ﬁnd a dispersion radial proﬁle in agreement with [2] results. References [1] J. Ruel, G. Bazin, M. Bayliss, M. Brodwin, R.J. Foley, B. Stalder, K.A. Aird, R. Arm- strong, M.L.N. Ashby, M. Bautz et al., ApJ792, 45 (2014), 1311.4953 [2] C. Sifón, N. Battaglia, M. Hasselﬁeld, F. Menanteau, L.F. Barrientos, J.R. Bond, D. Crichton, M.J. Devlin, R. Dünner, M. Hilton et al., MNRAS461, 248 (2016), 1512.00910 [3] S. Amodeo, S. Mei, S.A. Stanford, J.G. 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https://openalex.org/W4239870429	https://www.qeios.com/read/7VCATZ/pdf	English	null	Fainting	Definitions	2,020	cc-by	61	Fainting National Cancer Institute National Cancer Institute Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Qeios ID: 7VCATZ · https://doi.org/10.32388/7VCATZ Open Peer Review on Qeios Source National Cancer Institute. Fainting. NCI Thesaurus. Code C50857. National Cancer Institute. Fainting. NCI Thesaurus. Code C50857. Extremely weak; threatened with syncope. Qeios ID: 7VCATZ · https://doi.org/10.32388/7VCATZ 1/1
https://openalex.org/W3217782631	https://ris.utwente.nl/ws/files/268749640/applsci_11_11212_v2.pdf	English	null	Modelling Regime Changes of Dunes to Upper-Stage Plane Bed in Flumes and in Rivers	Applied sciences	2,021	cc-by	14,425	Citation: Duin, O.J.M.v.; Hulscher, S.J.M.H.; Ribberink, J.S. Modelling Regime Changes of Dunes to Upper- Stage Plane Bed in Flumes and in Rivers. Appl. Sci. 2021, 11, 11212. https://doi.org/10.3390/ app112311212 Keywords: river dunes; sediment transport; morphological modelling Academic Editor: Donatella Termini Article Modelling Regime Changes of Dunes to Upper-Stage Plane Bed in Flumes and in Rivers Olav J. M. van Duin 1,2, Suzanne J. M. H. Hulscher 1,* and Jan S. Ribberink 1 1 Department of Civil Engineering, Faculty of Engineering, Marine and Fluvial System, University of Twente, 7522 NB Enschede, The Netherlands; Olav.vanDuin@deltares.nl (O.J.M.v.D.); jsribberink@home.nl (J.S.R.) 2 Unit of Inland Water Systems, Department of Operational Water Management, Deltares, 2800 MH Delft, The Netherlands * Correspondence: s j m h hulscher@utwente nl * Correspondence: s.j.m.h.hulscher@utwente.nl Abstract: In this paper we derive a new morphological model, with an extended version of the sediment transport model for the mean step length (the average distance travelled by sediment particles), in which this mean step length depends on the mean bed shear stress. This model makes the step length increase with increasing ﬂow, in line with previous experimental results. To account for suspension and the large-scale turbulent structures in rivers, the step length also depends explicitly on water depth. This approach enabled modelling of the transition from dunes to the upper-stage plane bed. It was shown that by increasing the step length, the lag between shear stress and bed load transport rate increases, and the dunes eventually become smoother and lower, until ﬁnally the dunes wash out. The newly adopted model approach is tested successfully with a synthetic data set from the literature, where plane bed conditions are indeed reached in the model, similar to the results of a more advanced model. It is shown that with increasing discharge, the ﬂow increases, which leads to higher step length and to the washing out of the dunes. Although the present model still overestimates the dune height for river cases, the potential of the model concept for river dune dynamics, including the transition to upper-stage plane bed, is shown. The model results indicate that, if a transition to upper-stage plane bed occurs in a realistic river scenario, a reduction of the water depth of approximately 0.5 m can occur. applied sciences applied sciences applied sciences 1. Introduction This model is able to predict the evolution of dunes from small initial distur- bances up to equilibrium dimensions with limited computational time and good accuracy. In addition, this model has been coupled with an existing large-scale depth-averaged hydraulic model to form a ‘dynamic roughness model’ [22]. The coupled model clearly shows the expected hysteresis effects in dune roughness and water levels and different behaviour of sharp-peaked versus broad-peaked ﬂood waves within the dune regime [22]. p p p g [ ] As Nakagawa and Tsujimoto (1980) [23] have argued, a lag distance between ﬂow properties (and thereby bed shear stress) and sediment transport is the principal cause of bed instability and thereby regime transitions. One of the contributing factors is the step length of sediment particles, which is the distance travelled from dislodgement to rest according to Einstein (1950) [24]. This creates a phase-lag effect which is not taken into account in equilibrium transport formulas, such as that of Meyer-Peter and Müller (1948) [25]. The latter formula was used in the original model of Paarlberg et al. (2009) [21], which made it impossible to model a transition to upper-stage plane bed. The pick-up and deposition model, as proposed by Nakagawa and Tsujimoto (1980) [23], inherently allows a phase-lag effect over distance. The deposition of sediment away from the pick-up point is determined by using a probability distribution function that relies on the mean step length. This pick-up and deposition model has already been used in the dune evolution model of Shimizu et al. (2009) [14], with good results regarding prediction of generated dunes and upper-stage plane bed in ﬂumes. However, this model is far too complicated to allow for simulations of river dune dynamics, that is, at full river scale during a longer period (e.g., a ﬂood wave and afterwards, typically a month). Therefore, the Paarlberg et al. (2009) [21] model was extended to enable predictions of a transition to upper-stage plane bed. Van Duin et al. (2017) [26] have shown that replacing the transport formula of Meyer-Peter and Müller (1948) [25] with the pick-up and deposition model of Nakagawa and Tsujimoto (1980) [23] leads to improved predictions of dune dimensions in the dune regime. Furthermore, this model is in principle able to simulate the washing out of dunes as well, signifying the potential of this approach for the prediction of a transition to upper-stage plane bed. 1. Introduction Received: 6 October 2021 Accepted: 23 November 2021 Published: 25 November 2021 Hydraulic roughness values play an important role in correctly predicting water levels in rivers [1–3], which is critical for ﬂood management purposes. This especially holds for bifurcated rivers [4], due to feedback mechanisms between the downstream branches. River dunes increase the hydraulic roughness signiﬁcantly: their shapes cause form drag. Because of their signiﬁcant impact on hydraulic roughness, water level forecasts during a high river water discharge depend on accurate predictions of the evolution of river dune dimensions. One aspect of this is the correct prediction of a transition to upper-stage plane bed conditions. The general motivation of this paper is to contribute to the development of a relatively simple and physics-based dune evolution model that is able to capture proper dune dimensions and dynamic behaviour under transitional conditions. In effect, the goal is to model river dune behaviour under varying discharges between the lower-stage plane bed to the upper-stage plane bed. Such a model should work under ﬂume and ﬁeld conditions, and should predict transitions at the appropriate discharges. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2021 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/). In the past, many approaches have been used to model dune dimensions, varying from empirical equilibrium dune height predictors (e.g., [4–7]) to different forms of stability analyses (e.g., [8–11]). Recently, more advanced models have been developed that calculate the turbulent ﬂow ﬁeld over bedforms, in some cases in combination with morphological computations (e.g., [12–20]). These models are valuable to study detailed hydrodynamic https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 11212. https://doi.org/10.3390/app112311212 Appl. Sci. 2021, 11, 11212 2 of 25 processes, but are computationally intensive, which makes them unsuitable for the pre- diction of bedform evolution and roughness over full-scale river reaches during a longer period (e.g., that of a ﬂood wave). To enable efﬁcient predictions of dune dimensions over the time-scale of a ﬂood wave, Paarlberg et al. (2009) [21] have developed a model in which the ﬂow and sediment transport at the ﬂow separation zone is parameterized instead of using full hydrodynamic equations. 1. Introduction Manually selecting high values of the step length leads to the washing out of dunes within this model. However, the automatic selection of a physics-based step length, and the physics behind this, is still an issue. p y p g p y The model of Van Duin et al. (2017) [26] does not incorporate suspended sediment, which was clearly shown to be important for high flow conditions by Naqshband et al. (2017) [27]. Under the inﬂuence of turbulent action at the bottom of a ﬂume or channel, sediment may be entrained in the water column, and it may therefore be necessary to allow for step length values that are higher than those typically found for bed load. Depending on the ﬂow and turbulence structure there, sediment may be transported over a large distance in suspension. If this suspended transport occurs along dunes, the average distance travelled by sediment becomes far larger than for bed load alone (see also Yamaguchi et al. (2019) [20]). This has implications for the spatial lag between ﬂow and sediment transport, and thereby for the river dune morphology as well, neither of which have been investigated so far. Naqshband et al. (2017, 2014b) [27,28] used data from experiments and rivers to show that with an increasing suspension parameter, dunes ﬁrst get higher (i.e., a larger amplitude to length ratio), arrive at a maximum height, then become lower, and ﬁnally disappear for large suspension numbers. These results indicate that high suspension numbers are required for the dunes to wash out, and that alongside the inﬂuence of bed load lags, suspension lags should also be included in determining dune dimensions. Appl. Sci. 2021, 11, 11212 3 of 25 3 of 25 One of the mechanisms contributing to this dependence of dune morphology on sus- pended sediment has been studied by Naqshband et al. (2014a) [29]. These authors have shown that signiﬁcant portions of suspended load ‘escape’ the dune by not avalanching on the lee side of the dune but going over the ﬂow separation zone and depositing further away (also shown by Kostaschuk et al. (2010) [30]). The authors ﬁnd that while bed load and suspended load are comparable in magnitude, the gradient of bed load is larger than that of suspended load. 1. Introduction This implies that the general shape of these equilibrium dunes is mostly determined by bed load, and to a lesser extent by suspended load, which deposits more evenly along the dune. However, for strongly increasing ﬂow, turbulence becomes stronger, and suspended load can become more and more dominant. Then, the situation could arise that so much sediment is spread out evenly that dunes start decaying and may even disappear entirely (see also Engelund and Fredsøe (1974) [31], Fredsøe and Engelund (1975) [32], Smith and McLean (1977) [33], and Bridge and Best (1988) [34]). So far, these effects have not been incorporated in idealized dune evolution models (e.g., Paarlberg et al. (2009) [21]). In summary, no morphodynamic model is available that has the potential to capture the behaviour of full-scale river dunes during a ﬂood wave from moderate to extremely high discharges and afterwards. Therefore, in this paper we investigate the potential of the inclusion of a step length model (that combines bed load and suspended load processes, although the latter, as a ﬁrst step, in a strongly schematized way) with an idealized dune model such that it enables the modelling of dune dynamics during such a ﬂood wave. More precisely, the two research questions are: (1) To what extent can this new model replicate dune dynamics as they occur in ﬂume conditions under variable discharge, including upper-stage plane bed and bedform hysteresis effects? (2) To what extent can this type of model in principle describe dune dynamics, including upper-stage plane bed and hysteresis effects in ﬁeld conditions? The organization of the chapter is as follows. In Section 2 the set-up of the model is discussed, while step length models from the literature and a new step length model are discussed in Sections 3 and 4, respectively. Section 5 shows the model results for ﬂume conditions, while Section 6 shows the model results for river conditions. In Sections 7 and 8, the discussion and conclusion are presented. 2.1. General Set-Up The basis of the present model is the dune evolution model developed by Paarlberg et al. (2009) [21]. Paarlberg et al. (2009) [21] extended the process-based morphodynamic sand wave models of van den Berg et al. (2012) [35] and Hulscher (1996) [36], with a parameterization of ﬂow separation (see Figure 1), in order to enable simulation of ﬁnite amplitude river dune evolution. For more details on the similarities and differences of river dunes and sand waves, see Hulscher and Dohmen-janssen (2005) [37]. Figure 1. Schematization of a dune (ﬂow left to right). Figure 1. Schematization of a dune (ﬂow left to right). The model consists of a ﬂow module, a sediment transport module, and a bed evo- lution module, which operate in a decoupled way. The model simulates a single dune which is assumed to be in an inﬁnite train of identical dunes. Therefore periodic boundary conditions are used. Because varying discharge means the linear stability analysis as used by Paarlberg et al. (2009) [21] has to be done often during a model run (as each discharge leads a different most unstable mode, and therefore wavelength), this approach is com- putationally expensive. Paarlberg et al. (2009) [21] have shown that the dune length that follows from the numerical stability analysis is nearly linearly related to the water depth Appl. Sci. 2021, 11, 11212 4 of 25 (i.e., dune length = 7.3 water depth). This relationship is also in line with the empirical relations for dune height and length of Van Rijn (1984b) [7] as presented by Julien and Klaassen (1995) [38]. Therefore, for computational efﬁciency, this relation is also adopted in the present study. (i.e., dune length = 7.3 water depth). This relationship is also in line with the empirical relations for dune height and length of Van Rijn (1984b) [7] as presented by Julien and Klaassen (1995) [38]. Therefore, for computational efﬁciency, this relation is also adopted in the present study. 2.2. Flow Model In general the ﬂow is forced by the difference in water level along the domain. While the water depth at the start and end of domain are the same due to the periodic boundary conditions, the water level differs because the domain is sloped. The average bed level is taken as zero and has a slope (this average bed slope is an input parameter for the model). By solving the ﬂow equations with a certain average water depth, a discharge is found. The average water depth is adjusted until this discharge matches the discharge given as input. g p j g g g p The ﬂow in the model of Paarlberg et al. (2009) [21] is described by the steady two- dimensional shallow water equations in a vertical plane (2-DV), assuming hydrostatic pressure conditions. For small Froude numbers and small vertical ﬂow acceleration, the momentum equation in the vertical direction reduces to the hydrostatic pressure condition, and the time variations in the horizontal momentum equation can be dropped. The governing model equations that result are shown in Equations (1) and (2). u∂u ∂x + w∂u ∂z = −g ∂ζ ∂x + Av ∂2u ∂z2 + gi (1) ∂u ∂x + ∂w ∂z = 0 (2) (1) (2) The velocities in the x and z directions are u and w, respectively. The water surface elevation is denoted by ζ, i is the average channel slope, g is the acceleration due to gravity, and Av denotes the constant vertical eddy viscosity. Note that a steady ﬂow model is used to compute unsteady ﬂow of a ﬂood wave. This is a reasonable approach, because the length scale of a dune is small, so the remaining terms are large compared to the time derivatives. The computational domain is shown in Figure 2. Figure 2. The computational domain. In Figure 2 the symbol λ denotes the dune length (in m), h is the domain-averaged water depth (in m), and zb is the bed level relative to the x-axis (in m). The ﬂow is forced in the domain because the x-axis is actually at a slope i with regard to the real horizontal plane, creating a water level difference along the domain. Figure 2. The computational domain. Boundary Conditions The boundary conditions are deﬁned at the water surface (z = h + ζ) and at the bed (z = zb). The boundary conditions at the water surface, Equation (3) represents no ﬂow through the surface, and Equation (4) means no shear stress at the surface (so wind stress is assumed to be negligible). The kinematic boundary condition at the bed, Equation (5) yields that there is no ﬂow through the bed. u ∂ζ ∂x z=h+ζ = w (3) ∂u ∂z z=h+ζ = 0 (4) u ∂zb ∂x z=zb = w (5) u ∂ζ ∂x z=h+ζ = w (3) ∂u ∂z z=h+ζ = 0 (4) u ∂zb ∂x z=zb = w (5) (3) (4) (5) In order to close the turbulence model, a time- and depth-independent eddy viscosity is assumed. In order to represent the bed shear stress correctly for a constant eddy viscosity, a partial slip condition at the bed, Equation (6) is necessary. τb = Av ∂u ∂z z=zb = Sub (6) (6) In Equation (6), τb (m2/s2) represents the volumetric bed shear stress (i.e., without the density), ub (m/s) is the ﬂow velocity along the bed, and the resistance parameter S (m/s) controls the resistance at the bed. For more details about the model equations and numerical solution procedure, reference is made to Paarlberg et al. (2009) [21] and Van den Berg et al. (2012) [35]. For more details, the reader is referred to van Duin et al. (2016) [26]. 2.2. Flow Model In Figure 2 the symbol λ denotes the dune length (in m), h is the domain-averaged water depth (in m), and zb is the bed level relative to the x-axis (in m). The ﬂow is forced in the domain because the x-axis is actually at a slope i with regard to the real horizontal plane, creating a water level difference along the domain. In Figure 2 the symbol λ denotes the dune length (in m), h is the domain-averaged water depth (in m), and zb is the bed level relative to the x-axis (in m). The ﬂow is forced in the domain because the x-axis is actually at a slope i with regard to the real horizontal plane, creating a water level difference along the domain. Appl. Sci. 2021, 11, 11212 5 of 25 2.3. Bed Load Sediment Transport Model The pick-up and deposition model of Nakagawa and Tsujimoto (1980) [23] uses the following formulae to determine bed load transport. Pick-up of sediment (probability of a particle being picked up in s−1) is determined by: ps(x) = F0 s ∆g D50 θ(x) 1 − θc θ(x) 3 (7) (7) where F0 = 0.03, θ is the Shields parameter, θc is the critical Shields parameter, and ∆= ρs/ρ−1. The grain density ρs is set to 2650 kg/m3, and the density of the water ρ is set to 1000 kg/m3 The local, critical volumetric bed shear stress τc(x), corrected for bed slope effects, is given by the following equation: τc(x) = τc0 1 + η ∂zb ∂x r 1 + ∂zb ∂x 2 (8) (8) with τc0 the critical volumetric bed shear stress for ﬂat bed, deﬁned by Equation (9) and η = tan(ϕ)−1, in which the angle of repose ϕ = 30◦for sand. In this equation, θc0 is the critical Shields parameter for ﬂat bed, and D50 is the median grain size. τc0 = θc0g∆D50 (9) (9) Deposition at a location is determined by adding all the sediment that arrives at that speciﬁc location. Therefore, in order to determine the deposition at a certain location x, the distribution of picked-up sediment from all upstream locations is needed. The determination of deposition is done by applying the following formula: pd(x) = ∞ Z 0 px(x −s) f (s)ds (10) (10) Appl. Sci. 2021, 11, 11212 6 of 25 where the distribution f(s) determines the fraction of sediment that is deposited a distance s away from the pick-up point (x −s). The distribution function is deﬁned as follows: where the distribution f(s) determines the fraction of sediment that is deposited a distance s away from the pick-up point (x −s). The distribution function is deﬁned as follows: f (s) = 1 Λ exp −s Λ (11) (11) Herein Λ is deﬁned as the mean step length, which is further discussed in Section 2.4. By using this function, 99.3% of the sediment that has been picked up at certain location is deposited between that location and 5 times the step length in the downstream direction. The remaining 0.7% is deposited at x = 5Λ. Finally, the transport gradient is determined as follows: ∂q ∂qb ∂x = D50[ps(x) −pd(x)] (12) (12) To summarize, the entire sediment transport calculation process is as follows. 2.4. Step Length To calculate how the sediment is distributed moving away from each pick-up point, the mean step length of the sediment particles has to be calculated. Step length is deﬁned b ( ) f ll by Einstein (1950) as follows: (13) Λ = αD50 Λ = αD50 (13) where α is a non-dimensional step length parameter. Francis (1973) [39], Fernandez Luque and Van Beek (1976) [40], and Sekine and Kikkawa (1984) [41] have done experiments to determine the dependency of, among other things, bed load transport, particle velocity, and step length on various parameters with moving sand along a plane bed. This data shows a range of approximately 40 to 240 times the particle diameter, for values of u/ws (in which u = friction velocity and (u* = (τ/ρ)1/2), and ws = settling velocity) from about 0.18 to 0.35. From this data different step length models are derived by various authors. In Section 3, two step length models based on bed load from the literature are implemented, and results are discussed. Hereafter, Section 4 presents a third method in which, in addition to the models of Section 3, suspended load processes are also explicitly accounted for. In general, the step length is assumed to be constant along the dune, so it can vary only over time due to variation in dune-averaged ﬂow parameters. 2.5. Bed Evolution The bed evolution is modelled using the Exner Equation (17), where the sediment transport gradient is calculated with Equation (12), and εp = 0.4 is the bed porosity. 1 −εp ∂zb ∂t = −∂qb ∂x (14) (14) After each time step for the bed evolution, the model checks the angle of the bed be- tween every pair of neighbouring calculation points. If necessary, the model avalanches the ‘excess’ sand, so that the angle of the bed does not exceed the angle of repose (30◦) anywhere. 2.3. Bed Load Sediment Transport Model First, the dimensionless bed shear stress is determined from ﬂow characteristics. Then the pick-up of sediment along the dunes follows from bed shear stress. With an exponential decay function, the deposition of sediment away from each pick-up point is determined. The difference between sediment deposition and pick-up determines the net transport gradient along the dune. 3.2. Shimizu et al. (2009) Step Length Models Shimizu et al. (2009) [14] have used a minimum (αmin = 50) and maximum (αmax = 250) value of non-dimensional step length α in a conceptually derived relation between α and dimensionless grain shear stress θ′. For values of θ′ between 0 and 0.5 (the dune regime), α was assumed to be constant at the minimum value (αmin). For values of θ′ above 0.8 (the upper-stage plane bed regime), α was assumed to be at the maximum value (αmax). In the transitional regime (θ′ from 0.5 to 0.8), α was linearly interpolated. Besides the Shields parameter, there is no further dependency on sediment parameters. p g 3.1. Sekine and Kikkawa (1992) Step Length Models p g 3.1. Sekine and Kikkawa (1992) Step Length Models p g 3.1. Sekine and Kikkawa (1992) Step Length Models Sekine and Kikkawa (1992) [42] have used the data sets mentioned in Section 2 to verify a numerical model of the saltation of particles. They have found that all computed step length values are between two times larger or smaller than the observed values. Their predictions for the thickness of the saltating bed load layer closely match the data of Sekine and Kikkawa (1984) [41]; the particles remain within a few grain diameters from the bed. They further show in their calculations (1) that the mean step length varies between about 10 and about 250 times the particle diameter, (2) that it is directly proportional to Appl. Sci. 2021, 11, 11212 7 of 25 friction velocity u* (u* = (τ/ρ)1/2), and (3) that it is inversely proportional with the settling velocity ws. The suspension parameter u/ws ranges from about 0.15 to 0.28 in this set of calculations, so bed-load conditions were present (u/ws < 1). The relation between these parameters and the non-dimensional step length α is as follows: α = Λ D50 = α2 u∗ ws 3/2 1 −u∗c/ws uc/ws 3/2 (15) (15) where α2 = 3.0 × 103, and uc is the critical friction velocity (uc = (τc/ρ)1/2; note that this is volumetric bed shear stress, which has a unit of m2/s2). 3.3. Comparison of Step Length Models Both the conceptual model of Shimizu et al. (2009) [14] and the more physics-based model of Sekine and Kikkawa (1992) [42] are applied and tested in the dune model de- scribed in 2. For this test calculation scenario, A4 of Shimizu et al. (2009) [14] was used, which corresponds to a ﬂood wave in a ﬂume setting, see Figure 3. For the model runs, sediment with a D50 of 0.28 mm, a slope i of 2 × 10−3, and a hydrograph as presented below are used. Figure 3. Hydrograph of scenario A4 after Shimizu et al. (2009) [14]. This artiﬁcial scenario has not been physically simulated and measured, but corre- sponds to ﬂume conditions with regard to water depth and dune height. The goal of the present calculations is to investigate if transition to the upper-stage plane bed occurs in roughly the same way as the dune evolution model of Shimizu et al. (2009) [14] predicts, as well as the general qualitative behaviour of their model. This advanced dune evolution d l i id d ith k t b l l d l d t d ti diff i Figure 3. Hydrograph of scenario A4 after Shimizu et al. (2009) [14]. This artiﬁcial scenario has not been physically simulated and measured, but corre- sponds to ﬂume conditions with regard to water depth and dune height. The goal of the present calculations is to investigate if transition to the upper-stage plane bed occurs in roughly the same way as the dune evolution model of Shimizu et al. (2009) [14] predicts, as well as the general qualitative behaviour of their model. This advanced dune evolution model is provided with a k-ε turbulence closure model and a separate advection-diffusion model for suspension. Scenario A4 of Shimizu et al. (2009) [14] started from a ﬂat bed, and This artiﬁcial scenario has not been physically simulated and measured, but corre- sponds to ﬂume conditions with regard to water depth and dune height. The goal of the present calculations is to investigate if transition to the upper-stage plane bed occurs in roughly the same way as the dune evolution model of Shimizu et al. (2009) [14] predicts, as well as the general qualitative behaviour of their model. This advanced dune evolution model is provided with a k-ε turbulence closure model and a separate advection-diffusion model for suspension. Scenario A4 of Shimizu et al. 3.3. Comparison of Step Length Models (2009) [14] started from a ﬂat bed, and This artiﬁcial scenario has not been physically simulated and measured, but corre- sponds to ﬂume conditions with regard to water depth and dune height. The goal of the present calculations is to investigate if transition to the upper-stage plane bed occurs in roughly the same way as the dune evolution model of Shimizu et al. (2009) [14] predicts, as well as the general qualitative behaviour of their model. This advanced dune evolution model is provided with a k-ε turbulence closure model and a separate advection-diffusion model for suspension. Scenario A4 of Shimizu et al. (2009) [14] started from a ﬂat bed, and Appl. Sci. 2021, 11, 11212 8 of 25 showed dunes growing to a height of about 4 cm. A transition to upper-stage plane bed occurred at about 0.6 h, and a re-emergence of dunes occurred at about 1.7 h. Both step length models in the current model are tested with this scenario. The resulting develop- ment of the dune trough and crest positions (in the vertical) and water depth over time are shown in Figure 4. Figure 4. Dune crest and trough position (black lines) and water depth (blue line) over time with the step length model of (a) Sekine and Kikkawa (1992) [42] and (b) Shimizu et al. (2009) [14]. Figure 4. Dune crest and trough position (black lines) and water depth (blue line) over time with the step length model of (a) Sekine and Kikkawa (1992) [42] and (b) Shimizu et al. (2009) [14]. Using the Shimizu et al. (2009) [14] step length model leads to bedform growth, though no upper-stage plane bed occurs. Apparently, this step length model does not lead to strong enough lag in the present study; the dunes do not wash out at all but keep growing at the moment in time where the k-ε dune evolution model of Shimizu et al. (2009) [14] predicted a transition to upper-stage plane bed (0.6 h). Besides growing for too long, the dunes also become too high (10 cm). With the Sekine and Kikkawa (1992) [42] step length model, no dunes are able to grow at all, which is completely different from the results of the k-ε dune evolution model of Shimizu et al. (2009) [14]. 4. New Step Length Model The process of turbulent diffusion is represented by the friction velocity u, the settling process by the settling velocity ws. y g y In addition, Claudin et al. (2011) [46] derived a similar relation for the relaxation length of suspended sediment. There is a difference in how the spatial lag of bed-load and suspended load relate to the water motion: where the step length of bed load depends on (bed) friction velocity, the relaxation length of suspended sediment scales with friction velocity and the water depth (see Equation (17)). Therefore, as a ﬁrst step, it is assumed in the present study that the step length of particles α should also scale with the water depth h in order to simulate the inﬂuence of suspended sediment. A new model for non-dimensional step length α is proposed by the following equation, which depends on both non-dimensional grain shear stress and water depth. h α θ′, h = αg θ′ h hre f (18) (18) Here href is a reference water depth equal to the water depth at the start of the transitional regime of the case used to tune the step length model, scenario A4 of Shimizu et al. (2009) [14], of which the results can been seen in Figure 5. The value of the non-dimensional grain shear stress-dependent step length αg follows from a modified version of the Shimizu et al. (2009) [14] step length model, as can be seen in Figure 5. To reiterate, with this modiﬁcation the step length no longer depends on only bed shear stress or friction velocity as with bed load, but also on the water depth as with suspended transport. It represents processes inherent in the turbulent mixing of suspended material—namely, that larger water depth leads to larger turbulent vortices, which in turn leads to sediment higher in the water column, and due to the size of the vortices, a larger settling distance. Furthermore, in the new step length model the dimensionless step length keeps increasing for θ′ values above 0.8 (with the same slope as between θ′ = 0.5 and θ′ = 0.8) because formulae for the spatial lag or adjustment length of suspended sediment are generally not capped at a certain value (see e.g., [45,46]). 4. New Step Length Model Because using the Shimizu et al. (2009) [14] step length model leads to results closest to the results of the Shimizu et al. (2009) [14] dune evolution model in ﬂumes, it was taken as the basis for further extensions to enable river dune simulations. To compensate for not modelling suspended transport explicitly, and since u/ws > 1 for most of the scenario (it varied between 0.9 and 1.8), the parameter α needs to become much higher with the new step length model to implicitly take into account the effects of suspended transport. Suspended sediment load differs from bed load in the sense that turbulent vortices have the potential to move the sediment to higher parts in the water column. Due to the vertical mixing and settling process of suspended sediment, the suspended sediment load does not respond to variations in bed-shear stress (and thereby sediment pick-up) immediately, but with a spatial (and/or time) lag. To some extent, this is similar as bed-load, which also experiences a (smaller) spatial lag due to the step length of sediment grains. The turbulent mixing capacity εs is related to eddy viscosity νt, which for wall boundary layer ﬂows is related to the friction velocity u* and the water depth h as follows (see e.g., Van Rijn (1993) [43]): (16) εs ∼νt ∼u∗h (16) εs ∼νt ∼u∗h The suspension mixing height above the bed is controlled by the turbulent mixing capacity εs, and the settling by gravity (settling velocity ws) and scales with εs/ws = uh/ws (Rouse, 1937) [44]. Galappatti and Vreugdenhil (1985) [45] derived a depth-averaged model for suspended sediment based on the usual advection-diffusion equation. They show that the vertical processes of turbulent diffusion and settling of suspended sediment can be Appl. Sci. 2021, 11, 11212 9 of 25 translated into a relaxation or adjustment process of suspended sediment in ﬂow direction, involving an adjustment length (or spatial lag) of suspended sediment. A generalized expression is as follows: u translated into a relaxation or adjustment process of suspended sediment in ﬂow direction, involving an adjustment length (or spatial lag) of suspended sediment. A generalized expression is as follows: Λs;l = function u∗ ws ·h (17) h l l d l l h h d h h h (17) The equation shows that the spatial lag directly scales with the water depth h. 4. New Step Length Model Different values of αmin at θ′ = 0.5 and αmax at θ′ = 0.8 have been tested, and using αmin = 50 and αmax = 350 works best within the new dune evolution model compared to the dune evolution results of Shimizu et al. (2009) [14]. The water depth at the start of the transitional regime was 0.1166 m, which will be used as the reference water depth href. In Figure 5 the currently used model for αg is compared with the Shimizu et al. (2009) [14] step length model for αS. It should be noted that this ﬁgure is without the additional inﬂuence of the water depth as deﬁned in Equation (18). Combining that equation with the ﬁgure above and all other previous considerations leads to the following new equation for α: α θ′, h =    αmin h hre f , \|θ′ ≤0.5 h αmin + (θ′ −0.5) αmax−αmin 0.8−0.5 i h hre f , \|θ′ > 0.5 (19) (19) where αmin = 50, αmax = 350, and href = 0.1166 m. f By multiplying the result with the median grain diameter, the dimensional step length is found (as deﬁned by Equation (13)). By using this method, the value of α can become higher than the maximum observed value of α (250) found in the bed load experiments of Nakagawa and Tsujimoto (1980) [23]. By multiplying the result with the median grain diameter, the dimensional step length is found (as deﬁned by Equation (13)). By using this method, the value of α can become higher than the maximum observed value of α (250) found in the bed load experiments of Nakagawa and Tsujimoto (1980) [23]. With this modiﬁed method for step length calculation, the speciﬁc inﬂuence of sus- pended sediment on the spatial lag of sediment transport (i.e., through the water depth) is introduced in a strongly schematized way. This leads to the possibility of also applying the Appl. Sci. 2021, 11, 11212 10 of 25 10 of 25 model in the full-scale conditions of rivers (with larger water depths), in which suspended sediment is known to play a large role. Figure 5. The non-dimensional step length of the non-dimensional step length model of Shimizu et al. 4. New Step Length Model (2009) [14], using αS for bed load, and the currently used step length model Equation (19), using αg, which represents the grain-shear-stress-dependent part of non-dimensional step length in the new model. In this ﬁgure the depth is assumed to be equal to href in order to more clearly show the relation with the Shimizu step length model. model in the full-scale conditions of rivers (with larger water depths), in which suspended sediment is known to play a large role. model in the full-scale conditions of rivers (with larger water depths), in which suspended sediment is known to play a large role. Figure 5. The non-dimensional step length of the non-dimensional step length model of Shimizu et al. (2009) [14], using αS for bed load, and the currently used step length model Equation (19), using αg, which represents the grain-shear-stress-dependent part of non-dimensional step length in the new model. In this ﬁgure the depth is assumed to be equal to href in order to more clearly show the relation with the Shimizu step length model. 5. Results with Flume Conditions We start with testing whether the model is able to model a transition to upper-stage plane bed for ﬂume conditions, that is, the computational scenario A4 as presented by Shimizu et al. (2009) [14] (see also Section 3.3). With this scenario, Shimizu et al. (2009) [14] show that their model is able to predict transitions to the upper-stage plane bed. The model also clearly shows hysteresis effects; the relation between discharge and water depths is as signiﬁcantly different for the rising limb of the hydrograph as it is for the falling limb. The parameters of the scenario are typically equivalent to a ﬂume scenario. With the model runs presented here, the suspension parameter u/ws varied between 0.9 and 1.8 during the scenario, so with this sediment and this ﬂow regime the suspension regime is present for most of the time. Using scenario A4 from Shimizu et al. (2009) [14], the following development of the dune ﬁeld over time is found with the dune evolution model as developed in the present study. Here in Figure 6 the washing out and regrowth of the dunes can be clearly observed. From the same results, the development of the dune trough and crest positions (in the vertical) and water depth over time are shown in Figure 7. As can be seen in the beginning of the run, dunes start developing along with increasing discharge. At a certain high discharge, the shear stress and the step length become so high that the dunes are washed out. Due to the decrease in form drag and thereby total shear stress, the water level stabilizes temporarily, despite still rising discharge. The bed remains washed out until the discharge and step length become so low that dunes start developing again. 11 of 25 Appl. Sci. 2021, 11, 11212 11 Figure 6. Dune ﬁeld over time, using scenario A4 from Shimizu et al. (2009). Figure 7. Dune crest and trough position (black lines) and water depth (blue line) over time, u scenario A4 from Shimizu et al. (2009). Shimizu et al. (2009) [14] found roughly the same moments of washing out (0.6 h) an emergence of dunes (1.7 h). Furthermore, the dune evolution model of Shimizu et al. (2009) predicted a dune with a maximum height of about 4 cm, which corresponds rather w with the present model result (3.75 cm). The relation between the step length paramet Figure 6. 5. Results with Flume Conditions Dune ﬁeld over time, using scenario A4 from Shimizu et al. (2009). Figure 6. Dune ﬁeld over time, using scenario A4 from Shimizu et al. (2009). Figure 6. Dune ﬁeld over time, using scenario A4 from Shimizu et al. (2009). Figure 6. Dune ﬁeld over time, using scenario A4 from Shimizu et al. (2009). Figure 6. Dune ﬁeld over time, using scenario A4 from Shimizu et al. (2009). g , g ( ) Figure 7. Dune crest and trough position (black lines) and water depth (blue line) over time, using scenario A4 from Shimizu et al. (2009). Figure 7. Dune crest and trough position (black lines) and water depth (blue line) over time, using scenario A4 from Shimizu et al. (2009). Shimizu et al. (2009) [14] found roughly the same moments of washing out (0.6 h) and re- emergence of dunes (1.7 h). Furthermore, the dune evolution model of Shimizu et al. (2009) [14] predicted a dune with a maximum height of about 4 cm, which corresponds rather well with the present model result (3.75 cm). The relation between the step length parameter α and the speciﬁc discharge for the rising and falling stages of the hydrograph can be seen in Figure 8. 12 of 25 Appl. Sci. 2021, 11, 11212 12 of 25 Figure 8. Speciﬁc discharge versus non-dimensional step length α separated for the rising and falling stages of the hydrograph. Figure 8. Speciﬁc discharge versus non-dimensional step length α separated for the rising and falling stages of the hydrograph. Here a hysteresis effect is already observed, caused by the transition to upper-stage plane bed and then later returning to the dune regime. With the same discharge, dunes can be present in the rising stage and not in the falling stage. Because of the presence of dunes, a part of the ﬂow power is lost due to form drag. This means the shear stress acting on the sand grains (effective bed shear stress) is relatively lower, and a lower step length is selected than when there are no dunes present. The effect of hysteresis on water depth can be seen in Figure 9. Figure 9. Speciﬁc discharge versus water depth separated for the rising and falling stages of the hydrograph, using scenario A4 from Shimizu et al. (2009). Figure 9. 5. Results with Flume Conditions Speciﬁc discharge versus water depth separated for the rising and falling stages of the hydrograph, using scenario A4 from Shimizu et al. (2009). Appl. Sci. 2021, 11, 11212 13 of 25 13 of 25 In the rising part of the hydrograph, dunes are able to develop ﬁrstly at medium discharges, while in the falling part, the dunes start developing at the end of the ﬂood wave at lower discharges. This has a clear effect on the resulting water depths at the same discharge. For example, at a speciﬁc discharge of 0.12 m2/s, the water depth in the rising part is clearly higher than in the falling part. In the rising limb, the dunes have had a longer time to grow than in the falling limb, and are therefore higher. Because the dunes are higher, the water depth is higher despite the discharge being the same. Shimizu et al. (2009) [14] have clearly shown this hysteresis effect as well, though for their model it’s more pronounced. They have also reported in the order of 25% lower water depths than we found. 6. Results with River Conditions The bed conﬁguration of the ﬁrst ﬂood wave is used as input for the second ﬂood wave. The results of the second run are reported in Section 6.1. twice. The bed conﬁguration of the ﬁrst ﬂood wave is used as input for the second ﬂood wave. The results of the second run are reported in Section 6.1. To test if a transition to the upper-stage plane bed can be modelled for river conditions, the model is also run with a 5% higher discharge than for the scenario above. This leads to water depths between 9 and 11.4 m, which is about 5% higher than the water depths in Waal for the ﬂood of 1998 as reported by Julien et al. (2002) [47]. The duration of the ﬂood wave is the same as in the other scenario, and the calculation time is just under 35 min. This is less than before, because in this scenario dunes are washed out during a part of the run, which lets the model solve the ﬂow equations more efﬁciently. Again, to let the model adjust to the hydrodynamic conditions, the ﬂood wave is modelled twice. The results of the second run are reported in Section 6.2. 6. Results with River Conditions Although the new dune evolution model is still in a developing stage, from a practical point of view it is interesting to test how the model behaves under river conditions. For this purpose the model is run with similar conditions to the Dutch river Waal during the ﬂood of 1998. At the peak of this ﬂood wave, the total discharge was 6250 m3/s in the river Waal [47]. For simplicity, the dune evolution model is applied to only the main channel; interactions with the ﬂoodplains are not taken into account here. It is assumed that 60% of the peak discharge went through the main channel. The main channel is assumed to be 300 m wide, which corresponds well to the width of the main channel along the river Waal in the SOBEK model made by Deltares and used by Paarlberg (2012) [48]. This would make the speciﬁc discharge 12.5 m2/s, which is rounded to 13 m2/s for the current study. To simulate these conditions, a D50 of 1.2 mm (in the range presented by Giri et al., 2008 [49]), a slope i of 1 × 10−4 [50], and a hydrograph as presented in Figure 10 are used as input for the model. The settling velocity corresponding to the used D50 is ws = 0.13 m/s. This hydrograph leads to water depths between 8.5 and 11.1 m, which correspond well to the typical water depths in the Waal for the ﬂood of 1998 as reported by Julien et al. (2002) [47]. Figure 10. Hydrograph of the river scenario, which is based on the 1998 ﬂood in the river Waal. Figure 10. Hydrograph of the river scenario, which is based on the 1998 ﬂood in the river Waal. Compared to the ﬂood wave of 1998, as shown by Julien et al. (2002) [47], we kept the duration of the rising limb the same, while we shortened the duration of the falling limb somewhat in order to create a symmetric ﬂood wave. The entire duration of the ﬂood wave presented here is exactly 12 days or 288 h. The calculation time is very reasonable; with a time step of 2 min, the model goes through 12 days modelled time in under 55 min real time. To let the model adjust to the hydrodynamic conditions, the ﬂood wave is modelled Appl. Sci. 2021, 11, 11212 14 of 25 14 of 25 twice. 6.1. Results for the River Scenario The development of the dune ﬁeld, starting from an already developed dune ﬁeld, can be observed in Figure 11. Figure 11. Dune ﬁeld over time, using the river scenario. Figure 11. Dune ﬁeld over time, using the river scenario. Figure 11. Dune ﬁeld over time, using the river scenario. The dunes grow and shrink during the ﬂood wave, though in general the dune height variation is low. From the same results, the development of the dune trough and crest positions (in the vertical) and water depth over time are shown in Figure 12. As can be seen in the beginning of the run, dunes start developing slowly along with increasing discharge. This continues past the moment of peak discharge until about 155 h, after which the dune height decreases. Due to the increase in discharge, the water level still increases and decreases, with seemingly little inﬂuence of the dune height. 15 of 25 Appl. Sci. 2021, 11, 11212 Figure 12. Dune crest and trough position (black lines) and water depth (blue line) over time, using the river scenario. Figure 12. Dune crest and trough position (black lines) and water depth (blue line) over time, using the river scenario. During the ﬂood wave of 1998, dune heights in the Waal varied between 0.2 and 0.6 m on the centreline [47], while the model predicts values between 1.6 and 2.1 m. This is still a considerable difference, which indicates that further model improvements are necessary. Increasing the step length likely has a dampening effect on the dune height, but it also causes the dunes to wash out sooner. Therefore, care should be taken in adjusting the model settings. In Figure 13 the response of the water depth to the varying discharge can be seen. Figure 13. Discharge versus dune height for the rising and falling stages of the hydrograph, using the river scenario. The arrow signiﬁes the direction of development over time. Figure 13. Discharge versus dune height for the rising and falling stages of the hydrograph, using the river scenario. The arrow signiﬁes the direction of development over time. Appl. Sci. 2021, 11, 11212 16 of 25 16 of 25 The hysteresis loop observed here is in the same direction as for the ﬂood wave of 1998 as reported by Julien et al. (2002) [47], with dune heights in the falling limb being higher than in the rising limb (Figure 14). 6.1. Results for the River Scenario Here the dune height also keeps growing for a period of time after the discharge is already decreasing. The relation between the non-dimensional shear stress and the dune height can be seen in the following ﬁgure. The dunes do not wash out; however, they do decrease in height about 5 h after the maximum discharge has been reached. Figure 14. Non-dimensional shear stress θ versus dune height for the rising and falling stages of the hydrograph, using the river scenario. The arrow signiﬁes the direction of development over time. Figure 14. Non-dimensional shear stress θ versus dune height for the rising and falling stages of the hydrograph, using the river scenario. The arrow signiﬁes the direction of development over time. The relation between α and the speciﬁc discharge for the rising and falling stages of the hydrograph can be seen in Figure 15. Here again a hysteresis effect is observed, caused by the lag between dune development and discharge. It should be noted that now, much higher step lengths are reached than for the ﬂume scenario, up until α = 4750, which is a dimensional step length of about 6 m. This is about 2.6 times higher than that of the ﬂume scenario. Figure 15. Speciﬁc discharge versus non-dimensional step length α separated for the rising and falling stages of the hydrograph, using the river scenario. The arrow signiﬁes the direction of development over time. Figure 15. Speciﬁc discharge versus non-dimensional step length α separated for the rising and falling stages of the hydrograph, using the river scenario. The arrow signiﬁes the direction of development over time. 17 of 25 Appl. Sci. 2021, 11, 11212 The effect of hysteresis on water depth can be seen in Figure 16. Figure 16. Speciﬁc discharge versus water depth separated for the rising and falling stages of the hydrograph, using the river scenario. The arrow signiﬁes the direction of development over time. The effect of hysteresis on water depth can be seen in Figure 16. Figure 16. Speciﬁc discharge versus water depth separated for the rising and falling stages of the hydrograph, using the river scenario. The arrow signiﬁes the direction of development over time. In the rising part of the hydrograph, dunes increase in height, while in the falling part the dune height ﬁrst remains constant before decreasing later on. 6.1. Results for the River Scenario Because of the delay in dune height development compared to discharge, the water depth in the falling limb of the hydrograph is larger than in the rising limb, which agrees again in a qualitative sense with what is reported by Julien et al. (2002) [47] for the ﬂood wave of 1998. 6.2. Results for the More Extreme River Scenario With the artiﬁcial more extreme scenario, which includes an increase of the discharge of 5%, a transition to upper-stage plane bed is predicted. It should be noted that this transition was not observed in the river Waal in 1998 [47], nor in the Dutch Rhine Branches in 1995 when the discharge at Lobith was 25% higher than it was in 1998 (e.g., [51]). The computed development of the dune ﬁeld, starting from an already developed dune ﬁeld, can be observed in Figure 17. Here the growth and washing out of the dunes can be observed on the left, and the washing out itself can be observed more clearly on the right. From the same results, the development of the dune trough and crest positions (in the vertical) and water depth over time are shown below (Figure 18). As can be seen in the beginning of the run, dunes start developing along with increasing discharge. At around 137 h from the start of the model run, the dunes start washing out, and they’re completely gone at about 141 h, 5 h before the maximum discharge occurs. Due to the decrease in form drag and thereby total shear stress, the water level goes down, despite still-rising discharge. The bed remains washed out until the discharge is low enough for dunes to start developing again. For the ﬂood of 1998, dune heights in the Waal varied between 0.2 and 0.6 m on the centreline, while the model results indicate values between 0.2 and 2.2 m. In Figure 19 the response of dune height to changing discharge can be seen. The hysteresis loop observed here is in the opposite direction of the measured ﬂood wave of 1998 as reported by Julien et al. (2002) [47]. Due to the washing out of dunes during the ﬂood wave, dunes are now generally lower in the falling limb than in the rising limb. g y g g The relation between α and the speciﬁc discharge for the rising and falling stages of the hydrograph can be seen in Figure 20. 18 of 25 Appl. Sci. 2021, 11, 11212 Figure 17. Dune ﬁeld over time, using the more extreme river scenario. The right part zooms in on the dune ﬁeld in the period where the transition occurs. Figure 17. Dune ﬁeld over time, using the more extreme river scenario. 6.2. Results for the More Extreme River Scenario force on the particles, which increases step length because that is partly based on grain shear stress. The effect of hysteresis on water depth can be seen in Figure 21. Figure 21. Speciﬁc discharge versus water depth separated for the rising and falling stages of the hydrograph, using the more extreme river scenario. Figure 21. Speciﬁc discharge versus water depth separated for the rising and falling stages of the hydrograph, using the more extreme river scenario. In the rising part of the hydrograph dunes are able to develop ﬁrstly at lower dis- charges, while in the falling part the dunes start developing at higher discharges. This has a clear effect on the resulting water depths at the same discharge. The hysteresis loop is reversed compared to the results in Section 6.1. Of great importance for the river situation is the signiﬁcant drop in water levels when dunes are washed out. Within 4 h the water levels become about 0.25 m lower, while the discharge is still growing; if it assumed dunes keep growing instead, and we extrapolate from the point where the water depth is still rising, the difference in water levels is approximately 0.5 m. Though this effect will probably be mitigated in real life by the presence of ﬂoodplains, it is still a signiﬁcant effect. This example shows that knowing when the transition to upper-stage plane bed occurs is important, because it may have a large impact on the water levels and thereby on the dike heights needed to prevent ﬂooding. 6.2. Results for the More Extreme River Scenario The right part zooms in on the dune ﬁeld in the period where the transition occurs. Figure 18. Dune crest and trough position (black lines) and water depth (blue line) over time, using the more extreme river scenario. Figure 18. Dune crest and trough position (black lines) and water depth (blue line) over time, using the more extreme river scenario. 19 of 25 Appl. Sci. 2021, 11, 11212 Figure 19. Discharge versus dune height for the rising and falling stages of the hydrograph, using the more extreme river scenario. The arrow signiﬁes the direction of development over time. Figure 19. Discharge versus dune height for the rising and falling stages of the hydrograph, using the more extreme river scenario. The arrow signiﬁes the direction of development over time. Figure 20. Speciﬁc discharge versus non-dimensional step length α separated for the rising and falling stages of the hydrograph, using the more extreme river scenario. The arrow signiﬁes the direction of development over time. Figure 20. Speciﬁc discharge versus non-dimensional step length α separated for the rising and falling stages of the hydrograph, using the more extreme river scenario. The arrow signiﬁes the direction of development over time. Here again a hysteresis effect is observed, caused by the transition to upper-stage plane bed and back to the dune regime. It should also be noted that now, much higher step lengths are reached, up until α = 10,800, which is a dimensional step length of about 13 m. This is about 10 times the value of the ﬂume scenario. For the river scenario of Section 6.1, the maximum step length was only 2.6 times higher than the ﬂume scenario. This difference between the river scenarios mostly relates to the washing out of dunes, which only occurs for the more extreme river scenario. When the dune is washed out, water depth drops somewhat, but due to the lack of bedforms, the ﬂow can exert more Appl. Sci. 2021, 11, 11212 20 of 25 20 of 25 force on the particles, which increases step length because that is partly based on grain shear stress. The effect of hysteresis on water depth can be seen in Figure 21. force on the particles, which increases step length because that is partly based on grain shear stress. The effect of hysteresis on water depth can be seen in Figure 21. 7. Discussion (2009) [14] with αmax = 300 as well as αmax = 350 are shown in Figure 22. Here it can be seen that the bed is washed out at about 37 min with αmax = 300 instead of 35 min as with αmax = 350, and that the maximum dune height is 3.75 cm and 2.8 cm, respectively. This shows that the results are sensitive to the settings of the step length model, though there is no extreme effect on general model behaviour. Figure 22. Dune crest and trough position for the ﬁrst 45 min of scenario A4 of Shimizu et al. (2009) [14]) with (a) αmax = 300 and (b) αmax = 350. Figure 22. Dune crest and trough position for the ﬁrst 45 min of scenario A4 of Shimizu et al. (2009) [14]) with (a) αmax = 300 and (b) αmax = 350. Figure 22. Dune crest and trough position for the ﬁrst 45 min of scenario A4 of Shimizu et al. (2009) [14]) with (a) αmax = 300 and (b) αmax = 350 Figure 22. Dune crest and trough position for the ﬁrst 45 min of scenario A4 of Shimizu et al. (2009) [14]) with (a) αmax = 300 and (b) αmax = 350. To ensure that the model still represents the dune regime well, the model was run again for the experimental conditions of Venditti et al. (2005a, 2005b) [52,53] tested in the Section Results of van Duin et al. (2017) [26]. Compared to the previous results, the dune height is now generally slightly lower, though still represented well. Dune length is very similar to before, represented well except for the artiﬁcial defects of bedforms D and E. Migration rate and thereby transport rate are much higher than before, and are now generally overestimated instead of underestimated. This is in line with the results presented in Section 3.3 of van Duin et al. (2016) [26], where the migration rate and thereby transport rate increased strongly with step length. Overall the results are slightly worse than with the model version optimized for the dune regime, though dune dimensions are still represented well by the new model. The question arises whether the step length should be varied only due to the changing ﬂow regime as is done now, or along the dune as well (because of local variation in shear stress). Explorative experiments of Van Duin et al. 7. Discussion In the present study, under ﬂume conditions, differences have been observed in outcome between the new model and the k-ε model of Shimizu et al. (2009) [14], that is, step lengths have to become signiﬁcantly larger to reach the transition to an upper-stage plane bed, and there are differences of 25% in water depths. Of course, the models differ in signiﬁcant ways. Firstly, Shimizu et al. (2009) [14] have used a non-hydrostatic ﬂow model with a non-linear k-ε model for turbulence closure, whereas in the idealized dune evolution model, a hydrostatic ﬂow model with a constant eddy viscosity has been used. Secondly, their transport module also used a separate suspended sediment model, while in this research suspended sediment has been only modelled implicitly. Pinpointing the exact reason of the differences is hard because the A4 ﬂume case under review is a ‘synthetic’ case; there are no actually measured values as a reference to determine which model is closer to the truth. The Shimizu et al. (2009) [14] dune model is more physically complex, which suggest that its results should be better. However, it is still promising that the new relatively simple model presented in this research is able to represent similar bedform behaviour during a ﬂood wave as the more complex model in a qualitative sense. Appl. Sci. 2021, 11, 11212 21 of 25 21 of 25 During the research for this paper, it was found that the timing of a transition to upper-stage plane bed is sensitive to the value of α. As an example, the results of the ﬁrst 45 min for scenario A4 of Shimizu et al. (2009) [14] with αmax = 300 as well as αmax = 350 are shown in Figure 22. Here it can be seen that the bed is washed out at about 37 min with αmax = 300 instead of 35 min as with αmax = 350, and that the maximum dune height is 3.75 cm and 2.8 cm, respectively. This shows that the results are sensitive to the settings of the step length model, though there is no extreme effect on general model behaviour. During the research for this paper, it was found that the timing of a transition to upper-stage plane bed is sensitive to the value of α. As an example, the results of the ﬁrst 45 min for scenario A4 of Shimizu et al. 7. Discussion (2012) [26] suggest that the latter is not necessary, but this warrants further research. This also applies for more realistic non-linear relations of the step length model, as the current one (Equation (19)) is still relatively simple. y p Although the model results show hysteresis effects with regard to discharge and water depth caused by the development of dunes, it should be realized that the present model does not describe the hysteresis effects due to the phase lead of discharge with respect to water depth, which may be relevant for ﬁeld situations. This phase lead arises from the phenomenon that the pressure gradient of the front of the ﬂood wave is higher than in the tail of the ﬂood wave, as described by Jones (1915) [54] and later studied by various authors (e.g., [55–57]). The spatial phase lag of the sediment transport relative to the local bed shear stress is the crucial element of the model for representing the growth and washing out of bedforms. This sediment transport phase lag is not an explicit part of our model, but it is brought into the model through the step-length of grains. This stepping of grains leads to the situation that the local sediment transport along the dune is coupled to the bed shear stress (and grain pick-up) some distance upstream. The original Einstein step length model in principle is a bed load formulation, as it explicitly models grains to make a step at a distance of some grains (i.e., rolling or sliding a small distance). To incorporate the suspension effect, we are stretching the Einstein step length model towards much larger steps. For Appl. Sci. 2021, 11, 11212 22 of 25 22 of 25 suspended sediment, this phase-lag is caused by the relatively slow process of upward turbulent diffusion and downward settling of suspended sediment particles. The length scale of this suspension adjustment process is a function of the suspension numbers u*/ws and increases with the water depth h (see Equation (17)). Now, by incorporation of the depth in the step length formulation, we allow for much larger steps. Such larger steps can be thought of as overﬂowing the ﬂow separation zone in a dune, so that the sediment accumulates at the stoss side of the next dune. If this step becomes very large, suspended sediment will be able to pass even more than one dune crest. 7. Discussion p It could be interesting to use a separate suspended transport model similar to the bed load models used in this study, with the lower step lengths used for bed load and the higher step lengths used for suspended load. With such a dual method, it is important to know what the appropriate step lengths are for each of the transport modes. This could be done by either implementing more physics-based predictors of the step length for bed load and suspended load, or by calibrating the step length models using dune data sets from the ﬁeld, such as that of Sieben (2004) [50], as a benchmark. Another interesting exercise would be to test the Froude number effect in this model with laboratory and ﬁeld experiments (see e.g., Naqshband et al. (2014) [28]). In general, a well-calibrated version of the dune evolution model could then be applied within a model of a river system (replacing a part of the hydraulic roughness model) to better determine if and under which conditions a transition to upper-stage plane bed may occur. As far as we know for the Dutch rivers, washing out of dunes with a high discharge has never occurred, and it is unknown what will happen with the extreme (design) discharges that may occur in the future. In current practice, roughness values are calibrated by matching observed and modelled water levels [58,59]. This means that there is signiﬁcant uncertainty in roughness for extreme discharges that have not been observed yet. Using a dynamic roughness model which includes the dune dynamics may lead to more physically correct results. The river Waal case in this study for the 1998 ﬂood has shown that a relatively small increase in river discharge can lead to the occurrence of upper-stage plane beds together with a considerable drop in water depth in a relatively short time. This shows that the occurrence of upper-stage plane bed can be an unexpected and rapid occurrence. 8. Conclusions In this study the dune evolution model of Paarlberg et al. (2009) [21] was extended with an alternative sediment transport model using sediment pick-up, deposition, and step length models instead of the usual equilibrium transport formula. The model was devised in such a way that similar dune behaviour is obtained as with the more complex k-ε model of Shimizu et al. (2009) [14] for a ﬂood wave corresponding to ﬂume conditions. p g Step lengths have been allowed to become larger than with the step length model of Shimizu et al. (2009) [14]. In addition, higher step lengths than the maximum observed in the experiments of Nakagawa and Tsujimoto (1980) [23] have been adopted such that effects of suspended load are implicitly taken into account. We would like to note that this implicit formulation is a ﬁrst step to account for suspended sediment transport processes in a strongly schematized way, which needs to be extended and improved in further studies. It was shown that in this way, the model is able to predict a transition to upper-stage plane bed and clearly shows hysteresis effects due to the lag between bedform dimensions and discharge. While the simulated associated moments in time and the maximum dune height are close to the results of Shimizu et al. (2009) [14], the exact results in terms of water depth still showed differences of about 25%. A scenario corresponding to the 1998 ﬂood in the river Waal was modelled as well. While the qualitative behaviour of the dunes was represented well, the dune height was overestimated. This signiﬁes the need for further model improvement/calibration. For the river scenario, the step length is signiﬁcantly higher than the values used for the ﬂume conditions. It was shown that the Waal model is also computationally fast. By slightly Appl. Sci. 2021, 11, 11212 23 of 25 23 of 25 increasing the discharge of the original river scenario, a transition to upper-stage plane bed was also modelled. Although in 1995, for much higher discharges, no transition to upper-stage plane bed has occurred (Wilbers and Ten Brinke (2003) [51]), this does show the sensitivity of the dune dynamics in this transitional regime and also indicates the need for a well-validated dune evolution model for extreme discharges. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Acknowledgments: This study was carried out as part of the project ‘BedFormFlood’, supported by the Technology Foundation STW, the applied science division of now, and the technology programme of the Ministry of Economic Affairs. The authors thank Anke Wigger and Dominique van de Meché for their help in editing the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Casas, A.; Benito, G.; Thorndycraft, V.; Rico, M. The topographic data source of digital terrain models as a key element in the accuracy of hydraulic ﬂood modelling. Earth Surf. Process. Landf. 2006, 31, 444–456. [CrossRef] 2. Vidal, J.-P.; Moisan, S.; Faure, J.-B.; Dartus, D. River model calibration, from guidelines to operational support tools. Environ. Model. Softw. 2007, 22, 1628–1640. [CrossRef] f 3. Morvan, H.; Knight, D.; Wright, N.; Tang, X.; Crossley, A. 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J. Geophys. Res. Space Phys. 2005, 110. 8. Conclusions It should be noted that when a transition to upper-stage plane bed indeed occurs during extreme ﬂoods (i.e., during dike design conditions), this has the potential to greatly reduce the design water depth because the hydraulic roughness of the main channel would be signiﬁcantly lower. However, in a bifurcation river system such as the river Waal, balancing effects distribute this lowering of the water levels over both branches, if it would occur in one branch only [4]. For the model results presented here, the difference in water levels in one branch is approximately 0.5 m. Although in reality this effect would also be dampened due to the presence of ﬂoodplains, which are not accounted for here, this is still considered as a signiﬁcant effect. Author Contributions: Conceptualization, O.J.M.v.D., S.J.M.H.H. and J.S.R.; methodology, O.J.M.v.D., S.J.M.H.H. and J.S.R.; software, O.J.M.v.D.; resources, S.J.M.H.H.; writing—original draft preparation, O.J.M.v.D., S.J.M.H.H. and J.S.R.; writing—review and editing, O.J.M.v.D., S.J.M.H.H. and J.S.R.; visu- alization, O.J.M.v.D.; supervision, S.J.M.H.H. and J.S.R.; project administration, S.J.M.H.H.; funding acquisition, S.J.M.H.H. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by NWO-AES, grant number 10483. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. References Flow, sediment transport and bedform dynamics over the transition from dunes to upper-stage plane beds: Implications for the formation of planar laminae. Sedimentology 1988, 35, 753–763. [CrossRef] 33. Smith, J.D.; McLean, S.R. 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https://openalex.org/W2902468329	https://www.scielo.br/j/pd/a/SRK4rfvBhXc5s9Dd6WbTYhH/?lang=en&format=pdf	English	null	Integrated Approaches for Weed Suppression in Chickpea (Cicer arietinum) under Residual Moisture After Rice Crop	Planta Daninha	2,018	cc-by	5,725	ed approaches for weed suppression in chickpea (Cicer arietinum) under residual moisture af 6 (9 páginas) PROVA GRÁF PLANTA DANINHA SOCIEDADE BRASILEIRA DA CIÊNCIA DAS PLANTAS DANINHAS org> ISSN 0100-8358 (print) 1806-9681 (online) ISSN 0100-8358 (print) 1806-9681 (online) KHAN, I.A 1 KHAN, I.A 1 Article Keywords: herbicides, Stomp 330E, parthenium extract, residual moisture, weed control, seed yield. Article KHAN, I.A.1 KHAN, R.1 HASSAN, G.1* WAQAS, M.2 SHAH, S.M.A.1 KHAN, S.A.1 Abordagens Integradas para a Supressão de Plantas Daninhas no Grão-de-Bico (Cicer arietinum) sob Umidade Residual após o Cultivo de Arroz ABSTRACT - Three-year field studies were undertaken at Agricultural Research Institute, Dera Ismail Khan, Khyber Pakhtunkhwa Province, Pakistan from 2010-11 to 2012-2013 with the aim of assessing the efficacy of different management techniques on weed growth and yield of chickpea under rain-fed conditions on residual moisture of a previous rice crop. Treatments of the experiment consisted of recommended full and half doses of three herbicides, i.e., Stomp 330E, Dual Gold 960EC and Isoproturon 500EW and the plant extract of Parthenium hysterophorus. Hand weeding treatment was kept as positive control whereas weedy check was included as negative control treatment. The experiment was laid out in a Completely Randomized Block (CRB) design replicated thrice. Parameters of the investigation were weed density, fresh weed biomass, chickpea growth, biological yield and seed yield. The data revealed that the full dose of Stomp 330E proved superior in terms of weed suppression by giving only 84, 69 and 55 weeds m-2 as compared to weedy check for the years 2010- 11, 2011-12 and 2012-13, respectively. Likewise, the highest plant height and seed yield were also recorded for Stomp 330E (full dose) during the entire study, which was statistically at par with hand weeding. However, there was a reduction in overall chickpea yield during the second and third years of experimentation resulting from lower average rainfall. The extract of P. hysterophorus followed Stomp 330E in weed suppression, enhancing plant height as well as biological yield and seed yield. Moreover, the result also shows statistically similar results of the extract of P. hysterophorus for all other tested treatments. Based on the findings of the present research, it was found that herbicides and hand weeding showed superior results in terms of all tested parameters. However, both approaches are non-judicious: hand weeding is laborious while herbicides may cause environmental pollution, hence the herbicidal potential of P. hysterophorus needs to be encouraged in order to achieve sustainable weed management and high yield in an eco-friendly manner. * Corresponding author: <hassanpk_2000pk@yahoo.com> Received: February 20, 2017 Approved: September 25, 2017 * Corresponding author: <hassanpk_2000pk@yahoo.com> Received: February 20, 2017 Approved: September 25, 2017 Keywords: herbicides, Stomp 330E, parthenium extract, residual moisture, weed control, seed yield. Planta Daninha 2018; v36:e018176072 RESUMO - Estudos de campo foram realizados ao longo de três anos no Instituto de Pesquisas Agrícolas Dera Ismail Khan, província de Khyber Pakhtunkhwa, Paquistão, entre 2010-11 e 2012-2013, com o objetivo de avaliar a eficácia de diferentes técnicas de manejo no crescimento de plantas daninhas e rendimento do grão-de-bico sob sistema de irrigação pela chuva em umidade residual de cultivo prévio de arroz. Os tratamentos do experimento foram compostos de doses totais e meias doses, conforme recomendação do fabricante, de três herbicidas: Stomp Copyright: 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 that the original author and source are credited. Planta Daninha 2018; v36:e018176072 1 The University of Agriculture, Peshawar-Pakistan; 2 University, Jeddah-Kingdom of Saudi Arabia. Doi: 10.1590/S0100-83582018360100150 he University of Agriculture, Peshawar-Pakistan; 2 University, Jeddah-Kingdom of Saudi A Doi: 10.1590/S0100-83582018360100150 KHAN, I.A. et al. Integrated approaches for weed suppression in chickpea (Cicer arietinum) un 2 ed approaches for weed suppression in chickpea (Cicer arietinum) under residual moisture after ric 330E, Dual Gold 960EC e Isoproturon 500EW, além do extrato vegetal de Parthenium hysterophorus. O tratamento com capina foi mantido como controle positivo, enquanto o tratamento sem capina foi incluído como controle negativo. O experimento teve delineamento em blocos completamente casualizados com três repetições. Os parâmetros da investigação foram densidade de plantas daninhas, biomassa fresca de plantas daninhas, crescimento de grão-de-bico e rendimento biológico e de sementes. Os dados revelaram que a dose total de Stomp 330E foi superior em termos de supressão de plantas daninhas, com apenas 84, 69 e 55 plantas daninhas m-2, em comparação com o tratamento sem capina nos períodos de 2010-11, 2011-12 e 2012-13, respectivamente. Da mesma forma, também foram registrados maior altura das plantas e maior rendimento de sementes com o Stomp 330E (dose total) durante toda a duração do estudo – um resultado estatisticamente semelhante ao da capina de plantas daninhas. No entanto, durante o segundo e o terceiro ano do experimento, foi observada redução na produção total de grão-de-bico, proporcional à menor precipitação média. Assim como o Stomp 330E, o extrato de P. histerophorus também suprimiu as plantas daninhas, aumentando a altura das plantas e o rendimento biológico e de sementes do grão-de-bico. Além disso, os resultados obtidos pelo extrato de P. hysterophorus foram estatisticamente semelhantes aos de todos os outros tratamentos testados. INTRODUCTION Chickpea (Cicer arietinum L.) is one of the most important pulses as well as a vegetable crop in Pakistan. It contributes 75 percent to total pulses grown in Pakistan (Sibtain et al., 2015; Abbas et al., 2016). The area under chickpea cultivation in Pakistan during 2012-13 was 992 K ha with a production of 751.3 K tons with an average seed yield of 757 kg ha-1. Similarly, in Khyber Pakhtunkhwa, the area of chickpea cultivation was 37.9 K ha and the production was 20.1 K tons, while the average yield was 530 kg ha-1 (Pakistan Statistical Yearbook, 2016). Because chickpea is a rich and inexpensive source of energy and protein, it can help people to improve the quality of their daily nutrition (Esmaeilzadeh and Aminpanah, 2015; Tiwari and Meena, 2016). Moreover, it can also play an important role in the agricultural sustainability through its nitrogen ûxation ability (Khan et al., 2012). Although it is an important crop in terms of human nutrition and soil health, its yield is declining as compared to the maximum potential of cultivars as a result of various factors. One of the key constraints is weed infestation (Hussain et al., 2015). This crop is a poor competitor with weeds particularly at earlier growth stages because of its slow growth and limited leaf area development (Abbas et al., 2016). Weeds compete with crops for available moisture, nutrients, space and solar radiation. The most common and problematic weeds for chickpea crops include Chenopodium album, Asphodelus tenuifolius, Argemone mexicana, Carthamus oxyacantha, Cenchrus ciliaris, Cyperus rotundus, Fumaria sp., Polygonum sp., Lathyrus sp., Vicia sativa, Cynodon dactylon and Cirsium arvense (Khan et al., 2011). Annual broad-leaved weeds are more competitive to chickpea because they have a similar growth pattern to that of chickpea and severity also increases with advance in growth (Bhan and Kukula, 1987). In Pakistan, weed-induced yield losses in chickpea range between 24- 63% (Abbas et al., 2016). If weeds are not properly controlled within the critical growth period, yield loses may reach 88% (Bhalla et al., 1998). For an effective control of weeds in field crops, various mechanical, chemical and biological methods are applied (Silva et al., 2004). Mechanical methods such as hand weeding are the best and most effective but they are time-consuming and laborious (Ihsan et al., 2014). Likewise, in the chemical control method, herbicides are used to prevent yield losses caused by weeds. Planta Daninha 2018; v36:e018176072 Com base nos resultados desta pesquisa, observou-se que a aplicação dos herbicidas e a capina apresentaram resultados superiores em todos os parâmetros testados; no entanto, ambas as abordagens são pouco sensatas: a capina é trabalhosa, ao passo que os herbicidas podem causar poluição ambiental. Por isso, o potencial herbicida de P. hysterophorus precisa ser incentivado para se alcançar um manejo sustentável de plantas daninhas e um alto rendimento de forma ecologicamente correta. Palavras-chave: herbicidas, Stomp 330E, extrato de parthenium, umidade residual, controle de plantas daninhas, rendimento de sementes. INTRODUCTION The use of herbicides can provide effective and economic weed control and, consequently, give similar yield values or only slightly smaller values than those of weed free treatments Planta Daninha 2018; v36:e018176072 Planta Daninha 2018; v36:e018176072 Planta Daninha 2018; v36:e018176072 KHAN, I.A. et al. Integrated approaches for weed suppression in chickpea (Cicer arietinum) un 3 al. Integrated approaches for weed suppression in chickpea (Cicer arietinum) under residual moi roaches for weed suppression in chickpea (Cicer arietinum) under residual moisture after rice crop (Patel et al., 2006). However, in several cases of the chemical weed control method, herbicides residues have negatively affected the yield components and nodulation of susceptible varieties of chickpea (Waqas et al., 2016). (Patel et al., 2006). However, in several cases of the chemical weed control method, herbicides residues have negatively affected the yield components and nodulation of susceptible varieties of chickpea (Waqas et al., 2016). Similarly. among innovative techniques for crop management, allelopathy offers a unique and eco-friendly approach for weed management and growth regulation (Arora et al., 2015). Allelopathy involves the synthesis and release of bioactive chemical compounds that affect the growth and development of neighboring plants (Khan et al., 2014; Safdar et al., 2014). Putnam (1988) described six classes of allelochemicals: benzoxazinones, alkaloids, cyanogenic compounds, cinnamic acid derivatives, flavonoids and ethylene from different plants. These allelochemicals have the capability of damaging the normal growth of weeds by affecting their metabolic pathways (Weston and Duke, 2003). Nevertheless, it has been reported that foliar application of allelochemicals poses a significant impact on improving physiological processes and crop yield (Shehzad et al., 2016; Ashraf et al., 2017). Considering the importance of chickpea crops and weed-induced yield losses, the present study was designed to investigate the feasibility of using herbicides and plant extracts for control of weeds in chickpea. Study site and design Field studies were performed for three years at the Agricultural Research Institute, Dera Ismail Khan, Khyber Pakhtunkhwa-Pakistan. The mean maximum and minimum temperatures of the study are were 31.22 and 17.53 oC in 2011; 31.03 and 16.44 oC in 2012 and 31.49 and 16.93 oC in 2013, respectively. Likewise, average rainfall was 483 mm in 2011; it was reduced to 450 mm in 2012 and, in 2013, the recorded rainfall was 327.40 mm (Khyber Pakhtunkhwa, 2014). The experiments consisted of ten treatments replicated three times using a Randomized Completel Block design (RCBD). Data recording Weed density was recorded by randomly throwing a 33 cm x 33 cm quadrate three times in each plot. Weeds inside the quadrate were counted and identified. The mean was calculated and was then converted into density m-2. Fresh weed biomass was calculated by harvesting all weeds inside the quadrate and weighing them in g m-2. Similarly, for measuring various plant parameters such as plant height and number of branches per plant, ten representative plants were randomly chosen and tagged and their length was measured from base to tip in centimeters; subsequently, the means were computed. Biological yield and seed yield were measured in kg and then converted into kg ha-1. Statistical Analysis The data recorded for each trait individually underwent the ANOVA technique by using the computer software MSTATC. The parameters significant (P≤0.05) in ANOVA were subsequently subjected to the Least Significant Difference (LSD) test for comparison (Steel and Torrie, 1980). Treatments The size of each plot was 5 x 1.5 m2 with five rows. Row to row distance was maintained at 30 cm. The crop was raised on the residual moisture of a previously transplanted rice crop. The treatments are detailed in Table 1. Pre-emergence herbicides were sprayed with a knapsack backpack sprayer at recommended rates immediately after sowing whereas post-emergence treatments were foliarly applied after complete emergence (8 days after sowing). For herbicide application, first of all calibration was performed to find out the exact volume of water required for each plot. For comparison among treatments, hand weeding was kept as positive control while weedy check was kept as a negative control treatment. The chickpea cultivar Karak-III was used during the current course of experimentation. Table 1 - Detail of treatments applied in the experiment Treatment Time of application Rate Stomp 330 E (full dose) Pre-emergence 2.5 L ha-1 Stomp 330 E (half dose) Pre-emergence 1.25 L ha-1 Dual Gold 960 EC (full dose) Pre-emergence 1 L ha-1 Dual Gold 960 EC (half dose) Pre-emergence 0.5 L ha-1 Isoproturon 500 EW (full dose) Post emergence 3 kg ha-1 Isoproturon 500 EW (half dose) Post emergence 1.5 kg ha-1 Parthenium extract Post emergence 200 g L-1 Parthenium extract Post emergence 100 g L-1 Hand Weeding - - Weedy Check - - Table 1 - Detail of treatments applied in the experiment Planta Daninha 2018; v36:e018176072 roaches for weed suppression in chickpea (Cicer arietinum) under residual moisture after rice crop Crop husbandry Selection of the experimental site was based on the previous history of higher weed infestation. The field was ploughed twice, after harvesting of rice when the soil was suitable for ploughing. It was followed by planking by tractor mounted implements. Chickpea was planted at a seed rate of 60 kg ha-1 with a single row hand drill. No artificial irrigation was applied to the crop during its growth period. The soil of experimental site was silty loam in texture. The pH value of saturated soil paste was 7.8 and total soluble salts were 0.91 dS m-1. Soil was low in organic matter (0.78%), total nitrogen (0.08%), available phosphorus (8.2 ppm) and potassium (185 ppm). During seed bed preparation, a uniform basal dose of nitrogen and phosphorus at the rate of 40 kg ha-1 was applied. There was no disease or insect attack in all trials throughout the course of experimentation. At physiological maturity, the crop was manually harvested and all yield parameters were recorded. Extract preparation For extract preparation, mature Parthenium hysterophorus plants were collected and kept in oven for 48 hours at 65 oC for drying. The dried samples were then ground into powder, weighed on an electrical balance and soaked in distilled water for 24 hours at two different concentrations namely, 200 g L-1 and 100 g L-1 (w/v, dry basis). Afterwards, the mixture was filtered through muslin cloth and the obtained extracts were kept in bottles labelled with designated concentration. Weed density (m-2) The statistical analysis of the data revealed that weed density (m-2) was significantly affected by all the assigned treatments during the three-year studies (Figure 1). During the first year (2010-11), the lowest weed density (84 m-2) was found for Stomp 330E applied at full dose, followed by Dual Gold 960EC at full dose, while maximum weed density (171 m-2) was measured in weedy check plots. Similarly, during the second and third years (2011-12 and 2012-13), the same trend was found: minimum weed density (69.5 and 55 m-2) was recorded in experimental plots sprayed with the full dose of Stomp 330E. Dual Gold 960EC was ranked second in terms of weed control efficiency during the entire experimental duration. Treatment comparison showed that the full dose of all the tested herbicides resulted in maximum weed suppression. For plant extract, weed density was higher than full doses of herbicides but lower than the weedy check. However, the values of weed density for plant extract were statistically similar to the half doses of the applied herbicides (Figure 1). Planta Daninha 2018; v36:e018176072 Planta Daninha 2018; v36:e018176072 5 g pp pp p ( ) p Figure 1 - Effect of different weed control techniques on weed density (m-2) in chickpea. Figure 1 - Effect of different weed control techniques on weed density (m-2) in chickpea. There were differences in weed density among the study years which could be attributed to differential rainfall during three years of study. However, highly significant differences were found for all the weed control treatments. It was noted that pre-emergence herbicides showed a momentous effect towards weed suppression in comparison to post-emergence herbicides. Current results are in a great analogy with those found by Muhammad et al. (2011), who reported that pre-emergence herbicides, i.e. Stomp 330E and Dual Gold 960EC with application rate of 3.50 and 2.50 liter ha-1, control weeds up to 94.6 and 90%, respectively. Similarly, the potential of herbicides for weed suppression has also been stated by Patel et al. (2006). They found that application of herbicides significantly reduced weed density compared to weedy check. Low weed population in herbicide-treated plots was due to long persistence of chemicals in soil, which inhibited weed seed germination (Marwat et al., 2003). Furthermore, recent studies have been conducted in which maximum weed control and high yield were achieved with integration of hand weeding and pre-emergence herbicide application. Weed density (m-2) For example, the study of Rao et al. (2015) showed that pre-emergence application of Stomp 330E followed by hand weeding at 50 days after sowing resulted in maximum weed control and better crop yield than hand weeding alone. However, such type of integration approach depends on the socioeconomic conditions of growers. This approach can be advantageous in areas with cheap labor cost or high value crops. Fresh weed biomass (g m-2) Figure 2 shows the data on fresh weed biomass as affected by various weed management techniques in chickpea crop. The results show slight variations in fresh biomass accumulation during the three-year period of the study. It was found that, as with weed density, the least biomass accumulation (less than 500 g m-2) was found with Stomp 330E (full dose). These values were statistically similar to the positive control treatment, i.e., hand weeding. Fresh weed biomass in hand weeding was 405, 362 and 320 g m-2 for the years 2010-11, 2011-12 and 2012-13, respectively. Similarly, biomass accumulation as a result of plant extract application was found to be statistically similar in comparison to all other pre and post-emergence herbicides. This showed that the plant extract is capable of suppressing weed germination and growth. Ashraf et al. (2017) stated that plants have various types of bioactive compounds that can be used as herbicides for weed management. Likewise, the research study of Shah et al. (2016) reported that use of plant allelochemicals is an economical and ecofriendly solution for weed management. The present results are in close proximity with the findings of Patel et al. (2006), who reported the lowest weed biomass accumulation with hand weeding and Stomp 330E. Long persistence of Planta Daninha 2018; v36:e018176072 KHAN, I.A. et al. Integrated approaches for weed suppression in chickpea (Cicer arietinum) under residual moisture after rice crop roaches for weed suppression in chickpea (Cicer arietinum) under residual moisture after rice crop 6 Figure 2 - Effect of different weed control techniques on fresh weed biomass (g m-2) in chickpea. Figure 2 - Effect of different weed control techniques on fresh weed biomass (g m-2) in chickpea. herbicides on the soil negatively affects weed growth and weed biomass. Likewise, Avola et al. (2008) and Kumar et al. (2015) also reported that the lowest weed density and weed biomass were found after use of manual weeding and pre-emergence herbicides. The bare minimum biomass accumulation in hand weeding plots was due to poor growth of weeds as a result of regular weed removal whereas weedy check plots were left untreated throughout the growing season, which resulted in the highest weed density. Plant height (cm) and number of branches per plant Figure 3 - Effect of different weed control techniques on plant height (cm) in chickpea. Figure 4 - Effect of different weed control techniques on number of branches per plant in chickpea. Figure 4 - Effect of different weed control techniques on number of branches per plant in chickpea. Figure 4 - Effect of different weed control techniques on number of branches per plant in chickpea. Figure 4 - Effect of different weed control techniques on number of branch Plant height (cm) and number of branches per plant The data revealed that different treatments had a significant effect on plant height (Figure 3) and number of branches per plant (Figure 4). The analysis of the data showed that the plots treated with the full dose of Stomp 330E resulted in the highest plant height of 49.53, 53.09 and 56.66 cm and number of branches per plant during the duration of the study i.e. 2010-11, 2011- 12 and 2012-13, respectively. These values were found to be statistically similar to Dual Gold 960EC (Full dose), hand weeding and P. hysterophorus extract. However, the results of pre- emergence herbicides were statistically similar to those of the tested post-emergence herbicide for both crop growth parameters. Khan et al. (2012) also found a non-significant effect on growth under the application of pre and post-emergence herbicides. The overall results showed non-significant changes in chickpea plant height throughout the duration of the study while there was an increase in number of branches per plant in the third year of the research study. The higher plant height and the larger number of branches in herbicide-treated plots might be due to the fact that herbicides greatly reduced weed infestation, which really provides favorable growing conditions for better crop growth (Batish et al., 2007). These results are supported by the findings of Muhammad et al. (2011) and Emenky et al. (2010), who reported maximum plant height in plots where the crop was kept weed-free after use of herbicides and manual weeding. In addition to management techniques, the use of Karak-III cultivar also played a role behind the satisfactory results. It has been proved that the morphological characteristics of the cultivar Karak-III led to maximum branching, sufficient resource capturing capability, early maturity and weed tolerance capacity as compare to other chickpea cultivars (Waqas et al., 2016). Current investigations are in correspondence with the results of Gul et al. (2011), who found the best growth, maximum branches and higher seed yield in Karak-III as compared to other chickpea cultivars. Planta Daninha 2018; v36:e018176072 roaches for weed suppression in chickpea (Cicer arietinum) under residual moisture after rice crop Figure 3 - Effect of different weed control techniques on plant height (cm) in chickpea. Figure 3 - Effect of different weed control techniques on plant height (cm) in chickpea. Figure 3 - Effect of different weed control techniques on plant height (cm) in chickpea. Biological yield and seed yield (kg ha-1) The data in Figures 5 and 6 showed that different weed control measures had a remarkable effect on the biological yield and the seed yield of chickpea. The results showed that during the three years of the experiment, there was a slight change in biological yield of chickpea. However, through the entire study duration, maximum biological yield (4256.9, 3627.6 and 3208.3 kg ha-1) was recorded for Stomp 330E at full dose, followed by full dose of Dual Gold 960EC whereas the least biological yield was found in the weedy check (Figure 5). The biological yield for Stomp 330E was higher than the positive control (hand weeding), which showed that herbicides were more effective in terms of weed control and crop yield. Planta Daninha 2018; v36:e018176072 Planta Daninha 2018; v36:e018176072 Planta Daninha 2018; v36:e018176072 . Integrated approaches for weed suppression in chickpea (Cicer arietinum) under residual moisture after rice crop Figure 5 - Effect of different weed control techniques on biological yield (kg ha-1) of chickpea. Figure 6 - Effect of different weed control techniques on seed yield (kg ha-1) of chickpea. Similarly to biological yield seed yield was also significantly affected by various weed contr HAN, I.A. et al. Integrated approaches for weed suppression in chickpea (Cicer arietinum) under roaches for weed suppression in chickpea (Cicer arietinum) under residual moisture after rice crop 8 Integrated approaches for weed suppression in chickpea (Cicer arietinum) under residual moisture after rice crop Figure 5 - Effect of different weed control techniques on biological yield (kg ha-1) of chickpea. Figure 6 - Effect of different weed control techniques on seed yield (kg ha-1) of chickpea. Figure 5 - Effect of different weed control techniques on biological yield (kg ha-1) of chickpea. Figure 5 - Effect of different weed control techniques on biological yield (kg ha-1) of chickpea. Figure 6 - Effect of different weed control techniques on seed yield (kg ha-1) of chickpea. Figure 6 - Effect of different weed control techniques on seed yield (kg ha-1) of chickpea. Figure 6 - Effect of different weed control techniques on seed yield (kg ha-1) of chickpea. Figure 6 - Effect of different weed control techniques on seed yield (kg ha-1) of chickpea. Similarly to biological yield, seed yield was also significantly affected by various weed control strategies. However, there was a reduction in seed yield during the second and third years of experimentation (Figure 6). The probable reason behind this reduction was lower precipitation in the reported year (2012-13). During 2010-11, the highest seed yield (1522.2 kg ha-1) was recorded for Stomp 330E applied at full dose followed by Dual gold 960EC, Isoproturon 500EW and P. hysterophorus extract. Seed yield during 2011-12 was reduced to 1257.44 kg ha-1 while in 2012-13 it reached only 1008.7 kg ha-1, and there was a similar trend for seed yield for all the applied treatments (Figure 6). The highest biological yield and seed yield in Stomp 330E were due to low weed crop competition (low weed density) and optimal use of resources (nutrients, solar radiations, water and space) (Tewari and Tiwari, 2004; Daur et al., 2008). Findings of the current study agree with those of Chaudhary et al. (2011), Khan et al. (2012) and Planta Daninha 2018; v36:e018176072 Planta Daninha 2018; v36:e018176072 9 KHAN, I.A. et al. Integrated approaches for weed suppression in chickpea (Cicer arietinum) un Integrated approaches for weed suppression in chickpea (Cicer arietinum) under residual moistu roaches for weed suppression in chickpea (Cicer arietinum) under residual moisture after rice crop Chandrakar et al. (2015), who reported the highest biological yield and seed yield under the application of Stomp 330E and hand weeding. Chandrakar et al. (2015), who reported the highest biological yield and seed yield under the application of Stomp 330E and hand weeding. Moreover, the post-emergence application of plant extract and herbicides also affected the biological yield of chickpea. It was found that, in comparison to the full dose of Isoproturon 500EW, the highest biological yield and seed yield resulted in P. Integrated approaches for weed suppression in chickpea (Cicer arietinum) under residual moisture after rice crop Figure 5 - Effect of different weed control techniques on biological yield (kg ha-1) of chickpea. Figure 6 - Effect of different weed control techniques on seed yield (kg ha-1) of chickpea. hysterophorus (Figures 5 and 6). This showed that the plant extract of P. hysterophorus possesses the stimulatory capability of enhancing plant growth and development. This stimulatory effect, which results from foliar application of plant extracts, has been reported to improve crop growth and immunization against various biotic and abiotic stresses (Narwal, 2013). Foliar application of allelochemicals improves crop growth and yield by affecting various physiological processes, such as photosynthesis, cell division and elongation (Abbas et al., 2017). Moreover, plant extracts are harmless to grazing animals and beneficial insects (Jamil et al., 2009). They are more easily degradable than synthetic agrochemicals because of their short half-life with no toxic ring structures and low halogen substitution (Kordali et al., 2009; Jabran et al., 2010). Based on the findings of this 3 year research study, it is concluded that Stomp 330E proved most superior in controlling weeds and, consequently, achieving better crop growth and yield. However, the non-judicious use of synthetic herbicides for crop management can cause soil and water pollution. Thus, the herbicidal potential of different allelopathic plants should be utilized as observed in this study, in which the plant extract of P. hysterophorus showed momentous effects towards weed suppression and crop growth along with its additional benefit of environmental sustainability. REFERENCES Abbas G, Ahmed A, Amer M, Abbas Z, Rehman M, Hussain A et al. Impact of pre-emergence herbicides for the control of weeds in chickpea (Cicer arietinum L.) under hot arid climate. 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https://openalex.org/W4226278500	https://www.researchsquare.com/article/rs-1487276/latest.pdf	English	null	A comprehensive study of Iraqi marshlands, with the emphasis on the development possibilities	Research Square (Research Square)	2,022	cc-by	8,859	A comprehensive study of Iraqi marshlands, with the emphasis on the development possibilities Mina A. Alani Zhejiang University Jie Shen (  jshen@zju.edu.cn ) Zhejiang University Auwalu Faisal Koko Zhejiang University Mina A. Alani Zhejiang University Jie Shen (  jshen@zju.edu.cn ) Zhejiang University Auwalu Faisal Koko Zhejiang University Abstract Iraqi Marshes is one of the most important historical heritage in Iraq and in the middle east, it has a very important geographical location and unique architecture and landscape design. For many years, Iraqi marshlands was always appealing to the scholars from all around the word. In 2016, marshlands had been inscribed on the “World Heritage List” during the 40th session if the World Heritage Committee. Marshland’s Inhabits usually enjoy a very unique life style. This area have different kind of animal, birds, and plants. Back in the 90s, there was a destructive action that done by the former Iraqi government which involves drainage of the marshlands, that led the inhabitants to leave the area. After 2003 there was a refolding of the marshlands, however, despite the cultural and historical importance of the marshlands, until now this area is still neglected and not getting the enough attention for development, the paper focus on investigating and fully analyses these area using data collection from literature review for the possibilities of future development and emphasizing the importance of the environmental tourism that can play a major role in Iraq’s national income. The result of the research has shown a poor managements to the marshland, there is no any governmental plans to develop the marshlands. Thus we should shed a light on this area and put its development into consideration. Research Article Keywords: Iraq, Mesopotamia, Wetland restoration, Iraqi marshlands, Marsh Arabs, Tourism Posted Date: April 28th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1487276/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/22 Page 1/22 2.1. Location and Climate The Tigris and the Euphrates rivers that originate in the Taurus Mountain Range of Turkey. Fed by mountain snowmelt, those rivers flow into Iraq. The marshes formed at the concourse of Tigris and Euphrates rivers. [8]. 2. Background Studies This chapter mainly focused on the history and the origin of the marshland, in other words how this area created back in the history? This chapter mainly focused on the history and the origin of the marshland, in other words how this area created back in the history? The history of the marshlands dated back to the Sumerians who established one of the oldest civilizations of Ur. The Sumerians had their own language, and is considered one of the oldest languages on earth near, as presented in (Fig. 2.b). The ancient Iraqis built their civilization on the edge of rivers in the middle of water bodies. The Sumerians discovered the secrets of water and swamps, and were also pioneers in adapting to their environment. They built their temples and homes on reed swamp basins and built the first boats. They were created by the flooding of the Tigris and Euphrates rivers, which renewed the fertility of the land, nourished the marshes annually, and ensured the continuation of the life cycle, as presented in (Fig. 2.a). Their location on the flat low plains enabled sewage and surrounding canals to fill them to a depth of 10 feet. The area is unique because there are many islands above the water level, where rice farmers and buffalo breeders live. Palm trees line the shores of the marshes, extending over large areas along the banks of rivers and streams. The current inhabitants of the marshes (Marsh Arabs) are descendants of the Sumerians and are the living link between today's Iraqis and the ancient Sumerians. [6] Furthermore, there is a similarity between the traditional styles of the ancient Sumerians. The area has been inhabited since the dawn of civilization by the Sumerians around 6000 BC. A large number of carvings and figures have been documented since the Sumerian and Babylonian times. [7] 2.1.1 Geography The marshlands in southern Iraq are formed in a triangle shape, whose head is at the Amaraa city, and its base extends between two cities, Basra and Nasiriyah, and extends north to the city of Kut. As it mentioned above, the Tigris and Euphrates rivers are the main sources that feed the marshes, as presented in (Fig. 3). The water level of the marshes ranges from one to two meters above sea level and about 22 meters above sea level near the border with Iran.[9] 1. Introduction The Iraqi Marshlands are among the world's largest wetlands, covering an area of 15,000 to 20,000 square kilometers in the lower part of the Mesopotamian basin where the Tigris and Euphrates flow. It forms a large triangular region bounded by three major southern cities: Nasiriyah to the west, Amarah to the northeast, and Basra to the south. Marshlands contain distinct physical elements that give them a unique identity that can be clearly identified through the physical pattern. Due to the historical and cultural importance of this area, the marshlands has been listed it as United Nations Educational, Scientific and Cultural Organization (UNESCO) heritage site. The location of the marshlands is privileged because of its environmental factors, thus, the dwellings were built on artificial floating islands built with reeds and mud. For flood protection, more layers are added each year to strengthen the platform foundation, as presented in (Fig. 1.a). The marshes are home to of many animals, plants, and different species of birds and fish. The marsh people of who can referred as “the marsh Arabs”, have practiced sustainable traditional resource management for thousands of years, and developed an imaginative lifestyle that closely relates them to their wetland landscape. Traditionally, women played an active role both inside and outside the home, collecting reeds, producing handicrafts, helping to care for water buffaloes, working in the fields, and selling in the market. There was extensive destruction during the Iran- Iraq war. The destruction continued through the 1990s when the former Iraqi government systematically drained more than 90% of the marshes in a military action intended to crush its population. After 2003, small steps were taken to revive the marshes. But the area still needs more and more attention instead of being neglected and it is another disaster that has negatively affected this area, as presented in (Fig. 1.b). In short this area is like a hidden gem which needs to be discovered and taken care of and it deserves to be treated in the way which has highlighted the cultural and historical significance of this area.[1] [2] [3]. Page 2/22 Page 2/22 2.1.2 Climate Iraq's climate is continental and subtropical, with hot, dry summers and mild, wet winters. The rainy days during the year range from 40 to 60 days and the probability of heavy rain (1–10 mm) is only 25–27%, The average annual precipitation ranges from 42 to 185 mm, as presented in (Figs. 5 and 7). Within the marsh area, the air temperature within the area reaches over 50°C during the summer, and rarely drops Page 3/22 below zero during + the winter. It can be seen that the average annual temperature varies between 22.2 and 27.2°C. The absolute maximum and minimum are 49 and − 2.2–2.8°C, respectively, as presented in (Fig. 4). The total annual radiation is 525 mWh with its highest levels in June and July and lowest levels in December and January. Eight to nine hours of sunshine. [7]. below zero during + the winter. It can be seen that the average annual temperature varies between 22.2 and 27.2°C. The absolute maximum and minimum are 49 and − 2.2–2.8°C, respectively, as presented in (Fig. 4). The total annual radiation is 525 mWh with its highest levels in June and July and lowest levels in December and January. Eight to nine hours of sunshine. [7]. 3. The Marsh Arabs The Arabs of the Marshlands are allegedly a descendant of the Sumerians, and their way of life is considered one of the oldest living cultures, celebrated by Thesiger and other travelers for nearly half a century. Marsh Arab’s livelihood was adjusted to the flood environment and consisted of a mixture of fishing and rice farming mixed with the raising of buffalo cattle. Reed marshes was used to build houses and for centuries this self-sufficient way of life saw few opportunities. The Marsh Arabs have specific preferences for the areas they want to resettle, these preferences should be among the criteria for selecting the areas of the Marshes to be retaken; However, these areas must also meet the environmental conditions conducive to the re-establishment of wetland functions. [10] 3.1 Marsh Arab Culture, life style and social life Marsh Arabs enjoy a unique lifestyle adapted to their geographical, social and economic conditions. They keep buffalo, their main source of livelihood and move with them within the vicinity of their tribe, as presented in (Fig. 8). Since marshes are spaces dominated by water, with marshes, rivers and ditches, the inhabitants built their homes from reeds in the middle of the water on artificial islands. Each house has a small yard, usually made of reeds and mats. Building materials are plentiful, cheap and available to everyone. Besides traditional building methods and the age-old custom of working together to build their own homes, there is not a single family who does not have a dwelling of their own. These homes differ according to their function. Beside residence function there is the guest house (Al-Madhif), as presented in (Fig. 7.a). which is the focal point of the village. It is the home where guests are received and meetings are held. It was built with great care because it is like the parliament of the region, a place for meetings. Water is not an obstacle to mobility and can easily reach the cities on the edge of the marshes. They use different types of boats and even kids can manage the smallest of them, as presented in (Fig. 7.b and Fig. 9). [10],[11]. The main economic activities of the Marsh Arab are fishing, water buffalo breeding, collecting, processing reeds for buffalo fodder and building houses, as presented in (Fig. 11.a and Fig. 12). Marsh Arabs also Creating and selling handicrafts such as reed mats, baskets, fans and pigeon houses to earn a living, as presented in (Fig. 11.b). The ancient Mesopotamians used a variety of plants for a range of medicinal, culinary, and magical uses. In modern Mesopotamia, the Marsh Arabs also used plants from the marshes for medicinal and healing purposes. 3.2 Economical activities 3.2 Economical activities Page 4/22 The Marsh men practice many activities that enrich the economy of the country which will be mentioned bellow : The Marsh men practice many activities that enrich the economy of the country which will be mentioned bellow : The Marsh men practice many activities that enrich the economy of the country which will be mentioned bellow : ● Fishing: For most of the marshland area, fishing is no longer an important activity in which marsh dwellers can take part. Fish numbers in these areas have decreased dramatically due to environmental degradation, overfishing and invasive species, as presented in (Fig. 13.a and b). [17]. ● Agriculture: Since 2003, when Iraqis began to re-flood the marshes, many Marsh Arabs have started small-scale agricultural production for the first time when the land became available for agriculture. Most farming consists of small-scale farming or gardening on land adjacent to families' homes. In 2009, 22% of the former bog land was used to grow wheat or vegetables on land that was submerged, and is now dry. ● Agriculture: Since 2003, when Iraqis began to re-flood the marshes, many Marsh Arabs have started small-scale agricultural production for the first time when the land became available for agriculture. Most farming consists of small-scale farming or gardening on land adjacent to families' homes. In 2009, 22% of the former bog land was used to grow wheat or vegetables on land that was submerged, and is now dry. ● Market: Women works in the market, they sells dairy products, fish and buffalo in the main markets of urban cities, while some sell fish door-to-door in the suburbs. They also sell reed baskets, hand fans, and mats; The availability of reeds depends on the availability of water. Before the 1990s, young women were selling goods in the market; Now, most of the women who are selling in the market are older women, as presented in (Table 1) bellow: Table 1 Types of Professions of the Marsh Arab (males and females) in 2018. [16] Profession name Male % Female % Buffalo herders 15% 30% Hunting Birds and Fishes 35% 20% Reeds collectors 10% 25% Boats Makers 5% N/A% Farmers 30% 25% Tourist Guides 5% N/A% Table 1 ypes of Professions of the Marsh Arab (males and females) in 2018. [16] 4. Marshlands Transformation From Past To Present Wet lands in Southern Iraq has been exposed to important changes and this changes is due to two basic factors : The natural and political factors. The natural factors, closely related to the changes in the world’s climate, the politics factors related to some destructive action that has been done by the previous. Iraqi government and has significantly affected the marshlands in a negative way which we will get to this in details : 4.1 Marshlands Before the drainage The marshlands were greatly influenced by the hydraulic structure built during the 20th century, the construction of major hydraulic works played an essential role in flood control. Al-Hindiya dam were inaugurated in 1913 on the Euphrates River, while the Kut dam was established in 1938, which directed more water flow towards the Gharraf River to provide irrigation for field farming, thus reducing the amount of water flowing from the Tigris River to Iraq. In the 1970s, the Syrians built dams on the Euphrates, followed by Turkey in the 1990s, as presented in (Fig. 14.a and b). In addition, several oil fields were discovered in marshlands area. Drilling for oil meant draining part of the marshes. More than 1,000 square kilometers represent the major oil fields in southern Iraq, and that number is properly increasing. [19] 3.3 Collective Results Page 5/22 Marksmen have a unique life style due to their circumstances that they’ve lived in, life in mash is harsh and it required an extra effort to live with this environment, Thus marshmen adopt this unique life style that somehow self sufficient that helps them to adopt with the environment, this involves building their houses from marsh reeds, use hunting and fishing for food, buffalo for transportation and milk production from cow and goats, women usually work inside home and some work outside in collecting Page 5/22 Page 5/22 reeds and other chores but not as much as men due to culture and religion restrictions, marshmen could take an active role in supporting tourism in this area by educate them to work as a tour guide. 4.2 The drainage of the marshlands In early 1990s, the former Iraqi government drained the southern Iraqi marshes, by separating and blocking Tigris tributaries flowing into some areas in the marshes, as presented in (Fig. 18) And another dam was built to divide the marshes into smaller areas. This operation made a dire change to the historical and natural course of Euphrates with the purpose of eliminating of the river‘s feed to some marshes. Some parts in the marsh was completely disappeared between 1992 and 1994 and they transformed it into a desert, disturbing its ecological composition and leaving detrimental vestiges that pose serious challenges to its survival, as presented in (Fig. 15a and b, Fig. 16, Fig. 17). brought tragic effect on the marsh dwellers, animals and plants. Several initiatives had been taken by the Iraqi Government and others to restore this area. The ecology experts described the draining of the marshes as not only an ecological disaster, but also a loss of cultural life. In fact, the draining has caused the disappearance of the cradle of the Sumerian civilization and the end of a unique way of life that has lasted some 5,000 years, as presented in (Table 2). [22]. Page 6/22 Table 2 The changes in some traits of the marshes before and after the drying, source: [22] Trait Studied Before drying the marshes in 1991 After drying the marshes in 1991 Number of the Arab Marsh 300,000-500,000 (1,858) 75,000–85,000 (59) Water Discharge (m3/s) (60) Hwezeh 145 81 Hammar 231 21 The Central Marshes 253 0.97 Total Wetlands km2 (4) 8,926 1,296.9 Table 2 4.4 The current situation of the Marshlands Iraqi government with the help of other countries like United States, Canada, Japan, Denmark, Italy and some United Nations organizations, such as the United Nations Environment Program and the United Nations Development Program, tried to restore the marshland. Due post drainage changes that occurs in land use, changes and climatic changes as well as environmental fragmentation. Accordingly, many species as well as marsh dwellers were affected. Since 2012, the water has not been fresh, agricultural was in a bad situation due to the bad environment and sewage is not efficient; furthermore, this affects fishing stocks as it has been significantly decreased, as presented in (Fig. 20). 4.3 The Re-flooding of the Marshlands Immediately after the fall former Iraqi regime in April 2003, local farmers and water ministries began blowing up dams and the water start to flow. By February 2004, approximately 20% of the 15,000 square kilometers of the former marsh that had dried up had been re-flooded. It also be noted that analysis of the vegetation re-growth trend from January 2003 until September 2005 indicates that the vegetation cover is expanding at a rate of 800 square kilometers per year, as presented in (Table 3). Wetland restoration has been hailed as a rare success story in a country beset by conflict. But still, marshes today make up about 14 percent of what they were in the 1970s. Euphrates River, which prepared a plan to restore the marshes takes five years, but this requires large amounts of water, and this needs to increase the share of water entering the rivers, as presented in (Fig. 19), which prompts the necessity of concluding it and agreements with neighboring countries. [24] Page 7/22 Table 3 Marshes that Re-flooded after 2003, Source: [21] Marsh Name Total Area (Km2) The area that has been re-flooded (Km2) Marsh location Central Marshes 3000 600 West Tigris- Left Euphrates Hammar Marsh 3000 900 West Tigris- Right Euphrates Hwizah Marsh 2350 1850 East Tigris Total 8350 3350 40% Challenges that faces the restoration of the marshlands There are many difficulties that stand in the way of the completion of projects to restore the ecosystem of the marshes to its previous era, resulting from: - There are many difficulties that stand in the way of the completion of projects to restore the ecosystem of the marshes to its previous era, resulting from: - ● First, the current deteriorating security situation. ● First, the current deteriorating security situation. ● Secondly, the shortage of specialized human cadres to implement plans and programs related to the revitalization of the marshes. ● Third, there is a need for large caital to complete this project ● Fourth, the need for laws to regulate fishing in bodies of water and lakes, and law enforcement cadres, and to create penalties against those who abuse the environment by using pesticides. ● Fourth, the need for laws to regulate fishing in bodies of water and lakes, and law enforcement cadres, and to create penalties against those who abuse the environment by using pesticides. 5. Marshland Importance Of Iraqi National Income. The marshes have many values, including rich flora and fauna, cattle grazing fields, fish and other breeding grounds for wild animals. Marshes are important ecosystems, which influence and are also affected by many natural forces and human activities. The main recommendation of this study is vital for the importance of the restoration of the marshes, both in terms of their ability to produce and sustain life in these wetlands, as well as for their ability to convert human pollution from upper land towns and cities to less toxic substances due to retention, dilution, precipitation and weathering factors usually occurring in Marsh habitats. [25]. The importance of the Marshlands can be summarized as follows: The importance of the Marshlands can be summarized as follows: ● Storage and preservation of flowing water from the Tigris and Euphrates rivers and estuaries Tides. Page 8/22 ● Removing harmful pollutants from the water, by getting rid of minerals and materials Organic, where soil microbes break down organic waste to reduce Its harm. Page 8/22 ● Removing harmful pollutants from the water, by getting rid of minerals and materials Organic, where soil microbes break down organic waste to reduce Its harm. ● An environment with a unique biodiversity of fish, birds, wanders, and benthic fauna And others). ● A large warehouse for the raw materials of cane, papyrus and tree stumps Palm, which can be adopted in the paper industries, and in the industry Housing and in the manufacture of organic fertilizers and others. ● It contains energy sources such as oil, gas, reed, papyrus and dung Animals, solar energy and more. ● It contains energy sources such as oil, gas, reed, papyrus and dung Animals, solar energy and more. ● Affect and contribute to the reduction of desert encroachment and dust storms Soothing the atmosphere. ● Affect and contribute to the reduction of desert encroachment and dust storms Soothing the atmosphere. ● Globally recognized natural beauty, Iraqi cultural heritage and archaeological sites Timeless observatories for birds and wildlife and fisheries can be found Attracts tourists from different regions of the world. This paper will specifically focused on the importance of tourism on national income which will explain next. This paper will specifically focused on the importance of tourism on national income which will explain next. 5.1 Tourism The Marshlands region is one of the important tourist areas in Iraq, due it has a unique ecosystem that has lasted for thousands of years. This area is one of the richest areas in wild life, a home to a civilization, in addition to the potential economic and diverse wealthy abundant natural, making it a suitable area for tourism investment and the establishment of tourist resorts that attract many tourists. The marshes of Iraq have natural environmental features suitable for a tourist activity. They combine the natural beauty of the marshes, having large water bodies that are mostly green. A Tourist can enjoy a warm climate during the cold seasons, so it can be a tourist attraction in these seasons, and water bodies that works to alleviate the heat in the summer. Furthermore, its geographical location makes it easy to reach it through various transportation, as there are land roads that connect the marshes with the governorates of Iraq. So all of the above mentioned factors will make the marshland a great option for tourist attraction and for the national income, as presented in (Table 4) bellow: [18] Page 9/22 Table 4 The contribution of tourism revenues to national income, exports and local production for the years from (2000–2015), source: [18] Year Export revenue % Income revenue % Total domestic product revenue 2000 90.0 0.00008 0.06 2001 0.13 0.00025 0.24 2002 0.50 0.00043 0.42 2003 0.23 0.00025 0.39 2004 0.16 0.00012 0.12 2005 0.77 0.00057 0.65 2006 0.61 0.00037 0.39 2007 1.49 0.00079 0.87 2008 1.47 0.75 N/A 2009 3.01 1.495 1.37 2010 3.41 1.49 1.34 2011 2.07 1.04 0.91 2012 1.88 0.49 0.83 2013 0.24 0.11 0.09 2014 0.28 0.123 N/A 2015 0.35 0.032 0.21 Table 4 Table 4 The contribution of tourism revenues to national income, exports and local production for the years from (2000–2015), source: [18] 5.2 Collective Results As far as Iraq’s national income is concerned, the marshland of Iraq can be refer to as one of the richest area of Iraq due to many reason starting from the Animal wealth, plants and oil stock, all of those resources makes this area wealthy and self sufficient, However, the situation of this area and the residents are not as good as it seems to be, the suffer poverty and bad situation and sort of negligence, Thus this research focus on reserve and develop this area, redirect the attention to it and study the possibilities of using this area for Tourism which can also add many value and income to the country. 6. Marshland’s Development Possibilities Page 10/22 Iraqi marshland is of high importance due to its historical, cultural and geographical importance as it mentioned above. The challenges here is whether this area should be developed or not and if the answer is yes then how to develop it and which approaches to take, those two questions need to be studied and analyzed in order to be answered probably. 6.1 The importance of considering the development The uniqueness of the marshland area is priceless. The beautiful scenery and the charming nature, animal wealth and the marsh men, the unique life style they exhibit and the strategic geographical location are what make it special and attractive to many tourists and international organizations. However, the current situation is not good due to the lack of services, animal care, clean water and a proper accommodation for inhabitants or for the tourist to come as there isn’t any facilities for accepting tourist. Furthermore, the place is not safe enough to live and it definitely will decrease the tourist flow due to this issue. Those are basically the main factors why we should develop this area. If these factors didn’t be modified then this area will be an abandoned place and will gradually be forgotten, If we develop it based on the factors mentioned, that will have a positive effect for the image of Iraq. Furthermore, it will support the national income if it will be considered as a Tourist attraction, as presented in (Tables 5 and 6) bellow: The importance of marshlands development, Source: Author The importance of marshlands development, Source: Author Page 11/22 4. Animal Care • The marshland dwelling, even though it has a unique design that gives a unique identity to this area but it lake the essential services and infrastructure to cope with the severity of the hot, dry weather. • The marshlands lake many of the basic services like a constant flow of electricity, clean water and regiod infrastructure including sanitary services which makes it hard to live, even if the inhabitant could cope with this situation due to the long time they’ve baring this but its a different story for a tourist. • The lack of safety and security will make it hard for tourists to come and explore this area without risking their lives. However, some tourists go even though it’s a high-risk area, but those few courageous tourists seem willing to take the risk. Page 12/22 Page 12/22 Page 12/22 6.2 Collective Results Based on the previous data collection that has been mentioned in this research, it’s essential to study, investigate and analyze this data, extract the requirements and find out the problems and the challenges in order to take steps forward towards development. These results can be summarized as the followings. ● Iraqi marshland is of a high importance area, the results shows that it has a long history that dated back to the Sumerian civilization as it is shown in their clays. Although, there isn’t a specific theory of its emergence but this wetland is a heritage that should be reserved and developed and should be put into consideration by the government. This could take several approaches and we will discuss later in the research. ● Iraq and specially the southern part where the marshland located has a hot and dry weather in summer and relatively cool weather in winter. It’s important to consider that when it comes to developing this area, the hot weather is something uncomfortable for the residents and for the upcoming tourist who is curious to investigate this area. So that should be studies and focus on when reserve or redesign this area. We should create an atmosphere that can somehow fights the severity of the weather. ● Iraq and specially the southern part where the marshland located has a hot and dry weather in summer and relatively cool weather in winter. It’s important to consider that when it comes to developing this area, the hot weather is something uncomfortable for the residents and for the upcoming tourist who is curious to investigate this area. So that should be studies and focus on when reserve or redesign this area. We should create an atmosphere that can somehow fights the severity of the weather. Page 13/22 ● Marshmen have a unique life style due to their circumstances that they’ve lived in. Life in mash is harsh and it required an extra effort to live with this environment. Thus, marshmen adopt this unique life style that somehow self-sufficient that helps them to adopt with the environment. This involves building Page 13/22 ● Marshmen have a unique life style due to their circumstances that they’ve lived in. Life in mash is harsh and it required an extra effort to live with this environment. 1. Services 1. Services 1. Services Table 6 Marshland’s development aspects, Source: Author 2. Accommodation 3. Safety and Security Table 6 Marshland’s development aspects, Source: Author 2. Accommodation 3. Safety and Securit 6.3 The requirements of the development To develop this area there are some requirements that should be put into consideration. These requirements have been extracted based one several aspects, including the nature of the place, the nature of people, the cultural style, settlement patterns, the climate and the rule and regulation of the country. These requirements can be summarized as the following. ● The first requirement to develop any area is to have a sense of responsibility and awareness by the ministries and the organizations that involved reserve and develop the important historical and cultural area and to have the awareness to highlight the importance and the value of the heritage of Iraq. This work is not merely depend on a certain individual but it’s a cooperative work that involves many parties, including Architects, urban planning and governmental organizations, in order to make the development happens. ● The first requirement to develop any area is to have a sense of responsibility and awareness by the ministries and the organizations that involved reserve and develop the important historical and cultural area and to have the awareness to highlight the importance and the value of the heritage of Iraq. This work is not merely depend on a certain individual but it’s a cooperative work that involves many parties, including Architects, urban planning and governmental organizations, in order to make the development happens. ● The second is to analyze the nature of the place including the area, the location and the roads that leads to it from the other regions of Iraq, also to analyze the row material that is found there, the different type of animals and plants, the quality of water and the spices that lives under the water, also to analyses the different heights of the area. This data will help for future development approaches. ● The second is to analyze the nature of the place including the area, the location and the roads that leads to it from the other regions of Iraq, also to analyze the row material that is found there, the different type of animals and plants, the quality of water and the spices that lives under the water, also to analyses the different heights of the area. This data will help for future development approaches. 6.2 Collective Results Thus, marshmen adopt this unique life style that somehow self-sufficient that helps them to adopt with the environment. This involves building Page 13/22 their houses from marsh reeds, hunting and fishing for food, buffalo for transportation and milk production from cow and goats. Women usually work inside home and some work outside in collecting reeds and other chores but not as much as men due to culture and religion restrictions. Marshmen could take an active role in supporting tourism in this area by educate them to work as a tour guide. ● The drainage of the marshland that happens back in the 90s had a very negative impact to this area as it transformed this area from a charming wetland to a desert, which effect the economic situation of the residents as well as their being, that force them to move to other places. It also affects the animal, birds and the plants. After 2003 there was a reflooding project that helps a lot in restoring the life there to some extent even though it never gets back to its glory back in the 70s. This paper focus on not only to get it back to the 70s situation but to seek the development opportunities and highlight the importance of this area instead of neglecting it. ● As far as Iraq’s national income is concerned, the marshland of Iraq can be considered one of the richest areas of Iraq due to many reasons starting from the Animal wealth, plants and oil stock. All of those resources make this area wealthy and self-sufficient. However, the situation of this area and the residents are not as good as it seems to be the suffer poverty and bad situation and sort of negligence. Thus, this research focus on reserve and develop this area, redirect the attention to it and study the possibilities of using this area for Tourism which can also add many values and income to the country. 6.3 The requirements of the development ● The fifth is to study the settlements patterns, in terms of the material used, and the technique they used to build it, if there are varieties of settlement types and what’s the difference between them, and is it structurally rigid, and whether we can renovate it or redesign it and are there hotels or motels that can accept tourist or no, and are there services that feeds this area like restaurants, coffee shops, museum, shops, etc. ● The sixth is to carefully study the animals living there and see which one should be kept from hunting and which one is possible to hunt, furthermore to find out how we can maintain a better well-being for the animal and plant that located in this area, and to which extent this animal add value to this area. This will play a major role for the economics cherishment of the place if has been probably taking care of the valued. ● The sixth is to carefully study the animals living there and see which one should be kept from hunting and which one is possible to hunt, furthermore to find out how we can maintain a better well-being for the animal and plant that located in this area, and to which extent this animal add value to this area. This will play a major role for the economics cherishment of the place if has been probably taking care of the valued. ● And last but not least is to study the rules and regulations of the country, specially of the safety and security of this area and for the roads that leads to it from the major cities and airports, to analyze whether it is safe and secure to settle there or to invite tourist without having to risk their life for merely tourism purpose. This should be highly considered because no development is ever possible without maintaining strict safely rules. 6.3 The requirements of the development ● The Third is to analyze the climate of this throughout the year, like temperature, wind, etc.., and how we can use this dry hot climate for our benefits. At the same time, how to ease the hardship that the inhabitants are baring due to the extreme climate. A fully analyzed climate situation is extremely essential for the future development. ● The Third is to analyze the climate of this throughout the year, like temperature, wind, etc.., and how we can use this dry hot climate for our benefits. At the same time, how to ease the hardship that the inhabitants are baring due to the extreme climate. A fully analyzed climate situation is extremely essential for the future development. Page 14/22 Page 14/22 Page 14/22 ● The Forth is to study the nature of the people, because in this area people are living a conservative lifestyle due to religion and traditions. Thus, this thing should be put into consideration as there are many factors to consider like the separation between man and women and the cultural influences. This will play a role in designing or redesign the settlements. ● The Forth is to study the nature of the people, because in this area people are living a conservative lifestyle due to religion and traditions. Thus, this thing should be put into consideration as there are many factors to consider like the separation between man and women and the cultural influences. This will play a role in designing or redesign the settlements. ● The fifth is to study the settlements patterns, in terms of the material used, and the technique they used to build it, if there are varieties of settlement types and what’s the difference between them, and is it structurally rigid, and whether we can renovate it or redesign it and are there hotels or motels that can accept tourist or no, and are there services that feeds this area like restaurants, coffee shops, museum, shops, etc. 6.4 Development approaches Based on the data that has been collected and mentioned previously, and the comprehensive analyze that has been made to this area based on several factors, the results have shown that taking an approach to develop this area is vital to maintain the glory of this area and to cherish this important heritage. The paper suggest that the development must be done in a way to keep the same spirit of the place and don’t mess or contradict the scenery and the overall atmosphere. There are two main goals for the development: ● First is to provide a better life for residents (the marsh people) in terms of providing better services, better lifestyle and more convenient settlements for them. This is essential for this area to be cherished because better service means better life quality and that will affect the wellbeing of the inhabitants. This can take several ways including services, animal care and the settlements. The details can be found in the (Table 7) below: Table 7 The approaches to provide a better lifestyle for marsh people, Source: Author The approaches to provide a better lifestyle for marsh people, Source: Author Page 15/22 Page 15/22 Page 15/22 ● The Second goal for development is to encourage tourism. Tourism is such an important cultural activity for any country. It has so many benefits from increasing the exposure of the country worldwide not to mention positive effect that will add up to Iraq national income and many. Since Iraq has many important historical sightseeing that would be so appealing for tourist and will add a great value to the country. Unfortunately, these heritages were not being highlighted. It’s neglected for many years including the marshlands that is our main focus in this research. That negligence is reflected the lack of awareness and responsibility by whoever involves in reviving and maintaining the cultural heritage of Iraq. It’s in a very poor condition even though it has a significant appealing to international organizations like UNESCO as we mentioned before. Thus, in this research we took the responsibility of shedding the light on this issue. In order to make this area suitable for tourist attraction, there several factors that has to be taken into consideration and will be explained in details in the following (Table 8) below: Marshland’s main requirements for becoming a tourist attraction, Source: Author Marshland’s main requirements for becoming a tourist attraction, Source: Author Page 16/22 7.2 Recommendations ● Activating the international agreements to ensure sufficient water inflows in Tigris and Euphrates and monitoring water quality and water pollution. ● focus on the environmental tourism aspect and how the marshlands can play a major role In Iraq Tourism ● Encourage people to participate and work as a Tour guide who can speak at least English. ● Establish a safe and secure environment to come and enjoy this place without putting their life at risk. ● Developing informed programs for the optimal use of water resources to rehabilitate the Iraqi marshes. ● Establish a safe and secure environment to come and enjoy this place without putting their life at risk. ● Developing informed programs for the optimal use of water resources to rehabilitate the Iraqi marshes. ● Conducting regular survey for the populations and species of wetland communities of fish, birds and plants. ● Conducting regular survey for the populations and species of wetland communities of fish, birds and plants. Funding: Zhejiang University List of abbreviations: Not applicable Availability of data and materials: All research data obtained during this study are included in this article. Raw data are available on request. 7. Conclusion And Recommendation 7.1 Conclusion The Marshlands in southern Iraq play an important role in maintaining regional and global biodiversity. It’s a rich area and has a lot of potential when it comes to the diversity living stocks living, plants and extended to the oil stock. This area has a unique atmosphere and the residents inhabits a unique life style. They depend on them self in terms of food production, house construction and the transportation within the marshes area. The development can take several aspects from building new houses for them using natural material and keep the spirit of the place also hunting control, maintain safety and security, encourage the food production, fix the pollution problem, and also developing it for Tourism that can also plays a major role for national income. This requires a sense of responsibility and efforts to achieve that goal, as this area has been listed as UNESCO world heritage site. We can see that this area is valued and recognized by international organization rather than Iraq its self. The main point that has been concluded Page 17/22 Page 17/22 from this research is that Iraqi marshlands are a hidden gem that has been damaged, abounded in every shape and form throughout the past years and up until now. The main purposes of this research is to highlight the importance of reserving and developing this area as it can give a positive effect to Iraq. 7.2 Recommendations from this research is that Iraqi marshlands are a hidden gem that has been damaged, abounded in every shape and form throughout the past years and up until now. The main purposes of this research is to highlight the importance of reserving and developing this area as it can give a positive effect to Iraq. 7.2 Recommendations Competing interests: The authors declare that they have no conflict of interests. Authors' Contributions: Page 18/22 Mina Al Ani: Corresponding Author: topic choice, research, analyse and findings. Shen Jie: Supervision: Helps with all the guidance and supervise the whole research process. Faisal Koko: Helps in organizing the research and giving valuable advices in publishing process. Page 18/22 Mina Al Ani: Corresponding Author: topic choice, research, analyse and findings. Shen Jie: Supervision: Helps with all the guidance and supervise the whole research process. Faisal Koko: Helps in organizing the research and giving valuable advices in publishing process. Page 18/22 Faisal Koko: Helps in organizing the research and giving valuable advices in publishing process. Acknowledgements: The Author thanks Shen Jie, Faisal koko, Zhejiang university, and school of civil engineering and architecture stuff. References 1. Bedair, H., et al. (2006). "Iraq’s southern marshes something special to be conserved; A Case Study." Marsh Bulletin. 1. Bedair, H., et al. (2006). "Iraq’s southern marshes something special to be conserved; A Case Study." Marsh Bulletin. 1. Bedair, H., et al. (2006). "Iraq’s southern marshes something special to be conserved; A Case Study." Marsh Bulletin. 2. Al-Ansari, Nadhir, Sven Knutsson, and Ammar Ali. "Restoring the Garden of Eden, Iraq." Journal of Earth Sciences and Geotechnical Engineering 2, no. 1 (2012): 53-88 3. Al-Mudaffar Fawzi, N., et al. (2016). "Effects of Mesopotamian Marsh (Iraq) desiccation on the cultural knowledge and livelihood of Marsh Arab women." Ecosystem Health and Sustainability. 3. Al-Mudaffar Fawzi, N., et al. (2016). "Effects of Mesopotamian Marsh (Iraq) desiccation on the cultural knowledge and livelihood of Marsh Arab women." Ecosystem Health and Sustainability. 4. UNEP, H. (2001). "The Mesopotamian Marshlands: Demise of an Ecosystem." Division of Early Warning and Assessment, United Nations Environment Program (UNEP) Nairobi, Kenya 46. 4. UNEP, H. (2001). "The Mesopotamian Marshlands: Demise of an Ecosystem." Division of Early Warning and Assessment, United Nations Environment Program (UNEP) Nairobi, Kenya 46. 5. Richardson, C. J. and N. A. Hussain (2006). "Restoring the Garden of Eden: an ecological assessment of the marshes of Iraq." BioScience 56(6). 5. Richardson, C. J. and N. A. Hussain (2006). "Restoring the Garden of Eden: an ecological assessment of the marshes of Iraq." BioScience 56(6). 6. Marshlands Iraq’s Marshlands ”Eden Again”. Report: Walid Abdul-Amir Alwan Photos: Muhanad Al- Assadi Islamic Tourism – Issue 18 – July-August / 2005 For more information, visit our website www.islamictourism.com 6. Marshlands Iraq’s Marshlands ”Eden Again”. Report: Walid Abdul-Amir Alwan Photos: Muhanad Al- Assadi Islamic Tourism – Issue 18 – July-August / 2005 For more information, visit our website www.islamictourism.com 7. Al-Ansari, N. and S. Knutsson (2011). Possibilities of restoring the Iraqi marshes known as the Garden of Eden. Water and Climate Change in the MENA-Region: Adaptation, Mitigation and Best Practices 28/04/2011-29/04/2011. 7. Al-Ansari, N. and S. Knutsson (2011). Possibilities of restoring the Iraqi marshes known as the Garden of Eden. Water and Climate Change in the MENA-Region: Adaptation, Mitigation and Best Practices 28/04/2011-29/04/2011. 8. Theories of the formation of the marshes (a geographic study), Iyad Abdullah Salman Al-Shammari, University of Maysan-Faculty of Basic Education-Department of Geography (Translated Arabic). 8. e01207. 17. UNAMI (United Nations Integrated Water Task Force for Iraq). 2011. White Paper. Iraqi Marshlands. Managing change in the Marshlands; Iraqi’s critical challenge. United Nations Integrated Water Task Force for Iraq. FAO, UNAMI, UNDP, UNEP, UNESCO Office for Iraq, UN Habitat, UNICEF, and World Health Organization, Basrah, Iraq. 18. Faris, N. S. (2019). "the reality of investment tourism in the marshes of southern Iraq and its impact on the employment of manpower." Economic Sciences 13(52). 18. Faris, N. S. (2019). "the reality of investment tourism in the marshes of southern Iraq and its impact on the employment of manpower." Economic Sciences 13(52). 18. Faris, N. S. (2019). "the reality of investment tourism in the marshes of southern Iraq and its impact on the employment of manpower." Economic Sciences 13(52). 19. Partow H., 2001. The Mesopotamian Marshlands: Demise of an Ecosystem. UNEP/DEWA, Nairobi. http://www.grid.unep.ch/product/publication/download/mesopotamia. 19. Partow H., 2001. The Mesopotamian Marshlands: Demise of an Ecosystem. UNEP/DEWA, Nairobi. http://www.grid.unep.ch/product/publication/download/mesopotamia. 20. The Marshes of Iraq Three studies in the environment, animals and tourism, Abed Ali, Hussien, Khalid, Al-Rafidain Center For Dialogue, Beirut/Lebanon 2019. 20. The Marshes of Iraq Three studies in the environment, animals and tourism, Abed Ali, Hussien, Khalid, Al-Rafidain Center For Dialogue, Beirut/Lebanon 2019. 21. Changing the areas and environment of the marshes of southern Iraq, Maher, Aber, Dunia, Al- Mustansiriya University, College of Education. 21. Changing the areas and environment of the marshes of southern Iraq, Maher, Aber, Dunia, Al- Mustansiriya University, College of Education. 22. Al-Zaidy, K. J. and G. Parisi (2018). "Re Extrapolation For The Iraq Marshes Which Falling Within The World Heritage List (A Literature Review)." Al-Qadisiyah Journal for Agriculture Sciences 8(2). 22. Al-Zaidy, K. J. and G. Parisi (2018). "Re Extrapolation For The Iraq Marshes Which Falling Within The World Heritage List (A Literature Review)." Al-Qadisiyah Journal for Agriculture Sciences 8(2). 23. EDEN IN IRAQ: The Wastewater Garden Project Ecological and Cultural Restoration in the Mesopotamian Marshes, project of : Nature Iraq (NGO), nstitute of Ecotechnics, nvironmental engineers Dr. Mark Nelson and Dr. Davide Tocchetto, and Jassim al-Asadi of Nature Iraq (NGO). 23. EDEN IN IRAQ: The Wastewater Garden Project Ecological and Cultural Restoration in the Mesopotamian Marshes, project of : Nature Iraq (NGO), nstitute of Ecotechnics, nvironmental engineers Dr. Mark Nelson and Dr. Davide Tocchetto, and Jassim al-Asadi of Nature Iraq (NGO). 24. e01207. [UNEP] United Nations Environment Program, 2005, UNEP Project to Help Manage and Restore the Iraqi Marshlands.(6 April 2006) http://marshlands.unep.or.jp/ ). 24. [UNEP] United Nations Environment Program, 2005, UNEP Project to Help Manage and Restore the Iraqi Marshlands.(6 April 2006) http://marshlands.unep.or.jp/ ). 25. Foster, K. P. (1998). "Gardens of Eden: Exotic flora and fauna in the ancient Near East." Transformations of Middle Eastern Natural Environments: Legacies and Lessons 25. Foster, K. P. (1998). "Gardens of Eden: Exotic flora and fauna in the ancient Near East." Transformations of Middle Eastern Natural Environments: Legacies and Lessons. 25. Foster, K. P. (1998). "Gardens of Eden: Exotic flora and fauna in the ancient Near East." Transformations of Middle Eastern Natural Environments: Legacies and Lessons. 26. Dr.Malik, Administrative Technical college (2012). "Economical Resources of South Marshes of Iraq and how to invest," Tikrit Journal for Science and economy, issue 8, P.26. 26. Dr.Malik, Administrative Technical college (2012). "Economical Resources of South Marshes of Iraq and how to invest," Tikrit Journal for Science and economy, issue 8, P.26. References Theories of the formation of the marshes (a geographic study), Iyad Abdullah Salman Al-Shammari, University of Maysan-Faculty of Basic Education-Department of Geography (Translated Arabic). 9. CRIM, 2007, Study the rehabilitation of Al Huweizah marsh ecological system, Ministry of Water Resources, Center of Restoration of Iraqi Marshes, V.1 to 7.. 9. CRIM, 2007, Study the rehabilitation of Al Huweizah marsh ecological system, Ministry of Water Resources, Center of Restoration of Iraqi Marshes, V.1 to 7.. 10. Thesiger, W. (1958). "Marsh dwellers of southern Iraq." National geographic 113(2). 10. Thesiger, W. (1958). "Marsh dwellers of southern Iraq." National geographic 113(2). 11. Thesiger, W. (1954). "The Ma'dan or Marsh Dwellers of Southern Iraq." Journal of the Royal Central Asian Society 41(1). 11. Thesiger, W. (1954). "The Ma'dan or Marsh Dwellers of Southern Iraq." Journal of the Royal Central Asian Society 41(1). 12. Iraq’s vast marshes, reborn after Saddam, are in peril again, are in peril again, Nabil al-Jurani and Susannah George, 2007. 12. Iraq’s vast marshes, reborn after Saddam, are in peril again, are in peril again, Nabil al-Jurani and Susannah George, 2007. 13. Thesiger, W., 1957, “Marsh Dwellers of Southern Iraq” in National Geographic, Vol. CXIII, No. 204-239. 14. http://m.dw.com 13. Thesiger, W., 1957, “Marsh Dwellers of Southern Iraq” in National Geographic, Vol. CXIII, No. 204-239. 14. http://m.dw.com 14. http://m.dw.com 15. The Wastewater Garden Project Ecological and Cultural Restoration in the Mesopotamian Marshes, NGO& Institute of Ecotechnics. 15. The Wastewater Garden Project Ecological and Cultural Restoration in the Mesopotamian Marshes, NGO& Institute of Ecotechnics. Page 19/22 16. Al-Mudaffar Fawzi, N., et al. (2016). "Effects of Mesopotamian Marsh (Iraq) desiccation on the cultural knowledge and livelihood of Marsh Arab women." Ecosystem Health and Sustainability 2(3): Page 19/22 Figure 5 Rainfall distribution area (mm) [7]. Figures Figure 2 Figure 2 Page 20/22 (a) The Mudheef (built by reeds )in southern Iraq is shown in Sumerian Civilization. Source : [4] (b) Mesopotamia buffalo and its connection to the Sumerian era. Source : Sargón de Akkad, el primer emperador de la Historia (historiaeweb.com) (a) The Mudheef (built by reeds )in southern Iraq is shown in Sumerian Civilization. Source : [4] (b) Mesopotamia buffalo and its connection to the Sumerian era. Source : Sargón de Akkad, el primer emperador de la Historia (historiaeweb.com) (a) The Mudheef (built by reeds )in southern Iraq is shown in Sumerian Civilization. Source : [4] (b) Mesopotamia buffalo and its connection to the Sumerian era. Source : Sargón de Akkad, el primer emperador de la Historia (historiaeweb.com) Figure 3 A close look to the major Iraqi Figure 4 Geographical distribution of mean annual temperature (°C) [7]. Figure 4 Geographical distribution of mean annual temperature (°C) [7]. Figure 8 Professional fishermen, or Barbara, fishing with seine net from a balam, or double-ended canoe [11]. Professional fishermen, or Barbara, fishing with seine net from a balam, or double-ended canoe [11]. Page 21/22 Figure 11 (a) An imaginary painting of the marsh Arab building the reed housed and boats, Source : [14]; (b) Marsh man holding marsh birds, Source [15] (a) An imaginary painting of the marsh Arab building the reed housed and boats, Source : [14]; (b) Marsh man holding marsh birds, Source [15] Figure 12 Marshmen fishing in the marshes. Source : [10] Page 21/22 Page 21/22 Figure 19 The area of the marshes after the re-flooding, source: [21] Figure 20 Water buffalo and ducks gather in an island paddock during the sunset in the Chabaish marsh in Nasiriyah, about 200 miles (320 kilometers) southeast of Baghdad, Iraq. Source: [12] Page 22/22
W2216994467.txt	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0138303&type=printable	en		The Effectiveness of Interventions for Non-Communicable Diseases in Humanitarian Crises: A Systematic Review	PloS one	2,015	cc-by	8,430	RESEARCH ARTICLE The Effectiveness of Interventions for NonCommunicable Diseases in Humanitarian Crises: A Systematic Review Alexander Ruby1, Abigail Knight2, Pablo Perel3, Karl Blanchet2, Bayard Roberts1* 1 ECOHOST–The Centre for Health and Social Change, London School of Hygiene and Tropical Medicine, London, United Kingdom, 2 Faculty of Public Health and Policy, London School of Hygiene and Tropical Medicine, London, United Kingdom, 3 Centre for Global Non Communicable Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom * Bayard.Roberts@lshtm.ac.uk Abstract Background OPEN ACCESS Citation: Ruby A, Knight A, Perel P, Blanchet K, Roberts B (2015) The Effectiveness of Interventions for Non-Communicable Diseases in Humanitarian Crises: A Systematic Review. PLoS ONE 10(9): e0138303. doi:10.1371/journal.pone.0138303 Editor: Tatsuo Shimosawa, The University of Tokyo, JAPAN Non-communicable diseases (NCDs) are of increasing concern in low- and middle-income countries (LMICs) affected humanitarian crises. Humanitarian agencies and governments are increasingly challenged with how to effectively tackle NCDs. Reviewing the evidence of interventions for NCDs in humanitarian crises can help guide future policies and research by identifying effective interventions and evidence gaps. The aim of this paper is to systematically review evidence on the effectiveness of interventions targeting NCDs during humanitarian crises in LMICs. Received: June 11, 2015 Accepted: August 28, 2015 Published: September 25, 2015 Copyright: © 2015 Ruby 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. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The work for this systematic review was funded by the Research for Health in Humanitarian Crises (R2HC) Programme. The R2HC programme is funded equally by the Wellcome Trust and DFID, and managed by the Enhancing Learning and Research for Humanitarian Assistance (ELRHA). Competing Interests: The authors have declared that no competing interests exist. Methods A systematic review methodology was followed using PRISMA standards. Studies were selected on NCD interventions with civilian populations affected by humanitarian crises in low- and middle-income countries. Five bibliographic databases and a range of grey literature sources were searched. Descriptive analysis was applied and a quality assessment conducted using the Newcastle-Ottawa Quality Assessment Scale for observational studies and the Cochrane Risk of Bias Tool for experimental studies. Results The search yielded 4919 references of which 8 studies met inclusion criteria. Seven of the 8 studies were observational, and one study was a non-blinded randomised-controlled trial. Diseases examined included hypertension, heart failure, diabetes mellitus, chronic kidney disease, thalassaemia, and arthritis. Study settings included locations in the Middle East, Eastern Europe, and South Asia. Interventions featuring disease-management protocols and/or cohort monitoring demonstrated the strongest evidence of effectiveness. No studies examined intervention costs. The quality of studies was limited, with a reliance on PLOS ONE \| DOI:10.1371/journal.pone.0138303 September 25, 2015 1 / 16 Interventions for NCDs in Humanitarian Crises observational study designs, limited use of control groups, biases associated with missing data and inadequate patient-follow-up, and confounding was poorly addressed. Conclusions The review highlights the extremely limited quantity and quality of evidence on this topic. Interventions that incorporate standardisation and facilitate patient follow-up appear beneficial. However, substantially more research is needed, including data on costs. Introduction It is estimated that two-thirds of deaths worldwide are attributable to non-communicable diseases (NCDs), with cardiovascular disease, cancer, diabetes mellitus, and chronic lung disease comprising the largest burden of NCDs.[1] The increasing prevalence of NCDs in low- and middle-income countries (LMICs) has driven the recent increases in the global NCD burden, and importantly the probability of premature death due to NCD is higher in LMICs than in their high-income counterparts.[2] Even in Sub-Saharan Africa—where communicable and vector-borne diseases are still the largest killers—it is estimated that NCDs will become the leading cause of death by 2030.[3] There are around 50 million persons who have been forcibly displaced from their homes as refugees and internally-displaced persons (IDPs) due to humanitarian crises,[4] defined here as events stemming from armed conflict, natural disasters, or food insecurity that threaten the health and safety of a community. There are also many millions more who remain in areas impacted by humanitarian crises or have recently returned to them after being displaced. While low-income countries continue to suffer the largest burden of humanitarian crises, trends have shown an increase in middle-income countries affected by humanitarian crises, with examples being armed conflicts in Iraq, Libya, Syria, Ukraine, the Balkans, and the Caucasus.[5] These countries have a particularly high burden of NCDs.[6] In addition, humanitarian crises have become more protracted and so health providers are facing pressure to expand beyond the immediate basic primary care traditionally provided by relief agencies and address longer-term health conditions such as NCDs. Moreover, it is known that a number of characteristics related to humanitarian crises such as stress and disrupted access to treatment can exacerbate NCDs.[7] The rise of NCDs in LMICs and the recent trends in humanitarian crises mean that the burden of NCDs has likely risen among crisis-affected populations. Governments, humanitarian organisations, and international agencies are now increasingly challenged with how to effectively tackle NCDs.[8] While there are best clinical practices on key interventions for treating NCDs in stable settings,[9] there is extremely limited guidance on tackling NCDs in crisisaffected settings. It is unclear what NCD interventions are effective and feasible in such settings, how best to deliver them, and how well interventions are adhering to clinical best practice. As a result, there are increasing calls for a better understanding of NCDs and interventions for NCDs in humanitarian crises.[3, 5, 8] However, no systematic review has been published that examines the evidence on effectiveness of interventions targeting NCDs during humanitarian crises in LMICs. Such a review can help guide future research, policies, and programming by identifying effective interventions as well as evidence gaps.[10] The aim of this paper was to systematically review evidence on the effectiveness of interventions targeting NCDs during humanitarian crises in LMICs. The specific objectives were to: (i) describe the study characteristics; (ii) examine evidence on effectiveness of NCDs in humanitarian PLOS ONE \| DOI:10.1371/journal.pone.0138303 September 25, 2015 2 / 16 Interventions for NCDs in Humanitarian Crises crises; and (iii) assess the quality of the evidence on NCD interventions in humanitarian crises. The review forms part of a larger review of evidence on health interventions in humanitarian crises.[11] Methods This systematic review followed the reporting items for systematic reviews as described in the PRISMA statement.[12] Eligibility Criteria The populations of interest were civilians in LMICs affected by humanitarian crises, defined here as events stemming from armed conflicts, natural disasters, or food insecurity that threaten the health and safety of a community. These included populations remaining in areas affected by crises and those forcibly displaced from them as refugees and IDPs. Studies that focused on current or former military populations were excluded. High-income countries were excluded as the vast majority of humanitarian crises occur in LMICs and the resources available to tackle NCDs in LMICs are very different to those in high-income countries. The time periods of humanitarian crises included acute, chronic, and early recovery time periods. The interventions of interest were health interventions covering health promotion, prevention, treatment, or rehabilitation activities at the individual or population level specifically for outcomes of NCDs. The outcomes included morbidity/mortality due to NCDs and surrogate outcomes (e.g. blood pressure, blood glucose levels) at the individual or population level. In addition, we also included information on process outcomes (e.g. adherence to clinical treatment) and feasibility of interventions and measurement methods, if the study included data on changes in health outcomes. We did not include mental health outcomes as these have been reviewed elsewhere. [13] Information Sources and Search Strategy The following bibliographic databases were searched: MEDLINE, Embase, Global Health, PsychInfo, and IBSS. The search terms were: (i) disaster-related terms; AND (ii) research studyrelated terms; AND (iii) geographic terms; AND (iv) NCD terms. A search of the grey literature was also conducted across a range of humanitarian-related databases and standard search databases such as Google. The full search strategy is provided in S1 File. Studies published in any language between January 1980 and June 2014 were included. Study Selection and Data Extraction Citations from the search results were imported from the bibliographic databases into EndNote for screening for eligibility based on the eligibility criteria given above. Duplicates were removed and the remaining citations assessed by title or abstract, and a full text review then conducted. References of the remaining studies selected after the full text review were examined for potentially relevant articles based on the eligibility criteria. Analysis of the final selected studies was then conducted. This involved extracting data from the final selected studies into an Excel database, with key extraction variables including: author and date of publication, geographic setting, sample population characteristics, study objectives, NCD condition studied, intervention characteristics, outcomes measured, results of the intervention, study conclusions, study design, and quality. The data screening and extraction were conducted independently by two authors and any variances resolved between them. PLOS ONE \| DOI:10.1371/journal.pone.0138303 September 25, 2015 3 / 16 Interventions for NCDs in Humanitarian Crises Quality assessment A quality assessment was conducted, with the Newcastle-Ottawa Quality Assessment Scale (NOS) version for cohort studies used for the observational studies [14, 15]. This was selected as it is a convenient and widely used tool with proven validity and reliability and has been endorsed by Cochrane Reviews [15–17]. For the randomised controlled trial (RCT) study we applied the widely used and validated Cochrane Risk of Bias Tool[18]. The NOS assigns stars for methodological rigour based on three categories: study selection, comparability of study groups, and outcome assessment. Studies were initially assessed within each category using the coding manual for cohort studies provided by Wells et al (see [15] and http://www.ohri.ca/programs/clinical_epidemiology/nosgen.pdf), with letters and descriptions assigned describing how each study fulfilled each criterion. Stars were then assigned per the NOS assessment scale when the study achieved high quality within that category. Criteria which were not applicable to particular studies were listed as not applicable but factored into overall impressions regarding that study’s conclusions. The Cochrane Risk of Bias Tool was developed to promote the assessment of quality of trials based on their risk of biased conclusions rather than focussing on reporting and methodological constraints [18]. The RCT was therefore evaluated as being at either high, low, or unclear risk of bias in several domains (selection, performance, detection, attrition, reporting, and other bias), and a descriptive justification of each conclusion was provided. Neither NOS nor the Cochrane Risk of Bias Tool uses an established summary score or threshold of quality, with the quality assessment primarily used to assess strengths and weaknesses of each study rather than to rank studies or to screen them out. Synthesis of results As the studies were heterogeneous in setting, intervention, and outcome, single effectiveness summary statistics across studies were not considered appropriate and were not estimated. Instead, a descriptive analysis of study results was reported. Results Study Selection The bibliographic databases yielded 4919 citations after duplicates were removed. Of these, only 8 met the study inclusion criteria (Fig 1).[19–26] The main reasons for excluding the 4879 studies were they were: in high-income countries; not in humanitarian contexts or took place too long after a humanitarian crises; not intervention studies; did not report changes in health outcomes; or were not full papers (e.g. conference abstracts only). These reasons applied at each screening stage. Exploring references from these 8 studies did not reveal any further studies meeting eligibility criteria. No studies were identified in the grey literature. Study Characteristics Key characteristics of the final 8 selected studies are included in Table 1. The studies were published between 1997 and 2014, with 6 out of 8 published within the past five years. Seven of the studies were with populations affected by armed conflict, and the remaining study with a population affected by an earthquake.[25] Sample sizes of the study populations ranged from 28 patients included in the RCT [26] to 12,550 patients in a diabetes cohort study.[24] The studies were conducted in 5 different countries: Afghanistan,[19] Georgia,[21] India,[26] Jordan,[20, 22–24] and Turkey.[25] Four of the studies[20, 22–24] came from the same research group studying Palestinian refugees in Jordan, with their studies covering diabetes and hypertension. PLOS ONE \| DOI:10.1371/journal.pone.0138303 September 25, 2015 4 / 16 Interventions for NCDs in Humanitarian Crises Fig 1. Results of screening process. doi:10.1371/journal.pone.0138303.g001 These four studies and one other[26] took place in long-term, relatively stable refugee settings, while the remaining studies were in more acute- or early post-crisis settings. Of the 8 studies, 7 used observational study designs[19–25] and 1 was an RCT.[26] The observational studies consisted of 5 cohort designs,[20–24, 26] 1 case series[19] and 1 interrupted time series.[25] The RCT[26] was the only study to compare outcomes between two groups. None of the studies examined the cost of implementing the intervention or the costeffectiveness of the intervention. The studies examined a broad range of NCD conditions: arthritis,[26] chronic kidney disease,[25] diabetes,[22–24] heart failure,[21] hypertension,[20] and thalassaemia.[19] All studies examined outcomes at the individual patient level and were primarily focused on disease management rather than prevention or health promotion. Details of each intervention and key outcome measures, study results, and specific study conclusions are presented in Table 1. Quality Assessment The quality assessment identified a number of common weaknesses. The observational studies (assessed using NOS) were generally adequate in describing the study population and establishing exposure. Deficiencies common to the observational studies were predominantly related to comparability and follow-up. None had a defined comparison group or unexposed cohort. Study transparency was also noted to be a weakness common to the observational studies. No study addressed potential biases, nor did any study discuss how missing data were handled. Only three of the observational studies adequately reported follow-up periods of participants, and most studies inadequately described their follow-up procedures. Follow-up periods ranged from undefined[19] to three years[23], with studies from settings of chronic crisis demonstrating longer follow-up. Outcome assessment was also problematic. Most studies only provided self-reported outcomes; the outcomes reported by the Khader et al. papers were of slightly higher quality in that they were linked to electronic medical records, but those assessments were not described in a standardised way such as via the International Classification of Disease (ICD) codes. Only four studies[20–22, 24] partially discussed study limitations, and only one PLOS ONE \| DOI:10.1371/journal.pone.0138303 September 25, 2015 5 / 16 Setting General conﬂictaffected rural population in Afghanistan attending a US military hospital. Camp-based Palestinian refugees in Jordan attending Nuzha primary care clinic. General conﬂictaffected population in Georgia (1 urban hospital and 3 rural districts). Author, Date [Reference] Bolt et al., 2010 [19] Khader et al., 2012 [20] Hebert et al., 2011 [21] PLOS ONE \| DOI:10.1371/journal.pone.0138303 September 25, 2015 Heart Failure (400 adult heart failure patients). Hypertension (4130 patients diagnosed with HTN). Thalassaemia (45 paediatric patients aged 13mos-11yrs) NCD Type (study population) Assess clinical outcomes of a heart failure disease management programme (HFDMP). Cohort design. Assess clinical outcomes of HTN care using EMR system. Assess utility of cohort monitoring using EMR in refugee context. Cohort design. Assess effect of palliative thalassaemia treatment in crisis setting. Caseseries design. Study Objectives and Design 2-year HFDMP: physician training; salary support; equipment supplied; patient education; free outpatient care. Standardised hypertension algorithm, including: diet/lifestyle management; graduated anti-hypertensive medications; referral if HTN persists; screening for HTN complications and associated conditions (e.g. DM); quarterly follow-up appointments. Cohort monitored via EMR for up to 2.5-years. Palliative splenectomy (programme of undeclared duration). Intervention Change in: ejection fraction (EF) (mean); BP (mean); BMI (mean); smoking status; health services and medication usage; NYHA HF class. HTN clinical measures: BP, glucose, cholesterol, kidney function (creatinine) testing, medications used. Cohort monitoring: incidence/ prevalence of HTN; clinic attendance (%); missed appointments; loss to f/u. Change in mean Hgb/Hct; change in mean blood transfusion frequency; complications encountered. Outcomes Measured Table 1. Summary of studies examining effectiveness of interventions targeting NCDs during humanitarian crises. 400 patients studied: 337 complete f/u, 51 lost to f/u, 12 died in war. EF increase 4.1±2.6% (p<0.001); BP— SBP decrease 30.9±20.0 mmHg (p<0.001), DBP decrease 17.8±13.0 mmHg (p<0.001); BMI statistically unchanged; smokers decrease 18.3% (p<0.001); ER use decrease 40.7% (p<0.001); hospital admission decrease 52.5% (p<0.001); beta-blocker use increase 73.3% (p<0.001); NYHA HF class—increase in Class I (+13.7%) and Class II (+19.2%), decrease in Class III (-26.0%) and Class IV (-6.8%); patients lost to f/u more likely rural. 4130 patients with HTN registered in EMR (cumulative, 2.5 years): 76% remain in care; 74% of those had BP checked; 74% of those checked had BP <140/90 mmHg; 15% had 1+ complications. 226 patients assessed for 1215-month outcomes: 62% remain in care; 76% of those meeting BP target (<140/90 mmHg); 3% glucose (DM) screened; 100% cholesterol screened; 99% creatinine screened; 8% had 1 + complications. Hgb: 5.4g/L pre-op to 8.7g/ L post-op; Hct: 16.5% preop to 26.3% post-op; transfusion every 24 days pre-op to every~50 days post-op; complications—2 pre-op deaths, 1 post-op respiratory distress, 1 transfusion reaction, 1 case CHF post-transfusion. Results (Continued) HFDMP was able to affect clinical outcomes in a LMIC experiencing war. Mixed clinical results: approx. 3/4 of patients meeting BP targets; cholesterol, kidney function properly screened; DM poorly screened; unclear if clinical practice lacking or if data recording lacking. EMR-based cohort monitoring promising for assessing programme implementation and future needs. Curative options likely impossible during crisis; splenectomy may be the best palliative option. Study Conclusions Interventions for NCDs in Humanitarian Crises 6 / 16 Setting Camp-based Palestinian refugees in Jordan attending Nuzha primary care clinic. Camp-based Palestinian refugees in Jordan attending Nuzha primary care clinic. Camp-based Palestinian refugees in Jordan attending 6 primary care clinics. Author, Date [Reference] Khader et al., 2012 [22] Khader et al., 2014 [23] Khader et al., 2014 [24] Table 1. (Continued) PLOS ONE \| DOI:10.1371/journal.pone.0138303 September 25, 2015 Diabetes Mellitus (12550 patients with DM; focus on 288 newly registered cases). Diabetes Mellitus (119 patients with DM). Diabetes Mellitus (2851 patients with DM). NCD Type (study population) Assess new and cumulative patient characteristics and clinical outcomes of DM care using EMR system. Assess utility of cohort monitoring using EMR in refugee context across multiple primary care clinics. Design: cohort Assess 12-, 24-, and 36-month clinical outcomes and complications of DM care using EMR system. Assess 3-year utility of cohort monitoring using EMR in refugee context. Cohort design. Assess clinical outcomes of DM care using EMR system. Assess utility of cohort monitoring using EMR in refugee context. Cohort design. Study Objectives and Design Standardised DM algorithm, including: diet/ lifestyle management; graduated anti-DM medications, including insulin if necessary; screening for DM complications and assoc. conditions (e.g.: HTN); quarterly follow-up appointments. Cohort monitored via EMR across 6 clinics (up to 2 years at 5 clinics, 3.5 years at 1 clinic). Standardised DM algorithm, including: diet/ lifestyle management; graduated anti-DM medications, including insulin if necessary; screening for DM complications and associated conditions (e.g.: HTN); quarterly follow-up appointments. Cohort monitored via EMR for up to 3 years. Standardised DM algorithm, including: diet/ lifestyle management; graduated anti-DM medications, including insulin if necessary; screening for DM complications and associated conditions (e.g.: HTN); quarterly follow-up appointments. Cohort monitored via EMR up to 2.5 years. Intervention DM clinical measures: 2-hr post-prandial blood glucose; BP, cholesterol, kidney function (creatinine) testing; BMI; foot assessment; ophthalmology referral; DM complications and associated risk factors. Cohort monitoring: incidence/prevalence of DM; clinic attendance (%); missed appointments; loss to f/u. DM clinical measures: 2-hr post-prandial blood glucose; BP, cholesterol, kidney function (creatinine) testing; BMI; DM complications. Cohort Monitoring: baseline prevalence of DM; clinic attendance (%); missed appointments; loss to f/u. DM clinical measures: 2-hr post-prandial blood glucose; BP, cholesterol, kidney function (creatinine) testing; foot assessment; ophthalmology referral. Medications used. Cohort monitoring: incidence/ prevalence of DM; clinic attendance (%); missed appointments; loss to f/u. Outcomes Measured Mixed clinical results: success testing cohort widely; clinical goals not broadly met; high numbers with associated risk factors. EMR-based cohort monitoring useful to highlight programme effects and future needs. 12550 patients with DM registered in EMR (cumulative; 2 years at 5 clinics, 3.5 years at 1 clinic): 78% remaining in care; males more likely to be smokers (OR M:F = 7.4 (CI 6.6–8.2; p<0.001)) and inactive (OR M:F = 1.8 (CI 1.6–1.9; p<0.001)) and to have 1+ complications (OR M:F = 1.6 (CI 1.4–1.8; p<0.001)); females more likely obese (OR M: F = 0.34 (CI 0.32–0.37; p<0.001)); 99% had PPBG measured; 65% at goal (180 mg/dl); 99% had cholesterol measured; 63% at goal (<200 mg/dl); 99% had BP measured; 87% at goal (<140/90 mmHg); 100% had BMI measured; 40% non-obese (<30 kg/ m2). (Continued) Mixed clinical results: approx. one-quarter of patients consistently missing DM goals; loss to f/u and complications rise over time; data indicate more aggressive treatment may be necessary. EMRbased cohort monitoring useful to highlight programme effects and future needs. Mixed clinical results: >half of patients not receiving proper PPBG checks; half of those checked poorlycontrolled; cholesterol, kidney function properly screened; DM complications poorly screened; unclear if clinical practice lacking or if data recording lacking. EMR-based cohort monitoring promising for assessing programme implementation and future needs. Study Conclusions 119 patients with DM assessed at 12-, 24-, and 36-months: 72/64/61% remaining in care at 12-/ 24-/36-months (χ2 test-fortrend = 47.9; p<0.001); 9/ 19/29% lost to f/u at 12-/ 24-/36-months (χ2 test-fortrend = 43.5; p<0.001); 71/ 78/71% meeting DM goal (PPBG 180 mg/dl) at 12-/ 24-/36-months; 7/14/15% with 1+ complications at 12-/24-/36-months. 2851 patients with DM registered in EMR (cumulative, 2.5 years): 70% remain in care; 42% of those had 2h-PPBG checked; 50% of those checked had PPBG 180 mg/dl; 18% had 1 + complications. 117 patients assessed for 1215-month outcomes: 61% remain in care; 58% of those meeting DM target (180 mg/dl); 100% cholesterol screened; 99% creatinine screened; 3% foot checked; no data on ophthalmology referrals; 10% had 1+ complications. Results Interventions for NCDs in Humanitarian Crises 7 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0138303 September 25, 2015 General urban and rural population affected by earthquake in Marmara region of Turkey (8 HD centres). Tibetan refugee in non-formal refugee communities in northern India. Sever et al., 2004 [25] Ryan, 1997 [26] Arthritis (28 patients with arthritis (24 OA, 4 RA), in 14 matched pairs). Chronic Kidney Disease (8 HD centres responsible for 439 patients with chronic kidney disease). NCD Type (study population) Compare limb mobility in matched pairs of Tibetan refugees with arthritis after either traditional Tibetan treatment or Western medications. RCT design. Assess clinical outcomes and infrastructure changes of haemodialysis centres affected by earthquake damage. Interrupted time series design. Study Objectives and Design Traditional Tibetan arthritis treatment (3 months); herbal pills; dietary restriction; behavioural advice; Western arthritis treatment (3 months); Ibuprofen or Indomethacin. Haemodialysis Intervention Limb mobility assessed via praxis-based scale (0– 5) for active movement; pain assessed via Visual Analogue Scale. Clinical outputs of HD centres: total number of HD visits, % patients receiving weekly HD. Clinical outcomes: patient weight, BP. HD infrastructure: number of HD centres, machines, patients served. Outcomes Measured Limb mobility: Traditional Tibetan treatment led to greater improvement in 12/ 14 matched pairs; 2 pairs were a draw; Mean improvement 1.39 (SD 0.59) points using traditional Tibetan treatment; 0.57 (SD 0.33) points using Western treatment.) Pain—Western treatment led to better pain improvement (data not given). 8 HD centres assessed: HD machines: 95 preearthquake; 74 (1mo) and 79 (3mos) post-earthquake; HD personnel: 112 preearthquake; 86 (1mo) and 94 (3mos) post-earthquake; HD patients: 439 preearthquake; 175 (1wk), 239 (1mo), and 288 (3mos) post-earthquake; HD sessions: 1093/wk preearthquake; 520/wk (1wk), 616/wk (1mo), and 729/wk (3mos) post-earthquake; % weekly HD: 2.3% pre- to 7.2% 1wk-post-earthquake. Interdialytic weight gain: 2.9 ±1.1kg pre- to 2.6±1.1kg 1wk-post-earthquake; BP stable throughout. Results Traditional Tibetan treatment led to better arthritis improvement compared to Western treatment when assessed via limb mobility. RCTs are practicable in traditional settings. Infrastructure damage signiﬁcantly impairs HD treatment during disasters. Increase in once-weekly HD but interdialytic weight gain not increased. Patient education and disaster planning may prevent adverse outcomes. Study Conclusions doi:10.1371/journal.pone.0138303.t001 odds ratio; PPBG–post-prandial blood glucose; RA–rheumatoid arthritis; RCT–randomised controlled trial; Ref#—reference; SBP–systolic blood pressure; SD–standard deviation. room; f/u–follow-up; Hct–haematocrit; HD–haemodialysis; HFDMP–heart failure disease management programme; Hgb–haemoglobin; HTN–hypertension; LMIC–low/middleincome country; mmHg–millimetres of mercury; NCD–non-communicable disease; NYHA HF class–New York Heart Association heart failure classiﬁcation; OA–osteoarthritis; OR– Acronyms: BMI–body mass index; BP–blood pressure; DBP–diastolic blood pressure; DM–diabetes mellitus; EF–ejection fraction; EMR–electronic medical record; ER–emergency Setting Author, Date [Reference] Table 1. (Continued) Interventions for NCDs in Humanitarian Crises 8 / 16 Interventions for NCDs in Humanitarian Crises [23] gave any information on sources of funding, and even then only in the online version of the article. The RCT study from India[26] was assessed using the Cochrane Risk of Bias Tool and was judged to have a high risk of selection, performance, and detection bias, primarily due to the study’s lack of blinding. The study used an open enrolment process and all members of the research team appear to have had knowledge of patients’ treatment. Although outcome reporting was a strength of the study, there was an overall high risk of additional biases given that a single non-blinded researcher assessed the outcomes. Further deficiencies surrounded the reporting of the randomisation process, which was not described in any detail. Further details on the scoring for individual studies are given in S2 File and S3 File. Synthesis of results Cardiovascular diseases were assessed via two cohort studies–one in Georgia examining a heart failure disease management programme[21] and one in Jordan examining hypertension care among Palestinian refugees.[20] The contexts of these two studies differed in the sense that the study in Georgia was examining the effectiveness of a health programme that then experienced the outbreak of war during the intervention, while the study in Jordan took place in a longterm refugee setting that was relatively stable during the study period. Both studies focused on the implementation of disease management algorithms in settings of humanitarian crisis and attempted to highlight both the feasibility and challenges of such programmes. In the study in Georgia by Hebert et al.,[21] the heart failure disease management programme saw some success among its 400 patients by demonstrating a statistically significant increase in ejection fraction—the fraction of blood volume exiting the heart’s ventricles with each heartbeat, which tends to decrease in the most common types of heart failure—and a statistically significant decrease in blood pressure over the course of the 2-year programme. Ejection fraction improved by 4.1±2.6% (p<0.001) and systolic and diastolic blood pressures decreased by 30.9±20.0 mmHg and 17.8±13.0 mmHg, respectively (p<0.001 for both). The intervention also demonstrated a decrease in smoking rates and in emergency room visits and hospitalisations. Heart failure classification also improved. The study by Khader et al. on a hypertension management programme in Jordan had mixed clinical results, with approximately three quarters of patients meeting blood pressure targets.[20] The intervention focused on the method of cohort monitoring by using an electronic medical record system to enrol patients in a cohort that could be studied over time. The monitoring allowed researchers to also assess if goals of care were being met, both with respect to hypertension care goals such as blood pressure monitoring and with respect to associated diseases such as hypercholesterolaemia and diabetes. Results were mixed; among a sub-cohort of 226 patients assessed for 12–15 months, 100% were screened for high cholesterol but only 3% were screened for diabetes using a glucose blood test. The study authors concluded that the interventions were an improvement on baseline care in both settings. However, no comparison group was included. Three cohort studies, all by Khader et al., focused on diabetes care among Palestinian refugees in long-term refugee settings in Jordan.[22–24] All three studies conducted very similar interventions consisting of a standardised diabetes protocol and assessment of patient outcomes and programme outputs via electronic medical records-based cohort monitoring. The concept was very similar as well to the aforementioned study targeting hypertension in a similar patient population with the initial DM study essentially mirroring that design.[20] The subsequent two diabetes studies differed in terms of follow-up and scope, with one study[23] focusing on 12-, 24-, and 36-month outcomes, and the other on the expansion of the PLOS ONE \| DOI:10.1371/journal.pone.0138303 September 25, 2015 9 / 16 Interventions for NCDs in Humanitarian Crises programme from one clinic to six.[24] While the Khader et al. studies had similar designs and settings, it was confirmed via correspondence with the studies’ authors that the populations of each study differed. For this reason—and because this review featured descriptive analysis rather than meta-analysis—it was felt that inclusion of each study for analysis was appropriate. In general, the diabetes studies claimed an improvement in the programme over time. Earlier assessment of the programme [22] found that over half of patients were not receiving postprandial blood glucose checks and that those checked only demonstrated proper diabetes control (</ = 180mg/dl) half of the time. Subsequent assessment described in 2014[24] found that most programme outputs had improved, with nearly all patients attending clinic meeting the blood testing goals. However, other treatment goals, specifically foot examination and ophthalmology referral, that were problematic during the earlier study did not continue to be assessed in the subsequent studies. These studies also found that loss to follow-up rose over time (Γ2 test-for-trend = 43.5; p<0.001). Nevertheless, the study authors contend that having a monitored cohort using an electronic medical record-based system could allow for improved retention of patients through more proactive patient monitoring. Chronic kidney disease was assessed by one retrospective study from Sever et al. in the Marmara region of Turkey in the aftermath of an earthquake.[25] This study focused on both the infrastructure changes and clinical patient outcomes of providing haemodialysis to patients with severe chronic kidney disease. The study found that infrastructure for providing haemodialysis was affected by the earthquake, with an acute decrease in haemodialysis centres, machines available, personnel, and subsequently numbers of haemodialysis treatments provided. Gradually these numbers improved during follow-up. The initial earthquake also led to an increase in the number of patients receiving once-weekly (i.e., less frequent) haemodialysis. Despite the infrastructure challenges, the authors found that mean interdialytic weight gain—the amount of weight patients gain between treatments, typically fluid weight due to poor blood filtration and urine production—actually decreased from a pre-earthquake 2.9±1.1 kg to 2.6±1.1 kg 1-week post-earthquake, despite the increased numbers of patients receiving haemodialysis less frequently. Moreover, the blood pressures of patients studied remained stable throughout the study period. The authors contend that adequate patient education regarding disaster preparedness and fluid restriction likely helped mitigate poor patient outcomes, although no comparison between the baseline health status of patients able to seek care after the earthquake and the status of the larger number of patients receiving haemodialysis before the earthquake was conducted. The only RCT eligible for inclusion in this systematic review studied changes in limb mobility among 14 matched pairs of arthritis patients living in a stable Tibetan refugee setting in northern India.[26] In this open, non-blinded RCT, patients were randomised to receive three months of either traditional Tibetan arthritis treatments (herbal pills, dietary restriction, and behavioural advice) or Western medication (ibuprofen or indomethacin). In 12 of 14 pairs, the traditional Tibetan treatment led to greater improvement in limb mobility, and in the remaining 2 pairs the treatments performed equally well. Although they have not presented the data, the authors do suggest that pain control was better with the Western treatment than the traditional treatment. The authors state that a secondary objective of the study was to examine the process of conducting an RCT on traditional treatment options, although the authors do not comment specifically on the nuances of conducting an RCT in unstable settings. One case series study by Bolt et al. examined thalassaemia among 45 paediatric Afghan patients seeking care at a United States-managed military hospital in a chronic crisis setting in Afghanistan.[19] The research team provided the intervention—palliative splenectomy—with the rationale that more curative treatment (e.g. stem-cell transplantation) would not be feasible in the Afghan context. The study reported an improvement in anaemia with mean PLOS ONE \| DOI:10.1371/journal.pone.0138303 September 25, 2015 10 / 16 Interventions for NCDs in Humanitarian Crises haemoglobin levels rising from 5.4g/L pre-operatively to 8.7g/L post-operatively. Furthermore, frequency of blood transfusion decreased from every 24 days to approximately every 50 days before and after surgery. The authors state that families were pleased with the improvements during follow-up, although patient-specific data, confidence intervals, and specifics regarding follow-up were not provided. Discussion To the best of our knowledge, this is the first systematic review to examine the evidence of effectiveness of interventions targeting NCDs in humanitarian crises. It highlights major gaps in evidence on NCD interventions in humanitarian crises, with only eight studies meeting inclusion criteria. While the selected studies addressed a range of NCDs, there were some notable absences—particularly studies for cancer treatment and respiratory diseases. In the case of cancer, the challenges of financing and sustaining cancer care for Syrian refugees have been highlighted and further research is required on these issues.[27] In addition, none of the studies examined the effectiveness of NCD prevention activities despite prevention being central to global efforts to tackle NCDs,[9] the potential risk-factors for NCDs in crisis and fragile settings,[28] and humanitarian agencies noting the importance of NCD prevention activities.[29] Nor did any study prioritise preparedness for crises in relation to NCD management. Geographically, the studies predominantly focused on the Middle East (which is understandable given the greater burden of NCDs in the region), and studies in more resource poor settings with weaker health systems are required. There was also a high risk of bias in the identified studies. While it is unwise to draw any definitive conclusions from such a small body of evidence, there were a number of findings that warrant further discussion. First is the apparent success of algorithm-based interventions. In Georgia, the improved clinical outcomes and use of appropriate medication showed the effectiveness of the heart failure disease management programme there.[21] Diabetes care was also implemented using an algorithm in Palestinian refugee clinics in Jordan.[22–24] Here too, the advantage of specific clinical measures led to improvement in programmatic outputs over the years of follow-up. Alongside streamlined clinical measures, it may be beneficial to include certain NCD medications on essential medication lists to facilitate their accessibility and use during a crisis. Second, the benefit of cohort monitoring using electronic medical records was highlighted.[20, 22–24] These studies were originally derived from similar cohort monitoring research conducted with other chronic diseases such as tuberculosis and HIV,[30–33] and it has been suggested links between NCDs and other chronic disease programmes such as tuberculosis and HIV could facilitate this monitoring as well as hasten implementation of NCD-focussed programmes.[34] The systematic collection of baseline and routine NCD data over time should be strongly supported, and agencies such as UNHCR have now begun implementing a standardised health information system for refugees.[35] Ideally, this monitoring should be done electronically, and given the trend toward cheaper and more mobile electronic options, the incorporation of electronic medical record technology appears to hold promise for the rapid implementation of cohort monitoring during crisis. Third, the studies also highlight the importance of capacity-building and preparation of local health staff and patients in effecting good clinical practice,[21] monitoring processes,[24] and supporting medication adherence and adaptability among patients. In addition to the limited number of studies, the strength and quality of the existing evidence was also generally quite limited. Most of the studies used cohort study designs and while some were able to consistently follow-up over time in order to measure changes in NCD outcomes, none included a comparison group not receiving the tested intervention. This omission PLOS ONE \| DOI:10.1371/journal.pone.0138303 September 25, 2015 11 / 16 Interventions for NCDs in Humanitarian Crises therefore limits conclusions on the effectiveness of the intervention. Where logistically and ethically appropriate, it would be of considerable value to include some form of comparison group in order to formulate a more robust assessment of the intervention effectiveness. The use of stepped wedge designs may be a useful approach to follow in such settings.[36] Where the use of controls is not possible, statistical methods such as interrupted time-series analysis could prove useful.[37] Other common weaknesses include lack of discussion on how missing data were addressed, and also on other potential biases in study designs and analyses. For example, the haemodialysis study in Turkey[25] was prone to recall bias as each time point analysed was based on questionnaires sent six months after the earthquake. The RCT examining arthritis[26] was weakened in its claims by a lack of blinding. Adequate patient follow-up was another area of weakness. While loss to follow-up may be expected in the volatile and transient settings of humanitarian crisis, the lack of analysis to address it is problematic. Adjusting for potential cofounding was also not conducted (and this was further undermined by the lack of control groups). There are also issues regarding the appropriateness and generalisability of some of the studies for other humanitarian contexts. For example, while the thalassaemia study for civilians in Afghanistan provided an intervention that was tailored toward the resources and context of Afghanistan, it nevertheless took place in a well-resourced US military hospital.[19] A number of the studies[20, 22–24, 26] were conducted in long-term refugee settlements that were relatively stable and so there is little evidence from more insecure and volatile settings. The lack of cost considerations across all studies further limits the generalisability of the evidence. While cost-effective interventions targeting NCDs in LMICs have been developed,[9, 38] further work needs to be done to better understand the feasibility and cost of NCD interventions in humanitarian crises given their different resources and the inherent security and logistical constraints in such settings. Such information on costs and financing of NCDs is crucial to address operational and ethical issues relating to the sustainability of providing NCD care in such settings—particularly in relation to tension between commencing long-term NCD care and the shorter-term mandates of many humanitarian agencies. Most of the evidence identified in this review is from relatively stable settings. This highlights the challenges to implementing rigorous research during an acute crisis giving the security and resource constraints and rapid population movement. However, previous longitudinal research with conflict-affected populations in volatile contexts on treatment for chronic conditions such as HIV has shown that such research is possible.[39] Given the time constraints in such settings, planning research designs in advance, pre-approving protocols, and using innovative designs that can also be rapidly implemented is recommended (e.g. using routine NCD data for cohort designs or stepped wedge designs as services are rolled out). Ethical concerns regarding intervening on conditions that require long-term care when the humanitarian response may be brief must be considered within the humanitarian and research communities, but the ethical implications of withholding an intervention or researching its effectiveness must also be strongly considered. Humanitarian donor agencies should also consider longerterm funding cycles and greater financial support for impact evaluation in order to understand the actual effectiveness of the health interventions they fund. Operational humanitarian agencies should also give greater priority to research and impact evaluation for NCDs, following the example of agencies such as Médecins Sans Frontières who have placed a relatively strong emphasis on rigorous operational research. Stronger links should also be fostered between humanitarian agencies and academia to strengthen NCD research in humanitarian contexts, and recent initiatives on this such as R2HC are to be welcomed.[40] PLOS ONE \| DOI:10.1371/journal.pone.0138303 September 25, 2015 12 / 16 Interventions for NCDs in Humanitarian Crises Limitations Only descriptive analysis was used, but alternative methods such as meta-analysis were not appropriate because of the multiple outcomes, interventions, study types, and the limited number of studies. The review searched only quantitative studies as the focus was on the effectiveness of interventions, and only studies from 1980 onward were included. Analysis of qualitative research examining aspects such as health care provider and user perspectives on NCD interventions in humanitarian crises would be extremely valuable. There is also the possibility that humanitarian agencies may not have published all their existing research (either as published or grey literature), and it is difficult to ascertain the potential levels of such non-publication. We could have tried hand searching humanitarian agency reports to attempt to find additional studies. However, our prior experience and discussions with humanitarian agency staff suggest it would be extremely unlikely to yield any further studies that would not have been published in scientific journals. The NOS tool used for the quality assessment in this review has been criticized for limited inter-rater reliability.[41] We did not observe any substantial discrepancies between quality assessors for this review but did not calculate inter-rater reliability. Conclusions Research during humanitarian crises is inherently difficult. Researching NCDs is arguably even harder as their chronic nature tends to demand more substantial follow-up. Nevertheless, this review has highlighted an urgent need to substantially expand research on NCD interventions in humanitarian crises given their growing disease burden. Currently available studies represent an attempt to rectify this knowledge gap but are few in number and of relatively limited quality. The findings point toward the success of standardised algorithms that can be implemented consistently and monitored via patient tracking using electronic medical records. Key research needs include: a better understanding of NCD delivery models in more acute and early recovery settings; using comparison groups (where appropriate); analysing the costs and sustainability of interventions; and developing methods to minimize bias in setting where standard randomised control studies are not feasible. Such work would support the generalisability of NCD intervention findings and provide much needed guidance in this neglected field. Supporting Information S1 File. Table. Search terms. (DOCX) S2 File. Table. Quality assessment of observational studies using NOS criteria. (DOCX) S3 File. Table. Quality assessment of RCT study using Cochrane Risk of Bias Assessment Tool. (DOCX) S4 File. Table. PRISMA Checklist. (DOC) S5 File. Original dataset. (XLSX) PLOS ONE \| DOI:10.1371/journal.pone.0138303 September 25, 2015 13 / 16 Interventions for NCDs in Humanitarian Crises Author Contributions Conceived and designed the experiments: AR AK PP KB BR. Performed the experiments: AR AK PP BR. Analyzed the data: AR AK PP BR. Contributed reagents/materials/analysis tools: AR AK PP BR. Wrote the paper: AR AK PP KB BR. References 1. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. The Lancet. 2013; 380(9859):2095–128. 2. Kontis V, Mathers CD, Rehm J, Stevens GA, Shield KD, Bonita R, et al. 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https://openalex.org/W2996659356	https://josr-online.biomedcentral.com/track/pdf/10.1186/s13018-019-1505-2	English	null	The regulatory mechanism of p38/MAPK in the chondrogenic differentiation from bone marrow mesenchymal stem cells	Journal of orthopaedic surgery and research	2,019	cc-by	5,754	RESEARCH ARTICLE Open Access © 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: Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation and joint inflammation, in which growth factors are significantly involved. The extracellular signal-regulated p38 MAPK pathways play important roles in the regulation of osteogenic and chondrogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). However, the exact mechanism remains unclear. Methods: In this study, the chondrogenic differentiation of human BMSCs was initiated in micromass culture in the presence of TGF-β1 for 14 days. Quantitative RT-PCR and Western blot were performed to detect the transfection effect of shRNA-p38 interfering plasmid in BMSCs. The protein expressions of p/t-p38, SOX9, collagen II, Aggrecan, p/t-Smad1, and p/t-Smad4, as well as the kinase activities of p38/ERK/JNK pathway, were investigated using Western blot analysis. Additionally, the level of chondroitin sulfate and glycosaminoglycans (GAG) expression were measured by Alcian blue staining and GAG assay kit via qualitative and quantitative methods, respectively. Results: The results demonstrated that p38 pathway was activated in the chondrogenic differentiation of BMSCs induced by TGF-β1. Cartilage-specific genes and chondrogenic regulators, such as SOX9, collagen II, Aggrecan, and GAG, were upregulated by TGF-β1, which could be reversed by predisposed with shRNA-p38 interfering plasmid and p38-MAPK inhibitors (SB203580). Moreover, the activation of p38/ERK/JNK pathways in the presence of TGF-β1 was suppressed by shRNA-p38 and SB203580 treatment. Conclusion: Collectively, the activation of p38/ERK/JNK/Smad pathways plays a facilitated role in the chondrogenic differentiation induced by TGF-β1. After suppressing the p38 pathway, the chondrogenesis can be inhibited, which can be used to guide the treatment of osteoarthritis. Keywords: Osteoarthritis, Chondrogenic differentiation, p38-MAPK, BMSCs, TGF-β1 Keywords: Osteoarthritis, Chondrogenic differentiation, p38-MAPK, BMSCs, TGF-β1 Ma et al. Journal of Orthopaedic Surgery and Research (2019) 14:434 https://doi.org/10.1186/s13018-019-1505-2 Ma et al. Journal of Orthopaedic Surgery and Research (2019) 14:434 https://doi.org/10.1186/s13018-019-1505-2 The regulatory mechanism of p38/MAPK in the chondrogenic differentiation from bone marrow mesenchymal stem cells Ning Ma1, Xiao Teng1, Qi Zheng1 and Peng Chen2* Ning Ma1, Xiao Teng1, Qi Zheng1 and Peng Chen2* Introduction addition, bone marrow MSCs possess the ability of multi-differentiation, such as chondrocytes, osteoblasts, and adipocytes, and have been the common seed cells for clinical research and therapy in orthopedics field [8]. However, the chondrogenic differentiation was hardly spontaneous; thus, it is necessary for the researchers to reveal the mechanisms underlying the differentiation from MSCs to chondrocytes, which is an important con- tribution for cartilage injury repair in OA. Osteoarthritis (OA) is a chronic joint disorder, which has been the leading cause of disability worldwide, par- ticularly in the elderly suffering from pain and loco- motor limitation [1–3]. It was proved to primarily occur in the hip and knee and results from the increase of age, obesity, strain, and injury [4, 5]. Chondrogenesis is mainly inducted by mesenchymal stem cell (MSC) con- densation and differentiation into chondrocytes, which is regarded as the clinical treatment of OA [6, 7]. In Chondrogenic induction is conducted by forcing ag- gregation of mesenchymal cells or chondroprogenitor cells to generate a “micromass” or “pellet” culture in a special culture system in vitro [9–11]. TGF-β has been widely proven to regulate cell differentiation by setting * Correspondence: doctorchen51@163.com 2Department of Orthopedics, Yan Cheng Third People’s Hospital (Affiliated Yancheng Hospital of Southeast University Medical College), No.2 Xindu West Road, Yancheng 224001, Jiangsu, China Full list of author information is available at the end of the article Real-time quantitative PCR Total RNA was isolated from MPC pellets using the RNeasy Mini Kit (Qiagen, Valencia, CA) and quantified spectro- photometrically based on A260. After the first-strand cDNA synthesis using the SuperScript First-Strand Synthesis Sys- tem (Invitrogen), levels of specific mRNA transcripts were determined by real-time polymerase chain reaction (PCR) utilizing SYBR Green PCR Master Mix and an iCycler real- time PCR detection system (Bio-Rad Laboratories). The pri- mer sequences were as follows: p-p38 forward: 5′-CTGAAC AACATCGTCAAGTGCC-3′, reverse: 5′-CATAGCCGGT CATCTCCTCG-3′; p38 forward: 5′-, CCCGAGCGTT ACCAGAACC-3′, reverse: 5′- TCGCATGAATGATG GACTGAAAT-3′; GAPDH, forward: 5′-GCACCGTCAA GGCTGAGAAC-3′, reverse: 5′-TGGTGAAGACGCCA GTGGA-3′. The 2−ΔΔCt method was used in each sample as relative quantification [21]. Each experiment was performed three times. In the present study, we employed a micromass chon- drogenic culture system for a relatively long duration of 14 days. We sought to investigate the function of p38/ MEK/ERK/JNK signal pathway in TGF-β-induced chon- drogenesis via regulating cartilage-specific genes for colla- gen II, Aggrecan, Sox9, and sGAG, and cross-talk between p38 MAPK and Smad1/4 signals in this process. Cell transfection and treatment Transient transfections of primary chondrocytes were performed as described using Fugene (Roche) [20]. BMSCs were transfected when 60% of them were fused. The cells were transfected with a shRNA-p38 or an empty expression vector as shRNA-negative control (NC) (Invitrogen), or predisposed with the pharmaco- logical p38 MAPK inhibitors (SB203580, 10 μM) after TGF-β1 inducing for 14 days. All the transfections were performed with three independent cell isolations, each of which has quadruplicate replicates. TGF-β triggers a series of downstream pathways via a transmembrane serine/threonine kinase receptor complex, known to be quite complicated. Smads are regarded as central cytoplasmic mediators of TGF-β [16, 17]. Upon TGF-β activation, Smad2 and Smad3 are activated to form protein complexes with common mediator Smad4, and then phosphorylated and translocated into nucleus to regulate transcription of target genes such as Sox9, Col2, and Aggrecan [18]. Recently, it was reported that p38 and Smad1/5/8 can be simultaneously activated by TGF-β- activated kinase 1 (TAK1), resulting in the induction of chondrogenic differentiation of stem cells [19]. These findings implied that chondrogenesis initiated by TGF-β1 may be tightly regulated through interactions between Smads and p38 MAPK signals. However, the mechanism of p38 pathways in TGF-β-induced chondrogenesis has not been fully clarified. Ma et al. Journal of Orthopaedic Surgery and Research (2019) 14:434 Ma et al. Journal of Orthopaedic Surgery and Research (2019) 14:434 Ma et al. Journal of Orthopaedic Surgery and Research (2019) 14:434 Page 2 of 8 Page 2 of 8 Chondrogenesis induction in vitro off a large variety of signaling cascades and is tightly controlled by feedback mechanisms at different levels [12]. Mitogen-activated protein kinase (MAPK) family transduction involves a multistep kinase cascade with extracellular signal-regulated protein kinase (ERK), p38 kinase, and c-Jun N-terminal kinase (JNK) [13]. ERK and p38 have been confirmed to occupy core positions in mediating chondrocyte proliferation and related gene ex- pression [14]. Further, MAPKs have been reported as the most important signaling pathway protein kinases, implicated in TGF-β-mediated chondrocyte functions in- cluding cell proliferation, differentiation, apoptosis, and inflammatory responses [15]. However, the definite ef- fects and the potential mechanism of ERK and p38 in chondrogenesis are still unclear. Chondrogenic differentiation of BMSCs was initiated in a micromass culture system. hBMSCs (at passages 3–6) were dissociated for single-cell suspension stating at a density of 2.0 × 107 cells/ml. Ten-microliter droplets were seeded in culture dishes and allowed to form cell aggregates at 37 °C. In the experimental groups, recom- binant human TGF-β1 (10 ng/ml) was added to the chondrogenic medium. Further, micromass cultures were performed for 14 days, and culture media was re- placed every 3 days. Alcian blue staining To assess the presence of glycosaminoglycans, which are cartilage-specific matrix proteins, BMSCs were cultured for 14 days in chondrogenic medium and stained with Alcian blue. After removal of the culture medium, the cell-seeded hydrogels were fixed with 4% paraformalyde- hyde for 30 min, then washed twice with PBS before the addition of 0.1% stock solutions of Alcian blue. After 30 min incubation at room temperature, the dye solution was removed and the constructs were washed with dis- tilled water. Moreover, the staining results were re- corded under an inverted microscope (Nikon Eclipse TS100). Each experiment was performed three times. As shown in Fig. 1b, at day 0 after adding TGF-β1, BMSCs grew adherently to the wall, and the cells were triangular or polygonal in shape. From day 5 to day 14, cell morphology changed significantly and gradually pre- sented a typical paving stone shape with uniform size and shape. Western blot analysis Human bone marrow mesenchymal stem cells (BMSCs) were purchased from the American Type Culture Collec- tion and cultured in α-modified Eagle medium (Invitro- gen, Carlsbad, CA, USA) containing 15% FBS (Invitrogen), 100 mM L-ascorbic acid 2-phosphate (Wako Pure Chem- ical Industries, Osaka, Japan), 1% penicillin and strepto- mycin (Sigma), and 1% L-glutamine (Invitrogen). The cells were incubated at 37 °C in a humidified incubator with 5% CO2. When hBMSCs were 80–90% confluent in the cul- ture flasks, the adherent cells were digested with trypsin and subcultured. The BMSCs were collected and lysed at 48 h after treat- ment using RIPA buffer. Identical amounts of proteins were resolved by 10–15% SDS-PAGE and then trans- ferred to a PVDF membrane (Millipore Corporation, Bil- lerica, MA, USA). The membrane was incubated with specific antibodies, Collagen II, Aggrecan, Sox9, p-p38, p38, p-ERK, ERK, p-JNK, JNK, p-Smad1, Smad1, p- Smad4, Smad4, and GAPDH (all from Cell Signaling Technology Inc.) at 4 °C overnight, and peroxidase- conjugated secondary antibodies (KPL, Gaithersburg, Ma et al. Journal of Orthopaedic Surgery and Research (2019) 14:434 Page 3 of 8 Ma et al. Journal of Orthopaedic Surgery and Research (2019) 14:434 Page 3 of 8 Page 3 of 8 MD, USA). Chemiluminescence (Millipore Corporation) and densitometry analysis (ImageJ software) were ap- plied to measure the protein expression. Each experi- ment was performed three times. relatively high level until day 14 compared to day 0 (Fig. 1a, p < 0.05 at day 5, p < 0.01 at day 7, and p < 0.001 at day 14). The p-p38 expression in TGF-β-induced BMSCs was sig- nificantly increased in a time-dependent manner. However, the expression of p38 showed no significant difference dur- ing 14 days. Therefore, these results indicated that the p38 signal was activated in chondrogenic differentiation of the BMSCs induced by TGF-β1. Sulfated glycosaminoglycan quantification Colorimetric assessment was performed to determine the amount of the sulfated glycosaminoglycan (sGAG) using a blyscan sulfated glycosaminoglycan assay kit (Biocolor B1000 Std.) according to the manufacturer’s instructions. Briefly, 0.25 ml of 1,9-dimethyl-methylene blue (DMMB) dye reagent was added to 80 μl of papain- digested extracts and incubated for 30 min at room temperature. The unbonded dye was removed by centri- fugation. Then, the sGAG-bonded dye pellet was dis- solved in 0.25 ml of dissociation reagent buffer. A serial dilution of chondroitin 4-sulfate (200 to 1500 ng) was prepared to generate a standard curve according to the same procedure used for sample preparation. The ab- sorbance was recoded at 525 nm using a microplate reader (Multiskan Spectrum, Thermo Scientific). Each experiment was performed three times. Statistical analysis Statistical analyses were carried out using Prism 5 soft- ware. Each experiment was performed in triplicate and analyzed by one-way ANOVA, followed by Dunnett’s post hoc test. Student’s t test was used to compare the values of the test and control samples. The results were expressed as mean ± SD. In all cases, a p value of < 0.05 was considered to be statistically significant. In addition, a quantitative method was utilized to measure the amount of sulfated glycosaminoglycan (sGAG) deposited in the extracellular matrix (ECM) of the micromasses. The results showed a significant in- crease with TGF-β1 group in sGAG content compared to the control group (Fig. 3a, p < 0.001), while TGF-β1 in combination with shRNA-p38 or SB203580 exhib- ited a significant decrease in sGAG content compared Inhibition of p38 signals suppressed the chondrogenic differentiation in TGF-β1-induced BMSCs The overexpression of p-p38 in TGF-β-induced BMSCs in- dicated that p38 signal pathway might function as an enhan- cer of the chondrogenic differentiation. To test this hypothesis, we investigated whether inhibition of p38 affects chondrogenic differentiation in TGF-β-induced BMSCs. In Fig. 2 a and b, the results showed that the protein level of p- p38 and the mRNA of p38 were significantly decreased using Western blot and RT-qPCR after being transfected with p38 interfering plasmid (shRNA-p38) in TGF-β- induced BMSCs (p < 0.001). Interestingly, the protein ex- pression of p38 was almost unchanged (Fig. 2a), while its mRNA level decreased significantly in the shRNA-p38 group when compared with control and shRNA-NC groups (Fig. 2b, p < 0.05). Chondrogenic potential of BMSCs was confirmed by examining the expression of cartilage-specific gene coding for collagen II, Aggrecan, Sox9, p/t-smad1, and p/t-smad4 proteins with Western blot. TGF-β-treated BMSCs exhibited higher expression in all these cartilage- specific genes after 14 days compared with the untreated control group (Fig. 2c, p < 0.001). Treatment of shRNA-p38 and pretreatment of SB203580, TGF-β1-induced gene ex- pression of collagen II, Aggrecan, Sox9, p/t-smad1, and p/t- smad4 were notably decreased compared with the TGF-β1 group (Fig. 2c). Further, the reverse effects of transfection interference plasmids shRNA-p38 were better than that of p38 inhibitors SB203580. Thus, these results indicated that cartilage-specific gene expression was affected by p38 inhibi- tors in reverse patterns, confirming that p38 could regulate the process of chondrogenic differentiation. Results Morphological changes of BMSCs following TGF-β1 induced for 0, 5, 7, and 14 days were observed under an inverted microscope. On day 0, BMSCs grew adherently to the wall, and the cells were triangular or polygonal in shape. From day 5 to day 14, cell morphology changed significantly and gradually presented a typical paving stone shape with uniform size and shape. p < 0.05, p < 0.01, and *p < 0.001 vs. TGF-β1 (D0) to the TGF-β1 group at day 14 (Fig. 3a, p < 0.01). Therefore, GAG content was significantly regulated by the p38 pathway in TGF-β1-induced BMSCs. Accord- ing to the results of Alcian blue staining, the chondro- genic differentiation (central blue nucleus) of BMSCs was significantly enhanced in the TGF-β1 group, while suppression of p38 could inhibit the chondrogenic dif- ferentiation of BMSCs (Fig. 3b). involvement of intracellular signaling in the regulation of BMSC cartilage formation. The TGF-β1-induced micromass culture system was employed in order to analyze the roles of p38 in the chondrogenic differenti- ation of BMSCs. This study demonstrated that p38/ ERK/JNK pathway was activated by TGF-β1 in chondro- genesis of BMSCs, and played crucial roles in gene tran- scription of cartilage-specific markers coding for Aggrecan, SOX9, collagen II, Smad1/4, and sGAG. The study also provided evidences that p38 might regulate TGF-β1-induced chondrogenesis and directly interact with p38/ERK/JNK pathway. Inhibition of p38 signals suppressed the p38/ERK/JNK pathway in the presence of TGF-β1-induced chondrogenesis The activation of p38/ERK/JNK was assessed using Western blot after treating with p38 inhibitors in TGF- β1-induced BMSCs. The results in Fig. 4 indicated that the expressions of p-p38, p-ERK, and p-JNK were over- expressed compared with the control group (p < 0.001). Both shRNA-p38 and SB203580 could decrease the ex- pression of p-p38, p-ERK, and p-JNK in TGF-β1- induced BMSCs. Therefore, these results showed that p38 may promote chondrogenic differentiation by regu- lating p38/ERK/JNK signal pathways. In terms of osteogenic differentiation, previous studies in- dicated that p38 and ERK were involved in bone morpho- genetic factor-9-induced osteogenic differentiation of rat dental follicle stem cells [22]. Particularly, p38/MAPK was accepted as a positive regulator in TGF-β-induced BMSCs [23]. In the present study, p38 signal was activated in chon- drogenic differentiation of the BMSCs induced by TGF-β after 14 days. The results of Western blot revealed that both shRNA-p38 and SB203580 could decrease the p-p38 expres- sion in TGF-β-induced BMSCs. These results were consist- ent with previous conclusions. However, the function of the ERK signal remains complex and inconclusive. Prior study reported that cyclic tensile stress may enhance the osteo- genic differentiation of periodontal ligament cells via the ERK signaling pathway, and the inhibition of ERK may Results Activation of p38 in BMSCs after TGF-β1 stimulation Activation of p38 in BMSCs after TGF-β1 stimulation To investigate whether p38 signal was involved in chondro- genesis induced by TGF-β1, the expression of p38 was ex- amined by Western blot analysis. Results showed that the expression of phosphorylated (p)-p38 was exhibited at a low level on day 0 after adding TGF-β1 (10 ng/ml), but gradually increased as chondrogenesis proceeded, and reached at a Ma et al. Journal of Orthopaedic Surgery and Research (2019) 14:434 Page 4 of 8 Fig. 1 Expression of p-p38/p38 in TGF-β1-induced BMSCs and morphological observation. a Western blotting of p-p38 and p38 expression in BMSCs following TGF-β1-induced for 0, 5, 7, and 14 days. Relative protein expression of p-p38/p38 was elevated on a time-dependent manner under the TGF-β1 induction when compared with day 0. b Representative images of BMSCs. Morphological changes of BMSCs following TGF-β1 induced for 0, 5, 7, and 14 days were observed under an inverted microscope. On day 0, BMSCs grew adherently to the wall, and the cells were triangular or polygonal in shape. From day 5 to day 14, cell morphology changed significantly and gradually presented a typical paving stone shape with uniform size and shape. p < 0.05, p < 0.01, and p < 0.001 vs. TGF-β1 (D0) Fig. 1 Expression of p-p38/p38 in TGF-β1-induced BMSCs and morphological observation. a Western blotting of p-p38 and p38 expression in BMSCs following TGF-β1-induced for 0, 5, 7, and 14 days. Relative protein expression of p-p38/p38 was elevated on a time-dependent manner under the TGF-β1 induction when compared with day 0. b Representative images of BMSCs. Morphological changes of BMSCs following TGF-β1 induced for 0, 5, 7, and 14 days were observed under an inverted microscope. On day 0, BMSCs grew adherently to the wall, and the cells were triangular or polygonal in shape. From day 5 to day 14, cell morphology changed significantly and gradually presented a typical paving stone shape with uniform size and shape. p < 0.05, p < 0.01, and p < 0.001 vs. TGF-β1 (D0) Fig. 1 Expression of p-p38/p38 in TGF-β1-induced BMSCs and morphological observation. a Western blotting of p-p38 and p38 expression in BMSCs following TGF-β1-induced for 0, 5, 7, and 14 days. Relative protein expression of p-p38/p38 was elevated on a time-dependent manner under the TGF-β1 induction when compared with day 0. b Representative images of BMSCs. Discussion Adult human MSCs are considered as promising candi- date cell sources for use in cartilage tissue engineering applications. In this study, we further clarify the Ma et al. Journal of Orthopaedic Surgery and Research (2019) 14:434 Page 5 of 8 Fig. 2 Downregulation of p38 inhibited the expression of chondrocyte-specific genes, such as p-smad1/smad1 and p-smad4/smad4 in TGF-β1- induced BMSCs. a Western blotting indicated that the protein expression of p-p38 was sharply decreased in BMSCs after transfecting with shRNA- p38 with no change on the p38 protein level. b The mRNA level of p-p38 and p38 was decreased in the shRNA-p38 group when compared with control and shRNA-NC groups from the results of qRT-PCR assays. c Representative pictures and statistical analysis results of Western blot showed that the expression of Aggrecan, collagen II, p-smad1/smad1, p-smad4/smad4, and SOX9 notably reduced in the TGF-β1-induced BMSCs when compared with control, which were upregulated by shRNA-p38 and SB203580 significantly. p < 0.05, p < 0.01, and p < 0.001 vs. control; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. TGF-β1 Fig. 2 Downregulation of p38 inhibited the expression of chondrocyte-specific genes, such as p-smad1/smad1 and p-smad4/smad4 in TGF-β1- induced BMSCs. a Western blotting indicated that the protein expression of p-p38 was sharply decreased in BMSCs after transfecting with shRNA- p38 with no change on the p38 protein level. b The mRNA level of p-p38 and p38 was decreased in the shRNA-p38 group when compared with control and shRNA-NC groups from the results of qRT-PCR assays. c Representative pictures and statistical analysis results of Western blot showed that the expression of Aggrecan, collagen II, p-smad1/smad1, p-smad4/smad4, and SOX9 notably reduced in the TGF-β1-induced BMSCs when compared with control, which were upregulated by shRNA-p38 and SB203580 significantly. p < 0.05, p < 0.01, and p < 0.001 vs. control; #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. TGF-β1 inhibit osteogenic differentiation [24]. In another study, in- hibition of p38 may suppress the osteogenic differentiation of dental pulp stem cells (DPSCs), whereas inhibition of ERK demonstrated the opposite effect [25]. Discussion In general, dif- ferences between cell type, chondrogenic culture systems, chondrogenic developmental stages, and inhibitor concen- trations may be the reason that leads to variations between these results, and diverse methods for detection of chondro- cyte differentiation were also regarded as critical factors to explain conflicting results due to differences in accuracy and efficiency [26]. In this study, hBMSCs were derived from BM of healthy adults, and a micromass chondrogenic cul- ture system was employed. The methods have been vali- dated by previous studies in micromass cultures of chick limb bud mesenchyme, pellet cultures of adult MPCs, and mouse chondrogenic ATDC5 cells [27–29]. Prior study re- vealed that phosphorylation of p38 was relatively stable and prolonged in chondrogenesis stimulated by TGF-β1, but the ERK subtype was phosphorylated in a rapid and transient manner [26]. In our study, the hMSC detection was opti- mized for a relatively appropriate duration of 14 days. It was found that the expressions of p-ERK, p-p38, and p-JNK were overexpressed in TGF-β1-induced hBMSCs, which could be reversed by p38 inhibitors. This may explain why ERK phos- phorylation was relatively difficult to detect. Chondrogenesis progresses with the expression of a cascade of stage-specific markers. Sox9 protein has been proven to be a master regulatory factor for chondrocyte differentiation [30]. It can transcriptionally activate cartilage-specific marker gene coding for collagen II and regulate synthesis of Aggrecan [31]. In our study, TGF- β1-treated cultures exhibited higher expression in colla- gen II, Aggrecan, and SOX9 after 14 days compared with the untreated controls, which were decreased by pre- treating p38 inhibitors. Moreover, TGF-β1 induced the chondrogenic differentiation of MSCs both in monolayer Ma et al. Journal of Orthopaedic Surgery and Research (2019) 14:434 Page 6 of 8 Fig. 3 Effects of p38 inhibition on GAG level and the degree of chondrocyte differentiation in TGF-β1-induced BMSCs. a GAG assay and b Alcian blue staining showed that the GAG level and the number of blue-stained cells (the nuclei of chondrocytes can be blue-stained) in BMSCs were distinctly increased in TGF-β1-induced BMSCs, while p38 silence inhibited chondrogenic differentiation significantly. p < 0.001 vs. control; ##p < 0.01 vs. TGF-β1 Fig. 3 Effects of p38 inhibition on GAG level and the degree of chondrocyte differentiation in TGF-β1-induced BMSCs. Discussion a GAG assay and b Alcian blue staining showed that the GAG level and the number of blue-stained cells (the nuclei of chondrocytes can be blue-stained) in BMSCs were distinctly increased in TGF-β1-induced BMSCs, while p38 silence inhibited chondrogenic differentiation significantly. p < 0.001 vs. control; ##p < 0.01 vs. TGF-β1 Fig. 3 Effects of p38 inhibition on GAG level and the degree of chondrocyte differentiation in TGF-β1-induced BMSCs. a GAG assay and b Alcian blue staining showed that the GAG level and the number of blue-stained cells (the nuclei of chondrocytes can be blue-stained) in BMSCs were distinctly increased in TGF-β1-induced BMSCs, while p38 silence inhibited chondrogenic differentiation significantly. p < 0.001 vs. control; ##p < 0 01 vs TGF-β1 Fig. 4 Knockdown of p38 suppressed the activation of p38/ERK/JNK signal pathway in TGF-β1-induced BMSCs. Western blot showed that the expression of p-p38, p-ERK, and p-JNK was remarkably increased in the BMSCs under TGF-β1 induced for 14 days, while shRNA-p38 and SB203580 suppressed the activation of p38/ERK/JNK signal pathway in TGF-β1-induced BMSCs. The non-phosphorylated proteins of p38, ERK, and JNK were essentially unchanged in different groups. p < 0.01 and *p < 0.001 vs. control; ##p < 0.01 and ###p < 0.001 vs. TGF-β1 Fig. 4 Knockdown of p38 suppressed the activation of p38/ERK/JNK signal pathway in TGF-β1-induced BMSCs. Western blot showed that the expression of p-p38, p-ERK, and p-JNK was remarkably increased in the BMSCs under TGF-β1 induced for 14 days, while shRNA-p38 and SB203580 suppressed the activation of p38/ERK/JNK signal pathway in TGF-β1-induced BMSCs. The non-phosphorylated proteins of p38, ERK, and JNK were essentially unchanged in different groups. p < 0.01 and ***p < 0.001 vs. control; ##p < 0.01 and ###p < 0.001 vs. TGF-β1 Ma et al. Journal of Orthopaedic Surgery and Research (2019) 14:434 Ma et al. Journal of Orthopaedic Surgery and Research (2019) 14:434 Page 7 of 8 Page 7 of 8 and in the collagen-GAG scaffold. In previous study, the treatment of MSC-seeded collagen-GAG scaffolds with TGF-β resulted in an increase in [35S] sulfate incorpor- ation which was reduced upon inhibition of the p38 pathway [18]. In our study, inhibition of the p38 led to a significant reduction of sGAG synthesis by Alcian blue staining and sGAG assay detection, which were consist- ent with the previous studies. Competing interests h h d l h The authors declare that they have no competing interests. References l 1. Felson DT, Lawrence RC, Dieppe PA, Hirsch R, Helmick CG, Jordan JM, Kington RS, Lane NE, Nevitt MC, Zhang Y, Sowers M, McAlindon T, Spector TD, Poole AR, Yanovski SZ, Ateshian G, Sharma L, Buckwalter JA, Brandt KD, Fries JF. Osteoarthritis: new insights. Part 1: the disease and its risk factors. 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Conclusion In conclusion, the present study proved that p38 signals were activated in TGF-β-induced BMSCs, and inhibition of p38 regulated the chondrogenic-specific genes such as SOX9, collagen II, Aggrecan, and sGAG level, through p38/ERK/JNK pathway in TGF-β-induced BMSCs. Mean- while, it was observed that cross-talk mechanisms between p38 and Smad1/4 pathways were involved in transcrip- tional regulation of chondrocyte-specific genes stimulated by TGF-β1. These results will deepen our understanding of the mechanism of chondrogenesis process, which pro- vided clues for the application of multipotential stem cells and the treatment of osteoarthritis. 6. Tang C, Jin C, Xu Y, Wei B, Wang L. Chondrogenic differentiation could be induced by autologous bone marrow mesenchymal stem cell-derived extracellular matrix scaffolds without exogenous growth factor. Tissue Eng Part A. 2016;22:222–32. 7. Li P, Gong Z, Shultz LD, Ren G. Mesenchymal stem cells: from regeneration to cancer. 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Discussion Therefore, our study indi- cated that TGF-β1-treated BMSCs differentiated into chondrocytes with expression of chondrocyte-specific genes, and the p38 pathway delicately regulated chon- drogenesis induced by TGF-β1. Availability of data and materials 8. Fu X, Liu G, Halim A, Ju Y, Luo Q, Song AG. Mesenchymal stem cell migration and tissue repair. Cells. 2019;8(8):784. 7. Li P, Gong Z, Shultz LD, Ren G. Mesenchymal stem cells: from regeneration to cancer. Pharmacol Ther. 2019;200:42–54. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate 13. Zhang M, Su J, Zhang Y, Xu J, Zhang S. Conveying endogenous and exogenous signals: MAPK cascades in plant growth and defense. Curr Opin Plant Biol. 2018;45:1–10. 12. Ihara S, Hirata Y, Koike K. TGF-beta in inflammatory bowel disease: a key regulator of immune cells, epithelium, and the intestinal microbiota. J Gastroenterol. 2017;52:777–87. Author details 1 f Author details 1Department of Orthopedics, Zhejiang Taizhou Central Hospital (Affiliated Hospital of Taizhou University), No. 999 Donghai Avenue, Jiaojiang District, Taizhou 318000, Zhejiang, China. 2Department of Orthopedics, Yan Cheng Third People’s Hospital (Affiliated Yancheng Hospital of Southeast University Medical College), No.2 Xindu West Road, Yancheng 224001, Jiangsu, China. In previous study, only gene expression of Smad2/3 was examined, and further studies of Smad proteins should be in- vestigated for the interaction between MAPK and Smad signaling on TGF-β stimulation [32]. Interactions between p38 pathways and Smad1/4 were investigated in the process of chondrogenesis stimulated by TGF-β1. TGF-β-induced Smad1/4 overexpression was reduced by inhibition of p38 with shRNA-p38 or SB203580, leading to the downregula- tion of chondrocyte-specific genes such as collagen II and Aggrecan. Therefore, our findings confirmed that p38 MAPK signal might regulate chondrocyte-specific genes by mediat- ing interaction between TGF-β1 and Smad1/4 molecules. This may provide a new perspective on the control of TGF- β-dependent transcriptional activation in the process of chondrogenesis. Received: 15 August 2019 Accepted: 28 November 2019 10. Spitkovsky D, Hescheler J. Adult mesenchymal stromal stem cells for therapeutic applications. Minim Invasive Ther Allied Technol. 2008;17:79–90. 11. Sierra-Sanchez A, Ordonez-Luque A, Espinosa-Ibanez O, Ruiz-Garcia A, Arias- Santiago S. Epithelial in vitro differentiation of mesenchymal stem cells. Curr Stem Cell Res Ther. 2018;13:409–22. Funding Not applicable. Page 8 of 8 Ma et al. Journal of Orthopaedic Surgery and Research (2019) 14:434 18. 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Jiang X, Huang X, Jiang T, Zheng L, Zhao J, Zhang X. The role of Sox9 in collagen hydrogel-mediated chondrogenic differentiation of adult mesenchymal stem cells (MSCs). Biomater Sci. 2018;6:1556–68. 31. Xie WF, Zhang X, Sakano S, Lefebvre V, Sandell LJ. Trans-activation of the mouse cartilage-derived retinoic acid-sensitive protein gene by Sox9. J Bone Miner Res. 1999;14:757–63. 32. Madej W, Buma P, van der Kraan P. Inflammatory conditions partly impair the mechanically mediated activation of Smad2/3 signaling in articular cartilage. Arthritis Res Ther. 2016;18:146. Ma et al. Journal of Orthopaedic Surgery and Research (2019) 14:434 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. 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https://openalex.org/W1933569466	https://dash.harvard.edu/bitstream/1/16121033/1/4427074.pdf	English	null	Effect of alcaftadine 0.25% on ocular itch associated with seasonal or perennial allergic conjunctivitis: a pooled analysis of two multicenter randomized clinical trials	Clinical ophthalmology	2,015	cc-by	5,916	Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:16121033 Published Version doi:10.2147/OPTH.S80503 doi:10.2147/OPTH.S80503 Citation Ciolino, Joseph B, Eugene B McLaurin, Nicholas P Marsico, Stacey L Ackerman, Julia M Williams, Linda Villanueva, and David A Hollander. 2015. “Effect of alcaftadine 0.25% on ocular itch associated with seasonal or perennial allergic conjunctivitis: a pooled analysis of two multicenter randomized clinical trials.” Clinical Ophthalmology (Auckland, N.Z.) 9 (1): 765-772. doi:10.2147/OPTH.S80503. http://dx.doi.org/10.2147/OPTH.S80503. Published Version doi:10.2147/OPTH.S80503 Effect of alcaftadine 0.25% on ocular itch associated with seasonal or perennial allergic conjunctivitis: a pooled analysis of two multicenter randomized clinical trials This article was published in the following Dove Press journal: Clinical Ophthalmology 2 May 2015 Number of times this article has been viewed Joseph B Ciolino1 Eugene B McLaurin2 Nicholas P Marsico3 Stacey L Ackerman4 Julia M Williams5 Linda Villanueva5 David A Hollander5 Joseph B Ciolino1 Eugene B McLaurin2 Nicholas P Marsico3 Stacey L Ackerman4 Julia M Williams5 Linda Villanueva5 David A Hollander5 Purpose: Seasonal and perennial allergic conjunctivitis represent the majority of cases of ocular allergy. This analysis was designed to evaluate the efficacy and safety of once-daily alcaftadine 0.25% in preventing ocular itching associated with seasonal or perennial allergic conjunctivitis. Subjects and methods: Pooled data from two double-masked, multicenter, placebo-controlled studies using the conjunctival allergen challenge (CAC) model of allergic conjunctivitis were analyzed. Subjects randomized to receive treatment with alcaftadine 0.25% or placebo were challenged with seasonal (grass, ragweed, trees) or perennial (cat dander, cat hair, dog dander, dust mites, cockroach) allergens, 16 hours after treatment instillation. The primary efficacy measure was subject-evaluated mean ocular itching at 3 minutes post-CAC. Secondary measures included ocular itching at 5 and 7 minutes post-CAC. The proportion of subjects with minimal itch (itch score 1) and zero itch (itch score =0), and safety were also assessed. Results: A total of 189 subjects enrolled in the two studies were treated with alcaftadine or placebo. Overall, 129 subjects were challenged with seasonal allergens and 60 subjects were challenged with perennial allergens. Alcaftadine 0.25% achieved a statistically significant reduction in mean itch score at 3, 5, and 7 minutes post-CAC compared with placebo in sub- jects challenged with seasonal allergens (P0.0001 at all time points) and those challenged with perennial allergens (P0.0001 at all time points). A higher percentage of subjects treated with alcaftadine compared with placebo achieved minimal itch (P0.001 versus placebo at all time points) and zero itch (P0.05 at all time points except 7 minutes for perennial) when challenged with either seasonal or perennial allergens. No treatment-related or serious adverse events were reported. Subjects and methods: Pooled data from two double-masked, multicenter, placebo-controlled studies using the conjunctival allergen challenge (CAC) model of allergic conjunctivitis were analyzed. Subjects randomized to receive treatment with alcaftadine 0.25% or placebo were challenged with seasonal (grass, ragweed, trees) or perennial (cat dander, cat hair, dog dander, dust mites, cockroach) allergens, 16 hours after treatment instillation. 1Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA, USA; 2Total Eye Care, P.A., Memphis, TN, USA; 3East West Eye Institute, Los Angeles, CA, USA; 4Philadelphia Eye Associates, Philadelphia, PA, USA; 5Allergan, Inc., Irvine, CA, USA Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Clinical Ophthalmology Clinical Ophthalmology Dovepress open access to scientific and medical research O r i g i n a l R e s e a r c h Effect of alcaftadine 0.25% on ocular itch associated with seasonal or perennial allergic conjunctivitis: a pooled analysis of two multicenter randomized clinical trials The primary efficacy measure was subject-evaluated mean ocular itching at 3 minutes post-CAC. Secondary measures included ocular itching at 5 and 7 minutes post-CAC. The proportion of subjects with minimal itch (itch score 1) and zero itch (itch score =0), and safety were also assessed. Results: A total of 189 subjects enrolled in the two studies were treated with alcaftadine or placebo. Overall, 129 subjects were challenged with seasonal allergens and 60 subjects were challenged with perennial allergens. Alcaftadine 0.25% achieved a statistically significant reduction in mean itch score at 3, 5, and 7 minutes post-CAC compared with placebo in sub- jects challenged with seasonal allergens (P0.0001 at all time points) and those challenged with perennial allergens (P0.0001 at all time points). A higher percentage of subjects treated with alcaftadine compared with placebo achieved minimal itch (P0.001 versus placebo at all time points) and zero itch (P0.05 at all time points except 7 minutes for perennial) when challenged with either seasonal or perennial allergens. No treatment-related or serious adverse events were reported. Conclusion: Once-daily alcaftadine 0.25% ophthalmic solution was well tolerated and demonstrated effective relief of ocular itching in subjects challenged with allergens classic for triggering either seasonal or perennial allergic conjunctivitis. Keywords: lastacaft, seasonal allergen, perennial allergen, allergic rhinoconjunctivitis, conjunctival allergen challenge © 2015 Ciolino et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Clinical Ophthalmology 2015:9 765–772 765 Introduction Allergic conjunctivitis is a common ocular condition estimated to affect 15%–40% of the general population in developed nations.1–4 Ocular allergy is characterized by an inflammatory response primarily of the conjunctival mucosa that also may affect the cornea and eyelids. Allergic conjunctivitis encompasses a group of disorders that include seasonal allergic conjunctivitis (SAC), perennial allergic conjunctivitis Clinical Ophthalmology 2015:9 765–772 0)765 submit your manuscript \| www.dovepress.com Dovepress Dovepress Ciolino et al (PAC), as well as more severe and chronic conditions of vernal keratoconjunctivitis and atopic keratoconjunctivitis.5,6 Acute allergic conjunctivitis is primarily caused by IgE- mediated mast cell degranulation and the subsequent release of histamine and other inflammatory mediators. Release of histamine induces ocular itching (the hallmark symptom of ocular allergy), as well as other ocular signs and symptoms, including redness, eyelid swelling, chemosis, and tearing. These ocular symptoms are commonly accompanied by nasal symptoms or rhinitis, and often collectively referred to as allergic rhinoconjunctivitis.7–9 Ocular itching is directly related to the action of histamine on H1 receptors in the conjunctiva.10 Other allergic symptoms have been attributed to the action of H1 or H2 receptors on the vasculature.11,12 allergens: cat hair, cat dander, dog dander, cockroach, dust mites, grasses, ragweed, and trees. Subjects may have been challenged with any of these allergens during the course of the two studies. Pooled analysis of data from the two stud- ies demonstrated that alcaftadine 0.25% achieved lower overall ocular mean itching scores compared with placebo and olopatadine 0.1%, 16 hours after treatment instillation using the CAC model.25 The objective of the current analysis of pooled data from these two similarly designed studies was to evaluate the efficacy of alcaftadine 0.25% compared with placebo in the subgroups of subjects challenged with allergens characteristic of SAC or PAC. Materials and methods Study design SAC and PAC account for the majority of cases of ocular allergy.1,6,13 Both conditions result from IgE cross-linking on the surface of mast cells following ocular exposure to allergen and present with similar signs and symptoms. SAC and PAC may differ in the specific allergen(s) that cause the reaction in an individual and in the duration of symptoms. SAC, typically characterized by an acute to subacute onset triggered primarily by tree, grass, and weed pollen, is more common and occurs during periods of high pollen counts. In contrast, PAC tends to occur throughout the year, caused typically by indoor antigens such as dust, molds, or animal dander.4,6,13,14 Ocular symptoms experienced by individuals suffering from allergic conditions can significantly impact daily activities and quality of life.15–17 Data collected from two multicenter, double-masked, randomized, placebo-controlled trials, conducted between October 2011 and December 2012 (Clinicaltrials.gov iden- tifier: NCT01470118 and NCT01732757), in five centers across the United States were pooled for analysis.22–24 Both studies were reviewed and approved by an independent review board (Alpha IRB; San Clemente, CA, USA) and conducted in accordance with the Declaration of Helsinki and International Conference of Harmonisation guidelines for Good Clinical Practice. All subjects enrolled in the study provided written informed consent and signed authorization for the Health Insurance Portability and Accountability Act prior to initiation of any procedures or treatment. Alcaftadine 0.25% ophthalmic solution (Lastacaft®; Allergan, Inc., Irvine, CA, USA) is a once-daily, dual-action antiallergic that inhibits histamine receptor activation and stabilizes mast cells and has been approved for the preven- tion of itching associated with allergic conjunctivitis in the United States.18 In preclinical investigations, alcaftadine exhibited high affinity for both H1 and H2 receptors, as well as in vitro antagonism of H4 receptors.19 Alcaftadine 0.25% has been shown to be safe and effective in the prevention of ocular itching using the conjunctival allergen challenge (CAC) model of allergic conjunctivitis. In pivotal studies, when compared with placebo, alcaftadine 0.25% was superior at reducing ocular itching at 15 minutes and 16 hours after instillation.20,21 submit your manuscript \| www.dovepress.com Dovepress 766 submit your manuscript \| www.dovepress.com Dovepress Data analysis and statistical methods Data analysis and statistical methods All subjects who were randomized comprised the intent- to-treat population used for efficacy analyses. The safety population included all randomized subjects who received at least one dose of the study treatment. Both eyes of each subject challenged with seasonal or perennial allergens were used for statistical summaries and analyses. Categorical variables were summarized using frequencies and percent- ages, and continuous variables were summarized using descriptive statistics, including the number of observations, mean, standard deviation, median, minimum, and maximum values. Hypothesis testing, unless otherwise indicated, was performed at the 5% significance level of type I error for two-sided tests. The last observation carried forward method was used to handle missing and incomplete efficacy data for the primary measure. At the second visit (day -14±3) or confirmation visit, subjects still satisfying eligibility criteria were challenged with the final concentration of allergen from the first visit to obtain baseline data. Following CAC with seasonal or perennial allergens, subjects rated ocular itching at 3, 5, and 7 minutes and those meeting qualifying criteria (post- challenge bilateral itching 2 and bilateral conjunctival redness 2 at two of the three time points assessed by the investigator [7 minutes, 15 minutes, and 20 minutes]) con- tinued to the third visit. The third visit (day 0) occurred approximately 2 weeks after visit 2, and subjects were randomized (1:1:1) to receive treatment with alcaftadine 0.25% ophthalmic solution (Lastacaft®; Allergan, Inc.), olopatadine 0.2% ophthalmic solution (Pataday®; Alcon Laboratories, Inc., Fort Worth, TX, USA), and placebo (0.3% hydroxypropyl methylcellulose [Tears Naturale® II; Alcon Laboratories, Inc.]). Each eye received a single drop of the study medication. Subjects then returned 15.5 hours later and were challenged 16+1 hours, using the allergen (seasonal or perennial) and dose estab- lished to elicit an allergic response at visits 1 and 2. The primary efficacy measure of ocular itching was summarized by visit, time point, and treatment group using descriptive statistics. The differences in the means between treatment groups were calculated, and mean ocular itching scores for each of the treatments were compared using two- sample t-tests. Additionally, the nonparametric Wilcoxon rank-sum test at each time point and repeated measures analysis of covariance model accounting for treatment and repeated time measurements within each visit were per- formed. Study treatment and clinical assessments Study treatment and clinical assessments Subject assessments were conducted at three study visits that were identical in the two trials. These assessments were included in the pooled analysis. At the first visit (day -21±3) or titration visit, subjects were challenged with increasing concentration of allergens followed by the subject rating ocular itching severity after 10 minutes. Subjects were challenged with seasonal or perennial allergens to which they had known sensitivity; allergens used for conjunctival challenge in both studies included cat dander, dog dander, cockroach, dust mite, grass, ragweed, and trees. Cat hair was also included as a possible allergen in one of the two studies. A CAC reaction was considered positive when the score for both ocular itching and conjunctival vessel bed redness was 2.0. Itching was graded by the subject on a 0–4 scale, which allowed half increments for measurements, and conjunctival redness was scored by the investigator on a 0–4 scale for ocular redness. Clinical Ophthalmology 2015:9 Subject eligibility criteria Subjects were enrolled in the two clinical studies if they were at least 10 years of age, had a history of ocular allergies, and reacted positively in a skin test in the past 24 months to cat hair, cat dander, grasses, ragweed, dog dander, cockroach, dust mite, or trees. Eligible subjects had a best-corrected visual acuity of 0.6 or better on the logMAR scale in each eye, measured using the Early Treatment Diabetic Retinopathy Study chart, and displayed a positive bilateral CAC reaction (scores 2 for itching and redness in the conjunctival ves- sel bed) within the first 10 minutes of the last instillation of allergen on visit 1 and in at least two of the three time points at visit 2 as described. Key exclusion criteria included subjects displaying symp- toms of clinically active allergic conjunctivitis at the start of each visit, defined as itching or a redness score 1 in any vessel bed, and subjects with a clinically significant ocular or systemic condition, or infection, which may confound study data. Individuals who had ocular surgery within the Two similarly designed studies were conducted compar- ing the efficacy and duration of action of once-daily alcafta- dine 0.25% and olopatadine 0.2%, and placebo using the CAC model.22–24 Subjects in the two studies were required to have a history of ocular allergies and at least one positive skin test to one or more of the following either seasonal or perennial Clinical Ophthalmology 2015:9 766 Dovepress Alcaftadine in seasonal or perennial allergic conjunctivitis Dovepress last 3 months or refractive surgery within the last 6 months or had any known allergy, contraindications, or sensitivity to the study medications were also excluded from the studies. antiallergy therapies (including over-the-counter sleeping aids that contain an antihistamine and all antihistamines), all other topical ophthalmic preparations (including tear substi- tutes), and H1 antagonist antihistamines/mast cell stabilizers (ie, olopatadine) were not permitted up to 72 hours prior to the start of the study or during the course of the study. Systemic and/or topical corticosteroids were not permitted up to 14 days prior to the start of the study or during the course of the study. Efficacy and safety endpoints The primary efficacy measure was ocular itching, 16 hours after instillation of study medication, evaluated by the sub- ject at 3 minutes post-CAC (primary endpoint) and at 5 and 7 minutes post-CAC (secondary endpoints). For safety assessment, the incidence of adverse events was monitored and the severity and relationship to the study drug was determined by the Investigator. Adverse events were coded to system organ class and preferred terms using the Medical Dictionary for Regulatory Activities, version 13.1. Data analysis and statistical methods Predefined analyses on the primary efficacy measure included comparisons of the number of subjects in each group After all evaluations were completed at the end of each visit, the use of over-the-counter antiallergy eye drop Visine-A® was permitted. Systemic and/or topical nonsteroi- dal anti-inflammatory drugs, aspirin or aspirin-containing products, prescription, over-the-counter or homeopathic Clinical Ophthalmology 2015:9 767 Dovepress Dovepress Ciolino et al with minimal itch (defined as itch score 1), and the number of subjects with zero itch (defined as itch score =0). Fisher’s exact test was conducted for comparisons at each time point (3, 5, and 7 minutes) for alcaftadine versus placebo. In the current analysis, grass, ragweed, and trees were categorized as seasonal allergens, and cat dander, dog dander, cat hair, dust mites, and cockroach were categorized as perennial allergens. Safety findings were tabulated and summarized using descriptive statistics. Statistical analyses were per- formed using SAS® software, version 9.2 (SAS Institute Inc., Cary, NC, USA). events (two in the alcaftadine 0.25% group and one in the placebo group). Baseline characteristics were similar between the alcaftadine 0.25% and placebo treatments groups with respect to age, sex, ethnicity, and race, although some dif- ferences were observed in iris color (Table 1). Efficacy of alcaftadine on ocular itching induced by seasonal or perennial allergens Treatment with alcaftadine 0.25%, 16 hours prior to allergen challenge, significantly reduced mean ocular itch- ing compared with placebo at 3, 5, and 7 minutes post-CAC in subjects challenged with seasonal or perennial allergens (Figure 2). Differences in mean ocular itching scores between alcaftadine 0.25% and placebo were statistically significant at 3, 5, and 7 minutes post-CAC (P0.0001 for all time points) for both seasonal and perennial allergens subgroups (Table 2). Alcaftadine 0.25%-treated subjects consistently demonstrated greater percentage reduction in itching from baseline at 3, 5, and 7 minutes post-CAC (seasonal allergens: -79.1%, -73.0%, and -70.4%, respectively; peren- nial allergens: -85.0%, -76.6%, and -71.0%, respectively) compared with placebo-treated subjects (seasonal allergens: -26.0%, -24.8%, and -32.0%, respectively; peren- nial allergens: -18.2%, -22.4%, and -26.0%, respectively), Subject disposition and baseline characteristics A total of 284 subjects were randomized in the two pooled studies and 189 were treated with either alcaftadine 0.25% ophthalmic solution or placebo (Figure 1). Of the 96 subjects who were treated with alcaftadine 0.25%, 63 were challenged with seasonal allergens and 33 with perennial allergens. Of the 93 subjects who received placebo, 66 were challenged with seasonal allergens and 27 with perennial allergens. Overall completion rate was high among the two treatment groups (93.8% alcaftadine 0.25% and 97.8% placebo), and only three subjects withdrew from the study due to adverse Clinical Ophthalmology 2015:9 submit your manuscript \| www.dovepress.com Dovepress 768 1XPEHURIVXEMHFWVUDQGRPL]HGLQWKHWZRVWXGLHV 1 6XEMHFWVUHFHLYLQJDOFDIWDGLQHRUSODFHER Q &RPSOHWHGQ 'LVFRQWLQXHGQ $GPLQLVWUDWLYHUHDVRQVQ $GYHUVHHYHQWVQ &RPSOHWHGQ 'LVFRQWLQXHGQ $GPLQLVWUDWLYHUHDVRQVQ $GYHUVHHYHQWVQ $OFDIWDGLQHQ 6HDVRQDODOOHUJHQVQ 3HUHQQLDODOOHUJHQVQ 3ODFHERQ 6HDVRQDODOOHUJHQVQ 3HUHQQLDODOOHUJHQVQ Figure 1 Subject disposition. 1XPEHURIVXEMHFWVUDQGRPL]HGLQWKHWZRVWXGLHV 1 6XEMHFWVUHFHLYLQJDOFDIWDGLQHRUSODFHER Q 6HDVRQDODOOHUJHQVQ 3HUHQQLDODOOHUJHQVQ 6HDVRQDODOOHUJHQVQ 3HUHQQLDODOOHUJHQVQ $GPLQLVWUDWLYHUHDVRQVQ $GYHUVHHYHQWVQ Figure 1 Subject disposition. Clinical Ophthalmology 2015:9 768 submit your manuscript \| www.dovepress.com Dovepress Alcaftadine in seasonal or perennial allergic conjunctivitis Dovepress p Table 1 Baseline demographics and characteristics (pooled intent-to-treat population) Characteristica Alcaftadine 0.25% (n=96) Placebo (n=93) Age (years) Mean±SD 38.7±13.1 36.7±12.6 Min–max, range 12–70 14–68 Sex, n (%) Male 33 (34.4) 39 (41.9) Female 63 (65.6) 54 (58.1) Ethnicity, n (%) Hispanic or Latino 9 (9.4) 9 (9.7) Not Hispanic or Latino 87 (90.6) 84 (90.3) Race, n (%) African American 12 (12.5) 16 (17.2) Asian 19 (19.8) 20 (21.5) Caucasian 56 (58.3) 53 (57.0) Otherb 9 (9.4) 4 (4.3) Iris color, n (%) Brown 118 (61.5) 124 (66.7) Blue 38 (19.8) 22 (11.8) Green 28 (14.6) 22 (11.8) Hazel 4 (2.1) 14 (7.5) Black 4 (2.1) 2 (1.1) Gray 0 2 (1.1) Notes: aPercentages are based on the total number of subjects in each treatment group except for iris color, which is based on the total number of eyes in each treatment group. bIncludes American Indian or Alaskan Native and Native Hawaiian or Other Pacific Islander. Abbreviations: max, maximum; min, minimum; SD, standard deviation. Significantly greater proportions of alcaftadine 0.25%-treated subjects compared with placebo-treated subjects achieved criteria for minimal itch (P0.001; Figure 3A) and zero itch (P0.05; Figure 3B) in the seasonal allergens subgroup. Subject disposition and baseline characteristics Similarly, among subjects who were challenged with peren- nial allergens, significantly higher proportions of alcaftadine 0.25%-treated subjects compared with placebo-treated subjects achieved an itch score 1 (P0.001; Figure 3C) and itch score =0 (P0.05 except at the 7 minutes time point; Figure 3D) compared with subjects receiving placebo. Ocular itching was further evaluated by analyzing the distribution of raw subject-reported itch scores at baseline and 16 hours post-treatment. For this analysis, all itch score data of each eye were included for all time points (3, 5, and 7 minutes) – a leftward shift in frequency of scores indicates improvement in magnitude of relief and percentage of sub- jects who had their symptoms alleviated. In both seasonal and perennial allergens subgroups, a larger proportion of alcaftadine 0.25%-treated subjects reported lower itch scores compared with placebo-treated subjects 16 hours after treatment instillation, and a greater leftward shift was observed following treatment with alcaftadine than placebo (Figure 4). Discussion within minutes. Therefore, critical to improving a patient’s quality of life is a safe and effective treatment that has a rapid onset of action and is effective against both seasonal and perennial allergens. Both SAC and PAC can cause significant discomfort, reduce quality of life, and lead to a loss of productivity, particularly during the spring and fall allergy seasons.15–17 Effectively treating the signs and symptoms of allergic conjunctivitis26 and limiting the onset of future symptoms via minimizing exposure to specific allergens27 have been shown to improve quality of life in patients with ocular allergy. The ocular manifestations of both SAC and PAC are secondary to mast cell degranulation and subsequent allergic inflammation in sensitized individuals. Once sensitized, exposure to either a seasonal or perennial aller- gen may elicit a classic allergic conjunctivitis response Topical ophthalmic antihistamines are the primary treatment option for patients with allergic conjunctivitis. With antagonistic activity against H1, H2, and H4 receptors, alcaftadine is a dual-action antiallergic agent that directly inhibits histamine receptor activation and indirectly reduces allergic responses by stabilizing mast cells.28,29 The role of H4 receptors in allergic conjunctivitis has not been fully elucidated; in vitro studies suggest that histamine binding to H4 receptors may mediate eosinophil chemotaxis.30 In vivo $ PLQXWHV PLQXWHV PLQXWHV 6XEMHFWVZLWK PLQLPDOLWFK $OFDIWDGLQH 3ODFHER $ ' PLQXWHV PLQXWHV PLQXWHV PLQXWHV PLQXWHV PLQXWHV 6XEMHFWVZLWK ]HURLWFK 6XEMHFWVZLWK PLQLPDOLWFK 3HUHQQLDODOOHUJHQV $OFDIWDGLQH 3ODFHER % PLQXWHV PLQXWHV PLQXWHV 6XEMHFWVZLWK ]HURLWFK 6HDVRQDODOOHUJHQV $OFDIWDGLQH 3ODFHER & PLQXWHV PLQXWHV PLQXWHV 6XEMHFWVZLWK PLQLPDOLWFK $OFDIWDGLQH 3ODFHER $OFDIWDGLQH 3ODFHER Figure 3 Comparison of the percentage of subjects with minimal itch (itch score 1) and zero itch (itch score =0), 16 hours after treatment instillation at 3, 5, and 7 minutes, for alcaftadine 0.25% and placebo post-conjunctival allergen challenge with seasonal (A and B) and perennial (C and D) allergens. Notes: P0.001 versus placebo; P0.05 versus placebo; calculated using Fisher’s exact test. Safety outcomes In the pooled population, a total of 11 adverse events were reported among the 189 subjects receiving alcaftadine 0.25% and placebo. Four alcaftadine 0.25%-treated subjects and one placebo-treated subject experienced at least one adverse event during the course of the studies. There were no treatment- related adverse events recorded and no serious adverse events occurred in the two pooled studies. with similar efficacy demonstrated between those subjects challenged with seasonal allergens and those subjects chal- lenged with perennial allergens. Ocular itching was also assessed by comparing the proportion of subjects who met the criteria of minimal itch (itch score 1) and zero itch (itch score =0) at the three time points measured post-CAC (3, 5, and 7 minutes). $ % 6HDVRQDODOOHUJHQV 0HDQRFXODULWFKVFRUH 0HDQRFXODULWFKVFRUH 3HUHQQLDODOOHUJHQV PLQXWHV PLQXWHV PLQXWHV PLQXWHV PLQXWHV PLQXWHV $OFDIWDGLQH 3ODFHER $OFDIWDGLQH 3ODFHER Figure 2 Comparison of mean itch scores 16 hours after treatment instillation at 3, 5, and 7 minutes, for alcaftadine 0.25% and placebo post-conjunctival allergen challenge with seasonal (A) and perennial (B) allergens. Notes: P0.0001 versus placebo; calculated using two-sample t-test. $ 6HDVRQDODOOHUJHQV 0HDQRFXODULWFKVFRUH PLQXWHV PLQXWHV PLQXWHV $OFDIWDGLQH 3ODFHER % 0HDQRFXODULWFKVFRUH 3HUHQQLDODOOHUJHQV PLQXWHV PLQXWHV PLQXWHV $OFDIWDGLQH 3ODFHER $ % Figure 2 Comparison of mean itch scores 16 hours after treatment instillation at 3, 5, and 7 minutes, for alcaftadine 0.25% and placebo post-conjunctival allergen challenge with seasonal (A) and perennial (B) allergens. Notes: P0.0001 versus placebo; calculated using two-sample t-test. Clinical Ophthalmology 2015:9 769 Dovepress Dovepress Ciolino et al Table 2 Differences in mean ocular itching scores 16 hours post-treatment instillation Time point post-CAC Seasonal allergens Perennial allergens 3 min 5 min 7 min 3 min 5 min 7 min Alcaftadine 0.25%–placebo, difference -1.56 -1.46 -1.17 -1.58 -1.43 -1.18 P-valuea 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 Note: aP-values calculated using two-sample t-test. Abbreviations: CAC, conjunctival allergen challenge; min, minute. Table 2 Differences in mean ocular itching scores 16 hours post-treatment instillation Discussion & PLQXWHV PLQXWHV PLQXWHV 6XEMHFWVZLWK PLQLPDOLWFK $OFDIWDGLQH 3ODFHER $ & ' PLQXWHV PLQXWHV PLQXWHV 6XEMHFWVZLWK ]HURLWFK 3HUHQQLDODOOHUJHQV $OFDIWDGLQH 3ODFHER % PLQXWHV PLQXWHV PLQXWHV 6XEMHFWVZLWK ]HURLWFK 6HDVRQDODOOHUJHQV $OFDIWDGLQH 3ODFHER % ' Figure 3 Comparison of the percentage of subjects with minimal itch (itch score 1) and zero itch (itch score =0), 16 hours after treatment instillation at 3, 5, and 7 minutes, for alcaftadine 0.25% and placebo post-conjunctival allergen challenge with seasonal (A and B) and perennial (C and D) allergens. Notes: P0.001 versus placebo; **P0.05 versus placebo; calculated using Fisher’s exact test. Clinical Ophthalmology 2015:9 770 submit your manuscript \| www.dovepress.com Dovepress Alcaftadine in seasonal or perennial allergic conjunctivitis Dovepress $ 6XEMHFWV ,WFKVFRUHDWEDVHOLQH $OFDIWDGLQH 3ODFHER Figure 4 Distribution of itch scores of each eye at baseline (untreated) and 16 hours after treatment instillation with alcaftadine 0.25% or placebo, at all time points measured (3, 5, and 7 minutes) post-conjunctival allergen challenge with seasonal (A and B) and perennial (C and D) allergens. $ ' 6XEMHFWV ,WFKVFRUHDWEDVHOLQH ,WFKVFRUHKRXUVSRVWWUHDWPHQWLQVWLOODWLRQ 3HUHQQLDODOOHUJHQV 6XEMHFWV & 6XEMHFWV ,WFKVFRUHDWEDVHOLQH $OFDIWDGLQH 3ODFHER $OFDIWDGLQH 3ODFHER $OFDIWDGLQH 3ODFHER % 6XEMHFWV ,WFKVFRUHKRXUVSRVWWUHDWPHQWLQVWLOODWLRQ 6HDVRQDODOOHUJHQV $OFDIWDGLQH 3ODFHER & 6XEMHFWV ,WFKVFRUHDWEDVHOLQH $OFDIWDGLQH 3ODFHER & $ ' ,WFKVFRUHKRXUVSRVWWUHDWPHQWLQVWLOODWLRQ 3HUHQQLDODOOHUJHQV 6XEMHFWV $OFDIWDGLQH 3ODFHER % 6XEMHFWV ,WFKVFRUHKRXUVSRVWWUHDWPHQWLQVWLOODWLRQ 6HDVRQDODOOHUJHQV $OFDIWDGLQH 3ODFHER ' % Figure 4 Distribution of itch scores of each eye at baseline (untreated) and 16 hours after treatment instillation with alcaftadine 0.25% or placebo, at all time points measured (3, 5, and 7 minutes) post-conjunctival allergen challenge with seasonal (A and B) and perennial (C and D) allergens. Clinical Ophthalmology 2015:9 Discussion studies have also suggested that H4 receptors play a role in mediating inflammatory and pruritic responses.31 allergens and the remaining patients were challenged with perennial allergens. While reduction of itching by alcaftadine was similar in subjects challenged with either seasonal or perennial allergens, the smaller sample size in the perennial allergen population likely accounted for the lack of statisti- cal difference at the 7-minute time point in the percent of patients with zero itch analysis. In the current analysis of pooled data from two, large, similarly designed clinical studies, alcaftadine 0.25% was well tolerated and demonstrated significant reduction in ocular itching induced by both seasonal and perennial aller- gens at all time points (3, 5, and 7 minutes) measured after conjunctival challenge with allergen using the CAC model. In addition, the reduction in itching by alcaftadine was similar between those subjects challenged with seasonal allergens and those challenged with perennial allergens. Significantly higher proportions of subjects challenged with seasonal or perennial allergens achieved minimal itch (itch score 1) or zero itch (itch score =0) after treatment with alcaftadine 0.25% compared with placebo. Alcaftadine was shown to have a rapid onset of action upon challenge with both seasonal and perennial allergens, up to 16 hours post drop instillation. This rapid and sustained effect of alcaftadine may be because of its ability to prevent loss of epithelial tight junction proteins (zonula occludens-1, E-cadherin) and to reduce eosinophil infiltration, as has been shown in animal models of allergic conjunctivitis.19 Overall, alcaftadine is well tolerated and is effective in reducing ocular itching associated with allergic conjunctivitis in response to both seasonal and perennial allergens. Acknowledgments This study was sponsored by Allergan, Inc., Irvine, CA, USA. Writing and editorial assistance was provided to the authors by Kakuri Omari, PhD, of Evidence Scientific Solutions, and funded by Allergan, Inc., Irvine, CA, USA. All authors met the ICMJE authorship criteria. Neither honoraria nor payments were made for authorship. References 1. Butrus S, Portela R. Ocular allergy: diagnosis and treatment. Ophthalmol Clin North Am. 2005;18(4):485–492. 2. Katelaris CH, Bielory L. Evidence-based study design in ocular allergy trials. Curr Opin Allergy Clin Immunol. 2008;8(5):484–488. 21. Torkildsen G, Shedden A. The safety and efficacy of alcaftadine 0.25% ophthalmic solution for the prevention of itching associated with allergic conjunctivitis. Curr Med Res Opin. 2011;27(3):623–631. 3. Rosario N, Bielory L. Epidemiology of allergic conjunctivitis. Curr Opin Allergy Clin Immunol. 2011;11(5):471–476. 22. Ackerman S, D’Ambrosio F Jr, Greiner JV, Villanueva L, Ciolino JB, Hollander DA. A multicenter evaluation of the efficacy and duration of action of alcaftadine 0.25% and olopatadine 0.2% in the conjunctival allergen challenge model. J Asthma Allergy. 2013;6:43–52. 4. Wong AH, Barg SS, Leung AK. Seasonal and perennial allergic conjunctivitis. Recent Pat Inflamm Allergy Drug Discov. 2014;8(2):139–153. 5. Bielory L. Ocular allergy overview. Immunol Allergy Clin North Am. 2008;28(1):1–23,v. 23. McLaurin EB, Marsico NP, Ciolino JB, Williams JM, Hollander DA. Alcaftadine 0.25% versus Olopatadine 0.2% in the Prevention of Ocular Itching in Allergic Conjunctivitis. Baltimore, MD: Paper presented at: Annual Scientific Meeting; November 7–11, 2013; American College of Allergy, Asthma and Immunology; 2013. 6. La Rosa M, Lionetti E, Reibaldi M, et al. Allergic conjunctivitis: a comprehensive review of the literature. Ital J Pediatr. 2013;39:18. 7. Abelson MB, Smith L, Chapin M. Ocular allergic disease: mechanisms, disease sub-types, treatment. Ocul Surf. 2003;1(3):127–149. 8. Akdis CA, Blaser K. Histamine in the immune regulation of allergic inflammation. J Allergy Clin Immunol. 2003;112(1):15–22. 24. McLaurin EB, Marsico NP, Ciolino JB, Williams JM, Hollander DA. Alcaftadine 0.25% versus Olopatadine 0.2% in Prevention of Ocular Itching Due to Allergic Conjunctivitis in a CAC™ Model. New Orleans, LA: Paper presented at: Annual Meeting; November 16–19, 2013; American Academy of Ophthalmology; 2013. 9. Leonardi A, Bogacka E, Fauquert JL, et al. Ocular allergy: recognizing and diagnosing hypersensitivity disorders of the ocular surface. Allergy. 2012;67(11):1327–1337. 10. Udell IJ, Abelson MB. Animal and human ocular surface response to a topical nonimmune mast-cell degranulating agent (compound 48/80). Am J Ophthalmol. 1981;91(2):226–230. 25. McLaurin EB, Marsico NP, Ackerman SL, et al. Ocular itch relief with alcaftadine 0.25% versus olopatadine 0.2% in allergic conjunctivitis: pooled analysis of two multicenter randomized clinical trials. Adv Ther. 2014;31(10):1059–1071. 11. Abelson MB, Udell IJ. H2-receptors in the human ocular surface. Arch Ophthalmol. 1981;99(2):302–304. 26. Scoper SV, Berdy GJ, Lichtenstein SJ, et al. Disclosure Joseph B Ciolino is supported by a Career Development Award from Research to Prevent Blindness, Inc., and the National Eye Institute. Eugene B McLaurin has received research support from Aciex, Acucela, Alcon, Allergan, Inc., AstraZeneca, Bausch & Lomb, Inotek Pharmaceuti- cals, InSite Vision, and Lexicon Pharmaceuticals. Stacey L Ackerman has received research support from Aciex, Alcon, Allergan, Inc., and Bausch & Lomb. Julia M Williams, Limitations of this study are inherent in any pooled analy- sis, though the two studies had similar designs. In addition, there was no prespecified percentage of patients to be tested in either study with a specific type of allergen. Approximately two-thirds of patients in the alcaftadine and control groups were challenged with allergens categorized as seasonal submit your manuscript \| www.dovepress.com Dovepress 771 submit your manuscript \| www.dovepress.com Dovepress Dovepress Ciolino et al 18. Lastacaft (alcaftadine ophthalmic solution) 0.25% [Package insert]. Irvine, CA: Allergan Inc.; 2011. Linda Villanueva, and David A Hollander are employees of Allergan, Inc., Irvine, CA, USA. Nicholas P Marsico has no conflict to disclose related to this work. 19. Gallois-Bernos A, Thurmond RL. Pharmacology of alcaftadine, a new antihistamine for ocular allergy. Invest Ophthalmol Vis Sci. 2011; 52:e6426. 20. Greiner JV, Edwards-Swanson K, Ingerman A. Evaluation of alcaftadine 0.25% ophthalmic solution in acute allergic conjunctivitis at 15 minutes and 16 hours after instillation versus placebo and olopatadine 0.1%. Clin Ophthalmol. 2011;5:87–93. Clinical Ophthalmology Clinical Ophthalmology References Perception and quality of life associated with the use of olopatadine 0.2% (Pataday) in patients with active allergic conjunctivitis. Adv Ther. 2007;24(6):1221–1232. 12. Smith JA, Mansfield LE, de Shazo RD, Nelson HS. An evaluation of the pharmacologic inhibition of the immediate and late cutaneous reaction to allergen. J Allergy Clin Immunol. 1980;65(2):118–121. 27. Stillerman A, Nachtsheim C, Li W, Albrecht M, Waldman J. Efficacy of a novel air filtration pillow for avoidance of perennial allergens in symptomatic adults. Ann Allergy Asthma Immunol. 2010;104(5): 440–449. 13. Gomes PJ. Trends in prevalence and treatment of ocular allergy. Curr Opin Allergy Clin Immunol. 2014;14(5):451–456. 14. Offiah I, Calder VL. Immune mechanisms in allergic eye diseases: what is new? Curr Opin Allergy Clin Immunol. 2009;9(5):477–481. 28. Namdar R, Valdez C. Alcaftadine: a topical antihistamine for use in allergic conjunctivitis. Drugs Today (Barc). 2011;47(12):883–890. 15. Virchow JC, Kay S, Demoly P, Mullol J, Canonica W, Higgins V. Impact of ocular symptoms on quality of life (QoL), work productivity and resource utilisation in allergic rhinitis patients – an observational, cross sectional study in four countries in Europe. J Med Econ. 2011;14(3): 305–314. 29. Wade L, Bielory L, Rudner S. Ophthalmic antihistamines and H1-H4 receptors. Curr Opin Allergy Clin Immunol. 2012;12(5):510–516. 30. Ling P, Ngo K, Nguyen S, et al. Histamine H4 receptor mediates eosinophil chemotaxis with cell shape change and adhesion molecule upregulation. Br J Pharmacol. 2004;142(1):161–171. 16. Blaiss MS. Allergic rhinoconjunctivitis: burden of disease. Allergy Asthma Proc. 2007;28(4):393–397. 31. Thurmond RL, Gelfand EW, Dunford PJ. The role of histamine H1 and H4 receptors in allergic inflammation: the search for new antihistamines. Nat Rev Drug Discov. 2008;7(1):41–53. 17. Palmares J, Delgado L, Cidade M, Quadrado MJ, Filipe HP. Allergic conjunctivitis: a national cross-sectional study of clinical characteristics and quality of life. Eur J Ophthalmol. 2010;20(2):257–264. Dovepress submit your manuscript \| www.dovepress.com Publish your work in this journal PubMed Central and CAS, and is the official journal of The Society of Clinical Ophthalmology (SCO). The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors. Clinical Ophthalmology is an international, peer-reviewed journal covering all subspecialties within ophthalmology. Key topics include: Optometry; Visual science; Pharmacology and drug therapy in eye diseases; Basic Sciences; Primary and Secondary eye care; Patient Safety and Quality of Care Improvements. This journal is indexed on Submit your manuscript here: http://www.dovepress.com/clinical-ophthalmology-journal Clinical Ophthalmology 2015:9 772 Dovepress
https://openalex.org/W4391995669	https://revistes.ub.edu/index.php/segleXX/article/download/45903/41365	Spanish; Castilian	null	Menjar en temps de guerra. Joan Comorera front les necessitats alimentàries de la rereguarda	Segle XX	2,024	cc-by	12,740	Revista catalana d’història 16 (2023), 25 -44 Revista catalana d’història 16 (2023), 25 -44 /25 Recerques i Assajos Menjar en temps de guerra. Joan Comorera front les necessitats alimentàries de la rereguarda Rosa Toran Belver* Amical de Mauthausen resum A mesura que els colpistes conquerien territori, s’agreujaren els problemes de subministres a la rereguarda republicana, sent especialment crucial l’escassetat d’ali- ments. Des de les conselleries de Proveïments i d’Economia, Joan Comorera posà en marxa menjadors populars i restaurants econòmics. I, atesa la vulnerabilitat dels infants, organitzà menjadors infantils en els cines de Barcelona. L’experiència s’es- tengué a altres poblacions industrials i repercutí en les rutines de les dones, com- promeses en realitzar el treball dels homes que estaven al front. El sosteniment dels menjadors infantils corria a càrrec d’unitats de l’Exèrcit Popular, amb l’objectiu de vincular el front i la rereguarda, sense deixar de banda la vessant educativa i els propòsits revolucionaris de l’endemà. Paraules clau: guerra, rereguarda, Joan Comorera, menjadors populars, menja- dors infantils, restaurants econòmics Comer en tiempos de guerra. Joan Comorera frente a las necesidades de alimentación de la retaguardia A medida que los golpistas conquistaban territorio, se agravaron los problemas de suministros en la retaguardia republicana, siendo especialmente crucial la esca- sez de alimentos. Desde las consejerías de Abastecimientos y de Economía, Joan Comorera puso en marcha comedores populares y restaurantes económicos y, dada la vulnerabilidad de los niños, organizó comedores infantiles en los cines de Bar- celona. La experiencia se extendió a otras poblaciones industriales y repercutió en las rutinas de las mujeres, comprometidas en realizar el trabajo de los hombres que estaban en el frente. El sostén de los comedores infantiles corría a cargo de unidades del Ejército Popular, con el objetivo de vincular el frente y la retaguardia, sin dejar de lado la vertiente educativa y los propósitos revolucionarios del mañana. Palabras clave: guerra, retaguardia, Joan Comorera, comedores populares, restau- rantes económicos, comedores infantiles. Rosa Toran Belver \| Menjar en temps de guerra Recerques i Assajos ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Fecha de recepción: 20/06/2023 Fecha de aceptación: 23/09/2023 Article pòstum de Rosa Toran Belver, que va morir el 30 de juny de 2023 Article pòstum de Rosa Toran Belver, que va morir el 30 de juny de 2023 Fecha de recepción: 20/06/2023 Fecha de aceptación: 23/09/2023 ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 /26 Rosa Toran Belver \| Menjar en temps de guerra g Joan Comorera facing food needs at the rearguard As coup plotters conquered territory, problems with distribution of goods in the Republican rearguard increased, food products being specially scarce. From the departments of Supplies and Economic Affairs, Joan Comorera set in motion soup kitchens and cheap restaurants, and for the children, given their particu- lar vulnerability, special dining-rooms inside Barcelona cinemas. This experience spread to other industrial cities and had an effect on the routines of women who were compromised in doing the jobs of men at the front. The support of children’s dining-rooms was taken over by the People’s Army Units, their objective being to connect the front and the rearguard without forgetting the educational side and the future revolutionary purposes. Key words: War, Rearguard, Joan Comorera, Soup kitchens, Cheap restaurants, Children’s dining-rooms § § Tots els relats personals sobre la vida quotidiana en temps de guerra apunten a la fam, sobre- tot en els nuclis urbans, però existeix un buit historiogràfic sobre els mecanismes per pal·liar- la i sobre les estratègies polítiques des de les conselleries de Proveïments i Economia que es crearen de bell antuvi als municipis, no exemptes de polèmiques i de confrontacions en les formes d’obtenció d’aliments, sobretot a comarques, i també de subministres a l’altra banda de la frontera. A mesura que la guerra impactava de forma directa la rereguarda catalana, les carències ali- mentàries estigueren en primer pla, sent la població infantil la més afectada. Moltes foren les iniciatives, sorgides des de les pròpies institucions públiques, amb la cooperació de col·lectius nacionals i internacionals, que a partir de 1937, i de forma més intensa l’any següent, malda- ren per pal·liar aquest greu problema. A Catalunya i a la ciutat de Barcelona, a banda de les ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 Rosa Toran Belver \| Menjar en temps de guerra /27 Recerques i Assajos colònies escampades per tot el territori i acollidores de la infància evacuada, s’organitzaren menjadors des de la Comisión de Auxilio Femenino del Ministerio de Defensa, el Centre de Reserva i Especialització d’Artilleria (crea), el Consejo Nacional de Ayuda a la Infancia Evacuada o l’Ajut Infantil a la Rereguarda, alhora que es reobrien algunes cantines escolars sota la tutela de l’Ajuntament de Barcelona i del Consell de l’Escola Nova Unificada (cenu). Però no solament els infants esdevenien víctimes de les dures circumstàncies, sinó que també la població adulta estava subjecta a escassetat, a racionaments i a la mesquinesa dels estraperlistes, mentre la nova inserció de la dona en el món laboral modificava el seu rol domèstic. Aquestes realitats abocaven a la cerca de solucions cooperatives i dirigistes. En les següents pàgines, l’estudi se centra bàsicament en la capital catalana i arrenca de les informacions de la premsa de l’època, especialment el diari Treball, òrgan oficial del psu, ama- tent a difondre l’obra de Joan Comorera Soler, secretari general del partit, i les seves iniciatives per tal de satisfer les necessitats alimentàries de la població des de les conselleries d’Economia i Proveïments. 1 La Federació Gastronòmica de la Unió General de Treballadors (f.o.s.i.g.). Treball, 4-8-1936, 6. 2 Partit Socialista Unificat de Catalunya. Una visita a casa nostra. Treball, 31-7-1936, 6. § Nomenat conseller d’Economia en nom del psu des del 31 de juliol al 6 d’agost de 1936, passà a Proveïments en nom de la ugt des del 17 de desembre de 1936 fins al 4 d’abril de l’any següent, per tornar a ocupar la d’Economia en representació del psu des del 29 de juny de 1937 fins a la fi de la guerra. Va ser durant aquest segon i més llarg mandat que contribuí a desplegar tres propostes noves: els menjadors populars, els restaurants econòmics i els menjadors infantils. Rosa Toran Belver \| Menjar en temps de guerra Recerques i Assajos La proliferació i diversitat dels dits serveis, un cop superada l’etapa d’atenció prioritària als milicians, comportaren al cap de poc temps, el 15 d’agost, la creació de la Comissió de Con- trol de Menjadors, ja anomenats Menjadors Populars, sota control de la ugt i la cnt, que anà dictant normes per a la seva utilització.3 Els seus usuaris eren obrers en atur forçós i disposats a treballar per a l’economia revolucionària quan se’ls necessités, així com pobres de solem- nitat de caràcter vitalici. Després d’aquest primer pas, la Conselleria d’Abastiments, en mans del cenetista Josep Juan Domènech des del 26 de setembre, creà el Comitè Obrer de Control de la Indústria Gastronòmica. Per regular el règim de menjadors i evitar l’aprofitament per part de desaprensius, els sindicats i comitès de barriada havien d’emetre targetes familiars per als obrers en atur, vàlides per una setmana, amb indicació del menjador pertinent i l’hora de l’àpat; a més de la presentació de nòmina dels que treballaven als menjadors.4 A mitjan desem- bre de 1936, s’enumeraren les provisions aportades als menjadors populars: 700.000 quilos de pa, 600.000 de patates, 100.000 de mongetes i 170.000 dotzenes d’ous. Però calia afrontar els problemes de l’escassetat d’aliments tant al front com a la rereguarda, reflex un de l’altre, i el conseller cenetista Josep Juan Domènech, en els darrers dies del seu mandat a Abastiments, en una conferència radiada sobre “Les dificultats d’abastiment i com deuen comportar-se tots els ciutadans”, ja apel·là als sacrificis i a l’interès col·lectiu, al mateix temps que anunciava la targeta de racionament.5 En assumir Joan Comorera la Conselleria de Proveïments el 17 de desembre, cinc mesos desprès del cop d’Estat, ja s’havia consumit tot l’estoc emmagatzemat, no s’havia produït prou i les finances estaven exhaurides. De fet, la demanda de productes alimentaris superava l’oferta, amb el consegüent augment de preus dels més bàsics i la impossibilitat per bona part de la població de poder adquirir-los, situació agreujada per l’arribada de refugiats a Catalunya. Da- vant aquesta mancança, els ajuntaments crearen comissions d’abastiments que gestionaven els productes disponibles i en fixaven els preus, mesures que no evitaren l’aparició d’un mercat negre que eludia les normes establertes. En aterrar a la conselleria, Comorera es va trobar que hi havia comitès i consellers munici- pals amb molt poder i situacions de privilegi que dificultaven l’intercanvi camp-ciutat. 3 Avisos y comunicaciones. La Vanguardia, 15-8-1936, 6. 4 Consejo de Gastronomía. El control del personal. La Vanguardia, 21-10-1936, 1-2. 5 El abastecimiento de Cataluña. La Vanguardia, 13-12-1936, 2. 1. El combat contra les carències alimentàries Des dels primers dies de la revolució, quan la ciutat de Barcelona bullia per les lluites al carrer, la vida quotidiana va quedar totalment trastocada. Una de les necessitats peremptòries era l’atenció als milicians de la mateixa ciutat o arribats de diversos indrets per partir cap al front d’Aragó. Els voluntaris es queixaven que a les barricades només menjaven pa i xoriço extremeny, i foren els mateixos sindicats qui desplegaren la seva experiència per organitzar els serveis, des d’hospitals de sang fins a menjadors, atesos per milicians i milicianes, en centres, fàbriques i ateneus o en restaurants tancats o requisats. També les cantines dels grups escolars de l’ajuntament barceloní van convertir-se en menjadors populars, amb una notable audiència, fet que afegí més fragilitat a la població infantil que havien atès fins aleshores. Després del cop d’Estat, la recentment creada Federació Obrera de Sindicats de la Indús- tria Gastronòmica de Catalunya (fosig) ingressà a la ugt i de bell antuvi es posà al servei de les milícies, comptant amb l’experiència d’haver organitzat alguns serveis de proveïments de l’Olimpíada Popular.1 N’esdevingué un emblema l’Hotel Gastronòmic núm. 1 a l’Hotel Ritz, “Menjador Popular al servei de la Revolució”, gestionat per la cnt i la ugt, o l’Hotel Suís de la plaça de l’Àngel. I a un dels edificis més emblemàtics de la Barcelona benestant, el monu- mental Círculo Ecuestre, ubicat al passeig de Gràcia 38, requisat pel psu, a més de servir dos mil coberts diaris, s’hi disposava de dutxes, serveis sanitaris, barberia…2 ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 /28 Rosa Toran Belver \| Menjar en temps de guerra Rosa Toran Belver \| Menjar en temps de guerra El seu objectiu va ser aturar les ingerències dels comitès locals, amb una política implacable i rigorosa, mentre la població començava a manifestar signes de protesta i les dones s’esvalotaven a les cues dels mercats, en diversos episodis que havien començat el mes de novembre de 1936 i que continuaren fins al febrer de l’any següent. La cnt-fai titllà Comorera de “l’home de les cues”, mentre que des dels rengles del seu partit es cridava “menys comitès, més pa”. Dues realitats s’encavalcaven: la crònica animadversió entre Comorera i els anarquistes, l’augment de preus i l’escassetat i dificultats en la distribució del blat, els principals mercats del qual estaven en mans dels feixistes. La línia empresa a la conselleria no era aliena al govern de plens poders o d’unitat, encapçalat pel conseller en cap Josep Tarradellas des del 26 de setembre de 1936, que atorgava autoritat i donava mans lliures als consellers. Sanejar l’economia, augmentar la producció, estructurar les indústries de guerra i una nova política fiscal, a banda de la creació de l’Exèrcit Popular, foren objectius prioritaris del Govern català, i pel que fa als proveïments, centralització, amb la creació a cada comarca de la Delegació de la Conselleria de Proveïments ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 osa Toran Belver \| Menjar en temps de guerra /29 Recerques i Assajos i de dipòsits comarcals, amb amenaces de requisa pels productes adquirits de forma unilateral a l’estranger.6 Sense estocs de blat i farina, es planejà la seva adquisició a França i a l’urss, i l’es- tabliment de targetes de racionament la segona setmana de gener. En definitiva, hom tractava de restablir l’ordre a la rereguarda, objectiu que topà amb dificultats i derivà en la confrontació amb la cnt-fai, que jutjava el redreçament i control com a una desviació contrarevolucionària i revisionista. La reconstrucció de la unitat sota el paraigua del Front Popular, amb l’objectiu prioritari de guanyar la guerra, se sobreposava al de la unitat antifeixista, estratègia que es reflectí en el que fou el quart Govern de Companys, format el 29 de juny de 1937. Les conselleries econò- miques passaren de la cnt al psu. Rosa Toran Belver \| Menjar en temps de guerra Proveïments quedà en mans de Miquel Serra Pàmies i Co- morera ocupà Economia, que mantingué fins al gener de 1939, amb Estanislau Ruiz Ponseti com a sotssecretari, veritable inspirador de la política econòmica del psu, en la línia de dirigir i planificar l’economia, a través de la coordinació de les empreses col·lectivitzades, i d’encarar l’estancament de la producció, l’acaparament i l’especulació que derivaven en inflació. En aquest llarg mandat Comorera destacà per la seva gran capacitat de treball, en un con- text bèl·lic cada cop més complicat per a la República, que esperonà un nou comitè d’enllaç ugt-cnt, creat el 18 de març de 1938, davant l’amenaça del feixisme, i una cada cop major influència del pce i de les orientacions de Moscou, on ell mateix hi feu una llarga estada, de gener a març de 1938. p 8 Tribunales de subsistencias. La Vanguardia, 9-3-1938, 10. 6 Política de proveïments. Treball, 25-12-1936, 1. p , 5 93 , 7 Atenció amb els especuladors ambulants. Treball, 25-2-1938 6 Política de proveïments. Treball, 25-12-1936, 1. 7 Atenció amb els especuladors ambulants. Treball, 25-2-1938, 9. 8 Tribunales de subsistencias. La Vanguardia, 9-3-1938, 10. 9 Resolució política del Comitè Central del p.s.u, presa en la seva reunió plenària celebrada a Barcelona els dies 5, 6 i 7 de juny. Treball, 12-6-1938, 10. Resolució política del Comitè Central del p.s.u, presa en la seva reunió plenària celebrada a Barcelona els dies 5, 6 i 7 uny. Treball, 12-6-1938, 10. Rosa Toran Belver \| Menjar en temps de guerra Recerques i Assajos sig demanava i projectava crear grans menjadors populars a les grans fàbriques, sobretot a les indústries de guerra i barriades obreres de tot Espanya.10 A mesura que la guerra avançava i els fronts s’enduien cada cop més homes, les dones esdevenien una mà d’obra indispensable per a les indústries de guerra, fenomen que exigia deslliurar-les de les tradicionals responsabilitats culinàries i de l’atenció als infants. Així, la de- manda de menjadors populars i guarderies fou un dels punts clau en les conclusions de la I Conferència Nacional de Dones del psuc, tot argumentant que les dones no havien de tenir vergonya pel fet d’alliberar-se de les rutines i obligacions familiars i havien d’oblidar-se de la preocupació de cercar individualment el menjar.11 Aquesta reivindicació fou també plantejada en la Conferencia Nacional de Mujeres Antifascistas a València, en què es demanaren menja- dors col·lectius i guarderies a cada barriada obrera i al costat de cada fàbrica, per incorporar la dona al treball,12 i reiterada en el I Congrés Nacional de la Dona, celebrat els dies 6, 7 i 8 de novembre de 1937, del qual sorgí la Unió de Dones de Catalunya. Des de la Conselleria d’Economia, ben connectada amb la de Proveïments, que també ocu- pava un correligionari del psuc, Miquel Serra Pàmies, en els primers mesos de 1938 es dictaren un seguit de mesures per millorar el proveïment de pa, un dels motius de descontentament popular. La Comissió Interventora, integrada per la Conselleria de Finances i Economia, tot i les inicials prevencions dels industrials, va elaborar un cens dels forns de pa que va permetre les inscripcions acurades, amb la presentació dels rebuts de la contribució al dia. La regularització va permetre posar fi als abusos, especialment el de destinar bona part de les quantitats lliurades de farina a altres usos, de manera que es va poder suavitzar el racionament de pa a la població.13 Era l’hora d’adaptar la totalitat de la indústria gastronòmica a les necessitats del moment i d’acabar amb els especuladors. Els menjadors populars havien de complir l’objectiu de facilitar l’increment del treball a les indústries de guerra. El repte era de tal magnitud que calia una gran transformació de la indústria del ram, i Joan Comorera creà la Comissió Interventora de la Indústria Gastronòmica, el 19 de gener de 1938,14 i en nomenà president Agustí Coma. 15 Pròxim Congrés de la f.o.s.i.g. Treball, 20-2-1938, 13. 11 La primera Conferència Nacional de Dones del p.s.u.Treball, 16-3-1937, 8. g 16 Un objectiu immediat a complir pels obrers gastronòmics. Treball, 24 -3-1938, 7. 12 La Conferencia Nacional de Mujeres Antifascistas. La Vanguardia, 2-11-1937, 4. 13 Los restaurantes de Barcelona al servicio exclusivo de los trabajadores. La Vanguardia, 28-4-1938, 4. 14 DOGC, 20-1-1938, 255. 14 DOGC, 20-1-1938, 255. 10 Els sindicats abans i després del 19 de juliol. Una indústria planificada. Treball, 6-2-1938, 12. 2. El segon mandat de Joan Comorera a la Conselleria d’Economia. El problema dels abastiments Si el primer any de guerra les reserves i les fruites i verdures de pobles propers a les ciutats van esmorteir les carències, amb el pas del temps la manca de carn i sobretot de farina esdevin- gué molt greu i la proliferació d’especuladors i grans traficants ni tan sols s’aturà amb la creació del Tribunal Especial d’Abastiments i l’acció dels jutjats, ja que els beneficis obtinguts sobre- passaven en molt les multes incoades. A Barcelona es venia pels carrers a preus inaccessibles, i hom calculava en uns deu mil els venedors que, gairebé en sistema de monopoli, dificultaven la venda dels que tenien permís, i inclús es constatava que alguns obrers que usaven els menja- dors populars deixaven el seu treball per dedicar-se al tràfic il·legal.7 Es posava especial èmfasi en la necessària col·laboració ciutadana per denunciar els casos d’especulació, més enllà de les actuacions policials i judicials, ja que millorar l’abastiment era lluitar també contra l’enemic emboscat.8 La magnitud del problema feu que es presentés un informe al Ple del Comitè Cen- tral del psuc sobre l’organització del consum col·lectiu i la lluita contra l’especulació.9 Des dels rengles del comunisme s’emmirallaven en els menjadors públics de la pàtria dels treballadors, la Unió Soviètica, i les campanyes per a la seva multiplicació no cessaren. La fo- ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 /30 Rosa Toran Belver \| Menjar en temps de guerra 13 Los restaurantes de Barcelona al servicio exclusivo de los trabajadores. La Vangua 14 DOGC, 20-1-1938, 255. j 14 DOGC, 20-1-1938, 255. Rosa Toran Belver \| Menjar en temps de guerra L’en- càrrec prioritari era incrementar els menjadors populars, reorganitzar el sector d’acord amb les necessitats i acabar d’una vegada per sempre amb el desordre i el règim irritant de desigualtat.i La fosig fou una bona aliada en aquest propòsit, amb reiterades crides a intensificar els menjadors populars i les cantines escolars, en primer lloc a Barcelona, Tarragona i Lleida, on hi havia molta població flotant.15 El seu president, Emili Vilaseca, llençà la consigna d’aconse- guir que en vuit dies hi hagués cinquanta menjadors a les principals fàbriques de Catalunya, i d’aportar alimentació extra als treballadors de les indústries de guerra,16 tal com s’acordà en el ple del Comitè Nacional de la ugt: Sindicatos y Federaciones nacionales, en orden al problema de abastecimiento deben centrar su acción con la máxima intensidad en la constitución de Come- dores colectivos en los lugares de trabajo, al objeto de impedir motivos de pérdida de tiempo y energías en las labores de producción; al fomento del movimiento 10 Els sindicats abans i després del 19 de juliol. Una indústria planificada. Treball, 6-2-1938, 12. p j pi 11 La primera Conferència Nacional de Dones del p.s.u.Treball, 16-3-1937, 8. ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 ISSN: 1889-1152. Rosa Toran Belver \| Menjar en temps de guerra DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 /31 Rosa Toran Belver \| Menjar en temps de guerra cooperativo, teniendo por base los núcleos de trabajadores, como medida perma- nente más eficaz, para ir imponiendo una justa regulación en la distribución y una política de contención en la inflación de precios.17 Recerques i Assajos No era només el sindicat ugetista el que reclamava aitals mesures, ja que la cinquena con- clusió del Comitè d’Enllaç de la Indústria Gastronòmica ugt-cnt també demanava un ràpid increment dels menjadors populars,18 i l’Assemblea de Dones Activistes del psu del 19 d’abril reclamava tancar els hotels de luxe i obrir menjadors populars i jardins d’infants.19 En els mítings del Front Popular, la creació de menjadors populars, infantils i cantines era una de les consignes corejades, i, en el manifest del Primer de Maig del Front Popular i del Comitè d’Enllaç ugt-cnt, a la demanda de multiplicar els menjadors populars abastits per l’Estat s’hi sumava la de garantir a la població un mínim d’articles alimentaris a preus relacionats amb els salaris.20 El Comitè de Catalunya de la ugt felicità el conseller Comorera per la seva tasca persistent en l’organització de menjadors populars, i li oferia el seu concurs i col·laboració entusiasta per endegar projectes encaminats a solucionar, segons les possibilitats del moment, el problema de l’alimentació dels treballadors.21 En començar a actuar la Comissió Interventora, funcionaven quatre menjadors populars, amb una mitjana diària de 5.421 coberts, i en una operació d’abast propagandístic s’inaugurà un menjador popular a l’Hotel Colon per servir 5.000 coberts diaris, i dos més, als carrers d’Aragó i d’Urgell, que significaven un augment de 6.000 coberts diaris.22 El mes d’abril de 1938 se’n servien més de 17.000, i estaven en vies de creació sis menjadors més a barris i zones fabrils.23 A finals del mateix mes s’havia duplicat el nombre d’establiments, amb 22.000 serveis diaris, a 2,50 pessetes el cobert, situats al Café Central, Bar Arenas, Hotel Colon, Primer de Maig, Gran Metro, Restaurant Baixador, Restaurant Carbó i a l’interior de l’Hospital Clínic, a banda dels instal·lats dins de fàbriques, o els cooperatius, com el del Sindicat de l’Ensenyança o el dels treballadors de la Generalitat. 17 Acuerdos del Comité Nacional de la ugt. La Vanguardia, 9-4-1938, 3. 18 Els treballadors gastronòmics per la mobilització. Treball, 8-4-1938, 4. 19 La gran mobilització femenina del pròxim Primer de Maig. Treball, 20-4-1938, 4. 20 1º de Mayo. Manifiesto del Frente Popular y del Comité de Enlace u.g.t. y c.n.t. La Vanguardia, 30-4-1938, 3. 21 Acción Sindical. El Comité de Cataluña de la u.g.t. aprueba el pacto de unidad sindical con la c.n.t. La política de Abastos. La depuración de los abusos en los precios. La Vanguardia, 13-5-1938, 6. 22 Generalidad. La inscripción en los comedores populares. La Vanguardia, 15-2-1938, 5. 23 Los abastecimientos. La Vanguardia, 6-4-1938, 5. 24 Los restaurantes de Barcelona al servicio exclusivo de los trabajadores. La Vanguardia, 28-4-1938, 4. 17 Acuerdos del Comité Nacional de la ugt. La Vanguardia, 9-4-1938, 3. g treballadors gastronòmics per la mobilització. Treball, 8-4-193 ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 Rosa Toran Belver \| Menjar en temps de guerra /32 Recerques i Assajos 9.000. Segons Coma, en poc temps Barcelona podria solucionar les dificultats i es referí a la col·laboració entusiasta de la majoria d’establiments, amb poques excepcions.25 Prengueren especial rellevància la inauguració del menjador popular a l’Harmonia del Pa- lomar (Sant Andreu del Palomar), al carrer Eugenio Parareda núm. 176, que servia 900 dinars i 900 sopars, i la instal·lació d’un altre al Frontón Sol y Sombra, a l’avinguda de les Corts Ca- talanes, cantonada Marina, que podia atendre 3.000 comensals en cada àpat.26 No totes les opinions sobre el seu plantejament eren positives. Per a uns la dieta era repe- titiva i insuficient, a base de llenties, mongetes i pa, que alguns s’emportaven per menjar a casa, mentre que per a altres el servei de menges variades a base de bacallà, carn, arròs, cigrons, mongetes, ous i pasta per a sopa era injust, ja que aquests aliments rarament se subministraven als particulars.27 Una crítica ben significativa partí del VI Congrés, celebrat a Barcelona el 2 d’agost de 1938, de la Federació de Cooperatives de Catalunya, presidida per Felip Barjau Riera, un dels fun- dadors del psuc. En la comunicació adreçada al conseller d’Economia s’expressava el desig de 400.000 famílies de Catalunya que advocaven per la reducció al mínim dels menjadors popu- lars. Creien que suposaven una renúncia al goig dels dinars en comú i reclamaven l’atorgament de queviures directament a les famílies, que havien vist reduïda la ració que se’ls atorgava quan era usuari dels menjadors algun dels seus membres.28 Mentre la majoria de la població patia carències alimentàries, una minoria enriquida pels tripijocs del mercat negre podia gaudir de tota mena de luxes i no tenia mesura en les con- sumicions als restaurants. La situació fou denunciada pel Comitè d’Enllaç de la Indústria Gastronòmica ugt-cnt, que apostava per l’anul·lació dels menjadors de preu lliure i de luxe, catalogats com a vicis de feixistes. 25 Abastecimiento de la ciudad. El servicio de los comedores populares. La Vanguardia, 17-6-1938, 3. 26 Los abastecimientos. La Vanguardia, 23-10-1938, 5. 27 La industria gastronómica. La Vanguardia, 2-7-1938, 2. 28 Acció Cooperatista. Òrgan de la Federació de Cooperatives de Catalunya (797), 26-8-1938. 29 Adaptación de la industria gastronómica a las necesidades de la hora presente. La Vanguardia, 6-4-1938, 5. 30 Los restaurantes de Barcelona al servicio exclusivo de los trabajadores. La Vanguardia, 28-4-1938, 4. 31 Adaptación de la industria gastronómica a las necesidades de la hora presente. La Vanguardia, 6-4-1938, 5. Rosa Toran Belver \| Menjar en temps de guerra També es projectava habilitar quatre cinemes a les barriades, destinats als treballadors de les fàbriques controlades per la Sotssecretària d’Armament, i procedir a instal·lar una cuina monumental en un dels cafès més cèntrics i espaiosos de Barcelona, amb capacitat per servir 6.000 coberts diaris. L’actuació no es limitava a la capital catalana, sinó que també es preveia instal·lar-ne a Manresa, Terrassa i Sabadell, i a altres poblacions fabrils.24 El mes de juny de 1938 s’inauguraren nous menjadors i el president de la Comissió In- terventora de la Indústria Gastronòmica, Agustí Coma, demanava la col·laboració ciutadana, fent intransferible el carnet, única manera de controlar les necessitats de la població, i per tal d’esmenar-ho inspectors recorrien els menjadors i sancionaven i retiraven els carnets cedits a algú diferent del titular. Aquell estiu es treballava per posar en marxa un menjador al Cine Novedades per servir-hi de 4.000 a 5.000 coberts, amb una previsible ampliació de 8.000 a ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 Rosa Toran Belver \| Menjar en temps de guerra 25 Abastecimiento de la ciudad. El servicio de los comedores populares. La Vanguardia, 17-6-1938, 3. 26 L b i i L V di 8 Abastecimiento de la ciudad. El servicio de los comedores populares. La Vanguardia, 17-6-1938, 3. Los abastecimientos. La Vanguardia, 23-10-1938, 5. Rosa Toran Belver \| Menjar en temps de guerra Recerques i Assajos amb els guanys controlats i intervinguts per la Generalitat, amb un 50% aportat a la Caixa de Crèdit Industrial i Comercial. Podien utilitzar-los els posseïdors de carnet de treball útil a la rereguarda, convenientment controlat, amb preferència per aquells amb una certificació de la Sotssecretària d’Armament i pels privats de targeta de racionament, avalats per l’Institut Muni- cipal del Cens, amb una inscripció prèvia. La gent de comarques amb necessitat de traslladar-se a Barcelona havien de proveir-se d’un certificat de l’alcaldia respectiva, que acredités la seva afecció a la República i la justificació de la seva estada a la ciutat.32 A principis de maig s’inauguraren 74 restaurants econòmics a 5 pessetes i el 8 de maig altres amb un cost de 8 a 10 pessetes, entre ells l’Euskadi, el Baviera, el Café de la Rambla, el Glaciar, el Terminus i el Velòdrom, i s’anunciaven nous menjadors populars a 2,50 pessetes.33 El carnet era vàlid a qualsevol dels 74 establiments sense distinció de districte, mentre no s’haguessin servit el nombre de coberts que figuraven en els cartells de cada menjador. Rosa Toran Belver \| Menjar en temps de guerra L’horari quedava establert de 12 a 15 i de 18 a 20:45, amb la possibilitat d’anar a l’Hotel Orient de 9 a 12 i de 15 a 18 si el treball impedia complir l’horari general, i també es dictà la prohibició de portar menjar a casa.34 En pocs dies els restaurants econòmics atenyeren la xifra de 89 del tipus B, 18 del tipus A i 6 pels forans, amb 40.000 coberts que se sumaven als 25.000 dels menjadors populars, i l’1 de juny es preveia l’obertura de 64 més del tipus B.35 A finals de juny s’havien expedit 60.000 carnets de tipus B i l’extraordinària demanda deixà més de 25.000 sol·licituds sense atendre quan s’acabaren els terminis per inscriure’s.36 Per tal d’evitar fraus, duplicitat i carnets de distintes categories, es procedí a elaborar un fitxer de tots els posseïdors, i tampoc faltaren els incompliments de les normes per part dels propietaris dels restaurants, sobre els quals requeien multes i fins i tot alguna detenció, arribant a designar-se un inspector permanent als establiments reincidents i implantant-se una bústia de reclamacions a tots ells.37 A partir de l’1 de setembre, en virtut d’una ordre del Departament d’Economia s’unificaren els tipus A i B en una sola categoria, la B, i la Comissió Interventora de la Indústria Gastronò- mica arbitrà noves normes per a la seva utilització: limitació del dret de possessió de carnets de menjadors econòmics i populars als qui veritablement ho necessitessin, per tal de poder establir una mitjana normal d’assistents, assegurar-los el menjar corresponent i evitar una duplicitat de racionament en perjudici de la resta de la població; expedició per separat dels tiquets per dinar i sopar; utilització en exclusiva dels restaurants del districte del client; cost dels tiquets a 2 pessetes, deduïdes en el preu del cobert i amb pèrdua si no se’n feia ús; servei tan sols per als treballadors de les indústries de guerra i en les d’utilitat reconeguda, casos en què calia renunciar al sobreracionament assignat i a la ració de la targeta de racionament, en cas de fer els dos àpats. La resta de ciutadans els podien utilitzar si hi havia prou distància entre el lloc de treball i el domicili, i els forasters havien de presentar un certificat de l’alcalde de la localitat. 32 Los restaurantes de Barcelona al servicio exclusivo de los trabajadores. La Vanguardia, 28-4-1938, 4. 33 Els menjadors populars i econòmics al servei exclusiu dels treballadors. Treball, 1-5-1938, 10. 34 L’horari dels restaurants econòmics. Treball, 7-5-1938, 2. 35 L’obra del Departament d’Economia. Restaurants per als infants. Treball, 13-5-1938, 8. 36 Els menjadors econòmics.Treball, 9-8-1938, 4. 37 Multas a unos establecimientos de comidas. La Vanguardia, 10-12-1938, 4. 32 Los restaurantes de Barcelona al servicio exclusivo de los trabajadores. La Vanguardia, 28-4-1938, 4. 33 Els menjadors populars i econòmics al servei exclusiu dels treballadors. Treball, 1-5-1938, 10. 34 L’horari dels restaurants econòmics Treball 7 5 1938 2 Rosa Toran Belver \| Menjar en temps de guerra El projecte rebé de seguida el suport de les dues centrals sindicals, representades a la Comissió Assessora.29 Per mitigar les desigualtats, una ordre de la Conselleria d’Economia del 8 d’abril de 1938 autoritzava la Comissió Interventora “per adap- tar a les necessitats dels moments tots els establiments que siguin necessaris”, reorganitzava la indústria gastronòmica sobre la base dels restaurants econòmics i exigia la tinença d’un carnet expedit per la mateixa comissió per gaudir dels menjars a partir del dia 1 de maig.30 I, a par- tir de l’11 d’abril, quedaren obertes les llistes d’inscripció a les oficines de la citada Comissió Interventora a l’avinguda de les Corts Catalanes núm. 670, encarregada d’expedir els carnets.31 A partir d’aleshores, tots els restaurants, proveïts dels queviures lliurats per la Comissió In- terventora, quedaven dividits en les categories A i B, amb únicament dos tipus de coberts a deu i cinc pessetes respectivament, segons la qualitat i situació del local, el nombre d’obrers i empleats de la zona, etc., amb el pa i les postres inclosos i amb catorze models de menús per a cada categoria. Quedaven fora d’aquesta classificació un limitadíssim nombre d’establiments, Brasserie Gran Hotel, La Cala, Oro del Rhin, Hostal del Sol i Cau Ferrat, anomenats de “ca- tegoria especial”, per a hostes estrangers o atencions oficials. Tots ells pertanyien al Agrupa- miento de la Industria Gastronómica (aig), única empresa del ram col·lectivitzada, i per tant ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 Rosa Toran Belver \| Menjar en temps de guerra /33 j 37 Multas a unos establecimientos de comidas. La Vanguardia, 10-12-1938, 4. 35 L’obra del Departament d’Economia. Restaurants per als infants. Treball, 13-5-1938, 8 , 7 5 93 , 35 L’obra del Departament d’Economia. Restaurants per als infants. Treball, 13-5-1938, 8. 36 Els menjadors econòmics.Treball, 9-8-1938, 4. 36 Els menjadors econòmics.Treball, 9-8-1938, 4. 32 Los restaurantes de Barcelona al servicio exclusivo de los trabajadores. La Vanguardia, 28-4-1938, 4. Rosa Toran Belver \| Menjar en temps de guerra Pel que fa als restaurants, havien de fixar un cartell amb les quantitats de cada àpat i vetllar per la condimentació adequada, dins de les naturals limitacions en la variació dels menús, exigides ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 Rosa Toran Belver \| Menjar en temps de guerra /34 Recerques i Assajos per les circumstàncies.38 També es dictaren altres mesures de control, tant per als menjadors econòmics com per als populars, i es pretengué facilitar l’adquisició dels tiquets, per quinzenes o mensuals, alhora que les aglomeracions de públic a les oficines determinà el lliurament de tiquets en diferents dies per districtes, en un horari força ampli, de 8 a 12 i de 14 a 15.39 Amb aquest ventall de normes per als restaurants econòmics es pretenia corregir les defi- ciències constatades arran de les experiències dels menjadors populars. La Vanguardia havia re- collit les opinions de lectors que es queixaven d’haver d’anar en pelegrinatge d’establiment en establiment per poder menjar o de suportar cues interminables. En una entrevista als senyors Coma i Bertran, membres de la Comissió Interventora, aquests valoraven positivament la des- congestió del servei a partir de la distribució per districtes i anunciaven una mesura en estudi, l’assignació personal de restaurant, prevista pel mes de desembre de 1938.40 Fins al 24 de gener, en vigílies de l’ocupació de Barcelona, encara es lliuraren tiquets per als restaurants econòmics i se’n projectaren altres, com el del Casal del Metge, el patronat del qual rebé l’autorització de l’esmentada comissió per planificar el servei de menjador de tipus econòmic, i estava previst inaugurar-lo l’1 de gener de 1939.41 L’altre pilar de l’obra de Comorera foren els menjadors infantils, instal·lats als cinemes de Barcelona, primer, i poc temps després a altres localitats. De la mateixa manera que en el cas dels menjadors populars, eren nombroses les demandes de sindicats i associacions per tal de pal·liar les deficiències alimentàries dels aproximadament 200.000 infants a Barcelona, que es notaven en forma de raquitisme, absentisme i baix rendiment escolar. 38 Nova ordenació dels menjadors econòmics. Treball, 16-8-1938, 5. 39 Las subsistencias. Unificación de los comedores económicos de Barcelona. La Vanguardia, 16-8-1938, 4; Los abastecimientos. La renovación de tiquets de los restaurantes economicos. La Vanguardia, 18-12-1938. 5. 40 Abastecimientos. Reformas en el sistema de comedores económicos. La Vanguardia, 19-11-1938, 4. 41 Notas sobre abastecimientos. El comedor del “Casal del Metge”. La Vanguardia, 24-12-1938, 2. 42 Ayuntamiento de Barcelona. Mi Revista (40), 1-5-1938. 43 Los abastecimientos. Harina para la infancia. La Vanguardia, 21-7-1938, 2. 44 Primer congrés de la Federació Catalana de Treballadors de l’Ensenyament u.g.t. Treball, 22-1-1938, 11. 45 Cal anar ràpidament a la creació de Menjadors Infantils, Guarderies i Cases-Bressol. Treball, 8-3-1938, 3. ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 Rosa Toran Belver \| Menjar en temps de guerra /35 Recerques i Assajos de Dones de bcn del psu,46 a la II Conferència de Dones del psu i en el marc de la Unió de Dones de Catalunya.47 Fou en aquest context que Comorera va exposar un projecte singular, en paral·lel als menja- dors populars: l’habilitació de sales de cinema com a menjadors, que començà amb l’estudi de les condicions de deu cinemes de Barcelona, cedits per la Comissió Interventora d’Espectacles Públics, apadrinats i sostinguts per unitats de l’Exèrcit.48 Ben aviat arribaren les felicitacions a Comorera per la iniciativa, com la de la Federació Nacional de Pioners49 o la del I Congreso de los Metalúrgicos de Cataluña,50 i des d’unitats del front i des dels radis del partit s’oferien a finançar-los o a cedir els seus locals. La crida per fer apadrinaments va tenir força repercussió, com feren la Secretaria de Serveis Socials de la ugt a totes les federacions i sindicats51 o les divisions del mateix Exèrcit. Una carta al conseller Comorera del Comissariat de la Comandància Principal d’Artilleria del XV Cos de l’Exèrcit el felicitava “por la creación de los restaurantes infantiles y ofreciéndole un impor- tante lote de víveres, ahorrados de su racionamiento, para destinarlo a uno de los restaurantes infantiles de esta ciudad, demostrando con este rasgo, su amor a los niños y su deseo de colabo- rar eficazmente, a la solución de uno de los problemas más graves que la guerra nos plantea”.52 No faltaren algunes iniciatives dels sindicats, com la del Sindicat de Funcionaris de l’Ajun- tament de Barcelona, de la ugt, amb la col·laboració de la 224a Brigada de la 50a Divisió, amb vista a crear un menjador infantil per a fills dels seus afiliats, de sis a catorze anys, amb preferèn- cia pels infants de combatents i pels dels afiliats inscrits voluntàriament a files.53 La Federació de Cooperatives de Catalunya, sense menystenir la bona voluntat de la creació de menjadors infantils, es mostrà reticent al projecte, com ja ho havia fet respecte als menjadors populars en l’esmentada exposició al conseller d’Economia arran dels acords del VI Congrés. 46 Per la mobilització total de les dones de Catalunya. Treball, 27-9-1938, 3. 47 Ahir començaren les sessions de la Segona Conferència Nacional de Dones del p.s.u. Treball, 2-10-1938, 8. 48 El Conseller d’Economia, Camarada Comorera, continua rebent importants aportacions a favor dels menjadors infantils. Treball, 27-5-1938, 2. 49 La Federació Nacional de Pioners a la Conselleria d’Economia. Treball, 21-5-1938, 8. 50 Vida Sindical. El primer Congreso de los Metalúrgicos de Cataluña. La Vanguardia, 4-9-1938, 10. 51 Els Menjadors Infantils creats pel camarada Comorera. Treball, 3-6-1938, 2. 52 Los abastecimientos. Los comedores infantiles. La Vanguardia, 10-7-1938, 5. 53 Creación de un comedor infantil. La Vanguardia, 18-9-1938, 6. 54 Acció Cooperatista. Òrgan de la Federació de Cooperatives de Catalunya (797), 26-8-1938. 55 Restaurants per als infants. Treball, 13-5-1938, 8. 56 La industria gastronómica. Apertura de inscripciones para cinco nuevos comedores infantiles. La Vanguardia, 13-7-1938, 3. Rosa Toran Belver \| Menjar en temps de guerra En temps de raciona- ment estricte, a Barcelona hi havia registrats 51.000 nens menors de dos anys, per als quals la manca de llet líquida era un problema i calia subministrar-los llet condensada, com es feia amb els malalts,42 i també es distribuïa farina, mitjançant la presentació de recepta mèdica.43 L’arriba- da d’infants refugiats i la necessària incorporació de la dona al treball feien del tot peremptori arbitrar mesures que la Conselleria d’Economia impulsà al llarg de l’any 1938. En el I Congrés fete-ugt, celebrat del 28 al 30 de gener de 1938, la ponència núm. 1, “La fete i la guerra”, reclamava cantines escolars, guarderies, menjadors infantils, parvularis…,44 i en el III Congrés de la ugt es creà la Comissió Proalimentació de l’Infant, presidida pel secre- tari de Serveis Socials, amb delegats de la fosig, sindicats de l’alimentació (forners, rebosters i pastissers), sindicats del Vestit, Fabril i Tèxtil, Metal·lúrgia, Banca i Borsa, fete, Treballadors Sanitaris i Ajut Infantil a la Rereguarda. Entre les tasques urgents es citaven les guarderies i cases bressol, a prop de les fàbriques, i els menjadors infantils, almenys per garantir un àpat al dia als nens en edat escolar, amb un cost de 2 pessetes diàries per infant, a més de la inversió en les instal·lacions.45 En el camp femení, Dolors Piera s’expressà de forma semblant a l’Assemblea ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 Revista catalana d’història 16 (2023), 25 - 44 Rosa Toran Belver \| Menjar en temps de guerra 46 Per la mobilització total de les dones de Catalunya. Treball, 27-9-1938, 3. ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 Rosa Toran Belver \| Menjar en temps de guerra Recerques i Assajos La directora general del servei era Josepa Boira, que comptava amb els col·laboradors Magí Soler, Jordi Viola, Agustí Coma i Antoni Bertran i Valldeperes fins que se n’anà al front. Per al seu funcionament va ser cabdal la col·laboració de les dones de la fosig (Juanita Monreal ocu- pava la secretaria general del Comitè Femení de Barcelona). El nombre de les seves afiliades havia augmentat de 500 l’any 1936 a 3.200 a finals de 1938, i, a més dels menjadors infantils, estaven compromeses en el Socors Roig, visitaven hospitals, eren padrines de guerra i dona- dores de sang, i satisfeien una quota extraordinària per apadrinar batallons.57 En una primera tanda es posaren en funcionament cinc menjadors, sent el gracienc Verdi el pioner: Cinema Verdi (carrer de Verdi, 32). A finals de maig de 1938 s’obriren les inscripci- ons58 per donar menjar a 2.000 criatures, amb l’apadrinament de l’11a Divisió del V Cos de l’Exèrcit, que aportà 40.000 pessetes, 40 quilos de cigrons i 95 d’arròs i cinc llaunes de carn en conserva, de la denominada “carn russa”, ingredients base dels menús diaris.59 Inaugurat el 19 de juny en un acte popular amb els infants i les famílies amenitzat per la banda de la divisió, comptà amb la presència del conse- ller Comorera i de representants militars, i l’avinentesa s’aprofità per estimular els apadrinaments i mostrar els llaços entre l’Exèrcit i la població civil, entre el front i la rereguarda.60 famílies amenitzat per la banda de la divisió, comptà amb la presència del conse- ller Comorera i de representants militars, i l’avinentesa s’aprofità per estimular els apadrinaments i mostrar els llaços entre l’Exèrcit i la població civil, entre el front i la rereguarda.60 Cinema Bailèn (carrer de Bailèn, 205). El 10 de juliol s’inaugurà el núm. 2, amb la previsió de servir 2.000 àpats diaris i apadrinat per carrabiners.61 Cinema Pons i Gallarza (Harmonia del Palomar, carrer de Pons i Gallarza, 58). Inaugurat el 22 de juliol i apadrinat per la 27a Divisió, entrà en funcionament l’1 d’agost.62 Cinema Pons i Gallarza (Harmonia del Palomar, carrer de Pons i Gallarza, 58). Inaugurat el 22 de juliol i apadrinat per la 27a Divisió, entrà en funcionament l’1 d’agost.62 Cinema Galileu (carrer de Galileu, 60). Fou inaugurat el 4 d’agost, amb la presèn- cia de la 27a Divisió.63 Cinema Galileu (carrer de Galileu, 60). Rosa Toran Belver \| Menjar en temps de guerra Jutjava més necessari establir cantines a tots els grups escolars, amb el menjar que els corres- pondria si anessin als menjadors infantils, pels avantatges del tracte dels mestres i del coneixe- ment de l’espai i per evitar els desplaçaments.54 L’organització dels menjadors estava a càrrec de la Comissió Interventora de la Indústria Gastronòmica i per al seu sosteniment es comptava amb les trameses de la Direcció General d’Abastiments, dirigida per Trifón Gómez, i amb les aportacions d’unitats de l’Exèrcit que els apadrinaven, a més de donatius de l’estranger i de particulars, canalitzats a través del compte corrent de la Conselleria d’Economia de la Caixa de Crèdit Industrial i Comercial. Gratuïts per als fills d’orfes i vídues de guerra i de pares sense ingressos, s’establia un preu mòdic per a la resta, una pesseta,55 i calia confirmar dos anys de residència a Barcelona, certificada per l’alcaldia de barri.56 ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 /36 Rosa Toran Belver \| Menjar en temps de guerra 57 Les dones de la Indústria Gastronòmica. Treball, 8-10-1938, 7. 58 Atenció a la política de Proveïments. Treball, 26-5-1938, 3. 59 Donativos para los comedores infantiles. La Vanguardia, 22-7-1938, 2. 60 Inauguración del primer comedor para niños. La Vanguardia, 21-6-1938, 4. 61 Per als infants de Catalunya. Treball, 5-7-1938, 4; Inauguración de nuevos comedores infantiles. La Vanguardia, 10-7-1938, 1. 62 Un altre restaurant infantil. Treball, 30-7-1938, 4; Generalidad de Cataluña. Nuevo comedor infantil. La Vanguardia, 30-7-1938, 4. 63 Los abastecimientos. Comedores infantiles. La Vanguardia, 4-8-1938, 2. 64 Inauguración de un comedor infantil. La Vanguardia, 17-9-1938, 2. 65 Les activitats de la Comissió Interventora de la Indústria Gastronòmica. Treball, 2-7-1938, 1. 66 Els menjadors infantils. Treball, 1-6-1938, 2. 67 La industria gastronómica. Apertura de inscripciones para cinco nuevos comedores infantiles. La Vanguardia, 13-7-1938, 3. 68 El Radi III i la inauguració d’un menjador infantil. Treball, 30-10-1938, 5. 69 Los abastecimientos. Apertura de inscripciones para el comedor infantil n.º 14. La Vanguardia, 11-9-1938, 2. 70 Los abastecimientos. Nuevo comedor infantil. Apertura del período de inscripción. La Vanguardia, 26-7-1938, 2. 71 La obra de los comedores infantiles. La Vanguardia, 22-9-1938, 2. Rosa Toran Belver \| Menjar en temps de guerra Recerques i Assajos A començaments de juliol ja s’havia anunciat la propera obertura de deu nous menjadors infantils65 i s’ampliava l’edat per al seu ús, de quatre a catorze anys.66 En aquesta nova tanda es posaren en marxa els següents: Cinema Horta (carrer d’Horta, 31-33). Inaugurat el mes de setembre. Cinema Rivoli (Rambla del Triomf). Amb el núm. 5, s’inaugurà el mes d’octubre per a 2.500 infants, sota el patrocini de la 42a Divisió. Cinema Les Corts (carrer de Cabestany, 3). Concebut per alimentar 1.500 nens i apadrinat per la 3a Divisió del II Cos d’Exèrcit, s’inaugurà el 30 d’octubre, amb el núm. 8.67 Cinema Muntaner (carrer de Muntaner, 106). Inaugurat el mes d’octubre.68 Cinema Muntaner (carrer de Muntaner, 106). Inaugurat el mes d’octubre Cinema Cèntric (carrer del Peu de la Creu, 2). Amb el núm. 6, s’inaugurà el 6 de novembre. Cinema Cèntric (carrer del Peu de la Creu, 2). Amb el núm. 6, s’inaugurà el 6 de novembre. Cinema Entença (carrer d’Entença, 90-92). Inaugurat el 16 de desembre per a 6.000 nens, amb el núm. 14.69 Cinema Nou (carrer dels Obradors, 10-12). Amb el núm. 9, obrí les inscripcions el mes de juliol,70 però es desconeix la data de la seva inauguració. Joan Comorera s’esplaià en una llarga entrevista a finals de setembre de 1938,71 en què feia balanç dels primers cinc menjadors en funcionament a Barcelona, que acollien diàriament 14.000 nens, i altres 7.000 a les comarques catalanes, a Sabadell, Terrassa, Borjas (sic), Tarragona, Vilada i Berga. Llançà el projecte de duplicar el nombre de comensals en un torn de cinc a set de la tarda, als mateixos locals i amb els mateixos personal i utensilis, de manera que, en el termini d’un mes, 50.000 nens barcelonins podrien ser atesos perfectament amb una menjada diària. No podia deixar d’esmentar l’especial atenció amb què es realitzava l’obra, dirigida i admi- nistrada per la Conselleria d’Economia mitjançant la Comissió Interventora de la Indústria Gastronòmica i la col·laboració entusiasta de la Dirección General de Abastos, amb Trifón Gómez, per a l’adquisició de queviures, fet que solucionava una de les parts més importants de l’abastiment de la població civil, l’alimentació regular i eficient dels nens. Rosa Toran Belver \| Menjar en temps de guerra Fou inaugurat el 4 d’agost, amb la presèn- cia de la 27a Divisió.63 Cinema Cortes (avinguda de les Corts Catalanes, 794, davant la plaça de toros Cinema Cortes (avinguda de les Corts Catalanes, 794, davant la plaça de toros Monumental). Inaugurat el 17 de setembre, amb el núm. 4, estava apadrinat per l’Agrupació de la Indústria Gastronòmica.64 Cinema Cortes (avinguda de les Corts Catalanes, 794, davant la plaça de toros Monumental). Inaugurat el 17 de setembre, amb el núm. 4, estava apadrinat per l’Agrupació de la Indústria Gastronòmica.64 Els rituals de les inauguracions oficials es repetiren en les obertures successives amb l’assis- tència del mateix Comorera, altres autoritats i representants de l’Exèrcit, acompanyats d’un variat programa d’actuacions per als nens inscrits, cinema, atraccions, música a càrrec de bandes de l’Exèrcit o de la Banda Municipal… ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 /37 Rosa Toran Belver \| Menjar en temps de guerra 72 El éxito de los comedores escolares glosado por el sr. Comorera. La Vanguardia, 17-12-1938, 2. 73 Generalidad de Cataluña. La obra de los Comedores infantiles. Los excombatientes argentinos antes de marcharse visitan los comedores infantiles. La Vanguardia, 7-1-1939, 2. 74 Manifestacions del Conseller d’Economia camarada Comorera, Treball, 22-9-1938, 3. 75 Comarcas catalanas. Una iniciativa en favor de la infancia. La Vanguardia, 17-7-1938, 8. 76 Primer Congreso ordinario del Sindicato de Obreros Mineros de la comarca del Berguedà. La Vanguardia, 27-7-1938, 5. 77 La Secretaria de Serveis Socials del Secretariat de Catalunya de la u.g.t. i la creació de menjadors infantils. Treball, 25-5-1938, 7. 78 Inaguración de un nuevo comedor infantil. La Vanguardia, 25-10-1938, 3. Rosa Toran Belver \| Menjar en temps de guerra I tampoc deixava d’emfasitzar la col·laboració entre les divisions de l’Exèrcit Popular, lluitadores per la llibertat i la independència d’Espanya, i la rereguarda, tot remarcant les emotives cartes dels menuts als seus padrins i la seguretat dels combatents en constatar que els seus fills estarien degudament atesos, fet que els apujava la moral i els estimulava a efectuar subscripcions i a donar part de les seves racions. A banda de menjar, Comorera explicava que darrerament havia arribat algun ramat de bens i cabres, que degudament cuidades donarien cries i serien una reserva per sub- ministrar carn fresca als nens, i amb aquesta finalitat la Conselleria d’Agricultura havia cedit una granja situada en una localitat pròxima a Barcelona per concentrar el bestiar, tot esperant ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 Rosa Toran Belver \| Menjar en temps de guerra /38 Recerques i Assajos que les unitats militars enviessin animals vius, crida que feia extensiva als Sindicats Agrícoles i a camperols i ramaders. Fou un projecte d’envergadura planejat a curt i mitjà termini, que es va veure interromput per la ràpida caiguda de la ciutat en mans dels feixistes. Pocs dies abans de l’ocupació de Bar- celona, Comorera mostrava la seva satisfacció per l’èxit dels menjadors, que proporcionaven cada dia menjar a 50.000 nens, i confiava que en un mes s’arribaria a 100.000 i més endavant a 200.000.72 I també fou aquests dies quan Comorera rebé la visita d’una delegació dels excombatents argentins de les Brigades Internacionals, que abans de marxar al seu país havien visitat diversos menjadors infantils, acompanyats per Eloína Malasechevarria, aleshores cap del Servei dels Restaurants Infantils, i aprofità l’avinentesa per lliurar un missatge adreçat al Partit Socialista de l’Argentina, en demanda d’ajut contra el feixisme invasor.73 3. L’expansió a altres ciutats Tampoc no hi faltà la salutació d’una nena als represen- tants de la divisió, un soldat de la qual llegí diverses cartes de combatents adreçades als infants.79 A Gramenet del Besòs, el menjador infantil número 19 estava situat al passeig Durruti;80 el de Sabadell, amb 5.000 inscrits apadrinats per la 44a Divisió, no va ser inaugurat fins al mes de desembre,81 i un dels darrers que es posaren en marxa va ser el de Vilaboi, al local de l’Ateneu Santboià, apadrinat pel 7è Batalló de Transports i condicionat per l’ajuntament amb la col·labo- ració de totes les entitats polítiques i socials, amb l’alta xifra de 1.300 nens inscrits dels 1.800 de la població.82 Completen la nòmina dels darrers posats en marxa el núm. 34 a Arenys de Munt, per a 600 nens,83 i el de Caldes de Montbui al balneari Rius, també amb el mateix nombre d’infants.84 presentació de la Federació de Pioners. Tampoc no hi faltà la salutació d’una nena als represen- tants de la divisió, un soldat de la qual llegí diverses cartes de combatents adreçades als infants.79 3. L’expansió a altres ciutats Amb la vocació d’estendre el projecte per tot Catalunya, el mes de setembre de 1938 funci- onaven menjadors a Terrassa, Sabadell, Borjas (sic), Tarragona, Berga i Vilada, amb un servei de 7.000 àpats, i estaven en fase d’adaptació els de Gramenet del Besòs, Vilaboi, Granollers, Mataró, Girona, Manresa, Calella, Figueres, Palamós, Puig-reig i Arenys de Munt.74 Les peticions arribaven des de sindicats i ajuntaments. A Figueres la Comissió Proalimenta- ció de l’Infant en va ser la promotora.75 També ho fou la del Sindicato de Obreros Mineros de la Comarca de Berga,76 i a Sabadell, conscients dels estralls que patien els fills dels combatents, la Federación Local de Sindicatos ugt encetà una recaptació als consells d’empresa de fàbri- ques i tallers per lliurar diners a la Conselleria d’Assistència Social i invertir-los en menjadors exclusius per als nens.77 Igual que a la capital catalana, les inauguracions oficials esdevenien actes propagandístics, amb la presència de les autoritats i els batallons patrocinadors, convertits en hostes de la ciutat. A tall d’exemple, a Tarragona el primer menjador infantil, apadrinat per la 46a Divisió, fou inaugurat el mes de setembre, amb una delegació de combatents i la seva banda militar, i amb l’assistència de Comorera i de Coma, el comandant militar de la plaça, el president de l’Au- diència, el cap administratiu d’Hisenda, delegats d’Hisenda, d’Ordre Públic i de la Generalitat, així com una representació d’alumnes de l’Escola de Capacitació.78 I a Terrassa, al menjador infantil núm. 12, patrocinat per la 60a Divisió i inaugurat el 14 d’octubre al Cinema Alegria, se celebrà un acte grandiós, amb la presència del tinent coronel de l’escolta del president de la República, el conseller Comorera i el de Cultura de l’ajuntament, el comandant i el comissari de la divisió, a més d’altres personalitats, que reberen el regraciament per part d’un nen en re- ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 Rosa Toran Belver \| Menjar en temps de guerra /39 Recerques i Assajos presentació de la Federació de Pioners. , 939, 85 L’obra cultural dels menjadors infantils. Meridià (53), 14-1-1939. 81 La inauguració d’un restaurant infantil a Sabadell. Treball, 23-12-1938, 4. 84 Restaurants Infantils. Treball, 10-1-1939, 2. 6 El Conseller Joan Comorera és aclamat pels infants del Menjador Infantil número 4 i per les seves mares i familiars. Tr 2-11-1938, 8. 82 Los abastecimientos. La Vanguardia, 11-12-193 83 Ibid. 82 Los abastecimientos. La Vanguardia, 11-12-1938, 2. 83 Ibid. 85 L’obra cultural dels menjadors infantils. Meridià (53), 14-1-1939. 86 El Conseller Joan Comorera és aclamat pels infants del Menjador Infantil número 4 i per les seves mares i familiars. Treball, 22-11-1938, 8. 87 L’obra cultural d’Ajut Infantil a la Rereguarda. Treball, 22-11-1938, 2. 88 Joan Comorera i els infants. Treball, 5-1-1939, 4. 89 Entorn d’una obra exemplar. Treball, 24-9-1938, 7. 90 Los abastecimientos. La Vanguardia, 14-7-1938, 4. 91 Protección a la infancia. La Vanguardia, 25-9-1938, 2. 92 Los abastecimientos. La Vanguardia, 13-11-1938, 2. 93 Una valuosa aportació dels nostres diputats a l’obra dels Restaurants Infantils. Treball, 7-12-1938, 1. 94 Los abastecimientos. Donativos. La Vanguardia, 4-8-1938, 2. 95 Un donatiu del president de la Generalitat als menjadors infantils, Treball, 10-1-1939, 2; El president Companys ha fet un donatiu de queviures a favor dels menjadors infantils. Per la mateixa finalitat, combatents de les Brigades Internacional han lliurat al Departament d’Economia la quantitat de 918 pessetes. Treball, 17-7-1938, 4. Rosa Toran Belver \| Menjar en temps de guerra Recerques i Assajos infants catalans, s’inicia amb la descripció de les activitats escolars i acaba a la llar, i després continua amb quatre capítols: educació física, intel·lectual i tècnica, cívica-moral i estètica. Es va considerar un bon complement de l’obra dels menjadors, i de fet el mes de novembre se n’havien repartit mil exemplars als centres de l’Ajut Infantil a la Rereguarda.87 Manuel Vall- deperes, en l’article “Joan Comorera i els infants”,88 a partir del lema “Per damunt de tot, els infants”, definia l’autor com un “home dur, enèrgic i tenaç davant l’enemic”, però “sensible en presència dels seus dos grans amors: Catalunya i els infants! I és per això, perquè és un patriota irreductible, que sap lligar tan hàbilment el present amb el futur. Per alguna cosa no és possible oblidar que els infants d’avui han d’ésser els homes de demà!”. Pocs dies abans de la caiguda de Barcelona, les consignes alertaven que se seguia lluitant pel demà i que els infants eren la llavor de les transformacions, de manera que si l’obra dels menjadors infantils esdevenia efectiva en una situació de guerra, més hauria de ser-ho l’endemà, a l’hora de la reconstrucció nacional. 4. Obra educativa i propaganda als menjadors infantils Com s’havia fet des de primeries de segle a les cantines escolars de l’Ajuntament de Barcelona, el vessant educatiu era present als menjadors infantils, però les especials circumstàncies el feren de- cantar vers dosis de propaganda i enaltiment dels patrocinadors, ben detallats en reportatges mono- gràfics. Emissions radiades, concursos de cartells i segells, revistes, biblioteques, periòdics murals…, elaborats pels infants, al costat de festivals i de la celebració de les diades especials, com la Diada de l’Infant, en col·laboració amb la Comissió Pro Any Nou de l’Infant. Bandes de les unitats de l’Exèr- cit apadrinadores actuaven de forma periòdica, i els mateixos soldats enviaven joguines i materials i eren correspostos pels infants, amb l’elaboració de banderes o la tramesa de cartes i dibuixos. Sorprèn que, inclús amb els clars indicis que anunciaven la derrota, les crides a la resistència del psu arribessin també a l’obra dels menjadors, afirmant que el futur del país estava en mans dels nens. El 14 de gener de 1939 la revista Meridià oferia una llarga entrevista de Domènec Guansé a l’escriptor i secretari de la Institució de les Lletres Catalanes Francesc Trabal, cap de Relacions i Propaganda dels Serveis de Restaurats Infantils,85 en què presentava el projecte d’un setmanari infantil, titulat Marrasquí, nom de l’heroi infantil de Carles Riba. Sota la direc- ció de l’escriptor i poeta Cèsar August Jordana, amb experiència en adaptacions i contes per a infants, es comptaria amb col·laboradors de primer ordre (Clusellas, Nyerra de L’Esquella, Apa, Mora, Martí Bas, Joan Oliver, Benguerel…), i tindria espai per a escrits o dibuixos dels nens. Però la revista no era l’únic projecte. Hi havia en cartera la confecció d’un àlbum amb la his- tòria gràfica de Catalunya, amb catorze sèries de dotze cromos cadascuna, amb text de Ferran Soldevila i dibuixos de Junceda, Clusellas, Narro, Mora, Martí Bas i Gonyi.i En tots els actes s’enaltia l’obra de Comorera; al menjador del Cinema Cortes fins i tot s’inaugurà el Club Infantil Joan Comorera, i se li reté un homenatge per part dels clubs in- fantils de la ciutat.86 Fou el moment de rememorar L’avi, l’obra escrita durant la seva estada a la presó, després del 6 d’octubre de 1934. El llibre, concebut com el breviari educatiu dels 79 Generalidad de Cataluña. Nuevo comedor infantil. La Vanguardia, 29-9-1938, 2. 80 Ibid ISSN: 1889-1152. ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 4. Obra educativa i propaganda als menjadors infantils DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 Rosa Toran Belver \| Menjar en temps de guerra /40 96 Para nuestros niños. La Vanguardia, 27-7-1938, 5. 97 Los abastecimientos. Donativos. La Vanguardia, 4-8-1938, 2. 98 La Generalidad de Cataluña. Los comedores infantiles. LaVanguardia, 7-8-1938, 4. 99 Los abastecimientos. La Vanguardia, 23-9-1938, 2. 100 Inauguració del Menjador Popular patrocinat per “Crítica”. Treball, 26-6-1938, 2. 101 La ayuda internacional a la República española. La Vanguardia, 24-11-1938, 4. 102 200 grams diaris de farina per a cadascun dels nens dels Menjadors Infantils. Treball, 17-11-1938, 8. 103 La companya de Comorera en missió de propaganda pro Menjadors Infantils. Treball, 25-11-1938, 8. 104 Carne-Cort, R. Pels infants de Catalunya. Ressorgiment (270), 1-1939. 105 El Consejo Nacional de la Infancia Evacuada. La Vanguardia, 28-8-1938, 7. Rosa Toran Belver \| Menjar en temps de guerra Recerques i Assajos i Fortificacions núm. 25, lliurà 1.000 pans corresponents al seu racionament del dia 19 de juli- ol;96 la 44a Brigada, 374è Batalló de la 44a Divisió, 30 caixes de préssecs, 800 racions de pa i 100 litres d’oli;97 la Comandància d’Enginyers del XIII Cos de l’Exèrcit, integrada pels batallons d’obres i fortificacions núm. 37 i 10, Batalló de Sapadors núm. 3 i personal de la Comandància, 100 quilos de patates, 150 de llegums, 1.060 pans i 55 quilos de sucre;98 el Batalló de Transmis- sions de l’Exèrcit de l’Est, 517 pans i altres comestibles.99 Des de l’estranger arribaren ajudes destinades als infants des de bon començament, com feu el diari Crítica de Buenos Aires, que lliurà 8 tones de queviures a la Conselleria d’Assistència Social de l’ajuntament barceloní, en el marc de la crida duta a terme pel Centre Antifeixista Sud-americà;100 la Federació de les Joventuts Comunistes franceses, amb un donatiu de 40.000 francs per garantir la creació d’un menjador per a 5.000 nens,101 i els quàquers a través de l’or- ganisme Amics dels Nens d’Espanya,102 a més de les tones de llet que arribaven des de França. I quan ja estaven en marxa un nombre significatiu de menjadors, Rosa Santacana, l’esposa de Joan Comorera, viatjà a l’Argentina a la recerca d’ajuts.103 D’aquesta estada en sortí l’article de R. Carné-Cort “Pels infants de Catalunya”, publicat a la revista Ressorgiment, que descrivia amb dramatisme la situació dels nens a Catalunya, on més de 300.000 passaven gana, i que esti- mulava les iniciatives, com establir el Dia del Pot de Llet un cop al mes, a càrrec d’una comissió femenina. Santacana portà un missatge de Companys per als catalans residents, i el Casal Català donà 1.000 pesos i organitzà una festa per estimular la solidaritat.104 5. Les fonts de sosteniment Si bé el principal impuls provenia de les unitats militars, hi hagué altres fonts que contri- buïren al seu funcionament. Una de les més freqüents eren els festivals organitzats sovint pel mateix Exèrcit, amb una notable presència d’autoritats civils i militars, que tenien lloc a les poblacions, amb obres de teatre, balls folklòrics, sarsueles…, on col·laboraven artistes de diver- sos gèneres, com el baríton Marcos Redondo o el tenor Emili Vendrell. A més de les recaptacions obtingudes als festivals, hi hagué un seguit d’iniciatives corpo- ratives, des dels sindicats fins als radis del partit, i també donacions directes de particulars a la conselleria. Des que es posà en marxa el projecte, van ser la ugt i el psu qui van protagonitzar més campanyes per recaptar diners, com feu el Secretariat de Serveis Socials de la ugt,89 el Sindicat Obrer d’Indústries Gràfiques i Similars,90 el Sindicat de Cafès, Restaurants Econòmics, Tabernes i Similars (sicrets)91 o el de la Comissió Interventora d’Espectacles Públics92 i els ma- teixos diputats del partit.93 Tampoc faltaren donatius d’oficines de la Generalitat, de personal dels menjadors que aportava un dia de sou i els d’empreses col·lectivitzades, com F. Vehils Vidal, Casa Vilardell, Magatzems Pelayo, Calzados Minerva o els Magatzems Jorba S. C.94 Els particu- lars participaven amb aportacions mensuals o amb una única quantitat, i la llista de donatius era detallada a la premsa juntament amb els oferiments que arribaven per apadrinar un menjador, posant especial èmfasi en les ocasions en què el president Companys feu una aportació.95 L’Exèrcit, a més de lliurar les recaptacions dels festivals i les dutes a terme pels seus batallons, que arribaven a quantitats altes, oferia donatius en espècie. A tall d’exemple, el Batalló d’Obres ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 /41 Rosa Toran Belver \| Menjar en temps de guerra 96 Para nuestros niños. La Vanguardia, 27-7-1938, 5. Rosa Toran Belver \| Menjar en temps de guerra Recerques i Assajos La Comisión de Auxilio Femenino del Ministerio de Defensa, presidida per Dolores Ibárru- ri, organisme delegat del Comité Nacional de Mujeres Antifascistas, a més del sosteniment de colònies i guarderies per a nens orfes o fills de combatents i refugiats, també impulsava menja- dors infantils, amb l’objectiu de cobrir tots els barris de Barcelona.106 Prengué espacial volada el del carrer de les Camèlies, 19, a Gràcia, inaugurat el 13 de novembre de 1938, per a 300 nens, amb la cooperació de la Conselleria d’Assistència Social de l’Ajuntament de Barcelona i del Comitè Noruec de Solidaritat Antifeixista, destinat prioritàriament als refugiats, però també amb atenció als nens del barri. Ubicat en un edifici nou i assolellat amb composicions mu- rals dels populars personatges de les pel·lícules de Walt Disney, els nens hi rebrien instrucció primària i gaudirien de sessions de cinema i activitats culturals. En la inauguració estigueren presents representants dels governs d’Euskadi i Astúries i el conseller Marià Martínez Cuenca, en nom del consistori, que insistí en l’esforç perquè els nens, en tornar a casa seva, s’empor- tessin un bell record de la seva estada a la ciutat, i en la necessitat de vetllar per la higiene i l’alimentació de la generació sobre la qual s’havia de fundar la nova Espanya.107 També arran de la col·laboració entre la comissió esmentada i l’ajuntament barceloní s’ins- tal·là un menjador al Refugi Montseny, que oferia esmorzar i berenar a 500 nens, gràcies a l’abastiment de l’Office International pour l’Enfance,108 i un altre al Rincón Blanco del Re- fugio Pasionaria, a on hi anaven 290 infants, inaugurat amb gran solemnitat per la mateixa Ibárruri i representants d’entitats nacionals i estrangeres.109 Pablo Picasso no va faltar a la crida, enviant a la comissió des de Londres 200.000 francs destinats a dos menjadors infantils, un a Madrid i l’altre a Barcelona, que serien batejats com a Comedores Picasso.110 Fora de la capital, per iniciativa d’ambdós organismes ja se n’havia inaugurat un a Igualada, cerimònia en què feren acte de presència autoritats i esposes d’alts càrrecs, representants d’erc, ugt, cnt, psu i sri i d’entitats locals, amb els recurrents festivals.111 El Centre de Reserva i Especialització d’Artilleria núm. 106 Protección a la infancia. La Vanguardia, 25-9-1938, 2. 107 Los abastecimientos. La Vanguardia, 11-11-1938, 4; Sobre abastecimientos. La Vanguardia, 12-11-1938, 4; Otro comedor in- fantil. La Vanguardia, 15-11-1938, 2. 108 Los abastecimientos. Un nuevo comedor infantil. La Vanguardia, 22-11-1938, 4. 109 Inauguración de un comedor infantil en el Rincón Blanco del Refugio “Pasionaria”. La Vanguardia, 10-12-1938, 4. 110 Creación de los comedores “Picasso”. La Vanguardia, 6-12-1938, 5. 111 Los comedores infantiles. La Vanguardia, 20-9-1938, 7. 112 Inauguración de unos comedores y escuelas para hijos y huérfanos de combatientes. La Vanguardia, 9-8-1938, 8. 113 L’aval d’una causa. Treball, 13-8-1938, 6. 114 Ayuntamiento de Barcelona. Visita del alcalde a un cuartel. La Vanguardia, 21-8-1938, 7. 6. Altres iniciatives a profit dels menjadors infantils No tan sols els infants barcelonins estigueren en la diana de les preocupacions. En primer lloc va caldre atendre els nens evacuats de les zones amenaçades pels rebels, després va caldre actuar a les pròpies ciutats de la rereguarda, objectius que implicaven l’organització del ser- vei de menjadors a les colònies o a recintes específics. Va significar un gran impuls la creació, l’agost de 1937, del Consell Nacional de la Infància Evacuada (cnie) des del Ministerio de Instrucción Pública, dirigit pel comunista Jesús Hernández, i l’abril de 1938 per l’anarquista Segundo Blanco, que comptà amb la mestra catalana, també cenetista, Ester Antich Sariol, di- rectora general de Primera Ensenyança, per dirigir aquell organisme.105 Amb l’excepció dels menjadors per als bascos gestionats pel Govern d’Euskadi, el sosteniment dels menjadors anà a càrrec del Comitè d’Avituallament d’Assistència Social fins a la seva dissolució, el 12 d’octubre de 1937, i cada municipi rebia de l’Estat una part del finançament per traslladar-lo a les famílies o institucions acollidores. /42 Rosa Toran Belver \| Menjar en temps de guerra 106 Protección a la infancia. La Vanguardia, 25-9-1938, 2. 7. Des de l’ajuntament barceloní Recerques i Assajos Quedaria incompleta la panoràmica de l’atenció als infants sense esmentar les cantines esco- lars dependents de l’Ajuntament de Barcelona, que des de les primeres dècades del segle havia format part de les mesures innovadores sorgides des de la Comissió de Cultura de l’ajuntament barceloní. La guerra havia interromput el servei i agreujà el problema de l’absentisme escolar i el de la desnutrició infantil, enmig dels greus problemes d’abastiment. Convertides moltes de les instal· lacions en menjadors populars, va ser a l’estiu de 1937 quan es va començar a plantejar la seva reorganització, de la mà del pedagog cenetista Joan Puig Elias i de la mestra comunista Dolors Piera Llobera, però no fou fins al mes de juny de 1938 que es tornaren a posar en marxa quatre cantines, de les quals se’n beneficiaren gairebé 4.000 nens, de les nou que estava previst inau- gurar aviat.115 Les dificultats per ampliar el servei s’intentaren suplir amb el repartiment de llet i bescuit, procedents de l’ajut internacional, als centres que no gaudien de cantina. D’aquesta manera, en els mesos finals de la guerra, les cantines escolars començaven a recuperar la seva funció inicial després d’haver donat servei als milicians durant l’estiu i la tardor de 1936 i ha- ver-se convertit posteriorment en menjadors populars. Rosa Toran Belver \| Menjar en temps de guerra Rosa Toran Belver \| Menjar en temps de guerra /43 ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 8. A manera de cloenda La base de les anteriors pàgines és bàsicament hemerogràfica i centrada en la capital cata- lana, destacant les iniciatives de Joan Comorera i del seu equip a partir de l’estiu de 1938 i la seva fermesa en encetar projectes fins i tot quan l’amenaça dels revoltats estava a les portes de Barcelona. El treball, no obstant això, obre portes per continuar la recerca amb indagacions en premsa local i als fons de les conselleries de Proveïments i d’Economia dipositats a l’Arxiu Nacional de Catalunya i la documentació conservada a l’Arxiu Contemporani de l’Ajuntament de Bar- celona. Caldria analitzar més a fons la tasca de les diverses conselleries i la seva relació amb les uni- tats de l’Exèrcit per copsar la dinàmica entre front i rereguarda en relació amb les dificultats de proveïment. Alimentar els soldats i garantir el sosteniment dels seus era bàsic per mantenir-los alta la moral, més enllà de l’alliçonament dels comissaris polítics. Alhora, la nova realitat a què s’enfrontaven les dones com a caps de família i treballadores per la causa exigia mesures socials urgents. La demanda de menjadors i guarderies no era només una necessitat conjuntural, sinó també una proposta revolucionària de cara a l’avenir, inspirada en el model soviètic i concebu- da com a realitat consolidada després de la derrota del feixisme. Rosa Toran Belver \| Menjar en temps de guerra 1 (crea), que havia fundat a la ca- pital catalana una llar del soldat a la caserna Fermín Salvochea, va ser un dels més actius en el suport als menjadors infantils, com fou l’inaugurat a l’Harmonia del Palomar, el 7 d’agost de 1938, per a fills i orfes de combatents, amb gran solemnitat, ja que comptà amb l’assistència del comandant general de Catalunya, José Riquelme, i la seva esposa, Manuela Ruiz Juan, que serà vicepresidenta de la Creu Roja de la República espanyola a l’exili a França, i també del comissari de l’Exèrcit de l’Est, el socialista Crescenciano Bilbao. Tots els discursos emfatitzaren la unitat entre l’Exèrcit i el poble, i ho reblà Margarita Nelken, padrina del menjador,112 que també dedicà l’article “L’aval d’una causa” a l’obra dels menjadors, exposant la idea que l’Exèr- cit, que abans reprimia, ara protegia i estimava els nens.113 L’alcalde de Barcelona, Hilari Salvadó, visità la caserna en diverses ocasions i en recorregué totes les instal·lacions.114 ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 License This work is licensed under a Creative Commons Attribution-Non- Commercial-NoDerivatives 4.0 International License. The author who publishes in this journal agrees to the following terms: a. The author exclusively assigns all intellectual property rights to the publisher worldwide and for the entire duration of the applicable intellectual property rights. b. The publisher will distribute the texts under the Attribu- tion-NonCommercial-NoDerivs 4.0 International, which allows the work to be shared with third parties, as long as they ack- nowledge the authorship, the initial publication in this magazine and the conditions of the license. Llicència Aquesta obra està sota una llicència internacional Creative Com- mons Reconeixement-NoComercial-SenseObraDerivada 4.0. L'autor/a que publica en aquesta revista està d'acord amb els termes següents: a. L'autor/a cedeix en exclusiva tots els drets de propietat intel·lectual a l’editor/a per a tot el món i per a tota la durada dels drets de propietat intel·lectual vigents aplicables. b. L’editor/a difondrà els textos amb la Creative Commons Reconeixement-NoComercial-SenseObraDerivada 4.0, la qual permet compartir l’obra amb tercers, sempre que en reconeguin l’autoria, la publicació inicial en aquesta revista i les condicions de la llicència. § § 115 Todo para los niños. Las cantinas escolares recientemente inauguradas aseguran el alimento, higiene y esparcimiento de los niños. Solidaridad Obrera, 26-6-1938, 2; Ahir foren inaugurades nou cantines escolars. Treball, 24-6-1938, 8. ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44 /44 § Rosa Toran Belver (Manresa, 5 de novembrede 1947 - El Masnou, 30 de juny de 2023) va ser presidenta de l’Amical Mauthausen i comissària de les exposicions Mauthausen, l’univers de l’horror (2001), Resistents i deportades (2003) o Més enllà de Mauthausen: Francesc Boix, fotògraf (2015). Com a especialista en la deportació, empresonament i extermini dels republicans espan- yols als camps nazis va publicar Vida i mort dels republicans als camps nazis (2002), Mauthausen: crònica gràfica d’un camp de concentració (2002) i Els camps de concentració nazis: paraules contra l’oblit (2005). Va rebre el Premi Leonor Serrano i Pablo, d’història de l’educació, juntament amb Cèlia Cañellas, pel seu llibre Dolors Piera, mestra, política i exiliada (2003); el Premi Bages de Cultura (2015) a la seva trajectòria i recerca sobre la lluita dels republicans espanyols a França contra el nazisme; i el Premi Bones Lletres d’Assaig Humanístic, concedit per Grup 62 i l’Acadèmia de les Bones Lletres de Barcelona, novament junt a Cèlia Cañellas, pel seu llibre Hilda Agostini. Les armes de la raó d’una mestra republicana, protestant i maçona (2021). També va publicar obres sobre la història del Masnou, com Història contemporània del Masnou (2017). § § Licencia Esta obra está bajo una licencia internacional Creative Commons Atribución-NoComercial-SinDerivadas 4.0. El autor o la autora a que publica en esta revista está de acuerdo con los términos siguientes: a. El/la autor/a cede en exclusiva todos los derechos de propiedad intelectual al/la editor/a para todo el mundo y toda la duración de los derechos de propiedad intelectual vigentes aplicables. b. El/la editor/a difundirá los textos con la Atribución-No Comercial- No Derivada 4.0 International que permite compartir la obra con terceros, siempre que éstos reconozcan su autoría, su publicación inicial en esta revista y las condiciones de la licencia. Rosa Toran Belver \| Menjar en temps de guerra Rosa Toran Belver \| Menjar en temps de guerra Recerques i Assajos Referències Acció Cooperatista. Òrgan de la Federació de Cooperatives de Catalunya Diari Oficial de la Generalitat de Catalunya Meridià Mi Revista Ressorgiment Solidaridad Obrera Treball La Vanguardia Licencia Esta obra está bajo una licencia internacional Creative Commons Atribución-NoComercial-SinDerivadas 4.0. El autor o la autora a que publica en esta revista está de acuerdo con los términos siguientes: a. El/la autor/a cede en exclusiva todos los derechos de propiedad intelectual al/la editor/a para todo el mundo y toda la duración de los derechos de propiedad intelectual vigentes aplicables. b. El/la editor/a difundirá los textos con la Atribución-No Comercial- No Derivada 4.0 International que permite compartir la obra con terceros, siempre que éstos reconozcan su autoría, su publicación inicial en esta revista y las condiciones de la licencia. Licencia Esta obra está bajo una licencia internacional Creative Commons Atribución-NoComercial-SinDerivadas 4.0. El t l t bli t i t tá d d El autor o la autora a que publica en esta revista está de acuerdo con los términos siguientes: El/l t / d l i t d l d h d ISSN: 1889-1152. DOI: 10.1344/segleXX2023.16.2 Revista catalana d’història 16 (2023), 25 - 44
https://openalex.org/W3032122047	https://bmccomplementmedtherapies.biomedcentral.com/track/pdf/10.1186/s12906-020-02971-y	English	null	Herbal medication triggering lupus nephritis - a case report	BMC complementary medicine and therapies	2,020	cc-by	3,265	© The Author(s). 2020 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://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background Drug-induced lupus (DIL) is a rare adverse reaction to a variety of drugs. Over 80 drugs have been implicated in DIL - hydralazine, procainamide, quinidine, and minocy- cline being few of the most well described triggers [1]. Pa- tients typically present as early as 1–2 months after the drug exposure, with fever, weight loss and fatigue, along with musculoskeletal complaints, most frequently arthral- gia [2]. Rarely the kidneys are involved, although there have been isolated case reports describing occurrence of renal lupus-like syndrome after exposure to penicillamine [3] and propylthiouracil [4]. Herbal medications are also known to cause acute kidney injuries, [5, 6] however there Herbal medication triggering lupus nephritis - a case report Anna Misya’il Abdul Rashid1, Fauzah Abd Ghani2, Liyana Najwa Inche Mat1 and Christopher Thiam Seong Lim1* Keywords: Lupus nephritis, Drug-induced lupus, Herbal supplement Keywords: Lupus nephritis, Drug-induced lupus, Herbal supplement are no cases yet reported to trigger a plants a lupus-like syndrome involving the renal tissue. Abstract Background: Herbal medication is widely used in our region as a mode of alternative medicine. Its contents and combinations are often modified to suit the needs of different populations. These products are said to boost the immune system and may serve as a protective measure against many diseases including Systemic Lupus Erythematosus (SLE). Some even lay claims to be able to cure SLE. Although they are not without side effects, these medications are still preferred due to their widespread availability and affordability, compared to modern medications. However, to date, there have been no reported cases in which these traditional medications can trigger a lupus-like reaction, moreover one involving the kidneys. Case presentation: We report a patient who developed overt lupus nephritis after consuming a course of herbal supplement. Her renal status did not improve upon cessation of the offending drug, and she required immunosuppressive therapy. After one cycle of IV cyclophosphamide, we managed to get the patient into remission - she is now on tapering doses of steroids. Conclusion: We wish to highlight the possibility of consumption of herbal medication and the emergence of drug-induced lupus nephritis. A thorough anamnesis and high index of suspicion of drug-induced lupus nephritis is warranted when a patient on supplements presents with urinary abnormalities. Case report W We report a case of a 29-year-old female who presented to us in August 2015, presenting with green discolor- ation and frothy urine associated with lower limb edema (Fig. 1). These symptoms were not preceded by any in- fective episodes. She was previously well and was only admitted for previous childbirth of which all her blood investigations were normal in 2014. In addition, she did not have any extra-renal symptoms, such as arthritis, serositis, cutaneous, or hematologic involvement. She was not on any medications but admits to using an herbal supplement named ‘Super Kidney’ for the past 6 months, containing ginseng, plantaginis folium, orthosi- phonis, strobilanthi folium and retrofracti fructus, which * Correspondence: drchrislim@gmail.com 1Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia Full list of author information is available at the end of the article * Correspondence: drchrislim@gmail.com 1Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia Full list of author information is available at the end of the article * Correspondence: drchrislim@gmail.com 1Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia Full list of author information is available at the end of the article Abdul Rashid et al. BMC Complementary Medicine and Therapies (2020) 20:163 https://doi.org/10.1186/s12906-020-02971-y Abdul Rashid et al. BMC Complementary Medicine and Therapies (2020) 20:163 https://doi.org/10.1186/s12906-020-02971-y BMC Complementary Medicine and Therapies BMC Complementary Medicine and Therapies Putting the renal bi- opsy, laboratory parameters and clinical presentation into perspective, it is conceivable that this was likely an auto- immune lupus-like reaction triggered by the supplements that she had been taking. We withdrew the herbal medica- tions and treated her with a course of 1 mg/kg prednisol- one daily and low molecular weight heparin injection. Six weeks later, her edema significantly reduced, and her urine regained its normal colour. Her urine dipstick was still 4+, but her proteinuria improved to 3.8 g/24 h. Serum creatin- ine remained normal at 62 μmol/L and her albumin level improved to 15 g/L. Hydroxychloroquine and angiotensin converting enzyme inhibitors (ACEi) were added. Despite 9 weeks of intensive treatment, she was persistently pro- teinuric with serum creatinine 66 μmol/L, 24-h urine pro- tein of 5.6 g and albumin 17 g/L. We then decided to start her on IV cyclophosphamide induction. She tolerated 6 of rigid and thickened capillary walls (Fig. 2) with presence of subendothelial depositions (Fig. 3) and splitting of glomerular basement membrane in Masson Trichrome (MT) (Fig. 4). Focal subepithelial vacuolation (Fig. 5) and focal regions of mesangial hypercellularity were observed. Immunofluorescence studies showed granular capillary loop and milder degree of mesangial immunopositivity for IgG (3+), IgA (3+), C3 (2+), C1q (3+), Kappa (1+) and Lambda (1+). Additional C4d immunohistochemistry showed granular positivity along the capillary walls. At this juncture, lupus nephritis ISN/RPS (2003) of Class IV- S (A/C) and V, and secondary membranoproliferative glomerulonephritis were considered. Putting the renal bi- opsy, laboratory parameters and clinical presentation into perspective, it is conceivable that this was likely an auto- immune lupus-like reaction triggered by the supplements that she had been taking. We withdrew the herbal medica- tions and treated her with a course of 1 mg/kg prednisol- one daily and low molecular weight heparin injection. Six weeks later, her edema significantly reduced, and her urine regained its normal colour. Her urine dipstick was still 4+, but her proteinuria improved to 3.8 g/24 h. Serum creatin- ine remained normal at 62 μmol/L and her albumin level improved to 15 g/L. Hydroxychloroquine and angiotensin converting enzyme inhibitors (ACEi) were added. Despite 9 weeks of intensive treatment, she was persistently pro- teinuric with serum creatinine 66 μmol/L, 24-h urine pro- tein of 5.6 g and albumin 17 g/L. We then decided to start her on IV cyclophosphamide induction. She tolerated 6 courses of 5.5 g in the period of 6 months and had no un- desirable side effects. Her response was favorable; she achieved remission after the 6th dose. We continued her on oral prednisolone and managed to taper it to a main- tenance dose of 5 mg per day. Abdul Rashid et al. BMC Complementary Medicine and Therapies (2020) 20:163 Abdul Rashid et al. BMC Complementary Medicine and Therapies (2020) 20:163 Pag Page 2 of 6 Fig. 1 Green discoloration of urine (left) along with the offending drug (right) Fig. 1 Green discoloration of urine (left) along with the offending drug (right) serologies, ANA positive with 1:640 titer, C3 and C4 levels were low at 0.78 g/L and 0.14 g/L respectively, anti-smith antibody, anti-RNP antibody, anti-Jo antibody and anti-Scl 70 antibody were negative. However anti-SSA (anti Ro) antibody and anti-SSB (anti La) antibody were positive. We were not able to send anti-histone antibody due to the non-availability of reagent at that time. Unfortunately, we did not send the serum PLA2R antibodies. Her erythro- cyte sedimentation rate was high 120 mm/hr. but her C- reactive protein was normal 1.86 mg/dl. The renal biopsy showed diffuse membranoproliferative pattern composed are plants used traditionally for improving general well- being and diuresis. The supplement was not registered with the National Pharmaceutical Regulatory Agency (NPRA), thus its safety profile and detailed content was not available. On further questioning, the patient admit- ted that the supplement was brought from overseas. Dur- ing this visit however, urine dipstick revealed 4 + proteinuria and 24 h urine protein was 10 g. Her creatin- ine was normal at 47 μmol/L, albumin was low at 11 g/L and her peripheral blood counts were normal. Further in- vestigations revealed a negative hepatitis B, C, and HIV Fig. 2 Rigid and thickened glomerular basement membrane (Haematoxylin & Eosin (H&E), original magnification × 60) Fig. 2 Rigid and thickened glomerular basement membrane (Haematoxylin & Eosin (H&E), original magnification × 6 Abdul Rashid et al. BMC Complementary Medicine and Therapies (2020) 20:163 Page 3 of 6 of rigid and thickened capillary walls (Fig. 2) with presence of subendothelial depositions (Fig. 3) and splitting of glomerular basement membrane in Masson Trichrome (MT) (Fig. 4). Focal subepithelial vacuolation (Fig. 5) and focal regions of mesangial hypercellularity were observed. Immunofluorescence studies showed granular capillary loop and milder degree of mesangial immunopositivity for IgG (3+), IgA (3+), C3 (2+), C1q (3+), Kappa (1+) and Lambda (1+). Additional C4d immunohistochemistry showed granular positivity along the capillary walls. At this juncture, lupus nephritis ISN/RPS (2003) of Class IV- S (A/C) and V, and secondary membranoproliferative glomerulonephritis were considered. Authors contributions ARAM, LNIM, FAG, CTSL participate in the literature review and data collection, ARAM, LNIM, FAG, CTSL participate in the manuscript writing and data analyses, CTSL participate in the final editing of the approval. The author(s) read and approved the final manuscript. Discussion and conclusion It is difficult to pin- point the exact content or substrate that induces these changes owing to the fact that there are thousands of naturally occurring plants used for medicinal value worldwide. Some of these herbs are also used in combin- ation with other herbs, making identification almost im- possible. Furthermore, as they are marketed as health supplements, there was no strict criteria for surveillance of safety profile by the health authorities, making identi- fication of manufacturers and distributors almost impossible. types of injuries may be diverse, ranging from acute tubular necrosis, acute interstitial nephritis, fibrosis, chronic interstitial nephritis, malignancies, and several types of electrolytes disorders [5, 6]. It is difficult to pin- point the exact content or substrate that induces these changes owing to the fact that there are thousands of naturally occurring plants used for medicinal value worldwide. Some of these herbs are also used in combin- ation with other herbs, making identification almost im- possible. Furthermore, as they are marketed as health supplements, there was no strict criteria for surveillance of safety profile by the health authorities, making identi- fication of manufacturers and distributors almost impossible. This case highlights the possibility of herbal medica- tion exposing drug-induced lupus nephritis. We wish to highlight the atypical presentation course of our DIL nephritis whereby it mimics the natural course of idio- pathic lupus nephritis and needed dual immunosuppres- sive therapy for 6 months before achieving resolution. A thorough anamnesis and high index of suspicion of DIL is warranted when a patient on supplements presents with urinary abnormalities. It is also important to note the health implications of herbal medicines, warranting a stricter governance of these manufacturers and distribu- tors by local health authorities. Author details 1 f 1Department of Medicine, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia. 2Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia. Received: 24 March 2020 Accepted: 24 May 2020 Received: 24 March 2020 Accepted: 24 May 2020 Competing interests None declared. Most cases of DIL will resolve within weeks within stoppage of the offending drug. No treatment is gen- erally needed. We postulate that there might be a few explanations to the patient’s renal status not improv- ing despite discontinuation of the offending drug. Firstly, this may reflect an atypical presentation of DIL in which the renal function did not improve after stopping the drug, and secondly, the possibility of this patient having SLE, by chance, during the period of supplement ingestion or discontinuation cannot be to- tally excluded. This patient’s DIL of class V LN only responded to the initiation of cyclophosphamide as per American College of Rheumatology Guideline (ACR) guidelines [9]. Whether this is a true DIL or the herbal medication triggering a preexisting lupus in our patient, remains uncertain. Consent for publication p Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal. Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal. Acknowledgements None. Acknowledgements None. Discussion and conclusion Herbal medicine is a form of treatment based on the use of plants or plant extracts that may be eaten or applied to the skin to improve general well-being. Herbal medi- cine has been used by many different cultures through- out the world to treat illnesses, and to assist various bodily functions including SLE. Although DIL is well described in literature, there is still no uniform criteria to establish its diagnosis. Diag- nosis is primarily made based on the temporal associ- ation with drug administration and improvement of symptoms upon withdrawal of the offending drug. In most cases of DIL, symptoms are similar to SLE, but much less severe. Renal and central nervous system in- volvement in cases of DIL is very rare. Laboratory test- ing may detect the presence of auto antibodies but its presence is not a prerequisite for the diagnosis of DIL [7]. Herbal medications are known to cause acute kidney injury by virtue of naturally occurring acids that are nephrotoxic. The anatomy of the kidneys, with large endothelial surface allows toxins to be filtered through easily; making it vulnerable to toxic injuries [6]. The Fig. 3 Tram track along glomerular basement membrane (Periodic acid-silver methenamine (PAAG) silver stain, original magnification × 60) Fig. 3 Tram track along glomerular basement membrane (Periodic acid-silver methenamine (PAAG) silver stain, original magnification × 60) Abdul Rashid et al. BMC Complementary Medicine and Therapies (2020) 20:163 Abdul Rashid et al. BMC Complementary Medicine and Therapies (2020) 20:163 Page 4 of 6 Fig. 4 Subendothelial deposition with splitting along rigid capillary loops (Masson Trichrome (MT), original magnification × 60) ig. 4 Subendothelial deposition with splitting along rigid capillary loops (Masson Trichrome (MT), original magnification × 6 Fig. 5 Vacuolation along glomerular basement membrane (Periodic acid-silver methenamine (PAAG) silver stain, original magnification × 60) Fig. 5 Vacuolation along glomerular basement membrane (Periodic acid-silver methenamine (PAAG) silver stain, original magnification × 60) Abdul Rashid et al. BMC Complementary Medicine and Therapies (2020) 20:163 Abdul Rashid et al. BMC Complementary Medicine and Therapies (2020) 20:163 Page 5 of 6 types of injuries may be diverse, ranging from acute tubular necrosis, acute interstitial nephritis, fibrosis, chronic interstitial nephritis, malignancies, and several types of electrolytes disorders [5, 6]. Ethics approval and consent to participate Ethics approval and consent to participate Non -applicable. Availability of data and materials ll d d l d d y All data generated or analysed during this study are included in this published article. Abbreviations d Abbreviations DIL: Drug-induced lupus; SLE: Systemic Lupus Erythematosus; SLICC: Systemic Lupus International Collaborating Clinics Classification Criteria for Systemic Lupus Erythematosus; LN: lupus nephritis; ACR: American College of Rheumatology Guideline DIL: Drug-induced lupus; SLE: Systemic Lupus Erythematosus; SLICC: Systemic Lupus International Collaborating Clinics Classification Criteria for Systemic Lupus Erythematosus; LN: lupus nephritis; ACR: American College of Rheumatology Guideline DIL: Drug-induced lupus; SLE: Systemic Lupus Erythematosus; SLICC: Systemic Lupus International Collaborating Clinics Classification Criteria for Systemic Lupus Erythematosus; LN: lupus nephritis; ACR: American College of Rheumatology Guideline In our case, the temporal association between the onset of symptoms and the use of the herbal medication was definite. The patient previously had no abnormalities in the laboratory investigations and was asymptomatic; until the introduction of the offending supplement. The use of the supplement may have triggered a nephrotic syndrome that when further investigated, revealed an autoimmune etiology; as evidenced by positive antinuclear antibody with low levels of serum complement. Moreover, she did not develop extra-renal symptoms, such as arthritis, sero- sitis, cutaneous, or hematologic involvement. This by itself does not fulfill the usual Systemic Lupus International Collaborating Clinics Classification Criteria for Systemic Lupus Erythematosus (SLICC) [8] criteria for the diagno- sis of SLE, however the positive renal biopsy showing evi- dence of class V lupus nephritis (LN) is sufficient. The mechanism of autoimmunity is complex, but is hypothe- sized to be due to the capability of certain lupus inducing drugs or their metabolites to form stable complexes and directly stimulates lymphocytes, causing autoantibodies to develop as an immune response [2]. Some studies also suggest a genetic predisposition in slow acetylators with HLA-DR2, HLADR3 and HLA-DR4 haplotypes being more likely to be affected with DIL [7]. References 1. Borchers TB, Keen CL, Gershwin ME. Drug-induced lupus. Ann N Y Acad Sci. 2007;1108:166–82. 2. Rubin RL. Drug-induced lupus. Toxicology. 2005;209:135–47. 3. Donnelly S, Levison DA, Doyle DV. Systemic lupus erythematosus-like syndrome with focal proliferative glomerulonephritis during D-penicillamine therapy. Rheumatology. 1993;32(3):251–3. 4. Ramesh Prasad GV, Bastacky S, Johnson JP. Propylthiouracil-induced diffuse proliferative lupus nephritis: review of immunological complications. J Am Soc of Nephrol. 1997;8:1205–10. 5. Lord GM, Tagore R, Cook T, Gower P, Pusey CD. Nephropathy caused by Chinese herbs in the UK. Lancet. 1999;354:481–2. 1. Borchers TB, Keen CL, Gershwin ME. Drug-induced lupus. Ann N Y Acad Sci. 2007;1108:166–82. 2. Rubin RL. Drug-induced lupus. Toxicology. 2005;209:135–47. 3. Donnelly S, Levison DA, Doyle DV. Systemic lupus erythematosus-like syndrome with focal proliferative glomerulonephritis during D-penicillamine therapy. Rheumatology. 1993;32(3):251–3. 4. Ramesh Prasad GV, Bastacky S, Johnson JP. Propylthiouracil-induced diffuse proliferative lupus nephritis: review of immunological complications. J Am Soc of Nephrol. 1997;8:1205–10. 5. Lord GM, Tagore R, Cook T, Gower P, Pusey CD. Nephropathy caused by Chinese herbs in the UK. Lancet. 1999;354:481–2. Page 6 of 6 Abdul Rashid et al. BMC Complementary Medicine and Therapies (2020) 20:163 6. Akpan EE, Ekrikpo E. Acute renal failure induced by Chinese herbal medication in Nigeria. Case Rep Med. 2015;2015:150204. 7. Niklas K, Niklas AA, Majewski D, Puszczewicz M. Rheumatic diseases induced by drugs and environmental factors: the state of the art – part one. Rheumatologia. 2016;54(3):122–7. 8. Petri M, Orbai AM. Alarco n GS, Gordon C, Merrill JT, Fortin PR, et al. derivation and validation of the systemic lupus international collaborating clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum. 2012;64(8):2677–86. 9. Hahn BH, Mcmahon MA, Wilkinson A, Wallace WD, Daikh DI, Fitzgerald JD, Karpouzas GA, Merrill JT, Wallace DJ, Yazdany J, Ramsey-Goldman R. American College of Rheumatology guidelines for screening, treatment, and management of lupus nephritis. Arthritis Care Res. 2012;64(6):797–808. 6. Akpan EE, Ekrikpo E. Acute renal failure induced by Chinese herb Abdul Rashid et al. BMC Complementary Medicine and Therapies (2020) 20:163 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.
https://openalex.org/W4323544013	https://ai.jmir.org/2023/1/e41264/PDF	English	null	Developing an Inpatient Electronic Medical Record Phenotype for Hospital-Acquired Pressure Injuries: Case Study Using Natural Language Processing Models	JMIR AI	2,023	cc-by	11,035	Original Paper Developing an Inpatient Electronic Medical Record Phenotype for Hospital-Acquired Pressure Injuries: Case Study Using Natural Language Processing Models Elvira Nurmambetova1, BHSc; Jie Pan1,2, PhD; Zilong Zhang1, MSc; Guosong Wu1,2, PhD; Seungwon Lee1,2,3, MPH, PhD; Danielle A Southern1,2, MSc; Elliot A Martin1,3, PhD; Chester Ho4, MD; Yuan Xu1,2,5,6, MD, PhD; Cathy A Eastwood1,2, RN, PhD 1Centre for Health Informatics, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 2Department of Community Health Sciences, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 3Alberta Health Services, Edmonton, AB, Canada 4Department of Medicine, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada 5Department of Oncology, University of Calgary, Tom Baker Cancer Centre, Calgary, AB, Canada 6Department of Surgery, Foothills Medical Centre, University of Calgary, Calgary, AB, Canada these authors contributed equally Nurmambetova et al JMIR AI JMIR AI Nurmambetova et al Original Paper Developing an Inpatient Electronic Medical Record Phenotype for Hospital-Acquired Pressure Injuries: Case Study Using Natural Language Processing Models Developing an Inpatient Electronic Medical Record Phenotype for Hospital-Acquired Pressure Injuries: Case Study Using Natural Language Processing Models Elvira Nurmambetova1, BHSc; Jie Pan1,2, PhD; Zilong Zhang1, MSc; Guosong Wu1,2, PhD; Seungwon Lee1,2,3, MPH, PhD; Danielle A Southern1,2, MSc; Elliot A Martin1,3, PhD; Chester Ho4, MD; Yuan Xu1,2,5,6, MD, PhD; Cathy A Eastwood1,2, RN, PhD 1Centre for Health Informatics, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 2Department of Community Health Sciences, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 3Alberta Health Services, Edmonton, AB, Canada 4Department of Medicine, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada 5Department of Oncology, University of Calgary, Tom Baker Cancer Centre, Calgary, AB, Canada 6Department of Surgery, Foothills Medical Centre, University of Calgary, Calgary, AB, Canada these authors contributed equally Elvira Nurmambetova1, BHSc; Jie Pan1,2, PhD; Zilong Zhang1, MSc; Guosong Wu1,2, PhD; Seungwon Lee1,2,3, MPH, PhD; Danielle A Southern1,2, MSc; Elliot A Martin1,3, PhD; Chester Ho4, MD; Yuan Xu1,2,5,6, MD, PhD; Cathy A Eastwood1,2, RN, PhD 1Centre for Health Informatics, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 2Department of Community Health Sciences, Cumming School of Medicine, University of Calgary, Calgary, A 3Alberta Health Services, Edmonton, AB, Canada 4Department of Medicine, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada 5Department of Oncology, University of Calgary, Tom Baker Cancer Centre, Calgary, AB, Canada 6Department of Surgery, Foothills Medical Centre, University of Calgary, Calgary, AB, Canada these authors contributed equally 1Centre for Health Informatics, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 2Department of Community Health Sciences, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 3Alberta Health Services, Edmonton, AB, Canada 4Department of Medicine, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada 5Department of Oncology, University of Calgary, Tom Baker Cancer Centre, Calgary, AB, Canada 6Department of Surgery, Foothills Medical Centre, University of Calgary, Calgary, AB, Canada these authors contributed equally Corresponding Author: Elvira Nurmambetova, BHSc Centre for Health Informatics Cumming School of Medicine University of Calgary 3330 Hospital Dr NW Calgary, AB, T2N 4N1 Canada Phone: 1 4032206843 Email: elvira.nurmambetova@ucalgary.ca 1Centre for Health Informatics, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 2Department of Community Health Sciences, Cumming School of Medicine, University of Calgary, Calgary, AB, Ca 3Alberta Health Services, Edmonton, AB, Canada 4Department of Medicine, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, AB, Canada 5Department of Oncology, University of Calgary, Tom Baker Cancer Centre, Calgary, AB, Canada 6Department of Surgery, Foothills Medical Centre, University of Calgary, Calgary, AB, Canada these authors contributed equally KEYWORDS pressure injury; natural language processing; NLP; algorithm; phenotype algorithm; phenotyping algorithm; machine learning; electronic medical record; EMR; pressure sore; pressure wound; pressure ulcer; pressure injuries; detect pressure injury; natural language processing; NLP; algorithm; phenotype algorithm; phenotyping algorithm; machine learning; electronic medical record; EMR; pressure sore; pressure wound; pressure ulcer; pressure injuries; detect territorial governments [17]. Additional crown institutions at the provincial and federal-level monitor adverse events such as HAPI. For example, in Alberta, Canadian Institute for Health Information, the federal crown corporation, works with Alberta Health Services (provincial health care agency) to monitor PIs [18,19]. To date, there is no mandatory collection of PIs within Canadian acute-care facilities. Real-time PI evaluation and auditing using ICD codes are not possible as Canadian health data systems are set up such that ICD codes are assigned outside of providing care and have a few months lag in data extraction, transfer, and load [20]. Consequently, these agencies aim to monitor but are unable to conduct real-time auditing of PIs in Canada. Therefore, there is a need to develop EMR data-specific algorithm for identifying PIs for monitoring and auditing within Canadian acute-care facilities. Our objective was to create EMR data-specific algorithms for HAPIs. Availability and implementation of PI-specific algorithms within a clinical information system would allow the abovementioned federal and provincial agencies to conduct real-time surveillance of HAPIs, improving patient safety, enhancing the quality of care, and reducing the burden of costs associated with adverse events. The EMR phenotype case detection is evaluated via comparison with confirmed HAPIs status acquired in a clinical trial [21]. Study Design This is an EMR phenotyping study for enhancing HAPI identification using free-text notes. Obtained clinical trial data were linked to administrative and EMR data for model development and validation. The natural language processing (NLP) method’s performance was compared with results from the ICD validation study conducted in Alberta, Canada, by Wong et al [21]. Detailed information for HAPIs identification can be found in their study. Electronic medical records (EMRs) are used to track and organize patient information for efficient treatment of medical conditions in a secure system [7]. Free-text clinical notes in EMRs consist of detailed descriptions of patients' conditions and treatment. Additionally, clinical notes are typically written in a continuous manner across patients' interactions with health care systems, making clinical notes more real-time compared to diagnosis codes. Despite the rich information the clinical record may have, coders often cannot read every entry, given their limited time per chart and many patients have prolonged hospital stays. Recent studies suggest that using free-text in EMRs alone, or incorporating EMR data elements, can significantly improve the accuracy of case identification of specific comorbidities [8-16]. Xu et al compared the ICD algorithm with algorithms based on EMR keyword search, which achieved a high sensitivity of 0.655 (95% CI 0.601-0.710) [8]. The Canadian health system operates as a publicly funded single-payer insurance system by the federal, provincial, and JMIR AI Nurmambetova et al Conclusions: The EMR-based NLP phenotyping algorithms demonstrated improved performance in HAPI case detection over ICD-10-CA codes alone. Daily generated nursing notes in EMRs are a valuable data resource for ML models to accurately detect adverse events. The study contributes to enhancing automated health care quality and safety surveillance. Introduction Pressure injury (PI), also known as a pressure ulcer, is an injury of the skin and deep tissues caused by external pressures. Annually, PIs affect approximately 250,000 to 500,000 Canadians, with an estimated prevalence of 26.0% in health care institutions [1,2]. Hospital-acquired pressure injuries (HAPIs) are PIs developed during an inpatient hospital stay. HAPIs can significantly extend a patient’s hospitalization length of stay and cause severe secondary complications, such as muscle and profound tissue impairment [3]. HAPI is considered mostly preventable, and its prevalence has been reckoned as an acceptable indicator of the quality of care [4,5]. Collecting HAPI status using chart review is time and labor-intensive, thereby not suitable for large-scale population-based applications. Considering all the factors, there is a need for automated ways to accurately and timely identify HAPIs for analyzing large cohort studies that support quality improvement efforts and assisting unit managers with developing reliable patient safety programs. The International Classification of Diseases, 10th Revision, adapted to the Canadian health system (ICD-10-CA), can be used to estimate the prevalence of adverse events from administrative data. However, the coded administrative data are prone to miss positive cases: previous research demonstrated that the sensitivity of the ICD algorithm for identifying HAPI cases is around 30% compared to chart review [1]. In addition to the sensitivity issue, ICD codes are not generally assigned with a specific time when diseases occur. Therefore, they are unsuitable for reporting the time when HAPIs occur [6]. Thus, there is a need for more accurate HAPI detection. (JMIR AI 2023;2:e41264) doi: 10.2196/41264 (JMIR AI 2023;2:e41264) doi: 10.2196/41264 Abstract Background: Surveillance of hospital-acquired pressure injuries (HAPI) is often suboptimal when relying on administrative health data, as International Classification of Diseases (ICD) codes are known to have long delays and are undercoded. We leveraged natural language processing (NLP) applications on free-text notes, particularly the inpatient nursing notes, from electronic medical records (EMRs), to more accurately and timely identify HAPIs. Objective: This study aimed to show that EMR-based phenotyping algorithms are more fitted to detect HAPIs than ICD-10-CA algorithms alone, while the clinical logs are recorded with higher accuracy via NLP using nursing notes. Methods: Patients with HAPIs were identified from head-to-toe skin assessments in a local tertiary acute care hospital during a clinical trial that took place from 2015 to 2018 in Calgary, Alberta, Canada. Clinical notes documented during the trial were extracted from the EMR database after the linkage with the discharge abstract database. Different combinations of several types of clinical notes were processed by sequential forward selection during the model development. Text classification algorithms for HAPI detection were developed using random forest (RF), extreme gradient boosting (XGBoost), and deep learning models. The classification threshold was tuned to enable the model to achieve similar specificity to an ICD-based phenotyping study. Each model’s performance was assessed, and comparisons were made between the metrics, including sensitivity, positive predictive value, negative predictive value, and F1-score. Results: Data from 280 eligible patients were used in this study, among whom 97 patients had HAPIs during the trial. RF was the optimal performing model with a sensitivity of 0.464 (95% CI 0.365-0.563), specificity of 0.984 (95% CI 0.965-1.000), and F1-score of 0.612 (95% CI of 0.473-0.751). The machine learning (ML) model reached higher sensitivity without sacrificing much specificity compared to the previously reported performance of ICD-based algorithms. https://ai.jmir.org/2023/1/e41264 JMIR AI 2023 \| vol. 2 \| e41264 \| p. 1 (page number not for citation purposes) https://ai.jmir.org/2023/1/e41264 XSL•FO RenderX XSL•FO RenderX JMIR AI JMIR AI 2023 \| vol. 2 \| e41264 \| p. 2 (page number not for citation purposes) https://ai.jmir.org/2023/1/e41264 Clinical Trial Data Previously completed randomized controlled trial (RCT) data of 678 eligible consenting inpatients were obtained from an affiliated research team and were used as the reference standard [21]. The trial evaluated the efficacy of a pressure-sensing mattress in preventing interface pressure. A research nurse performed a clinical head-to-toe skin assessment for PI formation, and suspected deep tissue injuries were monitored throughout 3 days of enrollment [21]. Assessments were conducted within 24 hours of admission, on the day of trial enrollment, and the third day after enrollment, and documented in Allscripts Sunrise Clinical Manager (SCM) EMR (Figure 1). Three days were chosen as a length of time for the research https://ai.jmir.org/2023/1/e41264 JMIR AI 2023 \| vol. 2 \| e41264 \| p. 2 (page number not for citation purposes) XSL•FO RenderX JMIR AI Nurmambetova et al nurse to perform data collection and risk assessment for 3 reasons. First, this is the average length of stay in the local inpatient units, and a longer trial period may include varying nursing practice due to hospital discharge with a shorter length of stay, unit changes, and more nursing shift changes. Second, the dedicated investigation team deemed 3 days sufficient for pressure-related skin and soft tissue changes to develop. Lastly, as a continuous collection of interface pressure throughout the enrollment period leads to a large volume of data, 3 days allowed for optimal data collection while maintaining participant enrollment. agreed to participate in the trial right after being admitted to the hospital, the research nurse noted the PI status on admission. The following data elements were abstracted from the clinical trial data: record ID, medical unit, sex, first-skin assessment date, second-skin assessment date, presence of PIs, and other possible related conditions (cerebrovascular disease, diabetes mellitus, etc). The clinical trial measured and classified PIs into 6 stages: stage 1, stage 2, stage 3, stage 4, suspected deep tissue injury, and unstageable PIs [22]. Stages of PIs were identified according to the National Pressure Ulcer Advisory Panel’s pressure ulcer staging system [23]. Stage 1 PIs include sores. Stage 2 captures open wounds on the surface of the skin. Stage 3 PIs represent wounds extending beneath the skin and affecting fat tissue. At stage 4, PIs are deep and reach into muscles, bones, and tendons. The trial is registered at clinicaltrials.gov (NCT02325388). Additional details surrounding the clinical trial data were published by Wong et al [21]. Clinical Trial Data The research nurse, who measured pressure-related skin ulcerations, was trained as a wound care specialist in the provision of pressure ulcers, ostomy, and continence care [21]. The patients’ PI status check on admission was determined based on when the patient was admitted. The clinical trial team relied on the medical record if the patient had been admitted long before the study and consented to the study. If the patient Figure 1. Illustration of the clinical trial for assessment of PI status in the enrolled patient cohort (n=678) and the data input used for the development of classification models. PI: pressure injury. Study Cohort Inclusion and Exclusion Criteria During the RCT, eligible patients were at least 18 years old, were expected to have a length of stay of at least 3 days, and did not receive near-end-of-life care within 3 days of trial enrollment [21]. Participants were recruited from nursing units with a high risk for PI development including acute medical, neurosurgery, neurology, and intensive care [21]. For this study, patients were excluded if their data did not link to EMR data, had incomplete skin assessments, or included erroneous assessment or discharge dates. Patients with PIs on the day of admission were also excluded in order to track only PIs developed during hospitalization. Furthermore, intensive care unit (ICU) patients were excluded since their data were stored in another data warehouse with distinct data elements from those found in SCM and required restricted access. After careful selection, the final cohort of eligible patients was 280 (Figure 2). Figure 1. Illustration of the clinical trial for assessment of PI status in the enrolled patient cohort (n=678) and the data input used for the development of classification models. PI: pressure injury. patients were excluded if their data did not link to EMR data, had incomplete skin assessments, or included erroneous assessment or discharge dates. Patients with PIs on the day of admission were also excluded in order to track only PIs developed during hospitalization. Furthermore, intensive care unit (ICU) patients were excluded since their data were stored in another data warehouse with distinct data elements from those found in SCM and required restricted access. After careful selection, the final cohort of eligible patients was 280 (Figure 2). JMIR AI 2023 \| vol. 2 \| e41264 \| p. 3 (page number not for citation purposes) Data Linkage to Discharge Abstract Database and SCM EMR Deterministic data linkage was performed between the RCT data, administrative data from the discharge abstract database (DAD), and SCM EMR data [24]. SCM was the EMR system employed in Calgary hospitals at the time of the study. Data linkage steps followed a previously established methodology [25]. First, the PI RCT data were linked to the DAD using the provincial health number and admission date. Then, DAD variables were used to connect these data with SCM. Forward feature selection was used to determine the best combination of documents with 2 machine learning (ML) models: extreme gradient boosting (XGBoost) and random forest (RF) [26,27]. Forward feature selection is an iterative way to obtain the best subset of features [28]. The analyses began with no feature in the input of models. Then, in each iteration, new features were added and observed for improvements (Figure 3). The experiments were run with each feature from the list of all possible features, where the best predictor was then added to our feature set. This iteration ended when introducing a new feature did not significantly improve the targeted metric. In our experiments, the forward feature selection was performed for every document type. Instead of adding 1 feature in each iteration, all documents belonging to 1 type were added to the input of models. This feature selection stopped when adding a new document type did not increase the target metric. Due to the long convergence time, the forward feature selection was not conducted during the development of the deep learning model. Rather, the same optimal document set determined by ML models was used. Inclusion and Exclusion Criteria During the RCT, eligible patients were at least 18 years old, were expected to have a length of stay of at least 3 days, and did not receive near-end-of-life care within 3 days of trial enrollment [21]. Participants were recruited from nursing units with a high risk for PI development including acute medical, neurosurgery, neurology, and intensive care [21]. For this study, 2). JMIR AI 2023 \| vol. 2 \| e41264 \| p. 3 (page number not for citation purposes) JMIR AI 2023 \| vol. 2 \| e41264 \| p. 3 (page number not for citation purposes) https://ai.jmir.org/2023/1/e41264 https://ai.jmir.org/2023/1/e41264 XSL•FO RenderX XSL•FO RenderX JMIR AI Nurmambetova et al Figure 2. Flowchart of inclusion and exclusion criteria for the patient cohort based on trial completeness, PI status, and age (minimum age of 18 years old) for all controls in the panel. ICU: intensive care unit; PI: pressure injury; SCM: Sunrise Clinical Management. Figure 2. Flowchart of inclusion and exclusion criteria for the patient cohort based on trial completeness, PI status, and age (minimum age of 18 years old) for all controls in the panel. ICU: intensive care unit; PI: pressure injury; SCM: Sunrise Clinical Management. ) p p j y g and assistance with a meal. The remaining document types contained daily intake and output, physiological indicators, pain scale, and other related data. Discharge summaries, unit transfer notes, and inpatient triage reports were not written for most patients during the clinical trial because the trial was primarily conducted in the middle of the hospital stay. Document Types and Sequential Forward Type Feature Selection In total, 37 types of documents were noted for the included patients during the clinical trial. Nursing notes were the primary source of suitable HAPI information and constituted the largest proportion of the documents. Among the nursing notes, “Patient Assessment” contained the assessment of skin and wounds under the Integument section. The Integument section described skin integrity, bruises, wound formation, and exposure to air. The “Patient Assessment Neuro” document included the patient's neurological state, where the main components related to PIs were level of consciousness, communication, and sensory deficit. The “Patient Care” document included patients' hygiene, activity, exercise, and nutrition, such as mobility, positioning, JMIR AI 2023 \| vol. 2 \| e41264 \| p. 4 (page number not for citation purposes) https://ai.jmir.org/2023/1/e41264 https://ai.jmir.org/2023/1/e41264 XSL•FO RenderX XSL•FO RenderX JMIR AI JMIR AI Nurmambetova et al Figure 3. A visual illustration of the sequential forward selection process for identifying feature subsets that maximize the performance of the ML pipeline. The candidate feature subsets were evaluated by using 5-fold cross-validation. For each subset of document types, 40 experiments were conducted with all possible combinations of 5 folds, 2 vectorizers, and 4 ML models. FPR: false positive rate. ML: machine learning; TF-IDF: term frequency-inverse document frequency; TPR: true positive rate. q y q y; p in a context sequence. Publicly released BERT parameters are trained on corpora such as Wikipedia, which is formatted differently from clinical text. As such, ClinicalBERT, a language model specifically pretrained using clinical notes, was used for the text evaluation [35]. Medical language has been demonstrated to contain vast amounts of discipline-specific jargon, abbreviations, and acronyms while being a domain-specific and technical language [36]. Multiple studies have demonstrated that ClinicalBERT performs better than BERT [37,38]. Therefore, the decision to proceed with ClinicalBERT for our study was made. The ClinicalBERT embedding was not fine-tuned with clinical notes data due to a moderate sample size. Rather, the ClinicalBERT was downloaded and tested from a GitHub repo found in the study where Alzentzer et al observed performance improvements on three common clinical NLP tasks after training BERT models on clinical notes and discharge summaries [38,39]. The document embedding layer weights were not taken from ClinicalBERT. Bag of Words Preprocessing and ML All nursing notes of selected document types were merged into 1 text and converted into a bag-of-words (BOW) vector with the count of words or term frequency-inverse document frequency (TF-IDF) vectorizer by using a Python scikit-learn ML library [29-31]. A binary classification model was developed to identify HAPI cases by considering all patients who developed any stage of PI during a hospital stay as positive cases and patients without PIs as the negative cohort. The BOW matrices were used as the independent input for the models. RF and XGBoost classification models were trained to perform classification. These 2 models were chosen because they were representative of ensemble models: RF for bagging and XGboost for boosting. Ensemble models have been shown to display superior performance than a single classifier [32]. Two sets of hyperparameters were tried for each model. The 5-fold cross-validation was conducted to determine the most useful document types, high-performing ML model, and its hyperparameters. Document Types and Sequential Forward Type Feature Selection As the maximum sequence length of BERT limits it from handling text with more than 512 tokens, sentence embeddings generated by ClinicalBERT are fed to another transformer to obtain the “document embedding,” a highly abstracted vector capturing global information about the whole document [35,38]. HAPI status was classified based on this document embedding (Figure 4). The project-specific document embedding transformer was trained from the ground up through random initialization. Details of implementation and training of our HAN-BERT models are described in Figure S1 in Multimedia Appendix 1 [40,41]. Natural Language Processing https://ai.jmir.org/2023/1/e41264 JMIR AI 2023 \| vol. 2 \| e41264 \| p. 5 (page number not for citation purposes) Deep Learning Model A hierarchical attention network (HAN) structure with bidirectional encoder representations from transformers (BERT) was used to classify the text in the EMR clinical notes [33,34]. BERT is a contextualized word representation model that uses a masked language model that predicts randomly masked words https://ai.jmir.org/2023/1/e41264 JMIR AI 2023 \| vol. 2 \| e41264 \| p. 5 (page number not for citation purposes) XSL•FO RenderX JMIR AI Nurmambetova et al Figure 4. Composition of input sequence representations for text classification using BERT. The meaning of one sentence is summarized into the vector of [CLS] (classifier), an artifact token concatenated at the beginning of each sentence to become the sentence embedding. The sentence embedding is then fed to another transformer to generate the document embedding. An output layer with SoftMax activation provides the probability of text classification. BERT: bidirectional encoder representations from transformers. Model Evaluation We used 5-fold stratified cross-validation to split the 97 positive cases and 183 patient controls into 5 groups. Due to the fact both numbers were not divisible by 5, there was a minor difference in the distribution of cases and controls between groups although the effort was placed to retain the most similar a maximum score of 1 when both sensitivity and PPV are 1, and a minimum of 0 when either sensitivity or PPV is 0. We calculated the binomial proportion CIs for sensitivity, specificity, PPV, and NPV using the Statsmodels package in Python (Python Software Foundation) [42]. The CIs of the F1-score were from 5-fold cross-validation. a maximum score of 1 when both sensitivity and PPV are 1, and a minimum of 0 when either sensitivity or PPV is 0. We calculated the binomial proportion CIs for sensitivity, specificity, PPV, and NPV using the Statsmodels package in Python (Python Software Foundation) [42]. The CIs of the F1-score were from 5-fold cross-validation. a maximum score of 1 when both sensitivity and PPV are 1, and a minimum of 0 when either sensitivity or PPV is 0. We calculated the binomial proportion CIs for sensitivity, specificity, PPV, and NPV using the Statsmodels package in Python (Python Software Foundation) [42]. The CIs of the F1-score were from 5-fold cross-validation. Ethics Approval The study was approved by the Conjoint Health Research Ethics Board at the University of Calgary, Calgary, Alberta (REB13-0794). Model Evaluation We used 5-fold stratified cross-validation to split the 97 positive cases and 183 patient controls into 5 groups. Due to the fact both numbers were not divisible by 5, there was a minor difference in the distribution of cases and controls between groups, although the effort was placed to retain the most similar distribution between the 5 groups. Each time we selected 4 groups as a training set, the remaining group was used as a test set. The splitting was the same for ML and deep learning experiments. A comparison of different document type subsets was executed with the best model to determine which document type subset would yield the best performance of PI detection. To fairly compare our method with ICD-based PI identification algorithms, the classification threshold was tuned to achieve similarly estimated specificities (0.988 and 0.959) of 2 ICD-based algorithms developed and validated in a previous study by Ho et al [1]. The first case definition is more specific and yields greater detection precision. The second definition is more inclusive of nonspecific codes for wounds and is likely to capture a larger number of cases. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and F1-scores were calculated as target metrics. Additionally, PPV, NPV, sensitivity, and specificity of the 4 algorithms and 2 ICD algorithms were calculated with changing thresholds ranging from 0.05 to 0.95. F1-score is a measure of accuracy through a combination of sensitivity and PPV. F1 has Characteristics of Study Cohort The study included 280 eligible participants (Figure 2). Among the 280 patients, a research nurse identified 183 patients with no HAPIs, and 97 patients were found to have HAPIs. Table 1 provides demographic details of the patient cohort. The P values were calculated with MedCalc’s statistical calculators [43,44]. The median age was 68 (IQR 55-79) years. The cohort consisted of 127 (45.36%) females, and the median length of stay was 46 (IQR-79) days. A more detailed review of input data and linguistic inquiry and word count analysis for the number of words, sentences, and patients based on document types can be found in Table S1 in Multimedia Appendix 2. JMIR AI 2023 \| vol. 2 \| e41264 \| p. 6 (page number not for citation purposes) https://ai.jmir.org/2023/1/e41264 https://ai.jmir.org/2023/1/e41264 XSL•FO RenderX JMIR AI Nurmambetova et al Table 1. Descriptive statistics of patients (N=280). Characteristics of Study Cohort (60 (61.86%) patients with HAPI versus 82 (44.81%) patients without HAPI), and patients in the negative groups predominantly had “Patient assessment Neuro.” However, patients from both groups had a similar amount of “Patient care” during the trial (Table 2). JMIR AI 2023 \| vol. 2 \| e41264 \| p. 7 (page number not for citation purposes) Characteristics of Study Cohort P value Patients without pressure injury (n=183) Patients with pressure injury (n=97) All Characteristics .62 85 (46.45) 42 (43.30) 127 (45.36) Female, n (%) .10 67 (53-79) 68 (59-80) 68 (55-79) Age (years), median (IQR) .37 46 (19-109) 48 (28-96) 46 (22-104) Length of stay (days), median (IQR) .52 87 (47.54) 50 (51.55) 137 (48.93) Cerebrovascular disease, n (%) .02 28 (15.30) 26 (26.80) 54 (19.29) Chronic obstructive pulmonary disease, n (%) .04 36 (19.67) 30 (30.93) 66 (23.57) Congestive heart failure, n (%) .34 24 (13.11) 9 (9.28) 33 (11.79) Myocardial infarction, n (%) .79 17 (9.29) 10 (10.31) 27 (9.64) Dementia, n (%) .28 30 (16.39) 21 (21.65) 51 (18.21) Peripheral vascular disease, n (%) .57 80 (43.72) 39 (40.21) 119 (42.50) Hemiplegia or paraplegia, n (%) .62 1 (0.55) 1 (1.03) 2 (0.71) Leukemia, n (%) .50 2 (1.09) 2 (2.06) 4 (1.43) Lymphoma, n (%) .02 20 (10.93) 21 (21.65) 41 (14.64) Peptic ulcer disease, n (%) .005 27 (14.75) 28 (28.87) 55 (19.64) Moderate or severe renal disease, n (%) .99 15 (8.20) 8 (8.25) 23 (8.21) Liver disease, n (%) .045 52 (28.42) 39 (40.21) 91 (32.50) Diabetes mellitus, n (%) .58 31 (16.94) 19 (19.59) 50 (17.86) Solid tumor, n (%) .04 27 (14.75) 24 (24.74) 51 (18.21) Connective tissue, n (%) disease .30 73 (39.89) 45 (46.39) 118 (42.14) History of smoking, n (%) .50 26 (14.21) 11 (11.34) 37 (13.21) Currently smoking, n (%) .32 8 (4.37) 7 (7.22) 15 (5.36) History of illicit drug use, n (%) .85 5 (2.73) 3 (3.09) 8 (2.86) Currently use illicit drugs, n (%) Data Linkage and Extraction Table 2 shows the patient and document count and document word count for the patients eligible for this study. Most PI-positive patients had “Patient assessment” document type (60 (61.86%) patients with HAPI versus 82 (44.81%) patients without HAPI), and patients in the negative groups predominantly had “Patient assessment Neuro.” However, patients from both groups had a similar amount of “Patient care” during the trial (Table 2). Nurmambetova et al JMIR AI Table 1. Descriptive statistics of patients (N=280). (60 (61.86%) patients with HAPI versus 82 (44.81%) patients without HAPI), and patients in the negative groups predominantly had “Patient assessment Neuro.” However, patients from both groups had a similar amount of “Patient care” during the trial (Table 2). Patient care Word count per note, median (IQR) were also higher (0.855 (95% CI 0.767-0.943) vs 0.793 (95% CI 0.700-0.886) and 0.798 (95% CI 0.745-0.851 vs 0.746 (95% CI 0.646-0.846) than those detected by ICD algorithm respectively. The deep learning model underperformed with the area under the receiver operating characteristic curve (AUC-ROC) score of 0.68 (SD 0.04), compared to the RF with the highest area under the curve (AUC) score of 0.80 (SD 0.08), followed by XGBoost with the AUC score of 0.75 (SD 0.07; Figure 5). Considering the prevalence of 34.6% in our study, the baseline area under the precision-recall curve (AU-PRC) is 0.346. Figure 6 shows that an AU-PRC of 0.77 (SD 0.06) was achieved for the RF models using TF-IDF tokenization, 0.74 (SD 0.08) was achieved for the RF models using count tokenization, 0.67 (SD 0.04) was achieved for the XGBoost models, and 0.60 (SD 0.06) for the deep learning models. These results are greater than 0.346, and we conclude that these classifiers do not discriminate by random chance and perform well in finding positive HAPI cases without accidentally marking negative patients as positive. Figure S1 in Multimedia Appendix 3 shows the PPV, NPV, sensitivity, and specificity of the 4 algorithms and 2 ICD algorithms, with changing thresholds ranging between 0.05 and 0.95. Data Linkage and Extraction Table 2 shows the patient and document count and document word count for the patients eligible for this study. Most PI-positive patients had “Patient assessment” document type JMIR AI 2023 \| vol. 2 \| e41264 \| p. 7 (page number not for citation purposes) https://ai.jmir.org/2023/1/e41264 https://ai.jmir.org/2023/1/e41264 XSL•FO RenderX JMIR AI Nurmambetova et al Table 2. Characteristics of extracted documents, different components of nursing notes, and the average number of documents written by nurses. Patients without HAPI (n=183) Patients with HAPI (n=97) All (N=280) Document type Patient assessment 82 (44.81) 60 (61.86) 142 (50.71) Number of patients with this type of document, n (%) 0.00 (0.00-16.00) 12.00 (0.00-18.00) 1.00 (0.00-17.00) Number of notes per patient, median (IQR) 370.00 (303.00-421.00) 385.00 (311.00-441.00) 376.00 (306.00-430.00) Word count per note, median (IQR) Patient assessment neuro 102 (55.74) 39 (40.21) 141 (50.36) Number of patients with this type of document, n (%) 8.00 (0.00-13.00) 0.00 (0.00-13.00) 2.50 (0.00-13.00) Number of notes per patient, median (IQR) 430.00 (346.00-499.00) 424.00 (324.00-493.50) 428.00 (343.0-498.0) Word count per note, median (IQR) Patient care 183(100) 97 (100) 280 (100) Number of patients with this type of document, n (%) 16.00 (13.00-23.50) 16.00 (13.00-19.00) 16.00 (13.00-21.20) Number of notes per patient, median (IQR) 133.00 (66.00-176.00) 147.00 (91.00-185.00) 138.00 (72.00-179.00) Word count per note, median (IQR) JMIR AI 2023 \| vol. 2 \| e41264 \| p. 8 (page number not for citation purposes) Document Subset and Classification Models Across a subset of document types and all tested classification techniques, the combination of Patient Assessment, Patient Assessment Neuro, and Patient Care yielded the highest outputs in terms of target metrics. The TF-IDF vectorizer with RF classifier demonstrated the best performance in terms of sensitivity, PPV, and NPV when fixed at the specificity of 0.988 and 0.959 thresholds. The performance results are reported in Table 3. For a specificity of 0.988, the sensitivity of the (TF-IDF+RF) EMR-based model was 0.464 (95% CI 0.365-0.563), which surpassed the sensitivity 0.277 (95% CI 0.174-0.380) achieved by the ICD-based algorithm [1]. The PPV of our model had a mean of 0.938 (95% CI 0.869-1.000), which is higher than the reported 0.917 (95 % CI 0.854-0.980) of the ICD algorithm. The NPV was 0.776 (95% CI 0.722-0.830), which is also higher than the 0.739 (95% CI 0.638-0.840) reported in the ICD validation [1]. For a specificity of 0.959 achieved by the loose ICD-based algorithm, the EMR model sensitivity reached 0.546 (95% CI 0.447-0.645) compared to 0.328 (95% CI 0.220-0.436) found in ICD reporting [1]. Both PPV and NPV of EMR model JMIR AI 2023 \| vol. 2 \| e41264 \| p. 8 (page number not for citation purposes) https://ai.jmir.org/2023/1/e41264 XSL•FO RenderX JMIR AI Nurmambetova et al Nurmambetova et al JMIR AI 2023 \| vol. 2 \| e41264 \| p. 9 (page number not for citation purposes) https://ai.jmir.org/2023/1/e41264 Nurmambetova et al Table 3. The performance of NLPa methods on free-text electronic medical record documents at varying thresholds for the probability of pressure injury detection. The model was compared to ICDb algorithms such that the model was trained to mimic its specificity. F1-score % (95% CI) NPVd % (95% CI) PPVc % (95% CI) Specificity % (95% CI) Sensitivity % (95% CI) Model Specificity near 0.988 0.425 (0.312-0.538) 0.739 (0.638-0.840) 0.917 (0.854-0.980) 0.988 (0.963-1.013) 0.277 (0.174-0.380) ICD (Ho et al [1]) 0.612 (0.473-0.751) 0.776 (0.722-0.830) 0.938 (0.869-1.000) 0.984 (0.965-1.000) 0.464 (0.365-0.563) TF-IDFe+random forestf 0.550 (0.361-0.739) 0.758 (0.704-0.813) 0.909 (0.824-0.994) 0.978 (0.957-0.999) 0.412 (0.314-0.510) Count+random forest 0.450 (0.340-0.559) 0.727 (0.671-0.782) 0.857 (0.741-0.973) 0.973 (0.949-0.996) 0.309 (0.217-0.401) TF-IDF+XGBoostg 0.394 (0.207-0.580) 0.716 (0.660-0.772) 0.867 (0.745-0.988) 0.978 (0.957-0.999) 0.268 (0.180-0.356) Word Embedding+BERTh Specificity near 0.959 0.464 (0.350-0.578) 0.746 (0.646-0.846) 0.793 (0.700-0.886) 0.959 (0.913-0.100) 0.328 (0.220-0.436) ICD (Ho et al [1]) 0.665 (0.577-0.753) 0.798 (0.745-0.851) 0.855 (0.767-0.943) 0.951 (0.919-0.982) 0.546 (0.447-0.645) TF-IDF+random forest 0.546 (0.359-0.733) 0.758 (0.702-0.813) 0.837 (0.733-0.940) 0.956 (0.927 -0.986) 0.423 (0.324-0.521) Count+random forest 0.552 (0.404-0.699) 0.758 (0.702-0.813) 0.837 (0.733-0.940) 0.956 (0.927-0.986) 0.423 (0.324-0.521) TF-IDF+XGBoost 0.420 (0.280-0.560) 0.720 (0.663-0.776) 0.824 (0.695-0.952) 0.967 (0.941-0.993) 0.289 (0.198-0.379) Word embedding+BERT of NLPa methods on free-text electronic medical record documents at varying thresholds for the probability of pressure was compared to ICDb algorithms such that the model was trained to mimic its specificity. Table 3. The performance of NLPa methods on free-text electronic medical record documents at varying thresholds for injury detection. The model was compared to ICDb algorithms such that the model was trained to mimic its specificity. bICD: International Classification of Diseases. cPPV: positive predictive value. d dNPV: negative predictive value. eTF-IDF: term frequency-inverse document frequency. gXGBoost: extreme gradient boosting. hBERT: bidirectional encoder representations from transformers. JMIR AI 2023 \| vol. 2 \| e41264 \| p. 9 (page number not for citation purposes) https://ai.jmir.org/2023/1/e41264 https://ai.jmir.org/2023/1/e41264 XSL•FO RenderX JMIR AI JMIR AI Nurmambetova et al Figure 5. The ROC curves derived from the random forest with TF-IDF and word count, XGBoost, and deep learning models. AUC: area under the curve; ROC: receiver operating characteristic; TF-IDF: term frequency-inverse document frequency; XGBoost: eXtreme gradient boosting. Figure 6. The area under the precision-recall curve (AU-PRC) performance of 4 models: random forest with TF-IDF and word count, XGBoost, deep learning model. Limitations The study is not without limitations. First, the exclusion of ICU patients due to data elements being distinct from SCM led to a smaller sample size and a narrower clinical cohort. Nevertheless, the remaining data from the clinical trial represented a population at risk for HAPIs. Second, both the patient and nurse knew at the admission of a clinical trial measuring PI would be the trial goal, which may have impacted the data entry quality of PI and frequency of patients to report PI-related discomfort. Third, the model produced relatively modest sensitivity. However, this sensitivity is deemed valuable, given that the specificity was set to a high threshold, and the input was restricted to the first 72 hours after enrollment [16]. The sensitivity reported in similar studies used the whole or more extended hospitalization stay and more data elements [50-52]. In addition, the sensitivity of our study was obtained through a comparison to a clinical trial instead of chart review data. Chart review does not always capture all positive cases due to possible errors in the review process [53,54]. Fourth, the comparison with deep learning is not likely to be very fair because BERT-based models are usually applied to larger cohorts. Nevertheless, our result can be served as a reference for model selection for researchers working with similar sample sizes. Prabhakar et al applied ClinicalBERT to phenotype 10 diseases on a cohort consisting of 1610 discharge summaries [41]. When only using ClinicalBERT, they obtained a very similar F1-score (0.46) compared to our result [41]. The suboptimal performances of the advanced deep learning model may suggest that study needs to be more evolved before applying deep learning to free-text-based clinical phenotyping. Tree-based ML models are recommended for detecting adverse event conditions from noisy, moderately sized text samples. From the forward document type selection, we found that apart from the notes that directly document skin conditions in the patient assessment, the entries noting the patient’s consciousness, nutrition, and mobility were helpful in indicating HAPI. This makes clinical sense because the reduced level of consciousness, nutrition, and mobility are factors that may contribute to HAPI [45]. In addition, our findings align with several risk factors of the Braden Scale [46]. Nurmambetova et al AUC: area under the curve; PRC: precision-recall curve; TF-IDF: term frequency-inverse document frequency; XGBoost: eXtreme gradient boosting; Nurmambetova et al JMIR AI Figure 5. The ROC curves derived from the random forest with TF-IDF and word count, XGBoost, and deep learning models. AUC: area under the curve; ROC: receiver operating characteristic; TF-IDF: term frequency-inverse document frequency; XGBoost: eXtreme gradient boosting. Figure 6. The area under the precision-recall curve (AU-PRC) performance of 4 models: random forest with TF-IDF and word count, XGBoost, deep learning model. AUC: area under the curve; PRC: precision-recall curve; TF-IDF: term frequency-inverse document frequency; XGBoost: eXtreme gradient boosting; JMIR AI 2023 \| vol. 2 \| e41264 \| p. 10 https://ai.jmir.org/2023/1/e41264 (page number not for citation purposes) •FO JMIR AI 2023 \| vol. 2 \| e41264 \| p. 10 (page number not for citation purposes) https://ai.jmir.org/2023/1/e41264 https://ai.jmir.org/2023/1/e41264 JMIR AI Nurmambetova et al discharge summaries, achieving high specificity (0.9996) and low sensitivity (0.28). Our model results are in line with other studies that used free-text clinical notes to predict incidences of distinct adverse events [48-51]. Future Directions The present work focused on demonstrating ML models on cross-sectional EMR data can outperform the ICD-based PI identification algorithm. Future directions could include (1) leveraging cost-sensitive learning to assign various weights to assess the impact of misclassifying the patients with a PI, (2) identification of the potential risk features or predictors that may be associated with PI, (3) comparison of HAN-BERT against other novel NN structures, and (4) detailed ablation studies for assessing the performance of components on the designed models that will hopefully be integrated into a clinical decision-support system. These studies will require larger sample sizes than our current pilot study, but our current work can be used to create such a cohort. Compared to these previous studies that used EMR to automate phenotyping, our model achieved higher sensitivity while reporting comparable values for performance metrics such as PPV and NPV. Furthermore, our model can identify HAPIs with high specificity and improved sensitivity during the first three days in routine clinical practice settings. Melton et al [47] found NLP to be reliable and effective in detecting 16 out of 65 adverse events in 1000 manually reviewed charts. The model by Melton et al [47] then processed all inpatient cases with EMR Principal Findings Multiple methods were applied, and different combinations of clinical text were analyzed to determine the efficiency of NLP models in detecting HAPIs from nursing notes. The results of NLP models were compared with the ICD-based algorithm reported in the previous study [1]. An AUC of 0.80 (SD 0.07) of the ML model indicates fair accuracy in terms of produced sensitivity and specificity. The results demonstrate that different combinations of EMR data leverage NLP models to improve upon ICD-10–based HAPI case definitions. The TF-IDF with RF produced higher sensitivity at a strict specificity level. The satisfactory performance of ML models indicates that the free-text documented during hospitalization contains valuable information for HAPI detection. Developing algorithms using EMR data will facilitate the timely and accurate capture of HAPI incidences and measure the quality of nursing practice during patient hospitalization. Limitations Given that many factors of the Braden Scale are documented in nursing notes daily, it may be promising to use NLP to automatically extract the Braden Scale’s factors and achieve better PI detection or prediction upon the automatically rated Braden Scale [46]. The results showed that the XGBoost and RF methods perform better than the advanced deep learning models by a large margin. The joint effort of the TF-IDF vectorizer and tree-based classifier enabled the pipeline to stay robust to irrelevant vocabularies, even when the sample size was smaller than the feature size. The feature selection played a role in this task because a great part of the text in clinical documents was not relevant to HAPIs and only contributed noise for the classification task. On the other hand, deep learning models allowed every input word to contribute to the document embedding upon which the model judged the presence of HAPIs. The suboptimal performance of the deep learning model may have been avoided if the transformers' attention mechanisms had more training samples to converge. The noisy data and not a very large sample size were possibly the main factors that made the deep learning models perform poorly. However, this hypothesis needs further examination. Conflicts of Interest None declared. Multimedia Appendix 1 Implementation details. Implementation details. [DOCX File , 84 KB-Multimedia Appendix 1] Multimedia Appendix 3 ( ) g , g g [DOCX File , 103 KB-Multimedia Appendix 3] ( ) g [DOCX File , 103 KB-Multimedia Appendix 3] Multimedia Appendix 2 Linguistic inquiry and word count analysis. [DOCX File , 16 KB-Multimedia Appendix 2] Conclusions Our study revealed the feasibility of using inpatient clinical notes documented for 3 days to detect HAPIs with increased accuracy over ICD methods. NLP and ML application on inpatient clinical notes allowed better and more timely use of the clinical narratives compared to summarizing them into ICD https://ai.jmir.org/2023/1/e41264 JMIR AI 2023 \| vol. 2 \| e41264 \| p. 11 (page number not for citation purposes) XSL•FO RenderX JMIR AI Nurmambetova et al application of the model is particularly relevant for effectively mining clinical data that does not capture a large sample size for adverse effects phenotyping. 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Nurmambetova et al JMIR AI 2023 \| vol. 2 \| e41264 \| p. 15 (page number not for citation purposes) https://ai.jmir.org/2023/1/e41264 Abbreviations Abbreviations AUC: area under the curve AUC-ROC: area under the receiver operating characteristic curve AU-PRC: area under the precision-recall curve BERT: bidirectional encoder representations from transformers BOW: bag-of-words DAD: discharge abstract database EMR: electronic medical record HAN: hierarchical attention network Abbreviations AUC: area under the curve AUC-ROC: area under the receiver operating characteristic curve AU-PRC: area under the precision-recall curve BERT: bidirectional encoder representations from transformers BOW: bag-of-words DAD: discharge abstract database EMR: electronic medical record HAN: hierarchical attention network JMIR AI 2023 \| vol. 2 \| e41264 \| p. 14 (page number not for citation purposes) JMIR AI 2023 \| vol. 2 \| e41264 \| p. 14 (page number not for citation purposes) JMIR AI 2023 \| vol. 2 \| e41264 \| p. 14 (page number not for citation purposes) https://ai.jmir.org/2023/1/e41264 XSL•FO RenderX XSL•FO RenderX JMIR AI Nurmambetova et al HAPIs: hospital-acquired pressure injuries ICD: International Classification of Diseases ICU: intensive care unit ML: machine learning NLP: natural language processing NPV: negative predictive value PI: pressure injury PPV: positive predictive value RCT: randomized controlled trial RF: random forest SCM: Sunrise Clinical Manager TF-IDF: term frequency-inverse document frequency XGBoost: extreme gradient boosting K El Emam, B Malin; submitted 24.07.22; peer-reviewed by B Ru, T Zhang; comments to author 05.10.22; revised version 1.01.23; accepted 15.01.23; published 08.03.23 received 01.01.23; accepted 15.01.23; published 08.03.23 Please cite as: Nurmambetova E, Pan J, Zhang Z, Wu G, Lee S, Southern DA, Martin EA, Ho C, Xu Y, Eastwood CA Developing an Inpatient Electronic Medical Record Phenotype for Hospital-Acquired Pressure Injuries: Case Study Using Natural Language Processing Models JMIR AI 2023;2:e41264 URL: https://ai.jmir.org/2023/1/e41264 doi: 10.2196/41264 PMID: ©Elvira Nurmambetova, Jie Pan, Zilong Zhang, Guosong Wu, Seungwon Lee, Danielle A Southern, Elliot A Martin, Chester Ho, Yuan Xu, Cathy A Eastwood. Originally published in JMIR AI (https://ai.jmir.org), 08.03.2023. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR AI, is properly cited. The complete bibliographic information, a link to the original publication on https://www.ai.jmir.org/, as well as this copyright and license information must be included. https://ai.jmir.org/2023/1/e41264 XSL•FO RenderX
https://openalex.org/W4382042947	https://jurnal-umbuton.ac.id/index.php/taksonomi/article/download/2718/1429	Indonesian	null	HUBUNGAN KEDISIPLINAN SISWA TERHADAP MOTIVASI BELAJAR SISWA DI SEKOLAH DASAR	Taksonomi	2,022	cc-by-sa	2,714	HUBUNGAN KEDISIPLINAN SISWA TERHADAP MOTIVASI BELAJAR SISWA DI SEKOLAH DASAR Suardin 1 Pendidikan Guru Sekolah Dasar Universitas Muhammadiyah Buton Email: suardinmuh78@gmail.com Abstract: The achievement of the goals that have been set in the learning process that produces student discipline in the classroom. The goal is to discipline students, so students must comply with the rules or policies that apply at school. The temporary answer to this variable, namely student discipline, can have an impact on student learning motivation. The research location is SD Negeri 2 Nganganaumala, with 21 students as research subjects. The use of this research method is quantitative by using statistical or measurement techniques in a research. This type of research is used with expost facto research, which is intended to measure cause and effect. The results of this study indicate that the better the level of student discipline, the stronger the student's motivation to learn at SD Negeri 2 Nganganaumala in the learning process, in measuring the correlation test of students' discipline variables on students' learning motivation there is a very significant relationship. Taksonomi Jurnal Pendidikan Dasar Volume 2 Nomor 2 Tahun 2022 Halaman 71-76 E- ISSN 2798-947X Doi: https://doi.org/10.35326/taksonomi.v2i2.2718 The article is published with Open Access at: Taksonomi Jurnal Pendidikan Dasar Volume 2 Nomor 2 Tahun 2022 Halaman 71-76 E- ISSN 2798-947X Doi: https://doi.org/10.35326/taksonomi.v2i2.2718 The article is published with Open Access at: PENDAHULUAN Setiap aktivitas belajar yang dilakukan siswa, terlepas dari bentuk atau sifatnya, bahwa siswa harus melibatkan diri pada saat pembelajaran berlangsung, sehingga sesuatu dapat diketahui atau dipahami (Megawati & Kadarisman, 2021). Hal ini disebabkan karena belajar merupakan proses mengetahui sesuatu yang dapat meningkatkan rasa harga diri, martabat, dan pengetahuan seseorang (Elvira et al., 2021). Secara teoritis, interaksi yang berbeda yang menimbulkan keinginan untuk menjadi lebih baik melalui reaksi-reaksi yang dialami oleh individu yang bersangkutan itulah yang menyebabkan terjadinya interaksi antara stimulus dan respon yang berlangsung ketika pembelajaran terjadi (Maros & Juniar, 2016). Kedisiplinan siswa pada lingkungan sekolah akan mengarah pada pencapaian tujuan yang lebih jelas dalam proses pembelajaran yang dilakukan guru (Mukhlishina, 2022). Hal ini dimaksudkan agar kedisiplinan siswa, sehingga siswa harus mematuhi standar atau peraturan yang berlaku disekolah (Selviana Amu, Yulsy M. Nitte, 2021). Disiplin merupakan komponen utama dalam lingkungan sekolah baik dalam kelas maupun diluar kelas, sehingga Kedisiplinan siswa dapat berdampak pada motivasi belajar siswa (Irwani, 2020). Motivasi belajar sebagai keseluruhan kekuatan yang mendorong siswa untuk merancang, memelihara, dan mengarahkan kegiatan belajar dengan harapan mencapai tujuan (Rahma & Muhid, 2022). Motivasi belajar itu penting dan nilai manfaat dari kegiatan belajar sangat menarik bagi siswa untuk terlibat dalam kegiatan belajar (Akmaluddin & Haqiqi, 2019). Hal ini karena siswa sudah memahami betapa pentingnya motivasi belajar. Siswa yang tidak mempraktikkan kedisiplinan, memandang motivasi sebagai dorongan bagi seseorang. Namun, ada beberapa siswa yang terlebih dahulu didorong untuk disiplin, akhirnya memahami nilai motivasi belajar siswa (Handayani & Subakti, 2020). Disiplin adalah pengendalian diri untuk menyelesaikan semua tugas yang telah dikuasakan atau diakui sebagai kewajiban. Oleh karena itu, disiplin dapat dipandang sebagai kepatuhan atau ketaatan pada hukum atau aturan (Nenengkhoirunisa et al., 2022). Disiplin siswa adalah suatu keadaan ketertiban dan tata tertib yang ditaati oleh siswa, bebas dari segala pelanggaran yang dapat merugikan dirinya baik langsung ataupun tidak langsung (Chaerunisa & Latief, 2021). Proses belajar mengajar di kelas pada khususnya dan di sekolah pada umumnya bercirikan disiplin siswa (Endang Kristiani, 2021). Jika guru secara khusus menyadari perilaku disiplin, maka siswa akan mengembangkan perilaku disiplin tersebut dengan baik (Wahab et al., 2021). Seorang guru memiliki kewajiban untuk menjadi teladan bagi siswa. Sehingga, disiplin guru memiliki dampak yang signifikan pada bagaimana siswa mengembangkan kepribadian mereka dan menjadi termotivasi untuk belajar (Haryadi, 2020). Siswa secara alami mengikuti guru yang bertindak disiplin, seperti dalam hal waktu, penampilan, metode pengajaran, dan lain-lain (Afandi & Redjeki, 2021). Keywords: Relationship, Discipline, Motivation, Learning Abstrak: Tercapainya tujuan yang telah ditetapkan bahwa dalam proses pembelajaran yang menghasilkan kedisiplinan siswa di dalam kelas. Tujuannya adalah untuk mendisiplinkan siswa, sehingga siswa harus mematuhi aturan atau kebijakan yang berlaku di sekolah. Jawaban sementara pada variabel ini yaitu kedisiplinan siswa dapat berdampak pada motivasi belajar siswa. Adapun lokasi penelitian yaitu SD Negeri 2 Nganganaumala, dengan subjek penelitian sebanyak 21 siswa. Penggunaan metode penelitian ini yaitu kuantitatif dengan menggunakan teknik statistik atau pengukuran dalam sebuah peneltian. Jenis penelitian digunakan dengan penelitian expost facto, yang dimaksudkan untuk mengukur sebab dan akibat. Hasil penelitian ini menunjukan bahwa semakin baik tingkat Kedisiplinan siswa maka semakin kuat motivasi belajar siswa di SD Negeri 2 Nganganaumala dalam proses pembelajaran, pada pengkuran uji korelasi variabel kedisiplinan siswa terhadap motivasi belajar siswa terdapat hubungan yang sangat signifikan. Kata kunci: Hubungan, Kedisiplinan, Motivasi, Pembelajaran 71 METODE Metode penelitian ini yaitu metode kuantitatif dengan menggunakan teknik statistik atau pengukuran dalam sebuah peneltian (Arsy et al., 2021). Penelitian ini dilaksanakan di SD Negeri 2 Nganganaumala. Dalam penelitian ini, peneliti berusaha untuk memahami hubungan sebab akibat antara variabel yang mempengaruhi dan variabel yang terkena dampak, maka jenis penelitian yang digunakan adalah jenis penelitian Expost Facto, yang dimaksudkan untuk mengukur sebab dan akibat (Wahyuni et al., 2016). Variabel independen dan dependen digunakan dalam menganalisis uji koreasi antara kedua variabel (Amrizal et al., 2020). Kedisiplinan siswa (X) merupakan variabel terikat dalam penelitian ini, sedangkan motivasi belajar siswa (Y) merupakan variabel bebas. Populasi dalam penelitian ini adalah siswa kelas V SD Negeri 2 Nganganaumala sebanyak 21 siswa, 6 siswa laki-laki dan 15 siswa perempuan. Kuesioner adalah alat yang digunakan dalam penelitian ini untuk mengumpulkan data. Pengumpulan data angket digunakan untuk menilai kedisiplinan siswa, dan pengumpulan data. Kusioner digunakan untuk memperoleh informasi mengenai motivasi belajar siswa. Uji-t digunakan untuk mengevaluasi kekuatan uji korelasi antara variabel independen dan dependen ketika menganalisis hasil penelitian (Lestari & Miftakhul’Ulum, 2020). HASIL DAN PEMBAHASAN Tanggapan terhadap kuesioner yang diberikan kepada 21 siswa digunakan untuk menghasilkan data untuk penelitian ini. Dua variabel yang digunakan dalam penelitian ini yaitu variabel kedisiplinan siswa (X) dan variabel motivasi belajar siswa (Y). Untuk mendefinisikan dan mengevaluasi hubungan antara variabel independen dan variabel dependen dalam penelitian ini, akan diberikan deskripsi data. PENDAHULUAN Jika seorang pengajar melakukan hal ini secara konsisten dan pada akhirnya menjadi tertanam dalam budaya mereka, itu akan mencerminkan motivasi siswa untuk berpartisipasi dalam kegiatan belajar mengajar di sekolah. Pengamatan awal di SD Negeri 2 Nganganaumala, dari wawancara dengan guru kelas bahwa kedisiplinan siswa biasanya tidak menentu seperti datang terlambat, baju yang kurang rapi, dan hal ini berdampak pada cara siswa belajar, sehingga motivasi siswa dalam belajar selalu menjadi faktor utama seperti dalam kelas siswa masih banyak yang berbicara dengan temannya, mencoret-coret di atas kertas. Sehingga, Banyak siswa yang secara konsisten menerima nilai rendah dan tidak mencapai tingkat ketuntasan (KKM) yang dipersyaratkan. Terdapat 40% sampai 50% siswa pada setiap kelas menerima KKM di bawah rata-rata. Kedisiplinan siswa di rumah dan di sekolah secara langsung tercermin dalam tingkat motivasi belajar siswa. Untuk memperoleh hasil belajar terbaik, kedisiplinan siswa terhadap peraturan sekolah akan mendorong berkembangnya motivasi belajar yang efisien dan praktis. 72 1. Deskripsi Kedisiplinan Siswa 1. Deskripsi Kedisiplinan Siswa Deskripsi kedisiplinan siswa kelas V SD Negeri 2 Nganganaumala digunakan lembar angket dalam pengukuran statistik. Berdasarkan hasil analisis data penelitian diperoleh nilai frekuensi dan presentase untuk variabel tingkat disiplin siswa. Berikut ini adalah hasil dari kedisiplinan siswa: Tabel 1. Klasifikasi Data Kedisiplinan Siswa No. Kategori Rentang Skor Frekuensi Presentase 1. Sangat baik > 85 13 61,91 2. Baik 76–85 5 23,81 3. Cukup 65 – 75 2 9,52 4. Kurang <65 1 4,76 Tabel 1. Klasifikasi Data Kedisiplinan Siswa Tabel 1. Klasifikasi Data Kedisiplinan Siswa Kedisiplinan siswa didasarkan pada angket yang diberikan kepada siswa. Sehingga terdapat nilai nilai >85 dalam kategori sangat baik sebanyak 13 siswa (61,91%), yang mendapat nilai 76–85 dalam kategori baik sebanyak 5 siswa (23,81%), yang mendapat nilai 65–75 pada kategori cukup terdapat 2 siswa (9,52%), dan yang mendapat nilai 65 pada kategori kurang sebanyak 1 siswa (4,76%). 2. Deskripsi Motivasi Belajar Siswa Motivasi belajar siswa digunakan dalam variabel berdasarkan evaluasi klasifikasi data yang dihasilkan untuk variabel motivasi belajar siswa adalah nilai analisis statistik berdasarkan frekuensi dan persentase temuan. Motivasi belajar siswa digunakan dalam variabel berdasarkan evaluasi klasifikasi data yang dihasilkan untuk variabel motivasi belajar siswa adalah nilai analisis statistik berdasarkan frekuensi dan persentase temuan. 73 Tabel 2. Klasifikasi Data Motivasi Belajar Siswa Tabel 2. Klasifikasi Data Motivasi Belajar Siswa No. Kategori Rentang Skor Frekuensi Presentase 1. Sangat baik > 85 10 47,62 2. Baik 76–85 6 28,58 3. Cukup 65 – 75 4 19,04 4. Kurang <65 1 4,76 Persentase siswa yang memperoleh nilai >85 dalam kategori sangat baik adalah 10 siswa (47,62%), disusul dengan siswa yang memperoleh nilai 76–85 dalam kategori baik sebanyak 6 siswa (28,58%), diikuti oleh siswa yang mendapat nilai 65-75 dalam kategori cukup sebanyak 4 siswa (19,04%), dan siswa yang mendapat nilai 65 dalam kategori kurang sebanyak 1 siswa (4,76%). 3. Hubungan antara Kedisiplinan Siswa (X) terhadap Motivasi Belajar Siswa (Y) Di SD Negeri 2 Nganganaumala dilakukan pengujian hipotesis untuk mengetahui hubungan antara disiplin (X) dengan motivasi belajar siswa (Y). Hasil pengujian hipotesis ditunjukkan pada tabel berikut: a. Analisis Uji Korelasi a. Analisis Uji Korelasi a. Analisis Uji Korelasi Tabel 3. Uji Korelasi Variabel Tabel 3. Uji Korelasi Variabel Tabel 3. Uji Korelasi Variabel Kedisiplinan Siswa Motivasi Belajar Kedisiplinan Siswa Pearson Correlation Sig. (2-tailed) N 1 .000 21 .432 .000 21 Motivasi Belajar Pearson Correlation Sig. b. Analisis Uji t (t-test) j ( ) Uji-T digunakan untuk menentukan apakah variabel independen dan dependen berhubungan secara parsial: Tabel 4. Hipotesis Uji t 1. Deskripsi Kedisiplinan Siswa (2-tailed) N .432 .000 21 1 .000 21 Berdasarkan tabel 3 diatas, dapat diketahui hasil uji korelasi variabel bahwa nilai sig. yaitu 0.000 < α (0,5), yang berarti hipotesis Ho ditolak, dan hipotesis H1 diterima, sehingga hal ini menjelaskan bahwa terdapat hubungan yang positif antara variabel kedisiplinan siswa terhadap motivasi belajar siswa kelas V SD Negeri 2 Nganganaumala. b. Analisis Uji t (t-test) Tabel 4. Hipotesis Uji t Tabel 4. Hipotesis Uji t Model Unstandardized Coefficients Standardized Coefficients B Std. Error Beta t Sig. 1 (Constan t) 20.553 2.774 7.409 .000 Kedisiplinan Siswa 1.639 .142 .833 11.557 .000 a. Dependent Variabel: Motivasi Belajar Siswa 74 Berdasarkan tabel 4 di atas, Hipotesis yang diajukan adalah terdapat hubungan antara disiplin siswa (X) dengan motivasi belajar siswa (Y) di SD Negeri 2 Nganganaumala jika nilai thitung lebih besar dari ttabel sehingga nilai uji t sebesar 11.557 > 20.553 karena adanya signifikansi tingkat Kedisiplinan siswa(0,000 > 0,05) dan nilai ttabel = t (/2: n-1 = t (0,025: 21) = 0.001. Hal ini menunjukan bahwa terdapat hubungan yang substansial antara motivasi belajar siswa (Y) dengan variabel tingkat kedisiplinan (X). KESIMPULAN Berdasarkan hasil penelitian SD Negeri 2 Nganganaumala, beberapa siswa masuk ke kelas dan langsung duduk di depan guru, begitu juga sebaliknya. Siswa mengaku bahwa orang tua mereka jarang mengingatkan mereka untuk menjaga ketertiban dan disiplin di kelas ketika peneliti mempertanyakan apakah mereka sudah terbiasa. Setelah itu, peneliti memberikan angket kepada siswa untuk mengumpulkan data tambahan. Dari hasil angket disiplin siswa terlihat jelas bahwa terdapat perbedaan tingkat kedisiplinan siswa. Siswa yang disiplin selalu belajar dengan sungguh-sungguh, memperhatikan kelas, datang tepat waktu, dan mematuhi standar sekolah. Berdasarkan hasil analisis hipotesis penelitian, motivasi belajar siswa di SD Negeri 2 Nganganaumala berkorelasi signifikan dengan variabel tingkat kedisiplinan siswa. Namun, dibandingkan indikator lain berdasarkan sinyal kedisiplinan dalam kegiatan belajar di rumah, disiplin belajar belum mencukupi dalam penelitian ini. Hal ini disebabkan karena terkadang anak-anak sulit berkonsentrasi saat belajar di rumah, dan mereka lebih memilih bermain saat pulang sekolah daripada membaca ulang materi yang telah dipelajari. Selain waktu yang harus dihabiskan untuk belajar, siswa juga menikmati menonton Youtube di HP dan TV. Karena tingkat pengawasan orang tua yang biasa, siswa mungkin menjadi kurang disiplin. DAFTAR PUSTAKA Acoci, A. (2020). 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https://openalex.org/W4233583605	https://www.biogeosciences.net/14/1603/2017/bg-14-1603-2017.pdf	English	null	Overestimation of closed chamber soil CO&lt;sub&gt;2&lt;/sub&gt; effluxes at low atmospheric turbulence	null	2,016	cc-by	17,488	Correspondence to: Andreas Brændholt (andbr@env.dtu.dk) Received: 10 November 2016 – Discussion started: 11 November 2016 Revised: 1 March 2017 – Accepted: 7 March 2017 – Published: 28 March 2017 Received: 10 November 2016 – Discussion started: 11 November 2016 Revised: 1 March 2017 – Accepted: 7 March 2017 – Published: 28 March 2017 niques and erroneous data must be ﬁltered out to obtain un- biased diurnal patterns, accurate relationships to biotic and abiotic factors, and before estimating Rs ﬂuxes over longer timescales. Abstract. Soil respiration (Rs) is an important component of ecosystem carbon balance, and accurate quantiﬁcation of the diurnal and seasonal variation of Rs is crucial for a cor- rect interpretation of the response of Rs to biotic and abiotic factors, as well as for estimating annual soil CO2 efﬂux rates. factors, as well as for estimating annual soil CO2 efﬂux rates. In this study, we measured Rs hourly for 1 year by auto- mated closed chambers in a temperate Danish beech forest. The data showed a clear diurnal pattern of Rs across all sea- sons with higher rates during night-time than during daytime. However, further analysis showed a clear negative relation- ship between ﬂux rates and friction velocity (u∗) above the canopy, suggesting that Rs was overestimated at low atmo- spheric turbulence throughout the year due to non-steady- state conditions during measurements. Filtering out data at low u∗values removed or even inverted the observed diur- nal pattern, such that the highest efﬂuxes were now observed during daytime, and also led to a substantial decrease in the estimated annual soil CO2 efﬂux. 1 Introduction Soil respiration (Rs) in terrestrial ecosystems is the second largest ﬂux of CO2 after gross photosynthesis and was found to account for 63 % of ecosystem respiration on average in a study of 18 European forests (Janssens et al., 2001; Raich and Schlesinger, 1992). Rs may exhibit both a strong seasonal and diurnal pattern (e.g. Janssens et al., 2000; Tang et al., 2005) and accurate measurements of Rs at various timescales are thus important to correctly estimate this ﬂux component; i.e. periodic over- or underestimation of Rs may lead to huge errors in annual ecosystem CO2 budget estimates. By installing fans to produce continuous turbulent mix- ing of air around the soil chambers, we tested the hypothesis that overestimation of soil CO2 efﬂuxes during low u∗can be eliminated if proper mixing of air is ensured, and indeed the use of fans removed the overestimation of Rs rates dur- ing low u∗. Artiﬁcial turbulent air mixing may thus provide a method to overcome the problems of using closed-chamber gas-exchange measurement techniques during naturally oc- curring low atmospheric turbulence conditions. Other possi- ble effects from using fans during soil CO2 efﬂux measure- ments are discussed. In conclusion, periods with low atmo- spheric turbulence may provide a signiﬁcant source of er- ror in Rs rates estimated by the use of closed-chamber tech- CO2 produced in the soil must, in the long term, be emitted from the soil surface (Maier et al., 2011), although, in some ecosystems, leaching of dissolved organic and inorganic car- bon may occur (Kindler et al., 2011). Rs is therefore often measured as soil CO2 efﬂux by the closed-chamber method, which relies on Fick’s law of diffusion and steady-state dif- fusion rate conditions (Gao and Yates, 1998). Under these conditions, the ﬂux rate can be calculated from the increase in chamber CO2 concentration during the chamber deploy- ment period. To estimate annual Rs on a site, manual chamber measure- ments are often performed at regular intervals (e.g. twice a month) at multiple plots, which ideally encompass the tem- Biogeosciences, 14, 1603–1616, 2017 www.biogeosciences.net/14/1603/2017/ doi:10.5194/bg-14-1603-2017 © Author(s) 2017. CC Attribution 3.0 License. Biogeosciences, 14, 1603–1616, 2017 www.biogeosciences.net/14/1603/2017/ doi:10.5194/bg-14-1603-2017 © Author(s) 2017. CC Attribution 3.0 License. Overestimation of closed-chamber soil CO2 efﬂuxes at low atmospheric turbulence Andreas Brændholt1, Klaus Steenberg Larsen1,2, Andreas Ibrom1, and Kim Pilegaard1 1DTU Environment, Technical University of Denmark, Kgs. Lyngby, 2800, Denmark 2Department of Geosciences and Natural Resource Management, University of Copenhagen, Frederiksberg C, 1958, Denmark Correspondence to: Andreas Brændholt (andbr@env.dtu.dk) A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes 1604 poral and spatial variation in Rs in the studied ecosystem. For logistic reasons manual measurements are, however, most of- ten performed during daytime working hours and therefore do not capture the diurnal variation. To capture the diurnal variation, diurnal measurements campaigns at a few plots are often performed at a high temporal resolution (e.g. every 1– 2 h) but the number of campaigns over a full year may often be limited. Using campaign-wise diurnal cycle patterns for the entire year may cause biases, since the diurnal pattern of Rs may itself exert seasonal differences, which may not be captured during a limited number of diurnal measurement campaigns (Ruehr et al., 2010). act to either suppress or increase the CO2 efﬂux respectively (Kanemasu et al., 1974). To maintain ambient pressure in the chamber headspace, closed chambers often have a vent con- nected to the atmosphere (e.g. Hutchinson and Mosier, 1981; Savage and Davidson, 2003; Xu et al., 2006). High wind speeds moving over the vent tube may lead to a pressure drop in the chamber headspace, due to the Venturi effect, thereby leading to ﬂux overestimation (Conen and Smith, 1998). Cor- rect vent design has, however, been found to eliminate this effect (Xu et al., 2006). While the effects mentioned above are well described, the effect of atmospheric turbulence during closed-chamber measurements has received much less attention. Recently, a few studies have demonstrated that low friction velocity (u∗), a measure of atmospheric turbulence, can lead to overesti- mation of ﬂuxes measured by closed chambers, and that it is especially a problem during night-time where u∗is typically lowest (Görres et al., 2016; Juszczak et al., 2012; Lai et al., 2012; Schneider et al., 2009). It has been suggested that the overestimation of chamber ﬂuxes is because a stratiﬁed layer of CO2 builds up at the soil surface during periods of low u∗, but is broken down by the chamber movement at closure (Görres et al., 2016). The assumption behind the usage of closed chambers for estimating soil CO2 efﬂux may also be challenged and a number of potential biases have been identiﬁed that may cause an over- or underestimation of the true soil CO2 efﬂux (Davidson et al., 2002; Pumpanen et al., 2004; Rochette and Hutchinson, 2005; Ryan and Law, 2005). A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes Firstly, the increase in chamber headspace CO2 concentration during measure- ments decreases the concentration gradient between the soil and chamber headspace, thereby theoretically decreasing the apparent soil CO2 efﬂux according to Fick’s law of diffusion (Gao and Yates, 1998). Consequently, it has often been found that applying a linear ﬁt to the increase in chamber CO2 con- centration leads to a systematic underestimation of the mea- sured CO2 efﬂux compared to the true efﬂux (Anthony et al., 1995; Venterea, 2010). To correct for this methodological er- ror a non-linear ﬁt can be applied to the increase in chamber headspace CO2 concentration to estimate the CO2 efﬂux at time zero, where chamber headspace CO2 concentration is at ambient level. Though theoretically sounder, non-linear ﬁts have been found to increase the uncertainty of the ﬂux esti- mate (Venterea, 2010). The potential overestimation of soil CO2 efﬂuxes due to low u∗has become relevant, especially in recent years. High- frequency soil CO2 efﬂux measurements have become more widespread by the emergence of commercially available au- tomated closed-chamber systems, as well as increasing in- tegration and usage of chamber ﬂux technologies in inter- national research infrastructures, such as ICOS (Integrated Carbon Observation System), which aims to quantify the greenhouse balance using common approaches and protocols across multiple sites. However, insufﬁcient research has been done on the topic, and there is no consensus on how to ac- count for this effect to get unbiased measurements of soil CO2 efﬂuxes. Thus it is currently unknown what effect this bias has on upscaled annual soil CO2 efﬂux estimates for dif- ferent ecosystems, and how unbiased measurements can be performed during low u∗. Many chambers are placed on a soil collar permanently in- stalled a few centimetres into the soil to secure a good seal- ing of the chamber to the soil surface, thus preventing lat- eral transport of air into or out from the chamber in the top soil (Healy et al., 1996; Hutchinson and Livingston, 2001; Livingston and Hutchinson, 1995). However, the soil collar may cause a disturbance to the soil, e.g. by changing the mi- croenvironment, severing plant roots or even increasing root growth (Görres et al., 2016). These disturbances of the soil can potentially change the Rs inside the collar compared to undisturbed soil. Our study had two aims. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes The ﬁrst aim was to quantify the effect of u∗on automated closed-chamber soil CO2 ef- ﬂuxes in the short term (i.e. effect on diurnal ﬂuxes) and in the long term (i.e. effect on annual estimates of CO2 efﬂux). The second aim was to test the hypothesis that the overesti- mation of soil CO2 efﬂuxes during low u∗was due to insuf- ﬁcient mixing of the open air above the soil surface and to test whether unbiased soil CO2 efﬂux measurements could be achieved during low u∗by artiﬁcially inducing mixing of the air around the soil chambers by a fan. Placing the chamber on the soil before a measurement may also lead to a disturbance in the diffusion-driven soil CO2 ef- ﬂux. This can either lead to a ﬂush of CO2 from the soil pores into the chamber headspace resulting in a higher soil CO2 ef- ﬂux (Matthias et al., 1980; Hutchinson and Livingston, 1993) or lead to horizontal transport of gas in the soil resulting in a lower soil CO2 efﬂux (e.g. Conen and Smith, 2000; Kutzbach et al., 2007; Pedersen et al., 2010). The conditions of the atmosphere surrounding the cham- ber may also inﬂuence the measured CO2 efﬂux. An over- or under-atmospheric pressure in the chamber headspace can 2.1 Site description There were three gaps in the automatic data collection. From 12 November to 22 November an unknown system fail- ure occurred. From 3 February to 10 February measurements were stopped to prevent damage to the system due to accu- mulated snow. From 19 May to 23 July the system did not run due to a mechanical system failure, which required repair. This led to a data coverage of 76 % of the days (272 days out of 356 days). Chamber closure time was 90 and 150 s for the automated and manual chambers respectively, and the ﬁrst 20 s after chamber closure was discarded (the dead band). Soil temperature and soil moisture content were measured at a depth of 5 cm for both the manual and automated measure- ments. For the automated measurements, six soil thermome- ters were distributed close to the soil chambers, such that no soil chamber was further away than 10 m from an individual soil sensor. Measurements were performed at the Danish ICOS RI site DK-Sor at 40 m a.s.l. (55◦29′13′′ N, 55◦38′45′′ E). Measure- ments of tower-based eddy covariance have been running since 1996. The climate is temperate maritime with an an- nual average temperature of 8.5 ◦C and an annual average precipitation of 564 mm (Pilegaard et al., 2011). A dense forest covers the site, and the dominant tree species is European beech (Fagus sylvatica L.) with scattered stands of conifers such as Norway spruce (Picea abies (L.) Karst) and larch (Larix decidua Mill.) constituting 20 % of the forest (Wu et al., 2013). The stand of beech around the ﬂux tower was planted in 1921 and had an average height of 28 m and an average diameter at breast height of 42 cm in 2010. The average annual duration of canopy cover is 180 days with a peak LAI of 5.0, and the tree stem density is 266 per hectare. The understory is poorly developed due to the well-developed canopy, causing a sparsely vegetated forest ﬂoor. During spring, however, part of the forest ﬂoor is covered by wood anemone (Anemone nemorosa L.). Alongside the chamber measurements, wind speed in three dimensions was measured by a sonic anemometer (HS-50 Research Anemometer, Gill Instruments Limited, Lyming- ton, UK) at a height of 43 m above soil surface on the ﬂux tower from which u∗was calculated according to Stull (1988). 2.3 Fan experiment campaign During a 20 day campaign in July and August 2016 soil CO2 efﬂuxes were measured at the site with four of the 8100- 104 long-term CO2 ﬂux chambers and two of the 8100-101 long-term CO2 ﬂux chambers, with each chamber measur- ing soil CO2 efﬂuxes every 2 h using a chamber closure time of 5 min. A dead band of 60 s, longer than the 20 s used for the 1-year campaign, was required, as well as a longer clo- sure time, because an external gas analyser was attached to the LI-8100A during the fan experiment. The longer cham- ber closure time was used because the external analyser re- quired a larger difference in CO2 concentration during cham- ber measurements to achieve sufﬁcient precision. The longer dead band was used because the extra volume added to the system by the external gas analyser caused longer response times and therefore also longer time to achieve stability af- ter chamber closure. The aim of the campaign was to test if artiﬁcially increasing the mixing of air around the soil cham- ber would eliminate the bias of low u∗on measured chamber soil CO2 efﬂuxes. The artiﬁcial air mixing for each chamber was provided by 30 cm diameter table fans facing the cham- ber (Model 546601, HP Schou A/S, Kolding, Denmark) po- sitioned 3 m from the soil chamber and at a height of 30 cm from the soil surface to the middle of the fan. The fans pro- vided a wind speed of 1.2–1.5 m s−1 at the chamber collars. During the ﬁrst 10 days of the campaign, fans were installed at three chambers, resulting in three chambers with artiﬁcial air mixing and three chambers experiencing ambient condi- 2.1 Site description The soil is classiﬁed as Alﬁsols or Mollisols (depending on the base saturation) with an organic layer with a depth of 10–40 cm. The soil carbon pool (down to a depth of 1 m) is 20 kg m−2, with a C / N ratio of about 20 in the upper organic soil layers, dropping to about 10 in the lower mineral layers (Østergård, 2000). 2 Materials and methods vironmental, Lincoln, Nebraska USA). Both plots for auto- mated and manual measurements contained bare forest ﬂoor including litter but no shrubs or saplings. www.biogeosciences.net/14/1603/2017/ www.biogeosciences.net/14/1603/2017/ Biogeosciences, 14, 1603–1616, 2017 1605 www.biogeosciences.net/14/1603/2017/ 2.4 Data analysis All data analysis was done using R (R Core Team, 2014). Because the current paper focuses on the potential error of low turbulent air mixing and because ﬂuxes calculated us- ing non-linear regression ﬁtting may add additional aspects of uncertainty to the calculated ﬂuxes, we focused on CO2 efﬂuxes calculated on a time and area basis by applying lin- ear regression to the increase in chamber CO2 concentration during chamber closure time. However, non-linear efﬂuxes were calculated as well, and at four different dead bands of 10, 20, 30 and 40 s (see Supplement). The linear ﬂuxes were calculated in R (R Core Team, 2014) using the lm function and the non-linear ﬂuxes were calculated by ﬁtting the non- linear equation suggested by Hutchinson and Mosier (1981) with the nlsLM function (minpack.lm package) for model ﬁtting in R. Quality control for the automated chamber ef- ﬂuxes was done by removing ﬂuxes with an r2 < 0.95 of the linear regression before further analysis, equal to 17 % of the measurements for the 1-year campaign and 1 % of the measurements for the fan experiment campaign. For the man- ual measurements, the quality control was done in the ﬁeld directly following a measurement. If the coefﬁcient of vari- ance of the ﬂux (as provided by the LI-COR software imme- diately following a measurement) was higher than 1.4, the measurement was discarded and an extra measurement was performed on the soil collar. In addition to the annual soil CO2 efﬂuxes using all 24 h throughout the day, the daytime annual soil CO2 efﬂux was calculated in a similar manner, except that only measure- ments made between 09:00 and 15:00 CET were used. The manually measured soil CO2 efﬂuxes were used to parameterise the empirical model Rs = R283 exp −E0 1 Ts + 273.15 −T0 − 1 Ts −T0 , (1) of Lloyd and Taylor (1994), where Ts is soil temperature at 5 cm depth and R283 is the base respiration at a soil tempera- ture of 10 ◦C. T0 and E0 are ﬁtted parameters. The model was ﬁtted using non-linear least squares regression based on a Levenberg–Marquardt algorithm using nlsLM in the R pack- age minpack.lm (Elzhov et al., 2015). 2.4 Data analysis The model was used to form a continuous time series of mean daily soil CO2 ef- ﬂuxes throughout 1 year by using a continuous measurement of soil temperature measured at 5 cm depth at the site as in- put to the model. From the modelled daily soil CO2 efﬂuxes, monthly soil CO2 efﬂuxes were calculated as the sum of the daily soil CO2 efﬂuxes in the respective month. From this the annual soil CO2 efﬂux was calculated as the sum of all the 12 monthly CO2 efﬂuxes. The calculated efﬂuxes for the 1-year campaign were paired with u∗data from the eddy covariance system in the mast (43 m). The u∗values were used to create sub-datasets by a u∗threshold ﬁltering technique, where efﬂuxes mea- sured at u∗values lower than a speciﬁc threshold value had been ﬁltered out and removed from the dataset (Aubinet et al., 2000). Twelve different u∗threshold values were used, ranging from 0.1 to 1.2 m s−1, with a successive higher u∗ threshold value of 0.1 m s−1. Thus, 12 different sub-datasets each with a speciﬁc u∗threshold value were derived from the 1-year campaign soil CO2 efﬂuxes. The number of soil CO2 efﬂuxes was 43 505 for the unﬁltered dataset. For the 12 different sub-datasets going from a u∗threshold value of 0.1 to 1.2 m s−1, the number of soil CO2 efﬂuxes was 32 966, 26 848, 22 185, 18 557, 15 787, 13 449, 11 472, 9533, 7950, 6571, 5481 and 4571 respectively. A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes 1606 The annual soil CO2 efﬂux was obtained for each sub- dataset from the mean soil CO2 efﬂux for each hour of the day for each month. From this a daily mean was calculated for each month. Monthly soil CO2 efﬂuxes were calculated as the sum of the daily soil CO2 efﬂux in the respective month and the annual soil CO2 efﬂux was calculated. One period of data outage due to system failure (20 May to 22 June) was gap ﬁlled by linear interpolation between hourly values of mean diurnal patterns that were calculated from the adjacent periods of the data gap. tions. During the last 10 days of the campaign, the fans were moved to the other three chambers, thus providing a data set with 10 days of ambient air and 10 days with artiﬁcial air mixing on all six chambers. u∗at a height of 43 m above the soil surface was calculated similarly to the 1-year campaign. 2.2 One-year campaign of soil CO2 efﬂux and friction velocity measurements Soil CO2 efﬂux was measured automatically over 1 year from 10 October 2014 to 30 September 2015 (356 days) with ﬁve 8100-104 long-term CO2 ﬂux chambers and three 8100- 101 long-term CO2 ﬂux chambers in a multiplexed set-up with a LI-8100A Automated Soil CO2 Flux System and a LI-8150 Multiplexer (LI-COR Environmental, Lincoln, Ne- braska USA). Measurements were made on permanent, cir- cular soil collars (20 cm diameter) that were inserted 4 cm into the soil prior to the measurement period. The aver- age soil collar height was 5.2 cm with the individual collar heights ranging from 4.5 to 5.8 cm. The automated chambers each measured soil CO2 efﬂuxes once every hour and were positioned within 15 m of the ﬂux tower. Soil CO2 efﬂux was also measured manually at 12 additional circular plots with soil collars (10 cm diameter) installed 4 cm into the soil as for the automated chambers. The manual plots were posi- tioned close to the automated chamber plots (< 10 m) and also within 15 m of the ﬂux tower. Soil CO2 efﬂux at the 12 plots were each measured every 2 weeks during daytime be- tween 09:00 and 15:00 CET on days with little or no rain us- ing a portable 8100-102 10 cm survey chamber connected to a LI-8100A Automated Soil CO2 Flux System (LI-COR En- www.biogeosciences.net/14/1603/2017/ Biogeosciences, 14, 1603–1616, 2017 A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes www.biogeosciences.net/14/1603/2017/ 3.1 Friction velocity and soil CO2 efﬂuxes Soil CO2 efﬂux generally exhibited a diurnal pattern in- versely related to the diurnal pattern of u∗, with the highest efﬂuxes seen during night-time, when u∗was lowest (Fig. 1). During summer, autumn and spring, u∗showed a clear diur- nal pattern with highest values during daytime and lowest values during night-time (Fig. 2a, b, d), while this pattern was weaker during winter (Fig. 2c). Average hourly soil temperature for the automated soil chambers measured at 5 cm depth showed no diurnality dur- ing winter (Fig. 3c). However, for summer, autumn and spring a slight diurnal pattern was observed with lowest tem- peratures at 07:00–10:00 CET and highest temperatures late in the afternoon or early in the evening (Fig. 3a, b, d). For each of the sub-datasets, diurnal ensemble averages of soil CO2 efﬂux were calculated for each of the four dis- tinct seasons at the site: summer (July and August), autumn (September, October and November), spring (March, April and May) and winter (December, January and February). When comparing the soil CO2 efﬂuxes measured with the automated chambers during the 1-year campaign with u∗, we found a clear relationship with higher soil CO2 efﬂuxes at Biogeosciences, 14, 1603–1616, 2017 www.biogeosciences.net/14/1603/2017/ A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes 16 A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes 1607 0 2 4 6 8 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Friction velocity (m s −1) Soil CO2 efflux (µmol CO 2 m−2 s−1) Date 1 August 3 August 5 August 7 August 9 August 11 August Figure 1. Example of mean hourly soil CO2 efﬂuxes from the eight automated chambers (solid line) and mean hourly friction velocity (u∗) at 43 m above the soil surface (dashed line) for 10 days during August 2015. 0 2 4 6 8 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Friction velocity (m s −1) Soil CO2 efflux (µmol CO 2 m−2 s−1) Date 1 August 3 August 5 August 7 August 9 August 11 August Figure 1. Example of mean hourly soil CO2 efﬂuxes from the eight automated chambers (solid line) and mean hourly friction velocity (u∗) at 43 m above the soil surface (dashed line) for 10 days during August 2015. 3.1 Friction velocity and soil CO2 efﬂuxes G G G G G G G G G G G G G G G G G G G G G G G G 00:00 05:00 10:00 15:00 20:00 0.0 0.5 1.0 1.5 2.0 (a) Summer G G G G G G G G G G G G G G G G G G G G G G G G Friction velocity (m s−1) Time of day (h) G G G G G G G G G G G G G G G G G G G G G G G G 00:00 05:00 10:00 15:00 20:00 (b) Autumn G G G G G G G G G G G G G G G G G G G G G G G G Time of day (h) G G G G G G G G G G G G G G G G G G G G G G G G 00:00 05:00 10:00 15:00 20:00 (c) Winter G G G G G G G G G G G G G G G G G G G G G G G G Time of day (h) G G G G G G G G G G G G G G G G G G G G G G G G 00:00 05:00 10:00 15:00 20:00 (d) Spring G G G G G G G G G G G G G G G G G G G G G G G G Time of day (h) Figure 2. Mean (± standard deviation) diurnal pattern of friction velocity (u∗) at 43 m above the soil surface for summer (a), autumn (b), winter (c) and spring (d). Figure 2. Mean (± standard deviation) diurnal pattern of friction velocity (u∗) at 43 m above the soil surface for summer (a), autumn (b), winter (c) and spring (d). lower u∗values (Fig. 4), i.e. a signiﬁcant negative correlation between u∗and soil CO2 efﬂux for all seasons (P = < 0.001, r2 = 0.065). The relationship seemed, however, to level off at a u∗threshold value of approximately 0.7 m s−1. Further increasing the u∗threshold value only led to a small or no further decrease in estimated efﬂuxes. day, with only slightly lower efﬂuxes in the afternoon. The change in the diurnal pattern in response to an increased u∗ threshold value was also seen for the other seasons. 3.1 Friction velocity and soil CO2 efﬂuxes For win- ter and spring, the diurnal pattern of soil CO2 efﬂux became more uniform, with no apparent difference between night- time and daytime ﬂuxes (Fig. 5k to o and p to t respectively). For autumn, applying a u∗ﬁltering procedure reversed the diurnal pattern from the highest efﬂuxes being seen during night-time, to the highest efﬂuxes being seen during daytime. Based on this result, we then calculated the mean diur- nal pattern of soil CO2 efﬂux for each season during the 1-year campaign at different u∗threshold values (Fig. 5). With no u∗ﬁltering, the soil CO2 efﬂux showed a clear di- urnal pattern across all seasons with highest efﬂuxes during night-time. This was inversely related to the diurnal pattern of u∗(Fig. 2). The difference between night-time and day- time was most pronounced during summer, where night-time (21:00–03:00 CET) efﬂuxes were 35 % higher than daytime (09:00–15:00 CET) efﬂuxes. Applying a successively higher u∗threshold value decreased the difference between daytime and night-time efﬂuxes for all seasons. The most dramatic effect was seen between no u∗threshold value to a value of 0.3 and 0.5 m s−1. Increasing the u∗threshold value from 0.5 to 0.7 m s−1 only led to a slight change in the diurnal pat- tern. The u∗ﬁltering acted primarily by lowering the high night-time efﬂuxes and only by slightly lowering the day- time efﬂuxes. This uneven lowering of efﬂuxes across the day changed the distinct diurnal pattern with a large differ- ence between night-time and daytime efﬂuxes, i.e. at a u∗ threshold value of 0.5 and 0.7 m s−1, the diurnal pattern of soil CO2 efﬂux for summer was more uniform across the Looking at mean daily soil CO2 efﬂuxes from the auto- mated chambers revealed that they generally followed the soil temperature throughout the year (Fig. 6a). The same was found for the soil CO2 efﬂuxes based on the manual cham- ber measurements and the output of the empirical model (Fig. 6c). A high day to day variability was found throughout the year for the automated measurements, which was espe- cially pronounced during summer. Although following the same seasonal pattern, the modelled efﬂuxes based on the manual measurements were slightly lower than those from the automated measurements (summer: 2.97 for the man- ual vs. 4.10 µmol m−2 s−1 for the automated measurements, autumn: 2.36 for the manual vs. www.biogeosciences.net/14/1603/2017/ 3.3 Daytime soil CO2 efﬂuxes vs. daily efﬂuxes 0 2 4 6 8 0.0–0.1 0.1–0.2 0.2–0.3 0.3–0.4 0.4–0.5 0.5–0.6 0.6–0.7 0.7–0.8 0.8–0.9 0.9–1.0 1.0–1.1 1.1–1.2 Soil CO2 efflux (µmol CO2 m−2 s−1) u* binned group (m s−1) Figure 4. Box plot of mean hourly soil CO2 efﬂuxes for the 1-year campaign plotted against the binned groups of friction velocity (u∗). 0 2 4 6 8 0.0–0.1 0.1–0.2 0.2–0.3 0.3–0.4 0.4–0.5 0.5–0.6 0.6–0.7 0.7–0.8 0.8–0.9 0.9–1.0 1.0–1.1 1.1–1.2 Soil CO2 efflux (µmol CO2 m−2 s−1) u* binned group (m s−1) To assess the consequences of using only daytime soil CO2 efﬂux data, instead of data for the entire day, when upscal- ing to annual soil CO2 efﬂux, we calculated annual soil CO2 efﬂuxes for the 1-year campaign at different u∗thresh- old values using only ﬂuxes measured between 09:00 and 15:00 CET, and compared these to the annual soil CO2 ef- ﬂuxes calculated using efﬂuxes for the entire day at different u∗threshold values (see Sect. 3.2). At no u∗ﬁlter, the annual daytime soil CO2 efﬂux was 13 % lower than the annual entire-day soil CO2 efﬂux (808.9 vs. 703.3 g C m−2 year−1 for the annual entire-day and day- time soil CO2 efﬂuxes respectively) (Fig. 8). Increasing the u∗threshold value decreased the difference between annual daytime soil CO2 efﬂuxes and entire-day efﬂuxes, with a steep decrease from 13 to 4.5 % observed between no u∗ ﬁltering to a u∗threshold value of 0.3 m s−1 (705.4 vs. 673.7 g C m−2 year−1 for the annual entire-day and daytime soil CO2 efﬂuxes respectively). Further increasing the u∗ threshold value only resulted in a minor change in the rela- tionship between the annual daytime soil CO2 efﬂux and the entire-day efﬂux. However, they were almost identical at a u∗ threshold value of 1.2 m s−1 (596.9 vs. 595.5 g C m−2 year−1 for the annual entire-day and daytime soil CO2 efﬂuxes re- spectively). Figure 4. Box plot of mean hourly soil CO2 efﬂuxes for the 1-year campaign plotted against the binned groups of friction velocity (u∗). erally decreased the daily mean soil CO2 efﬂuxes, as well as the day to day variability (Fig. 6b). The biggest decrease was seen between no u∗ﬁlter to a u∗threshold value of 0.7 m s−1. Further increasing the u∗threshold value only led to minor additional decreases in the mean daily soil CO2 ef- ﬂuxes. 3.1 Friction velocity and soil CO2 efﬂuxes Seasonally averaged diurnal pattern of soil temperature (± standard deviation) at 5 cm depth measured by six soil thermometers close to the eight automated chambers for summer (a), autumn (b), winter (c) and spring (d). 3.3 Daytime soil CO2 efﬂuxes vs. daily efﬂuxes At a u∗threshold value of 0.7 m s−1 the mean daily soil CO2 efﬂuxes for summer, autumn, winter and spring was lowered with 25 % (to 3.09 µmol m−2 s−1), 17 % (to 2.37 µmol m−2 s−1), 19 % (to 0.492 µmol m−2 s−1) and 18 % (to 0.856 µmol m−2 s−1) respectively in comparison to when no u∗ﬁlter was applied. 3.1 Friction velocity and soil CO2 efﬂuxes 2.86 µmol m−2 s−1 for the automated measurements, winter: 0.485 for the manual vs. 0.608 µmol m−2 s−1 for the automated measurements), ex- cept for spring where the manual measurements were the highest (1.19 for the manual vs. 1.04 µmol m−2 s−1 for the automated measurements). Applying a successively higher u∗threshold value to the automated soil CO2 efﬂuxes gen- www.biogeosciences.net/14/1603/2017/ Biogeosciences, 14, 1603–1616, 2017 1608 A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes G G G G G G G G G G G G G G G G G G G G G G G G 0 5 10 15 20 (a) Summer G G G G G G G G G G G G G G G G G G G G G G G G Soil temperature (oC) Time of day (h) G G G G G G G G G G G G G G G G G G G G G G G G (b) Autumn G G G G G G G G G G G G G G G G G G G G G G G G Time of day (h) G G G G G G G G G G G G G G G G G G G G G G G G (c) Winter G G G G G G G G G G G G G G G G G G G G G G G G Time of day (h) G G G G G G G G G G G G G G G G G G G G G G G G (d) Spring G G G G G G G G G G G G G G G G G G G G G G G G Time of day (h) 00:00 05:00 10:00 15:00 20:00 00:00 05:00 10:00 15:00 20:00 00:00 05:00 10:00 15:00 20:00 00:00 05:00 10:00 15:00 20:00 Figure 3. Seasonally averaged diurnal pattern of soil temperature (± standard deviation) at 5 cm depth measured by six soil thermometers close to the eight automated chambers for summer (a), autumn (b), winter (c) and spring (d). A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes 1608 Figure 3. A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes 0 1 2 3 4 5 6 7 GGGGGGGG GGGGGGGGGG G G GGGG Summer u* > 0 m s−1 (a) GGGGG GGGGG G GGGGGG G GGG GGG (b) u* > 0.1 m s−1 GGGGGGGGG GGGGGGGGGGGGGGG (c) u* > 0.3 m s−1 GGGGG GGGG GGGGGGGGGGG GGGG (d) u* > 0.5 m s−1 GGGGG GGG GGGGGGGGGGGGG GGG (e) u* > 0.7 m s−1 0 1 2 3 4 5 6 7 GGGGGGGGGGGGGGGGGGGGGGGG Soil CO2 efflux (µmol CO2 m−2 s−1) (f) Autumn GGGGGGGGGGGGGGGGGGGGGGGG (g) GGGGG GGGGGGGGGGGGG GGGGGG (h) GGGGG GGGGGGGGGGGGGGG GGGG (i) GG G GGG GGGG GGGGGGGGG GG GG G (j) 0.0 0.4 0.8 GGGGGGGGGGGGGGGGGGGGGGGG (k) Winter GGGGGGGGGGGGGGGG GGGGGGGG (l) GGGGGGGGGGGGGGGGGGGGGGGG (m) GGGGGGGGGGGGGGGGGGGGGGGG (n) GGGGGGGGGGGGGGGGGGGGGGGG (o) 00:00 05:00 10:00 15:00 20:00 0.0 1.0 2.0 3.0 GGGGGGG GGGGGGGGGGG GGGGGG Time of day (h) Soil CO2 efflux (µmol CO2 m−2 s−1) (p) Spring GGGGGGGGGGGGGGGGGGGGGGGG Time of day (h) (q) GGGGGG G GGGGGGGGGGGGGGGGG Time of day (h) (r) GGGGGGGGGGGGGGGGGGGGGGGG Time of day (h) (s) GGGG G GGGGGGGGGGGGG G GGGGG Time of day (h) (t) 00:00 05:00 10:00 15:00 20:00 00:00 05:00 10:00 15:00 20:00 00:00 05:00 10:00 15:00 20:00 00:00 05:00 10:00 15:00 20:00 Figure 5. Seasonally averaged diurnal patterns of soil CO2 efﬂux (± standard deviation), at different friction velocity (u∗) threshold values, measured by the eight automated chambers for each of the four seasons. From the top, the four rows show the diurnal patterns for summer, autumn, winter and spring respectively. From the left, the ﬁve collars show the diurnal patterns for each season at no u∗ﬁltering, a u∗ threshold value of 0.1 m s−1, a u∗threshold value of 0.3 m s−1, a u∗threshold value of 0.5 m s−1 and a u∗threshold value of 0.7 m s−1 respectively. Figure 5. Seasonally averaged diurnal patterns of soil CO2 efﬂux (± standard deviation), at different friction velocity (u∗) threshold values, measured by the eight automated chambers for each of the four seasons. From the top, the four rows show the diurnal patterns for summer, autumn, winter and spring respectively. A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes From the left, the ﬁve collars show the diurnal patterns for each season at no u∗ﬁltering, a u∗ threshold value of 0.1 m s−1, a u∗threshold value of 0.3 m s−1, a u∗threshold value of 0.5 m s−1 and a u∗threshold value of 0.7 m s−1 respectively GGGGG GG G G G GG GG GGG G G GG G G G GG G GG GG G G GGGGGGGG GGG GGGGGGGGGGGGGG GG GGGGGG GGGG G GGGGGGGGGGGGGGGGG GG G GG GG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGG GGGGGGGG G GGG G GGG GGGGGGGG GGG GGGGGGGGGGGG GG G G G G GG G G G G GG G G GG G G G G G G G G G G G G G G G G G G G G GG G G G G G G GG G G G G G G GG GGG G GGG GG G G G G G G GG GG GG G G G GGG GG G GGG G G G GGGG G 0 1 2 3 4 5 6 7 GGGGG GG G G G GG GG GGG G G GG G G G GG G GG GG G G GGGGGGGG GGG GGGGGGGGGGGGGG GG GGGGGG GGGG G GGGGGGGGGGGGGGGGG GG G GG GG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGG GGGGGGGG G GGG G GGG GGGGGGGG GGG GGGGGGGGGGGG GG G G G G GG G G G G GG G G GG G G G G G G G G G G G G G G G G G G G G GG G G G G G G GG G G G G G G GG GGG G GGG GG G G G G G G GG GG GG G G G GGG GG G GGG G G G GGGG G (a) Soil CO2 efflux (µmol CO2 m−2 s−1) Date (month) Nov Jan Mar May Jul Sep G G G GGGGGG GG G G G GGGG GGGGGG G GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGG G G GGGGGGGGGGGG GGGG GGGGGGG GG GG GG G G GG GG G G G GGG GGGG G G G GGG G G G G GG G G G G GG GGG G G GG G G G GG G GGGGGGGG G G G G G G G Date Soil CO2 flux (µmolCOm2s) G G G GGGGGG GG G G G GGGG GGGGGG G GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGG G G GGGGGGGGGGGG GGGG GGGGGGG GG GG GG G G GG GG G G G GGG GGGG G G G GGG G G G G GG G G G G GG GGG G G GG G G G GG G GGGGGGGG G G G G G G G Date (month) Nov Jan Mar May Jul Sep (b) Date (month) Nov Jan Mar May Jul Sep (c) 0 5 10 Index z.hr GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG 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G GG GGGGGGGG GGGGGGGGG GGGG GG G GGGGGGGGGG GGGGGGGGGGGG GG GGGGGGGGGGGG GGGGGGGGGGG G GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGG GGGGGGG GG GGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGG GGGGGGG GGGGGGGGGGGGG GGGGGGGGG GGG GGGGGGGGGGGGG GGGGGGG GGG GGGGGGGGGGG GGGGGGG GGG GGGGGGGGGGG GGGGGGGGGGGG GGG GGGGGGGG GGGGG GGG GGGGGGGGGGGGGGGGGGGGGGG GGGGGGG GGG GGGGGGGGGGG GGGGGGGG GGGG GGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGG GGGGG GGGGGGGGGGGG GGGGGGGG GGGGG GGGGGG GGGGG GGGGGGGGGGGGGGGGGGGGGG GG GGGGGGGGGGGGGGGGGGGGG GGG GGGGGGGGGGGGGGGGGGGGGG GG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGGGGGG GGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGG GGGG GGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGG Date (month) Nov Feb May Aug Soil temp. 4 Discussion and b). However with fans, the opposite diurnal pattern with highest efﬂuxes during daytime was seen. The change in di- urnal pattern when using a fan was primarily due to a de- crease of the high night-time efﬂuxes (50 % lower for ef- ﬂuxes measured at 21:00–03:00 CET). However, a decrease in daytime efﬂuxes was also observed when using the fans (26 % lower for efﬂuxes measured at 09:00–15:00 CET). A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes Date (month) n Mar May Date (month) a ay J Figure 6. Comparison of the courses of the mean daily soil CO2 efﬂux throughout the year. Panels (a, b) show mean daily soil CO2 efﬂuxes (± standard deviation) throughout the year measured by the eight automated soil chambers, without friction velocity (u∗) ﬁltering and with a u∗threshold values of 0.7 respectively. (c) The black dots show the mean soil CO2 efﬂuxes for each of the manual soil chamber campaigns, and the solid line shows the output of the empirical model based on these manual measurements. The course of soil temperature throughout the year at 5 cm depth measured at the eight automated chambers is shown on the inset in (c). Figure 6. Comparison of the courses of the mean daily soil CO2 efﬂux throughout the year. Panels (a, b) show mean daily soil CO2 efﬂuxes (± standard deviation) throughout the year measured by the eight automated soil chambers, without friction velocity (u∗) ﬁltering and with a u∗threshold values of 0.7 respectively. (c) The black dots show the mean soil CO2 efﬂuxes for each of the manual soil chamber campaigns, and the solid line shows the output of the empirical model based on these manual measurements. The course of soil temperature throughout the year at 5 cm depth measured at the eight automated chambers is shown on the inset in (c). 4.1 Friction velocity and soil CO2 efﬂuxes Soil CO2 efﬂuxes generally followed the seasonal changes in soil temperature, being highest in summer and lowest in winter. Our 1 year measurement series with a total of 52 131 individual soil CO2 efﬂux measurements from eight auto- 3.2 Annual soil CO2 efﬂux The observation that u∗ﬁltering profoundly affected esti- mated soil CO2 efﬂuxes led us to design a simple fan ex- periment to test whether the bias of low atmospheric turbu- lent mixing on the measured chamber soil CO2 efﬂuxes could be eliminated by ensuring adequate mixing of air around the soil chamber. When no fan was installed, a signiﬁcant nega- tive relationship was found between soil CO2 efﬂux and u∗ (r2 = 0.040, P = < 0.001, slope = −0.377, data now shown) comparable to the 1-year campaign (Fig. 4). However, with fans installed the negative relationship changed into a signiﬁcant positive relationship (r2 = 0.080, P = < 0.001, slope = 0.353), clearly indicating a strong effect of installing fans. With no fans, the soil CO2 efﬂux showed a clear diur- nal pattern with highest efﬂuxes during night-time (Fig. 9a Annual soil CO2 efﬂuxes, based on the automated chamber ﬂux data for the 1-year campaign, were calculated with the exclusion of data at different u∗threshold values (Fig. 7). The highest annual soil CO2 efﬂux was found when no u∗ ﬁlter was used (808.9 g C m−2 year−1). Increasing the u∗ threshold value decreased the annual soil CO2 efﬂux, with the largest decrease of 21 % observed between unﬁltered data to a u∗threshold value of 0.7 m s−1 (from 808.9 to 641.7 g C m−2 year−1). Between a u∗threshold value of 0.7 to 1.2 m s−1, only a small decrease in annual soil CO2 efﬂux of 7 % was seen (from 641.7 to 596.9 g C m−2 year−1). The annual soil CO2 efﬂux from the empirical model based on the manual chamber measurements was 666.6 g C m−2 year−1. Biogeosciences, 14, 1603–1616, 2017 www.biogeosciences.net/14/1603/2017/ 1609 A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes GGGGG GG G G G GG GG GGG G G GG G G G GG G GG GG G G GGGGGGGG GGG GGGGGGGGGGGGGG GG GGGGGG GGGG G GGGGGGGGGGGGGGGGG GG G GG GG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGG GGGGGGGG G GGG G GGG GGGGGGGG GGG GGGGGGGGGGGG GG G G G G GG G G G G GG G G GG G G G G G G G G G G G G G G G G G G G G GG G G G G G G GG G G G G G G GG GGG G GGG GG G G G G G G GG GG GG G G G GGG GG G GGG G G G GGGG G 0 1 2 3 4 5 6 7 GGGGG GG G G G GG GG GGG G G GG G G G GG G GG GG G G GGGGGGGG GGG GGGGGGGGGGGGGG GG GGGGGG GGGG G GGGGGGGGGGGGGGGGG GG G GG GG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGG GGGGGGGG G GGG G GGG GGGGGGGG GGG GGGGGGGGGGGG GG G G G G GG G G G G GG G G GG G G G G G G G G G G G G G G G G G G G G GG G G G G G G GG G G G G G G GG GGG G GGG GG G G G G G G GG GG GG G G G GGG GG G GGG G G G GGGG G (a) Soil CO2 efflux (µmol CO2 m−2 s−1) Date (month) Nov Jan Mar May Jul Sep G G G GGGGGG GG G G G GGGG GGGGGG G 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G GG GGGGGGGG GGGGGGGGG GGGG GG G GGGGGGGGGG GGGGGGGGGGGG GG GGGGGGGGGGGG GGGGGGGGGGG G GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGG GGGGGGG GG GGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGG GGGGGGG GGGGGGGGGGGGG GGGGGGGGG GGG GGGGGGGGGGGGG GGGGGGG GGG GGGGGGGGGGG GGGGGGG GGG GGGGGGGGGGG GGGGGGGGGGGG GGG GGGGGGGG GGGGG GGG GGGGGGGGGGGGGGGGGGGGGGG GGGGGGG GGG GGGGGGGGGGG GGGGGGGG GGGG GGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGG GGGGG GGGGGGGGGGGG GGGGGGGG GGGGG GGGGGG GGGGG GGGGGGGGGGGGGGGGGGGGGG GG GGGGGGGGGGGGGGGGGGGGG GGG GGGGGGGGGGGGGGGGGGGGGG GG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGGGGGG GGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGG GGGG GGGGGGGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGGGGGGGGGGGG GGGGGGGGGGG Date (month) Nov Feb May Aug Soil temp. A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes Figure 6. Comparison of the courses of the mean daily soil CO2 efﬂux throughout the year. Panels (a, b) show mean daily soil CO2 efﬂuxes (± standard deviation) throughout the year measured by the eight automated soil chambers, without friction velocity (u∗) ﬁltering and with a u∗threshold values of 0.7 respectively. (c) The black dots show the mean soil CO2 efﬂuxes for each of the manual soil chamber campaigns, and the solid line shows the output of the empirical model based on these manual measurements. The course of soil temperature throughout the year at 5 cm depth measured at the eight automated chambers is shown on the inset in (c). A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes Estimates of annual soil CO2 efﬂux in response to increasing the friction velocity (u∗) threshold values for the automated chamber measurements during the 1-year campaign. The straight dashed line shows the annual soil CO2 efﬂux of 666.6 g C m−2 year−1 based on the manual measurements. Figure 7. Estimates of annual soil CO2 efﬂux in response to increasing the friction velocity (u∗) threshold values for the automated chamber measurements during the 1-year campaign. The straight dashed line shows the annual soil CO2 efﬂux of 666.6 g C m−2 year−1 based on the manual measurements. G G G G G G G G G G G G G 0.0 0.2 0.4 0.6 0.8 1.0 1.2 −15 −10 −5 0 ustarfilter bias Relative difference (%) u* threshold value (m s−1) Figure 8. Relative difference between the estimates of annual soil CO2 efﬂux based on daytime (09:00–15:00 CET) data at different friction velocity (u∗) threshold values, compared to the estimates of annual soil CO2 efﬂux based on data for the entire day at different friction velocity (u∗) threshold values. G G G G G G G G G G G G G 0.0 0.2 0.4 0.6 0.8 1.0 1.2 −15 −10 −5 0 ustarfilter bias Relative difference (%) u* threshold value (m s−1) ing of the layer of air above the surface (Brooks et al., 1997). This lowers the concentration gradient of CO2 from the soil to the atmosphere, causing a build-up of CO2 in the soil (Wohlfahrt et al., 2005; Flechard et al., 2007). This acts to suppress the apparent soil CO2 efﬂux, and may also lead to a higher apparent soil CO2 efﬂux once turbulence is re- established, because of the subsequent release of the CO2 that has been build up in the soil (Massman et al., 1997). Our chamber measurements, however, show higher and not lower soil CO2 efﬂuxes during low u∗. It is unlikely that u∗has a di- rect effect on the biological activity of bacteria, fungi and/or plant roots in the soil, and thus it seems evident that the ap- parent increase in soil CO2 efﬂux measured during low u∗is a measurement bias of the closed-chamber technique. Only a few studies have demonstrated the potential problem of using closed chambers during periods with low atmospheric turbu- lence (Görres et al., 2016; Koskinen et al., 2014; Lai et al., 2012; Schneider et al., 2009). Görres et al. A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes G G G G G G G G G G G G G 0.0 0.2 0.4 0.6 0.8 1.0 1.2 500 600 700 800 900 ustarfilter budget.dband20.LM Annual soil CO2 efflux (g C m−2 yr−1) u* threshold value (m s−1) G G G G G G G G G G G G 00:00 05:00 10:00 15:00 20:00 0 1 2 3 4 G G G G G G G G G G G G G G G G G G G G G G G G Soil CO2 efflux (µmol CO2 m−2 s−1) Time of day (h) (a) G G G G G G G G G G G G G G G G G G G G G G G G 00:00 05:00 10:00 15:00 20:00 G G G G G G G G G G G G G G G G G G G G G G G G Time of day (h) (b) G G G G G G G G G G G G Time of day (h) Time of day (h) ustarfilter u* threshold value (m s−1) Figure 9. Diurnal pattern of soil CO2 efﬂux measured by the au- Figure 9. Diurnal pattern of soil CO2 efﬂux measured by the au- tomated chambers during the fan experiment, based on bi-hourly means (± standard deviation). Panel (a) shows the diurnal pattern for half of the chambers with and without fans, where the ﬁrst 10 days were with fans (ﬁlled circles) and the last 10 days were without fans (open circles). Panel (b) shows the diurnal pattern with and without fans for the other half of the chambers, where the ﬁrst 10 days were without fans (open circles) and the last 10 days were with fans (ﬁlled circles). Figure 9. Diurnal pattern of soil CO2 efﬂux measured by the au- tomated chambers during the fan experiment, based on bi-hourly means (± standard deviation). Panel (a) shows the diurnal pattern for half of the chambers with and without fans, where the ﬁrst 10 days were with fans (ﬁlled circles) and the last 10 days were without fans (open circles). Panel (b) shows the diurnal pattern with and without fans for the other half of the chambers, where the ﬁrst 10 days were without fans (open circles) and the last 10 days were with fans (ﬁlled circles). Figure 7. www.biogeosciences.net/14/1603/2017/ Biogeosciences, 14, 1603–1616, 2017 A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes 1610 G G G G G G G G G G G G G 0.0 0.2 0.4 0.6 0.8 1.0 1.2 500 600 700 800 900 ustarfilter budget.dband20.LM Annual soil CO2 efflux (g C m−2 yr−1) u* threshold value (m s−1) Figure 7. Estimates of annual soil CO2 efﬂux in response to increasing the friction velocity (u∗) threshold values for the automated chamber measurements during the 1-year campaign. The straight dashed line shows the annual soil CO2 efﬂux of 666.6 g C m−2 year−1 based on the manual measurements. G G G G G G G G G G G 00:00 05:00 10:00 15:00 20:0 0 1 2 3 4 G G G G G G G G G G G G G G G G G G G G G G Soil CO2 efflux (µmol CO2 m−2 s−1) Time of day (h) (a) G G G G G G G G G G G Figure 9. Diurnal pattern of soil tomated chambers during the fan means (± standard deviation). Pan for half of the chambers with an 10 days were with fans (ﬁlled ci without fans (open circles). Panel ( G G G G G G G G G G G G G 0.0 0.2 0.4 0.6 0.8 1.0 1.2 500 600 700 800 900 ustarfilter budget.dband20.LM Annual soil CO2 efflux (g C m−2 yr−1) u* threshold value (m s−1) Figure 7. Estimates of annual soil CO2 efﬂux in response to increasing the friction velocity (u∗) threshold values for the automated chamber measurements during the 1-year campaign. The straight dashed line shows the annual soil CO2 efﬂux of 666.6 g C m−2 year−1 based on the manual measurements. A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes G G G G G G G G G G G G 00:00 05:00 10:00 15:00 20:00 0 1 2 3 4 G G G G G G G G G G G G G G G G G G G G G G G G Soil CO2 efflux (µmol CO2 m−2 s−1) Time of day (h) (a) G G G G G G G G G G G G G G G G G G G G G G G G 00:00 05:00 10:00 15:00 20:00 G G G G G G G G G G G G G G G G G G G G G G G G Time of day (h) (b) G G G G G G G G G G G G Figure 9. Diurnal pattern of soil CO2 efﬂux measured by the au- tomated chambers during the fan experiment, based on bi-hourly means (± standard deviation). Panel (a) shows the diurnal pattern for half of the chambers with and without fans, where the ﬁrst 10 days were with fans (ﬁlled circles) and the last 10 days were without fans (open circles). Panel (b) shows the diurnal pattern with and without fans for the other half of the chambers, where the ﬁrst 10 days were without fans (open circles) and the last 10 days were with fans (ﬁlled circles). 4.2 Diurnal pattern of soil CO2 efﬂuxes The hourly measurements of soil CO2 efﬂuxes showed a clear diurnal pattern, generally with the highest efﬂuxes dur- ing night-time, when no u∗ﬁlter was applied to the data (Fig. 5a, f, k, p). Soil temperature at 5 cm depth, however, did not show a diurnal pattern for winter, and only a slight diurnal pattern for the other seasons with the highest temper- atures late in the afternoon or early in the evening (Fig. 3). This could indicate a hysteresis between soil CO2 efﬂux and soil temperature for summer, autumn and spring. A few stud- ies with automated closed chambers have found a similar hysteresis between soil CO2 efﬂux and soil temperature (e.g. Phillips et al., 2011; Savage et al., 2013; Tang and Baldocchi, 2005). The hysteresis has been explained as a result of prim- ing of the soil bacteria by carbon exudates from plant roots (Kuzyakov and Gavrichkova, 2010). During daytime in the growing season, plants assimilate carbon via photosynthesis. Parts of the assimilated carbon go to the roots via the phloem, and is released into the rhizosphere (Kuzyakov, 2002). Once in the soil, this carbon is readily consumed by bacteria lead- ing to an increase in Rs. Even though photosynthesis takes place during daytime, the increase in Rs has been found to lag after photosynthesis, because the photo assimilates need to be transported from the leaves to the roots via the phloem. Varying lag times have been found, with the shortest times for short plants such as grasses, and lag times of up to 4– 5 days for mature trees (Kuzyakov and Gavrichkova, 2010). However, when applying a successively higher u∗threshold value, the diurnal patterns of soil CO2 efﬂux across all sea- sons changed, mostly due to removal of the overestimated night-time efﬂuxes. For our measurements, the overestima- tion of chamber efﬂuxes due to low u∗thus works as a se- lective systematic error that mostly applies to night-time of the diurnal pattern. This is in agreement with other studies (Görres et al., 2016; Koskinen et al., 2014; Lai et al., 2012; Schneider et al., 2009). During summer, we observed that the difference between night-time and daytime was smaller than during spring and autumn, and only the ﬂuxes between 10:00 and 20:00 CET were slightly lower than the night-time ﬂuxes. 4.2 Diurnal pattern of soil CO2 efﬂuxes It is possible that this pattern is due to an increase in the night-time soil respiration due to priming of the soil or- ganisms via carbon ﬂow from the roots into the rhizosphere A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes and soil. This was, however, not tested in the present study. For the other seasons priming is not as likely, since only low or no photosynthesis takes place during this time. Dur- ing winter and spring, the diurnal patterns changed from a clear diurnal pattern with highest night-time ﬂuxes to no dif- ference between night and daytime ﬂuxes when applying the u∗ﬁltering. During autumn, it changed from a clear diurnal pattern with highest night-time ﬂuxes to a pattern where the ﬂuxes seem to be slightly higher at daytime. and u∗were not confounded by such potential differences in the different subsets of observations. We also showed that this error of overestimation of soil CO2 efﬂuxes during low u∗had a dramatic effect on both the diurnal pattern of soil CO2 efﬂux, and on the estimate of annual soil CO2 efﬂux, as we will discuss this in the following sections. An alternative and/or additional reason for the stable conditions above the soil surface during calm periods may be the stratiﬁcation of the air caused by cooling due to longwave net radiation loss from the soil as suggested by Riederer et al. (2014). This as- pect was, however, not addressed in the current study. Our results show that overestimation of soil CO2 efﬂuxes at low u∗can change the apparent diurnal pattern of soil CO2 efﬂuxes, and they highlight the importance of taking this source of error into account, since negligence of the problem may lead to misinterpretation of the relationship between Rs and its physical drivers like temperature and soil humidity, as well as lead to erroneous estimation of lag times between Rs rates and ﬂow of carbon from recent plant assimilates. Much research has been done on time lags between inputs of carbon via photosynthesis and Rs using closed chambers (Högberg et al., 2001; Tang et al., 2005). We expect that it is possible that overestimates of soil CO2 efﬂuxes at low u∗ may have inﬂuenced the interpretation of the diurnal pattern in some previous studies that have not taken overestimation of efﬂuxes during low u∗into account. It could therefore be valuable to re-evaluate these studies by including data of u∗ if such data are available. A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes (2016) suggested that during low atmospheric turbulence, the chamber causes a disturbance and/or mixing of the stratiﬁed layer of still air above the soil surface, as the chamber moves on to the soil at the beginning of a measurement, while Lai et al. (2012) ac- credited the error to a similar effect by the internal chamber fan. In both cases, the CO2-rich air just above the soil surface is instantly mixed with less CO2-rich air from above. This causes a sudden drop in the CO2 concentration just above the soil surface, which in turn increases the concentration gradient between the soil and the atmosphere, thus leading to an apparent high CO2 efﬂux being measured by the cham- ber (Görres et al., 2016). Our results support this hypothesis. In addition, we observed that soil temperatures were similar in the data sets with different u∗threshold values (data not shown), indicating that the relationship between ﬂux rates Figure 8. Relative difference between the estimates of annual soil CO2 efﬂux based on daytime (09:00–15:00 CET) data at different friction velocity (u∗) threshold values, compared to the estimates of annual soil CO2 efﬂux based on data for the entire day at different friction velocity (u∗) threshold values. matic chambers clearly showed a negative relationship with the level of atmospheric turbulence (u∗), which was espe- cially visible during periods with similar soil temperature, where apparent rates of soil CO2 efﬂuxes were clearly higher at lower compared to higher u∗values. For closed-chamber measurements to represent real Rs, we assume steady-state diffusion from the source in the soil to the atmosphere according to Fick’s law. Accordingly, there must be a constant concentration of atmospheric CO2, for the ﬂux to be stable, and the physical application of a closed chamber should not change or break down any CO2 gradi- ents. Otherwise advection may take place. Our data strongly indicate that these assumptions are likely not met during low u∗, when the high soil CO2 efﬂuxes were observed. u∗typically varies on a daily basis with low u∗during calm nights and high u∗during daytime (Stull, 1988). This is in agreement with our results (Fig. 2). The low atmospheric turbulence during calm nights has been found to cause a build-up of CO2 above the soil, because of improper mix- Biogeosciences, 14, 1603–1616, 2017 www.biogeosciences.net/14/1603/2017/ 1611 4.3 Annual soil CO2 efﬂuxes The u∗ﬁltering had a considerable effect on the annual soil CO2 efﬂux estimates based on the automated cham- ber measurements by decreasing the annual efﬂux in re- sponse to increasing the u∗threshold value. This could be expected from the observed negative relationship between soil CO2 efﬂux and u∗. The annual soil CO2 efﬂux mod- elled from the manual daytime-only chamber measurements measured at 12 soil collars within 10 m from the auto- mated chambers was 666.6 g C m−2 year−1. During daytime (09:00–15:00 CET) u∗was usually high (Fig. 2). The risk of overestimation of a fraction of the manual soil CO2 ef- ﬂux measurements due to low u∗can therefore be viewed as minor. The automated chamber annual soil CO2 efﬂux in- cluding all data was 808.9 g C m−2 year−1, i.e. a value much higher than the 666.6 g C m−2 year−1 from the manual cham- bers. However, the annual soil CO2 efﬂux estimate from the automated chamber measurements decreased to 663.4 and 596.8 g C m−2 year−1 at a u∗threshold value of 0.5 and 1.2 m s−1 respectively, i.e. much closer to the annual soil CO2 efﬂux from the manual measurements (Fig. 7). Thus, it seems that the automated measurement and the manual day- time measurements provide comparable estimates of annual soil CO2 efﬂuxes, when the effect of low u∗is accounted for, as part of the quality checking procedure for the auto- mated measurements. This seems to increase the conﬁdence in the much less frequent manual measurements, and poten- www.biogeosciences.net/14/1603/2017/ 4.4 Fan experiment Mixing of the air around the soil chambers with fans had a considerable effect by decreasing the measured soil CO2 ef- ﬂuxes (Fig. 9). The biggest difference was seen during night- time, but even at daytime a smaller effect was observed. This selective lowering of efﬂuxes changed the apparent diurnal pattern, such that the highest ﬂuxes now were measured dur- ing daytime. By using a fan we hypothesised that the effect of breaking the stratiﬁed layer by the chamber during low u∗ would not occur because the artiﬁcially induced wind contin- uously ensured proper mixing of the air around the chamber and thus likely prevented a build-up of a stratiﬁed layer of CO2. We believe that the assumption for a steady-state rate of diffusion of CO2 out of the soil is closer to being fulﬁlled with a fan, since the proposed mechanism for the apparent higher soil CO2 efﬂux at low u∗can no longer take place due to mixing of the air by the fan prior to the measure- ment. Thus chamber soil CO2 measurements are no longer overestimated at low u∗, which is also seen by there being no negative relationship between soil CO2 efﬂuxes and u∗ when a fan was used. However, when using the fans we also observed an apparent decrease in the measured daytime soil CO2 efﬂuxes, when u∗was generally high. We suggest three potential causes of this difference in the daytime efﬂuxes. Firstly, the fan experiment campaign ran over 20 days; i.e. 10 of the days were with a fan installed and 10 days were without a fan. Thus a comparison for a chamber is made at different times, and differences in environmental conditions may have caused differences in the rates of Rs between the two periods. Indeed, a difference in the mean soil tempera- ture at 5 cm depth of 1 ◦C was found between the two peri- ods. However, the difference in soil CO2 efﬂuxes was found for both groups of chambers, i.e. both for the ones with a fan installed during the cold period and for those with a fan installed during the warm period, and thus the potential di- rectional bias of differences in soil temperature during the two periods can be ruled out. Secondly, the slight decrease of daytime efﬂuxes when fans were installed could be be- A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes However, the rate of diffusion of a gas out of the soil is known to be highly sensitive to wind speed at the soil sur- face, and higher wind speeds can increase the diffusion rate or even cause advective transport out of the soil (Janssens et al., 2000; Roland et al., 2015). This effect is well known to cause severe overestimation of soil CO2 efﬂuxes when soil chambers are equipped with a heavy internal fan because the resulting wind speed inside the chamber is much higher than outside (e.g. Hanson et al., 1993; Hooper et al., 2002; Le Dantec et al., 1999). The LI-8100A chambers used in our ex- periments do not have an internal fan. Instead they rely on the air movement that is created when air is pumped to and from the chamber in combination with the spherical shape of the chamber for adequate mixing of chamber air (LI-COR Biosciences, 2015). The wind speed in the LI-8100A cham- ber may therefore be fairly low compared to outside condi- tions. It is therefore possible that we see the opposite effect of what is seen in chambers with an internal fan, namely that the wind speed in the chamber is lower than outside, resulting in a lower rate of diffusion and consequently a lower mea- sured soil CO2 efﬂux, and that this effect increases with the installation of fans that increase the wind speed outside the chamber. A similar effect has also been found to occur during natural conditions outside the chamber where soil efﬂuxes measured by micrometeorological methods have been found to increase with increasing ambient wind speed (Denmead and Reicosky, 2003). To eliminate this potential bias due to difference in wind speed, a closer matching of the chamber wind speed with the ambient wind speed can be attempted, as suggested by Rochette and Hutchinson (2005). It is pos- sible that a lower wind speed induced by a fan would be ad- equate to eliminate the effect of low u∗. This would poten- tially lower the difference in wind between the outside and inside of the chamber, thus minimising the potential bias due to different wind speeds. This was, however, not tested in the current study. A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes 1612 tially shows that daytime only measurements, at least in our case, was not a major source of error for the upscaling to an annual estimate of soil CO2 efﬂux. This is in agreement with Juszczak et al. (2012), who found that there was no dif- ference between daytime and night-time efﬂuxes when the correct u∗threshold value had been applied. To exemplify this, we compared the annual soil CO2 efﬂuxes from the au- tomated chambers with estimates of annual soil CO2 efﬂuxes based on only daytime (09:00–15:00 CET) soil CO2 efﬂuxes from the automated chambers. The daytime annual soil CO2 efﬂux was 13 % lower than the annual entire-day soil CO2 ef- ﬂux with no u∗ﬁlter, and decreased to 4.5 % at a u∗ﬁlter of 0.3 m s−1 (Fig. 8), i.e. resulting in more comparable annual soil CO2 efﬂuxes when the overestimation of u∗is accounted for. cause low u∗was sometimes observed also during daytime, although to a much lesser degree than during night-time. A similar small decrease of the daytime efﬂuxes for the 1-year campaign when increasing the u∗threshold value was also observed (Fig. 5), highlighting that there may still be such an effect even during daytime. Thirdly, it is possible that, al- though a fan eliminated the bias of low u∗on chamber ef- ﬂuxes, the wind induced by the fan introduced another po- tential bias to the measurement. Steady-state diffusion rate of CO2 out of the soil with and without a chamber is a re- quirement for unbiased chamber measurements of soil CO2 efﬂux. However, the rate of diffusion of a gas out of the soil is known to be highly sensitive to wind speed at the soil sur- face, and higher wind speeds can increase the diffusion rate or even cause advective transport out of the soil (Janssens et al., 2000; Roland et al., 2015). This effect is well known to cause severe overestimation of soil CO2 efﬂuxes when soil chambers are equipped with a heavy internal fan because the resulting wind speed inside the chamber is much higher than outside (e.g. Hanson et al., 1993; Hooper et al., 2002; Le Dantec et al., 1999). The LI-8100A chambers used in our ex- periments do not have an internal fan. www.biogeosciences.net/14/1603/2017/ www.biogeosciences.net/14/1603/2017/ Biogeosciences, 14, 1603–1616, 2017 A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes Instead they rely on the air movement that is created when air is pumped to and from the chamber in combination with the spherical shape of the chamber for adequate mixing of chamber air (LI-COR Biosciences, 2015). The wind speed in the LI-8100A cham- ber may therefore be fairly low compared to outside condi- tions. It is therefore possible that we see the opposite effect of what is seen in chambers with an internal fan, namely that the wind speed in the chamber is lower than outside, resulting in a lower rate of diffusion and consequently a lower mea- sured soil CO2 efﬂux, and that this effect increases with the installation of fans that increase the wind speed outside the chamber. A similar effect has also been found to occur during natural conditions outside the chamber where soil efﬂuxes measured by micrometeorological methods have been found to increase with increasing ambient wind speed (Denmead and Reicosky, 2003). To eliminate this potential bias due to difference in wind speed, a closer matching of the chamber wind speed with the ambient wind speed can be attempted, as suggested by Rochette and Hutchinson (2005). It is pos- sible that a lower wind speed induced by a fan would be ad- equate to eliminate the effect of low u∗. This would poten- tially lower the difference in wind between the outside and inside of the chamber, thus minimising the potential bias due to different wind speeds. This was, however, not tested in the current study. cause low u∗was sometimes observed also during daytime, although to a much lesser degree than during night-time. A similar small decrease of the daytime efﬂuxes for the 1-year campaign when increasing the u∗threshold value was also observed (Fig. 5), highlighting that there may still be such an effect even during daytime. Thirdly, it is possible that, al- though a fan eliminated the bias of low u∗on chamber ef- ﬂuxes, the wind induced by the fan introduced another po- tential bias to the measurement. Steady-state diffusion rate of CO2 out of the soil with and without a chamber is a re- quirement for unbiased chamber measurements of soil CO2 efﬂux. A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes The results could, however, not explain the con- tinuous decrease in soil CO2 efﬂux. Thus it remains an open question as to why this decrease is still seen. One possible ex- planation is that the u∗used in this experiment was measured above the canopy at a height of 43 m. The soil chambers, however, were positioned at the soil surface and it is possible that the measured turbulence at 43 m was not well coupled to u∗above the soil surface surrounding the soil chambers (Thomas and Foken, 2007). A second anemometer installed closer to the soil surface (see Thomas et al., 2013) might have provided a clearer u∗threshold value. Although we have here shown that a u∗ﬁltering proce- dure seems to have a good potential to be used for quality control of closed-chamber measurements of soil CO2 efﬂux, it ultimately leads to data gaps that need to be ﬁlled using the appropriate gap ﬁlling techniques, e.g. based on established empirical relationships between measurement bias and u∗ (Lai et al., 2012). Data gaps, however, equal loss of data and thus methods should be developed to be able to observe un- biased chamber measurements also during periods with low atmospheric turbulence. Valid chamber measurements dur- ing periods with low u∗are especially important since this coincides with periods of data gaps in ﬂuxes estimated by the eddy covariance technique. Thus it is clear that cham- ber measurements during these periods cannot be used as ground truth for the eddy covariance ﬂux estimates (Görres et al., 2016). Our results from the fan experiment have shown promising results in terms of removing the negative relation- ship between u∗and soil CO2 efﬂuxes. This is, to the best of our knowledge, the ﬁrst time this has been attempted, and we expect that this method has a future potential. A few oth- ers have touched upon how to get unbiased chamber efﬂuxes during low u∗. Lai et al. (2012) found that closed-chamber CH4 and CO2 efﬂuxes were overestimated at low u∗. How- ever, by increasing the chamber closure time to 30 min, they found that ﬂuxes were no longer inﬂuenced by u∗after 13 min of chamber closure time, thus yielding reliable ﬂuxes. A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes value was used to quality check the data, indicating that even during low atmospheric turbulence the increase in chamber CO2 concentration often followed the expected pattern. High r2 values and near-linear increases in chamber CO2 concen- tration during chamber deployment alone can therefore not be used as the only means of quality checking, as this may still lead to falsely accepting overestimated measurements during low turbulence. tiﬁed or even eliminated through methodological and tech- nical advances. Here we showed that the bias of soil CO2 efﬂux measurements at low u∗can be accounted for by us- ing a u∗ﬁltering procedure. However, we do see a number of challenges with this methodology that need to be addressed. One challenge is to choose the correct u∗threshold value. A similar challenge has faced the eddy covariance method, where multiple methods have been developed to determine the right u∗threshold value (Gu et al., 2005). One method is to subjectively choose the appropriate level, e.g. based on vi- sual inspection of scatter plots of night-time ﬂuxes versus u∗ (Gu et al., 2005). For our data, this would mean choosing a u∗threshold value, where further increasing the u∗threshold value would no longer change the observed mean soil CO2 efﬂuxes. When looking at the diurnal patterns in Fig. 5, only a small further change in the diurnal pattern is seen between a u∗threshold value of 0.5 to 0.7 m s−1, thus indicating that a u∗threshold value between 0.5 and 0.7 m s−1 would be ap- propriate. A slightly higher u∗threshold value seems to apply to the mean hourly soil CO2 efﬂuxes aggregated into groups based on u∗values (Fig. 4), where no decrease in efﬂuxes is seen after the binned group with u∗values of 0.7–0.8 m s−1. Interestingly, a similar levelling off of the decrease in the es- timated annual soil CO2 efﬂux in response to an increasing u∗threshold value was not seen (Fig. 7). Instead, the annual soil CO2 efﬂux continued to decrease slightly even after the big decrease, which was seen up to a u∗threshold value of 0.7 m s−1. This indicates that efﬂuxes are still to some ex- tend inﬂuenced by u∗at these high levels. We analysed the data to see if the u∗ﬁltering had an inﬂuence on soil tem- perature, which potentially could selectively remove higher efﬂuxes measured at higher temperatures, leaving only mea- surements in colder periods at high u∗threshold values (data not shown). A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes However, it can be argued that longer chamber closure times introduce other potential biases for most closed-chamber sys- tems, because the chamber headspace concentration of CO2 or CH4 changes more and therefore affects the concentration gradient between soil and atmosphere, which can ultimately lead to an underestimation of efﬂux rates instead. We only used a chamber closure time of 90 s for the 1-year campaign, because this time was sufﬁcient to adequately measure an increase in chamber headspace CO2 concentration over time. We did, however, test whether ﬂux estimates were affected by using different dead bands of 10, 20, 30 and 40 s respectively, or by using linear or non-linear ﬂux calculation methods (see Supplement). In all cases we observed a similar pattern of overestimation of soil CO2 efﬂuxes during low u∗as reported here for linearly calculated efﬂuxes with a dead band of 20 s. Thus neither the dead band nor the ﬂux calculation method could eliminate the effect of low u∗on soil CO2 efﬂuxes. E h h d l d h b b d Another method to determine the u∗threshold value is to use objective statistical methods such as the moving point test also commonly used in eddy covariance data analysis (Gu et al., 2005). We suggest testing some of these methods for determination of the u∗threshold value for chamber CO2 efﬂuxes in future studies. We tried to see if using a stricter ﬂagging of data based on the r2 value of the linear ﬁt to the increase in CO2 concen- tration during chamber closure could also be used to remove overestimated efﬂuxes (data not shown). A similar effect of u∗on efﬂuxes could, however, be seen even when a higher r2 Even though automated closed-chamber systems are based on the same principle of passive, steady-state diffusion of gases between the soil and the chamber headspace during chamber closure, they come in a wide variety of designs with Biogeosciences, 14, 1603–1616, 2017 4.5 Towards unbiased soil CO2 efﬂuxes Chamber-based measurements of soil efﬂux of CO2 and other greenhouse gases play an important role in constraining the global carbon cycle. Thus it is crucial to obtain unbiased measurements that are as close to the true rates of emissions or uptake as possible. Many biases have already been quan- Biogeosciences, 14, 1603–1616, 2017 www.biogeosciences.net/14/1603/2017/ 1613 5 Conclusions The study had two major results. Firstly, we showed that soil CO2 efﬂuxes measured by automated closed chambers were overestimated at low u∗throughout 1 year in a Dan- ish beech forest and that this overestimation considerably bi- ased the diurnal patterns of soil CO2 efﬂuxes and led to an overestimation of the annual soil CO2 efﬂux. We recommend that any analysis of soil CO2 efﬂux measured by automated closed chambers must consider overestimation of efﬂuxes at low atmospheric turbulence, to yield unbiased estimates of soil CO2 efﬂuxes. This is crucial when investigating tem- perature responses of Rs and biological links in ecosystems between CO2 production and Rs on a short timescale, but also for correct estimation of annual soil CO2 efﬂuxes. We have shown that a u∗ﬁltering procedure can be used to re- move data that are inﬂuenced by insufﬁcient turbulence. The drawback of this postprocessing method is, however, a loss of data and thus a loss of information during atmospheric condi- tions where the eddy covariance method cannot be applied. Our analysis highlights the need for methodological devel- opments, which will allow for unbiased chamber measure- ments to be made also during low atmospheric turbulence. We see the results from our fan experiments as a signiﬁcant step along the way. Here we showed that ensuring continuous mixing of air around the soil chamber by a fan eliminated the overestimation of soil CO2 efﬂuxes due to low u∗, thus en- abling reliable chamber measurements even at low u∗, even though the continuous mixing of air may have introduced a Anthony, W. H., Hutchinson, G. L., and Livingston, G. P.: Cham- ber measurement of soil-atmosphere gas exchange: linear vs diffusion-based ﬂux models, Soil Sci. Soc. Am. J., 59, 1308– 1310, 1995. Brændholt, A., Steenberg Larsen, K., Ibrom, A., and Pilegaard, K.: Raw data for “Overestimation of closed-chamber soil CO2 efﬂuxes at low atmospheric turbulence” [Data set], Zenodo, doi:10.5281/zenodo.438015, 2017. Brooks, J. R., Flanagan, L. B., Varney, G. T., and Ehleringer, J. R.: Vertical gradients in photosynthetic gas exchange characteris- tics and reﬁxation of respired CO2 within boreal forest canopies, Tree Physiol., 17, 1–12, 1997. Conen, F. and Smith, K. A.: A re-examination of closed ﬂux chamber methods for the measurement of trace gas emissions from soils to the atmosphere, Eur. J. Soil Sci., 49, 701–707, doi:10.1046/j.1365-2389.1998.4940701.x, 1998. Conen, F. and Smith, K. A. Brændholt et al.: Overestimation of closed-chamber soil CO2 efﬂuxes 1614 new bias during chamber measurements. Additional studies are needed to further explore this approach. various shapes and sizes. We expect that chamber design may inﬂuence the effect of u∗, although it remains to be tested. Görres et al. (2016), however, also argued that automated chambers have the potential to provide unbiased chamber ﬂuxes during low atmospheric turbulence by fulﬁlling cer- tain design criteria that ensure that the stable atmospheric layer of air above the soil surface is not broken up during a measurement. These design criteria include a low chamber height of less than 20 cm and a low chamber closing speed in the horizontal plane, both aiming at keeping the chamber in the same horizontal plane, where there is no steep CO2 gradient. This approach is directly in contrast with the ap- proach we used with fans, where we ensured mixing of the air above the soil surface at all times, such that any stratiﬁca- tion was eliminated before the beginning of a measurement. The proposed design criteria may lead to a lower disturbance of the air column above the soil surface, but it is uncertain if it is possible to eliminate disturbance of the air completely. One of the fundamental principles of closed-chamber mea- surements is mixing of air inside the chamber. Even with a low chamber height we suspect that mixing of stratiﬁed air just a few centimetres from the soil surface may lead to an overestimated ﬂux during low u∗. Data availability. The LI-COR chamber raw data and the meteorological data used in this study can be found at doi:10.5281/zenodo.438015 (Brændholt et al., 2017). References Aubinet, M., Grelle, A., Ibrom, A., Rannik, U., Moncrieff, J., Fo- ken, T., Kowalski, A. S., Martin, P. H., Berbigier, P., Bernhofer, C., Clement, R., Elbers, J., Granier, A., Grunwald, T., Morgen- stern, K., Pilegaard, K., Rebmann, C., Snijders, W., Valentini, R., and Vesala, T.: Estimates of the Annual Net Carbon and Water Exchange of Forest: The EUROFLUX Methodology, Adv. Ecol. Res., 30, 114–173, 2000. The Supplement related to this article is available online at doi:10.5194/bg-14-1603-2017-supplement. Competing interests. 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https://openalex.org/W746082963	https://ojs.ihar.edu.pl/index.php/biul/article/download/1045/915	Polish	null	Wybrane właściwości ekstrudowanych preparatów powstałych na bazie wycierki ziemniaczanej	Biuletyn Instytutu Hodowli i Aklimatyzacji Roślin	2,012	cc-by-sa	4,018	WIOLETTA DROŻDŻ HANNA BORUCZKOWSKA TOMASZ BORUCZKOWSKI EWA TOMASZEWSKA-CIOSK MACIEJ BIENKIEWICZ Katedra Technologii Rolnej i Przechowalnictwa Uniwersytet Przyrodniczy we Wrocławiu Chosen properties of extruded preparations obtained from potato pulp Celem pracy było otrzymanie nowych preparatów z mieszanin suszonej wycierki i skrobi ziemniaczanej na drodze ekstruzji oraz zbadanie ich właściwości. Do rozmrożonej i wysuszonej wycierki ziemniaczanej dodano skrobię w ilościach 10%, 20% oraz 30%. Mieszaniny nawilżono do wilgotności 24% i poddano ekstruzji. Proces przeprowadzono w trzech wariantach temperaturowych 60–70–80°C, 90–100–120°C, 120–130–160°C, przy zastosowaniu następujących parametrów: obroty ślimaka 180 min-1, obroty dozownika 30 min-1, średnica dyszy 3 mm, sprężenie ślimaka 2:1. Określono właściwości mechaniczne (maksymalna siła działająca na wytworzone preparaty, praca konieczna do ich przecięcia) otrzymanych ekstrudatów. Zbadano również rozpuszczalność mierzoną w temperaturze 80°C oraz zawartość błonnika pokarmowego w rozdrobnionych ekstrudatach. Otrzymano 12 preparatów różniących się właściwościami. Wielkość oraz kierunek zmian zależał zarówno od temperatury ekstruzji jak i od zawartości skrobi w mieszaninach poddanych procesowi. Wzrost zawartości skrobi ziemniaczanej w ekstrudowanych mieszaninach spowodował zmiany właściwości mechanicznych uzyskanych preparatów. Wraz ze zwiększającym się udziałem skrobi w mieszaninach odnotowano wzrost rozpuszczalności wytworzonych ekstrudatów oraz zmniejszenie zawartości błonnika pokarmowego. Podwyższenie temperatury ekstruzji wpłynęło na właściwości mechaniczne ekstrudatów, im wyższa była temperatura procesu, tym większa była maksymalna siła potrzebna do przecięcia ekstrudatów, a mniejsze wydłużenie przy tej sile. Wraz ze wzrostem temperatury ekstruzji odnotowano wzrost rozpuszczalności ekstrudatów oraz spadek zawartości błonnika pokarmowego ogółem. DOI: 10.37317/biul-2012-0020 DOI: 10.37317/biul-2012-0020 BIULETYN INSTYTUTU HODOWLI I AKLIMATYZACJI ROŚLIN 2 NR 266 Słowa kluczowe: ekstruzja, wycierka ziemniaczana The aim of the experiment was the production of new preparations from the mixtures of dried potato pulp and starch by extrusion method and to investigate their properties. Dried potato pulp and starch were mixed in the quantities of 10, 20 and 30%. The mixtures, moistened up to 24%, became subjected to extrusion process within three different temperature variants: 60–70–80°C, 90–100–120°C, 120– 130–160°C and the following extrusion parameters: screw rotation speed 180 min-1, batcher rotation 205 Wioletta Drożdż ... 30 min-1, die diameter 3 mm and screw pressure 2:1 were used. The mechanical properties (maximal force acting on the produced preparations, work necessary for their cutting) of obtained extrudates were defined. We determined also solubility, measured in the temperature of 80°C and dietary fibre content of crushed extrudates. We obtained 12 preparations differing in their properties. The extent and direction of observed changes depended on extrusion temperature as well as on the content of starch in prepared mixtures subjected extrusion process. The increase of the level of starch content in extruded mixtures caused changes of mechanical properties of obtained preparations, it means the increase of maximal force acting on processed extrudates and necessary work for their cutting. Alongside with the increase of starch share in the mixtures also solubility of obtained extrudates was elevated, however a decrease of dietary fibre content was observed. The increase of extrusion temperature affected mechanical properties of extrudates. Alongside with the higher temperature of the process the higher maximal power necessary for cutting of samples was stated and parallel smaller sample elongation. With the increase of extrusion temperature, the samples became more soluble and the content of total dietary fibre decreased. Key words: extrusion, potato pulp WSTĘP Przemysł spożywczy wytwarza ogromne ilości produktów ubocznych bogatych w składniki bioaktywne: m.in. polifenole, witaminy, nienasycone kwasy tłuszczowe i błonnik (Laufenberg i in., 2003; Serena i in., 2007). Obecnie, ze względu na występowanie tych wartościowych związków, wzrasta zainteresowanie wykorzystaniem odpadów przemysłu spożywczego przez producentów żywności. Jednym z przemysłów generujących znaczne ilości obciążających środowisko pro- duktów odpadowych jest przemysł krochmalniczy. Głównym produktem ubocznym powstającym w dużych ilościach podczas przerabiania ziemniaków w krochmalni jest wycierka ziemniaczana, składająca się głównie z wody sokowej (powyżej 90% masy). Przemysł skrobiowy próbuje sprzedawać mokrą lub częściowo odwodnioną wycierkę jako karmę dla bydła. Jednakże zapotrzebowanie rolników na wycierkę ziemniaczaną jest ograniczone, głównie ze względu na jej niską dla zwierząt wartość pokarmową oraz malejące pogłowie bydła. W zasadzie wycierka ziemniaczana pozbawiona jest tłuszczu i białek, składa się w większości ze skrobi (ok. 35% w s.m.), jednak jej głównym skład- nikiem są substancje błonnikowe (ponad 50% w s.m.) (Mayer, 1998). Na błonnik ziemniaczany składają się głównie celuloza, hemiceluloza, pektyny, ligniny, a także skrobia oporna. Wycierka jest materiałem łatwo dostępnym w dużych ilościach oraz tańszym od innych materiałów roślinnych, z których wytwarzany jest błonnik (np. owoce cytrusowe). Charakteryzuje się niską alergennością (jest produktem bezglutenowym), a także stabilnością zarówno na niskie pH, jak i wysoką i niską temperaturę (sterylizacja, zamrażanie). Z powodu małego zainteresowania wycierką ze strony rolników, w ostatnich latach próbuje się znaleźć inne niż paszowe, kierunki zużytkowania wycierki ziemniaczanej. Jednak zastosowanie wycierki do celów niepaszowych wiąże się z wcześniejszym jej odwodnieniem, które jest procesem nader energochłonnym. Dlatego też problem ten staje się coraz częściej przedmiotem badań naukowców (Obidziński, 2009; Lotz i in., 2008). Duża zawartość błonnika pokarmowego w wycierce ziemniaczanej, jak również jego 206 Wioletta Drożdż ... zdolność wiązania wody i zawiesin wodno-tłuszczowych, powoduje wzrastające zainte- resowanie tym produktem ubocznym producentów żywności. Podejmowane były próby zastosowania wycierki jako składnika recepturowego do wytwarzania pasztetów (Kaack i in., 2006 a), kiełbas (Bengtsson i in., 2011), ciastek (Kaack i in., 2005) czy do wypieku chleba (Kaack i in., 2006 b). Wycierka ziemniaczana mimo wielu prób zastosowań nadal stanowi ogromny problem. Jednym ze sposobów zagospodarowania tego uciążliwego produktu odpadowego może okazać się proces ekstruzji, który z powodzeniem został wykorzystany do przetwarzania produktów ubocznych przemysłu owocowo-warzywnego. Altan i in. zastosowali wytłoki z pomidorów (Altan. i in., 2008 a) i winogron (Altan. i in., 2008 b) jako dodatki do chrupek, otrzymując produkt o dobrych właściwościach organoleptycznych i funkcjonalnych. y Proces ekstruzji Sporządzono mieszaniny suszonej wycierki i skrobi ziemniaczanej. Skrobię dodawano w ilościach 10%, 20% oraz 30%. Mieszaniny nawilżono wodą destylowaną do wilgotności 24%, następnie przesiano, zamknięto w szczelnym worku i poddano kondycjonowaniu przez 24 h. Ekstruzję przeprowadzono w jednoślimakowym ekstruderze laboratoryjnym typu 20 DN firmy Brabender, przy zastosowaniu następujących parametrów: obroty ślimaka 180 min-1, obroty dozownika 30 min-1, obciążenie ślimaka 5,5–6 A, średnica dyszy 3 mm, sprężenie ślimaka 2:1. Ekstruzję przeprowadzono w trzech wariantach temperaturowych: wariant I — 60–70–90°C, wariant II — 90–100–120°C, wariant III — 130–150–180°C. Metodyka analiz WSTĘP Z dobrym efektem wykorzystano również odpady kalafiora jako dodatek do chrupek (Stojceska i in., 2008). Ekstruzji poddano także wysłodki z buraków cukrowych (Rouilly, 2006) oraz wytłoki z pomarańczy (Larrea i in., 2005 b). Jednak w dostępnej literaturze brak jest doniesień na temat zastosowania wycierki ziemniaczanej jako surowca do wytwarzania preparatów błonnikowych metodą ekstruzji. Celem pracy było otrzymanie nowych preparatów z suszonej wycierki ziemniaczanej oraz mieszanin wycierki i różnej ilości skrobi ziemniaczanej na drodze ekstruzji w trzech wariantach temperaturowych oraz zbadanie ich wybranych właściwości. MATERIAŁ I METODY Materiał badawczy stanowiła wycierka ziemniaczana wyprodukowana przez Przedsię- biorstwo Przemysłu Ziemniaczanego S.A w Niechlowie oraz skrobia ziemniaczana wy- produkowana przez Przedsiębiorstwo Przemysłu Spożywczego PEPEES S.A w Łomży. Wycierkę ziemniaczaną zamrożono i przechowywano w temperaturze -18°C. Następnie rozmrożono i poddano suszeniu w suszarce z owiewem powietrza w temperaturze 40°C przez 48 h. Wysuszoną wycierkę zmielono w obrotowym młynie laboratoryjnym typu B/4- MO-01 880806 firmy Brabender do granulacji Ø 1mm. Metodyka analiz Właściwości mechaniczne Sposób opracowania wyników Uzyskane wyniki zostały poddane obliczeniom statystycznym, na podstawie dwu- czynnikowej analizy wariancji, przy poziomie istotności α = 0,05 przy wykorzystaniu programu Statistica 9.0. Przy pomocy testu Duncana wyniki zostały zestawione w grupy jednorodne. Właściwości mechaniczne Oznaczeniu poddano nierozdrobnione ekstrudaty. Oznaczenie wykonano przy użyciu maszyny wytrzymałościowej INSTRON 5544 stosując przystawkę pomiarową Bend 207 Wioletta Drożdż ... Fixture. Obciążenie głowicy wynosiło 2kN, a prędkość przesuwu głowicy 4,16 mm/s. Wyniki oznaczenia zostały opracowane za pomocą programu komputerowego Table Curve 2D v. 5.01, który umożliwił graficzne przedstawienie uzyskanych danych. Z wykresów obrazujących zależność siły działającej na ekstrudat od przesunięcia głowicy, odczytano wielkości, opisujące właściwości mechaniczne ekstrudatów: maksymalną siłę (N) (siła powodująca przecięcie ekstrudatu) oraz pracę (J) (praca jakiej trzeba użyć do całkowitego przecięcia ekstrudatu). Rozpuszczalność (Richter, 1968) Rozpuszczalność (Richter, 1968) Otrzymane ekstrudaty zmielono w laboratoryjnym młynku nożowym typu WŻ-1. Z rozdrobnionych ekstrudatów sporządzono 500 g 1% zawiesiny wodnej, z uwzględ- nieniem suchej masy. Próbę wstawiono na 30 min do łaźni wodnej z wytrząsarką, o tem- peraturze 80°C, a następnie schłodzono. Odparowaną podczas ogrzewania wodę uzupeł- niono wodą destylowaną do 500 g. Do 6 zważonych i wytarowanych naczynek wirów- kowych wlewano po 50 g roztworu i wirowano w wirówce Biofuge 28 RS przez 30 min przy prędkości 14500 min-1. Supernatant oddzielono i oznaczono jego suchą masę susząc przez 12 h w temperaturze 60°C, a następnie 3 h w 105°C. Rozpuszczalność obliczano ze wzoru: 100 ⋅ = B A R 100 ⋅ = B A R gdzie: R — rozpuszczalność (%), A — sucha masa supernatantu (%), B — sucha masa kleiku (%). Zawartość błonnika pokarmowego ogółem Oznaczenie wykonano metodą enzymatyczno-grawimetryczną zgodnie z międzynaro- dową normą Association of Official Analytical Chemists AOAC Metod 985.29. Sposób opracowania wyników WYNIKI I DYSKUSJA Do badań użyto rozmrożoną, wysuszoną, zmieloną wycierkę ziemniaczaną, którą mieszano w różnym stopniu ze skrobią ziemniaczaną i poddano ekstruzji w trzech wariantach procesu. Otrzymano 12 nowych preparatów. Zastosowanie różnego dodatku skrobi jak również różnych temperatur procesu wpłynęło na właściwości produktów gotowych. W wycierce ziemniaczanej oznaczono zawartość skrobi, błonnika pokarmowego ogółem oraz jej rozpuszczalność w wodzie w temperaturze 80°C. Średnia zawartość skrobi w badanej wycierce wynosiła 30,05%, natomiast zawartość błonnika pokarmowego ogółem 45,66%. Wartości te odpowiadają podawanym w literaturze zawartościom skrobi 208 Wioletta Drożdż ... (ok. 30%) oraz błonnika (ok. 50%) (Mayer, Hildebrandt, 1997; Mayer, 1998). Badana wycierka charakteryzowała się rozpuszczalnością wynosząca 10,19%. Na rysunkach 1 i 2 przedstawiono wpływ temperatury ekstruzji oraz zawartości skrobi w mieszaninie na maksymalną siłę działającą na wytworzone ekstrudaty oraz pracę konieczną do ich przecięcia. Temperatura ekstruzji jak również dodatek skrobi ziemnia- czanej miały istotny wpływ na właściwości mechaniczne otrzymanych produktów. Wartość maksymalnej siły potrzebnej do przecięcia ekstrudatu była największa w przy- padku najniższych temperatur ekstruzji (ponad 2-krotnie większa od dwóch pozostałych wariantów ekstruzji). Ekstrudaty otrzymane w najniższych temperaturach ekstruzji charakteryzowały się najtwardszą strukturą, przez co najtrudniej było je przeciąć. Wskazuje na to również wartość pracy koniecznej do ich zniszczenia. Podobne wyniki uzyskali Altan i in. (2008 b) badając wpływ temperatury ekstruzji na właściwości mechaniczne ekstrudatów otrzymanych z mieszaniny jęczmienia i wytłoków z winogron. Rys. 1. Wpływ temperatury ekstruzji oraz zawartości skrobi w mieszaninie na maksymalną siłę powodującą przecięcie ekstrudatu Fig. 1. Influence of extrusion temperature and starch content in mixture on maximum force needed for cutting of extrudates NIR=142,87 LSD=142,87 0 100 200 300 400 500 600 I II III wariant ekstuzji extrusion variant siła maksymalna [N] force max [N] a a b NIR=164,97 LSD=164,97 0 100 200 300 400 500 600 0 10 20 30 dodatek skrobi [%] starch addition [%] siła maksymalna [N] force max [N] a a a b NIR=164,97 LSD=164,97 0 100 200 300 400 500 600 0 10 20 30 dodatek skrobi [%] starch addition [%] siła maksymalna [N] force max [N] a a a b Rys. 1. Wpływ temperatury ekstruzji oraz zawartości skrobi w mieszaninie na maksymalną siłę powodującą przecięcie ekstrudatu Fig. 1. WYNIKI I DYSKUSJA Influence of extrusion temperature and starch content in mixture on maximum force needed for cutting of extrudates Ekstrudaty wytworzone z samej wycierki oraz z mieszanin wycierki i skrobi zawierających 10% i 20% skrobi charakteryzowały się podobnymi wartościami siły maksymalnej oraz pracy koniecznej do ich przecięcia, istotnie niższymi w porównaniu do ekstrudatów zawierających największą ilość skrobi. Na twardość produktów ekstradowa- nych duży wpływ ma zawartość substancji błonnikowych. Obecność cząstek błonnika pokarmowego prowadzi do przedwczesnego rozerwania ścian komórkowych w ekstru- datach, zanim pęcherzyki powietrza osiągną maksymalny stopień rozszerzenia, co powoduje, że otrzymany produkt posiada niski stopień ekspansji, natomiast jego gęstość i twardość wzrastają (Lue i in., 1991). Podobny wpływ błonnika na właściwości ekstrudatów został zaobserwowany również przez innych autorów (Jin i in., 1994; Yanniotis, 2007). Jednak decydujący wpływ na twardość ekstrudatów miała obecność w mieszaninie skrobi. Colona i Marciel (1983) dowodzą, że częściowo skleikowane gałeczki skrobi przylegają do ścian celulozowych, prowadząc do powstawania ściany złożonej z 209 Wioletta Drożdż ... celulozy, i skleikowanej skrobi co ogranicza możliwość ekspansji produktu, jednocześnie podnosząc jego twardość. Rys. 2. Wpływ temperatury ekstruzji oraz zawartości skrobi w mieszaninie na pracę wykonaną podczas przecięcia ekstrudatu Fig. 2. Influence of extrusion temperature and starch content in mixture on work needed for cutting of extrudates NIR=270,34 LSD=270,34 0 100 200 300 400 500 600 700 I II III wariant ekstruzji extrusion variant praca [J] work [J] b a a NIR=312,16 LSD=312,16 0 100 200 300 400 500 600 700 800 0 10 20 30 dodatek skrobi [%] starch addition [%] praca [J] work [J] a a a b celulozy, i skleikowanej skrobi co ogranicza możliwość ekspansji produktu, jednocześnie podnosząc jego twardość. NIR=270,34 LSD=270,34 0 100 200 300 400 500 600 700 I II III wariant ekstruzji extrusion variant praca [J] work [J] b a a NIR=312,16 LSD=312,16 0 100 200 300 400 500 600 700 800 0 10 20 30 dodatek skrobi [%] starch addition [%] praca [J] work [J] a a a b Rys. 2. Wpływ temperatury ekstruzji oraz zawartości skrobi w mieszaninie na pracę wykonaną podczas przecięcia ekstrudatu Fig. 2. Influence of extrusion temperature and starch content in mixture on work needed for cutting of extrudates W rozdrobnionych ekstrudatach oznaczono zawartość błonnika pokarmowego oraz rozpuszczalność w wodzie w temperaturze 80°C. Odnotowano niewielki spadek zawartości błonnika pokarmowego ogółem oznaczonego w ekstrudowanej wycierce ziemniaczanej (43,69%) w porównaniu do wycierki nie poddanej ekstruzji (45,66%). WYNIKI I DYSKUSJA Zawartość błonnika pokarmowego w próbach, do których dodano skrobię, wahała się w granicach 8,2–29,38%, zmniejszająca się ilość błonnika spowodowana była zmniejszającą się zawartością wycierki w materiale wyjściowym (rys. 3). Zawartość błonnika pokarmowego w otrzymanych ekstrudatach zmniejszała się wraz ze zwiększającą się temperaturą procesu. Już we wcześniejszych badaniach stwierdzono, że proces ekstruzji, szczególnie prowadzony w wysokich temperaturach, wpływa destrukcyjnie na polisacharydy nieskrobiowe. Obniżeniu ulega zawartość błonnika pokarmowego całkowitego i jego frakcji nierozpuszczalnej przy jednoczesnym, znacznym wzroście zawartości frakcji rozpuszczalnej. Intensywna obróbka termoplastyczna wpływa na rozpad polisacharydów nieskrobiowych do postaci oligosacharydów. Podczas ekstruzji następuje również rozerwanie wiązań kowalencyjnych i niekowalencyjnych w węglowodanach, co prowadzi do powstania krótszych i bardziej rozpuszczalnych fragmentów tych węglowodanów (Larrea i in., 2005 a; Wolf, 2010). Znajduje to również potwierdzenie w przypadku rozpuszczalności otrzymanych ekstrudatów (rys. 4). 210 Wioletta Drożdż ... Rys. 3. Wpływ temperatury ekstruzji oraz zawartości skrobi w mieszaninie na zawartość błonnika w ekstrudatach Fig. 3. Influence of extrusion temperature and starch content in mixture on dietary fibre content in extrudates NIR=0,83 LSD=0,83 0 4 8 12 16 20 24 28 I II III wariant ekstruzji extrusion variant błonnik pokarmowy [%] dietary fibre [%] a a b NIR=0,96 LSD=0,96 0 5 10 15 20 25 30 35 40 45 0 10 20 30 dodatek skrobi [%] starch addition [%] błonnik pokarmowy [%] dietary fibre [%] a b c d NIR=0,96 LSD=0,96 0 5 10 15 20 25 30 35 40 45 0 10 20 30 dodatek skrobi [%] starch addition [%] błonnik pokarmowy [%] dietary fibre [%] a b c d NIR=0,83 LSD=0,83 0 4 8 12 16 20 24 28 I II III wariant ekstruzji extrusion variant błonnik pokarmowy [%] dietary fibre [%] a a b Rys. 3. Wpływ temperatury ekstruzji oraz zawartości skrobi w mieszaninie na zawartość błonnika w ekstrudatach Fig. 3. Influence of extrusion temperature and starch content in mixture on dietary fibre content in extrudates Rys. 4. Wpływ temperatury ekstruzji oraz zawartości skrobi w mieszaninie na rozpuszczalność ekstrudatów Fig. 4. WYNIKI I DYSKUSJA 4). Wcześniejsze badania potwierdzają, że proces ekstruzji skrobi powoduje zwiększanie zawartości substancji rozpuszczalnych w ekstrudatach, co jest spowodowane przemianami jakie zachodzą w ekstrudowanym materiale, kleikowaniem i degradacją skrobi do niżej cząsteczkowych polisacharydów (Obuchowski, Chalcarz, 2006). był udział skrobi, tym wyższa była rozpuszczalność wytworzonych preparatów. (rys. 4). Wcześniejsze badania potwierdzają, że proces ekstruzji skrobi powoduje zwiększanie zawartości substancji rozpuszczalnych w ekstrudatach, co jest spowodowane przemianami jakie zachodzą w ekstrudowanym materiale, kleikowaniem i degradacją skrobi do niżej cząsteczkowych polisacharydów (Obuchowski, Chalcarz, 2006). PODSUMOWANIE Na podstawie przeprowadzonych badań stwierdzono, że poddając odpad przemysłowy jakim jest wycierka ziemniaczana ekstruzji można otrzymać całkiem nowe preparaty. Proces ekstruzji spowodował wzrost rozpuszczalności uzyskanych preparatów, przy niewielkiej stracie składnika funkcjonalnego jakim jest błonnik pokarmowy. Dodatek skrobi do ekstrudowanych mieszanin spowodował wprawdzie obniżenie zawartości błonnika w gotowych preparatach, jednakże znacznie przyczynił się do zwiększenia rozpuszczalności wytwarzanych ekstrudatów, co daje możliwość łatwiejszego wprowa- dzenia wycierki modyfikowanej do produktów żywnościowych. Dzięki wysokiej zawartości błonnika w wycierce, przy zwiększonej jej rozpuszczalności ten uciążliwy produkt krochmalni może znaleźć zastosowanie w produkcji, a także wzbogacaniu żywności. WYNIKI I DYSKUSJA Influence of extrusion temperature and starch content in mixture on solubility of extrudates 0 4 8 12 16 20 I II III wariant ekstruzji extrusion variant błonnik pokarmowy dietary fibre [% 0 5 10 15 20 25 30 0 10 20 30 dodatek skrobi [%] starch addition [%] błonnik pokarmow dietary fibre [% a b c NIR=1,04 LSD=1,04 30 35 40 45 50 55 60 I II III wariant ekstruzji extrusion variant rozpuszczalność [%] solubility [%] a b b NIR=1,20 LSD=1,20 0 10 20 30 40 50 60 70 80 0 10 20 30 dodatek skrobi [%] starch addition [%] rozpuszczalność [%] solubility [%] a b c d Rys. 3. Wpływ temperatury ekstruzji oraz zawartości skrobi w mieszaninie na zawartość błonnika w ekstrudatach Fig. 3. Influence of extrusion temperature and starch content in mixture on dietary fibre content in extrudates NIR=1,04 LSD=1,04 30 35 40 45 50 55 60 I II III wariant ekstruzji extrusion variant rozpuszczalność [%] solubility [%] a b b Rys. 4. Wpływ temperatury ekstruzji oraz zawartości skrobi w mieszaninie na rozpuszczalność ekstrudatów Fig. 4. Influence of extrusion temperature and starch content in mixture on solubility of extrudates NIR=1,04 LSD=1,04 30 35 40 45 50 55 60 I II III wariant ekstruzji extrusion variant rozpuszczalność [%] solubility [%] a b b NIR=1,20 LSD=1,20 0 10 20 30 40 50 60 70 80 0 10 20 30 dodatek skrobi [%] starch addition [%] rozpuszczalność [%] solubility [%] a b c d NIR=1,20 LSD=1,20 0 10 20 30 40 50 60 70 80 0 10 20 30 dodatek skrobi [%] starch addition [%] rozpuszczalność [%] solubility [%] a b c d Rys. 4. Wpływ temperatury ekstruzji oraz zawartości skrobi w mieszaninie na rozpuszczalność ekstrudatów Fig. 4. Influence of extrusion temperature and starch content in mixture on solubility of extrudates Ekstruzja wycierki i mieszanin wycierki ze skrobią spowodowała znaczny wzrost ich rozpuszczalności. Podczas gdy rozpuszczalność wycierki niemodyfikowanej wynosiła 10,19%, rozpuszczalność prób po ekstruzji wahała się w granicach 54,54%–59,24% w zależności od temperatury ekstruzji oraz 36,84%–73,96% w zależności od zawartości wycierki w wytworzonych ekstrudatach. Wzrost temperatur ekstruzji powodował zwiększenie rozpuszczalności gotowych produktów, ponieważ wraz ze wzrostem temperatury zwiększa się stopień zniszczenia struktury gałeczek skrobi ziemniaczanej (Fornal, 1998; Śmietana i in., 1996). Jednak o rozpuszczalności otrzymanych preparatów w największym stopniu decydowała ilość skrobi w ekstrudowanej mieszaninie. Im wyższy 211 Wioletta Drożdż ... był udział skrobi, tym wyższa była rozpuszczalność wytworzonych preparatów. (rys. LITERATURA Altan A., McCarthy K. L., Maskan M. 2008 a Evaluation of snack foods from barley-tomato pomace blends by extrusion processing. J. Food Eng. 84: 231 — 242. p g g Altan A., McCarthy K. L., Maskan M. 2008 b Twin-screw extrusion of barley-grape pomace blends: Extrudate characteristics and determination of optimum processing conditions. J. Food Eng. 89: 24 — 32. Altan A., McCarthy K. L., Maskan M. 2008 b Twin-screw extrusion of barley-grape pomace blends: Extrudate characteristics and determination of optimum processing conditions. J. Food Eng. 89: 24 — 32. Bengtsson H., Montelius C., Tornberg E. 2011. Heat-treated and homogenised potato pulp suspensions as p p g g Bengtsson H., Montelius C., Tornberg E. 2011. Heat-treated and homogenised potato pulp suspensions as additives in low-fat sausages. Meat Sci. 88: 75 — 81. g Colona P., Marciel C. 1983. Macromolecular modifications of manioc starch components by extrusion cooking with and without lipids. Carbohydr. Polym. 3: 87 — 108. Fornal Ł. 1998. Ekstruzja produktów skrobiowych — nowe wyroby, Pasze Przemysłowe 3: 7 — 14. Fornal Ł. 1998. Ekstruzja produktów skrobiowych — nowe wyroby, Pasze Przemysłowe 3: 7 — 14. Hać-Szymańczuk E. 2006. Wykorzystanie preparatów błonnikowych w przemyśle spożywczym. Przem. Spoż. 10, 56: 34 — 36. Hać-Szymańczuk E. 2006. Wykorzystanie preparatów błonnikowych w przemyśle spożywczym. Przem. Spoż. 10, 56: 34 — 36. Jin Z., Hsieh F., Huff H. E. 1994. Extrusion cooking of corn meal with soy fiber, salt and sugar. Cereal Chem. 71: 227 — 234. Kaack K., Laerke H. N., Meyer A. S. 2006 a. Liver pate enriched with dietary fibre extracted from potato fibre as fat substitutes. Eur. Food Res. Technol. 223: 267 — 272. Kaack K., Pedersen L. 2005. Low energy chocolate cake with potato pulp and yellow pea hulls. Eur. Food Res. Technol. 221: 367 — 375. Kaack K., Pedersen L., Laerke H. N., Meyer A. 2006 b. New potato fibre for improvement of texture and colour wheat bread. Eur. Food Res. Technol. 224: 199 — 207. Larrea M. A., Chang Y. K., Martinez-Bustos F. 2005 a. Effect of some operational extrusion parameters on the constituents of orange pulp. Food Chem. 89: 301 — 308. Larrea M. A., Chang Y. K., Martinez-Bustos F. 2005 b. Some functional properties of extruded orange pulp and its effect on the quality of cookies. LWT, 38: 213 — 220. Larrea M. A., Chang Y. K., Martinez-Bustos F. 2005 b. LITERATURA Some functi and its effect on the quality of cookies. LWT, 38: 213 — 220. and its effect on the quality of cookies. LWT, 38: 213 — Laufenberg G., Kunz B, Nystroem M. 2003. Transformation of vegetable waste into value added products: (A) the upgrading concept; (B) practical implementations. Bioresour. Technol. 87: 167 — 198. 212 Wioletta Drożdż ... Lotz M., Jahn M., Buntrock P., Eggengoor G. 2008. Potato fibres, methods of preparing them and their use. United States. Patent Application Publication pub. No.: US 2008/0226807 A1. pp p Richter M., Augustat S., Schierbaum F. 1968. Ausgewählte Methoden der Stärkechemie. VEB Fachbuchverlag, Leipzig. Rouilly A., Jorda J., Rigal L. 2006. Thermo-mechanical processing of sugar beet pulp. I. Twin-screw extrusion process. Carbohydr. Polym. 66: 81 — 87. Lue S., Hsieh F., Huff H. E. 1991. Extrusion cooking of corn meal and sugar beet fiber: Effects on expansion properties, starch gelatinization, and dietary fiber content. Cereal Chem. 68, 3: 227 — 234. Lue S., Hsieh F., Huff H. E. 1991. Extrusion cooking of corn meal and sugar beet fiber: Effects on expansion properties, starch gelatinization, and dietary fiber content. Cereal Chem. 68, 3: 227 — 234. Mayer F. 1998. Potato pulp: properties, physical modification and applications. Polym. Degrad. Stab. 59: 231 — 235. Mayer F. 1998. Potato pulp: properties, physical modification and applications. Polym. Degrad. Stab. 59: 231 — 235. Mayer F., Hillebrand J. 1997. Potato pulp: microbiological characterization, physical modification and application of this agricultural waste product. Appl. Microbiol. Biotechnol. 48: 435 — 440. Obidziński S. 2009. Badania procesu zagęszczania wycierki ziemniaczanej. Acta Agroph. 14 (2): 383 — 392. Obuchowski W Chalcarz A 2006 Wpływ surowca na zawartość substancji rozpuszczalnych oraz kierunek i Obidziński S. 2009. Badania procesu zagęszczania wycierki ziemniaczanej. Acta Agroph. 14 (2): 383 — 392. Obuchowski W., Chalcarz A. 2006. Wpływ surowca na zawartość substancji rozpuszczalnych oraz kierunek i i lk ść i h dó i k t ji P l d Zb ż Mł ki 4 16 18 p gę y j g p ( ) Obuchowski W., Chalcarz A. 2006. Wpływ surowca na zawartość substancji rozpuszczalnych oraz kierunek i wielkość przemian sacharydów w procesie ekstruzji. Przegląd Zbożowo-Młynarski 4: 16 — 18. Stojceska V., Ainsworth P., Plunkett A., Ibanoglu E., Ibanoglu S. 2008. Califlower by-products as a new source of dietary fibre, antioxidants and proteins in cereal based ready-to-eat expanded snacks. J. Food Eng. LITERATURA 87: 554 — 563. Serena A., Bach Knudsen K. E. 2007. Chemical and physicochemical characterization of co-products from the vegetable food and agro industries. Anim. Feed Sci. Technol. 139: 109 — 124. 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https://openalex.org/W2051178783	https://www.nepjol.info/index.php/JNPS/article/view/5914/6246	English	null	Congenital Posterior Urethral Diverticulum in a Male Child Case Report	Journal of Nepal Paediatric Society	2,013	cc-by	1,198	September-December, 2012/Vol 32/Issue 3 doi: http://dx.doi.org/10.3126/jnps.v32i3.5914 September-December, 2012/Vol 32/Issue 3 doi: http://dx.doi.org/10.3126/jnps.v32i3.5914 Case Report Introduction urine volume (Figure 1). Cystourethroscopy revealed a diverticulum in the region of posterior urethra distal to verumontanum. Intra venous urography showed both kidneys normal without any dilatation of upper tract. Diverticulum was approached through midline perineal incision. Cystoscope was advanced to locate extent of lesion thereby facilitating dissection. Dorsally placed diverticulum was rotated ventrally by hooking it with infant feeding tube. Diverticulectomy and urethroplasty was done over the infant feeding tube with the help of vicryl 5-0 suture along with reinforcement of corpous spongiosum (Figure 2). Post operatively patient was voiding with good urinary stream with insignifant post void residual urine. In one year follow up in outpatient department the patient was asymptomatic. P osterior urethral valve causing bladder outlet obstruction with back pressure changes is known in children but similar picture may be caused by congenital posterior urethral diverticula. We report a case of six year old boy who had features of bladder outfl ow obstruction. Micturating cystourethrogram revealed posterior urethral diverticulum which was suspected to have posterior urethral valve on ultrasonography, subsequently reconfi rmed on cystourethroscopy. P Abstract Bladder neck and posterior urethra are common sites for obstructive uropathy in children. Diverticula of posterior urethra are rare cause of obstruction in children. A six year old boy presented with features of bladder outflow obstruction since birth. Ultrasound findings were suggestive of posterior urethral valve. Micturating cystourethrogram and endoscopic examinations revealed posterior urethral diverticulum which was placed dorsally. Diverticulectomy and reconstruction of urethra was performed by midline perineal incision. This report emphasizes that a posterior urethral diverticulum may be considered in those cases where features are suggestive of posterior urethral valve bladder outflow obstruction. Key words: Posterior urethral valve, Diverticulum, Diverticulectomy J. Nepal Paediatr. Soc. Address for correspondence: Dr. Zaheer Hasan, E-mail: drzaheerhasan@yahoo.com Abstract Bladder neck and posterior urethra are common sites for obstructive uropathy in children. Diverticula of posterior urethra are rare cause of obstruction in children. A six year old boy presented with features of bladder outflow obstruction since birth. Ultrasound findings were suggestive of posterior urethral valve. Micturating cystourethrogram and endoscopic examinations revealed posterior urethral diverticulum which was placed dorsally. Diverticulectomy and reconstruction of urethra was performed by midline perineal incision. This report emphasizes that a posterior urethral diverticulum may be considered in those cases where features are suggestive of posterior urethral valve bladder outflow obstruction. Key words: Posterior urethral valve, Diverticulum, Diverticulectomy <257> J. Nepal Paediatr. Soc. Hasan Z1, Kumar Bindey2, Kumar P3 1Dr. Zaheer Hasan, MBBS, MS, MCh. Assistant Professor, 2Dr. Bindey Kumar, MBBS, MS, MCh, Additional Professor, 3Dr. Prem Kumar, MBBS, MD Radiology, Additional Professor. All from the department of Surgery, Indira Gandhi Institute of Medical Sciences, Patna Bihar. Address for correspondence: Dr. Zaheer Hasan, E-mail: drzaheerhasan@yahoo.com The Case A six year old male presented with complaints of straining while micturition, dribbling and sensation of incomplete emptying of urine with nocturnal enuresis for three years. There was no history of operative intervention. Physical examination was unremarkable. Laboratory evaluation revealed normal hemogram, blood urea 50 mg/dl, serum creatinine 1.5 mg/dl. Microscopic examination of urine revealed pus cells and growth in urine culture. Ultrasonography revealed thickened, trabeculated bladder and dilated posterior urethra. The patient was stabilized with urethral catheter and antibiotic cover. Micturating cystourethrogram revealed cystic dilatation in the region of posterior urethra which was placed dorsally resulting in bladder outfl ow obstruction with large postvoid residual Fig 1: Photograph, MCU showing posterior urethral diverticulum. Fig 1: Photograph, MCU showing posterior urethral diverticulum. <257> Congenital Posterior Urethral Diverticulum in a Male Child Case Report Fig 2: Photographs showing posterior urethral wall diverticulum hooked by Infant feeding tube urethral diverticulum may present at birth and in grown up children. Saurabh Agrawal et.al has reported posterior utethral diverticulum even in adult patient4. Similar case was reported by Shahram Mousavi et,al in a nineteen year old male patient5. Presenting features of posterior urethral diverticulum vary from asymptomatic patient to diff erent features of lower urinary tract symptoms. Some neglected cases may present with calculi, persistent infection and even adenocarcinoma. In most cases of congenital posterior urethral diverticulum, diverticulum is placed ventrally6 but in the present case it was placed dorsally which is unique. Discussion Urethral diverticulum is defi ned as saccular envaginations of urethral mucosa. In contrary to anterior urethral diverticulum, posterior urethral diverticulum is generally acquired in origin. In a review of 95 diverticulae of posterior urethra, only six were of congenital origin1. Trauma of diff erent types in an elderly multiparous female give rise to acquired posterior urethral diverticulum2. Etiology for congenital posterior urethral diverticulum is proposed that it is due to faulty or incomplete fusion of a segment of urethral plate. Congenital etiology is supported on the basis of back pressure changes in the urinary bladder without any predisposing factor. Proposed etiopathogenesis of posterior urethral diverticulum is that it can swell up signifi cantly during micturition compressing bladder neck and urethra from posterior aspect resulting in outlet obstruction and its antecedent complications3. Conclusion A high index of suspicion is required in order to diagnose posterior urethral diverticulum especially in bladder outlet obstruction cases. It is always should be kept in diff erential diagnosis where the diagnosis of posterior urethral valve is made. Fig 2: Photographs showing posterior urethral wall diverticulum hooked by Infant feeding tube J. Nepal Paediatr. Soc. How to cite this article ? Hasan Z, Kumar Bindey, Kumar P. Congenital Posterior Urethral Diverticulum in a Male Child Case Report. J Nepal Paediatr Soc 2012;32(3):257-258. 6. Gerald H.Jordan,Steven M Schlossberg in surgery of the penis and urethra :In Walsh,Retik,Vaughan,Wein Campbells’Urology. Eighth’s edition 2002 pp3902. References 1. Wachsberg RH, Sebastiano LL, Sullivan BC, Irwin R. Posterior urethral diverticulum presenting as a midline prostatic cyst: Sonographic and MRI appearance. Abdomen Imaging 1995;20:70–1. 1. Wachsberg RH, Sebastiano LL, Sullivan BC, Irwin R. Posterior urethral diverticulum presenting as a midline prostatic cyst: Sonographic and MRI appearance. Abdomen Imaging 1995;20:70–1. 2. Pate VA, Bunts RC. Urethral diverticula in paraplegics. J Urol 1951;65:108 –23. 2. Pate VA, Bunts RC. Urethral diverticula in paraplegics. J Urol 1951;65:108 –23. 3. Nghiem HT, Kellman GM, Sandberg SA, Craig BM. Cystic lesions of the prostate. Radiographics 1990;10:635–5. 3. Nghiem HT, Kellman GM, Sandberg SA, Craig BM. Cystic lesions of the prostate. Radiographics 1990;10:635–5. 4. Saurabh Agrawal, M. S. A. Ansari, R. Kapoor, and D. Dubey. Congenital posterior urethral diverticula causing bladder outlet obstruction in a young male. Indian J Urol 2008;24(3):414-415. 5. Shahram Mousavi, Abdolrasoul Mehrsai, Mohammadreza Nikoobakht, Amir Reza Abedi, Sepehr Salem, Gholamreza Pourmand. A Giant Congenital Posterior Urethral Diverticulum Associated with Renal Dysplasia. Urol J 2006;4:247-9. We have noticed that posterior urethral diverticulum was defi cient of corpous spongiosum. Common diff erential diagnosis of posterior urethral diverticulum are mullerian duct cyst and utricular cyst. The diff erentiating point of posterior urethral diverticulum with mullerian duct cyst and utricular cyst are opening which was paramedian and occupies distal to verumontanum in contrast to midline position in the later cysts. Congenital posterior 6. Gerald H.Jordan,Steven M Schlossberg in surgery of the penis and urethra :In Walsh,Retik,Vaughan,Wein Campbells’Urology. Eighth’s edition 2002 pp3902. <258> J. Nepal Paediatr. Soc. <258>
https://openalex.org/W4390915732	https://vestnikdnu.dp.ua/index.php/ifnit/article/download/162/170	Ukrainian	null	Феномен права в сучасному суспільстві споживання	Doslìdžennâ z ìstorìï ì fìlosofìï nauki ì tehnìki	2,024	cc-by	5,014	ФЕНОМЕН ПРАВА В СУЧАСНОМУ СУСПІЛЬСТВІ СПОЖИВАННЯ E-mail: pavlova_tatyana@ukr.net ORCID: 0000-0001-7178-3573 E-mail: r.pavlov.dnu@gmail.com ORCID: 0000-0001-7629-2730 E-mail: pavlova_tatyana@ukr.net ORCID: 0000-0001-7178-3573 E-mail: r.pavlov.dnu@gmail.com ORCID: 0000-0001-7629-2730 Анотація. Сучасне суспільство споживання потребує свого визначення і наукової розробки, а також розуміння специфіки феномену права як одного з його соціальних регуляторів. Тому необхідно визначити раціональні, онтологічні та аксіологічні аспекти цього феномену і специфіку їхнього прояву в сучасному суспільстві, в якому споживання стає ключовим моментом. Метою статті є визначення особливостей права як феномену в сучасному суспільстві споживання. Методологія роботи включає соціально-філософський аналіз, діалектику, феноменологічний метод, логічний та системно-структурний аналіз. Поєднан ня цих методів дозволяє розкрити сутнісні характеристики феномену права і його буття в соціальній реальності відповідно до змін, які постійно відбуваються і вимагають свого регулювання. Опис основних результатів. У процесі дослідження виявлено, що раціо нальність права, його філософські виміри визначають правову реальність як сукупність різноманітних і водночас пов’язаних між собою правових феноменів. Визначено форми буття і реалізації права в суспільстві, а також особливості їх функціонування сьогодні. Розглянуто право як комунікацію і семіотику, що є надзвичайно актуальним з урахуван ням сучасних культурних процесів, у яких комунікація набуває нових форм, а семіотика постійно змінюється. З’ясовано важливість філософської рефлексії щодо права, оскільки виключно юридичний підхід не розкриває його сутності як феномену. Показано онтоло гічний та аксіологічний виміри права, завдяки чому були розкриті буттєві та ціннісні його конотації. Саме через своє буття, реалізацію в суспільстві право стає реальністю і може реалізувати свою сутність – свободу. А завдяки своїм ціннісним вимірам воно здатне бути загальнозначущим, універсальним суспільним регулятором, що несе цінності і їх захищає. Показано, як змінюються цінності людини в сучасному суспільстві і як право повинно на це реагувати. Розкрито кореляцію змін у свідомості сучасних людей, що відбувається під впливом стратегії виробників товарів – підвищення продажів, і це зазвичай має глибинні системні і структурні наслідки. Розглянуто низку важливих соціально-економічних відно син, які повинне регулювати сучасне право в суспільстві споживання, де основним можна визначити питання відносин між тими, хто товар виробляє, і тими, хто його споживає. Визначено, що право сьогодні має залишатися актуальним і швидко змінюватися за потреби, з огляду на тенденції соціального розвитку. Наукова новизна. Визначено, що феномен права, незважаючи на суспільні зміни, залишається ключовим ціннісним виміром буття суспільства та соціальним регулятором, що може забезпечити реалізацію індивідуальних людських прагнень та загального суспільного інтересу як цілого. Соціально-економічні процеси, що відбуваються в суспільстві, тісно пов’язані з загальнокультурними, і зміни відбуваються як на рівні об’єктивного світу, так і на рівні свідомості окремих людей і ко лективної свідомості, а право в цьому процесі відіграє одну з ключових ролей. Висновки. Т. С. Павлова, Р. А. Павлов Дніпровський національний університет імені Олеся Гончара, Дніпро, Україна ФЕНОМЕН ПРАВА В СУЧАСНОМУ СУСПІЛЬСТВІ СПОЖИВАННЯ E-mail: pavlova_tatyana@ukr.net ORCID: 0000-0001-7178-3573 E-mail: r.pavlov.dnu@gmail.com ORCID: 0000-0001-7629-2730 ІSSN 2617-1929 (print) · Studies in History and Philosophy of Science and Technology · Vol 32 (2) · 2023 ІSSN 2617-1929 (print) · Studies in History and Philosophy of Science and Technology · Vol 32 (2) · 2023 DOI: 10.15421/272318 УДК 340.12:330.16 DOI: 10.15421/272318 УДК 340.12:330.16 THE PHENOMENON OF LAW IN MODERN CONSUMER SOCIETY Abstract. Modern consumer society needs its definition and scientific development, as well as an understanding of the specifics of the phenomenon of law as one of its social regulators. Therefore, it is necessary to determine the rational, ontological and axiological aspects of this phenomenon and the specifics of their manifestation in modern society, in which consumption becomes a key moment. The purpose of the article is to determine the features of law as a phenomenon in the modern consumer society. The methodology of the work includes socio-philosophical analysis, dialectics, phenomenological method, logical and system-structural analysis. The combination of these methods allows revealing the essential characteristics of the phenomenon of law and its existence in social reality in accordance with the changes that are constantly occurring and require their regulation. Description of the main results. In the process of research, it was determined that the rationality of law, its philosophical dimensions determine legal reality as a set of diverse and at the same time interconnected legal phenomena. The forms of existence and implementation of law in society are defined, as well as the peculiarities of their functioning today. Law is considered as communication and semiotics, which is extremely relevant in view of modern cultural processes in which communication takes on new forms, and semiotics is constantly changing. The importance of philosophical reflection on law is clarified, since an exclusively legal approach does not reveal its essence as a phenomenon. The ontological and axiological dimensions of law are shown, thanks to which its essential and valuable connotations were revealed. It is because of its existence, implementation in society that law becomes a reality and can realize its essence – freedom. And thanks to its value dimensions, it is capable of being a general, universal, social regulator that carries values and protects them. It is shown how human values change in modern society and how the law should respond to this. The correlation of changes in the consciousness of modern people, which occurs under the influence of the strategy of product manufacturers – to increase sales, and this, of course, has profound systemic and structural consequences. A number of important socio-economic relations, which should be regulated by modern law in a consumer society, are considered, where the main issue can be defined as the relationship between those who produce goods and those who consume them. T. S. Pavlova, R. A. Pavlov Oles Honchar Dnipro National University, Dnipro, Ukraine T. S. Pavlova, R. A. Pavlov Oles Honchar Dnipro National University, Dnipro, Ukraine ІSSN 2617-1929 (print) · Дослідження з історії і філософії науки і техніки · Том 32 · № 2 · 2023 ІSSN 2617-1929 (print) · Дослідження з історії і філософії науки і техніки · Том 32 · № 2 · 2023 ФЕНОМЕН ПРАВА В СУЧАСНОМУ СУСПІЛЬСТВІ СПОЖИВАННЯ E-mail: pavlova_tatyana@ukr.net ORCID: 0000-0001-7178-3573 E-mail: r.pavlov.dnu@gmail.com ORCID: 0000-0001-7629-2730 З’ясовано, що право сьогодні може розглядатися лише в широкому, філософському сенсі як феномен, оскільки в сучасному суспільстві споживання воно відіграє роль вагомого соціального регулятора, а також відображає найважливіші цінності та соціальні зміни, що відбуваються на рівні буття суспільства. лючові слова: феномен права, суспільство, держава, споживання, онтологія, гносеологія. 19 THE PHENOMENON OF LAW IN MODERN CONSUMER SOCIETY It was determined that today’s law must remain relevant and quickly change as needed, based on the trends of social development. Scientific novelty. It was determined that the phenomenon of law, despite social changes, remains a key value dimension of society and a social regulator that can ensure the realization of individual human aspirations and the general public interest as a whole. Socio-economic processes occurring in society are closely related to general cultural ones, and changes occur both at the level of the objective world and at the level of consciousness of individuals and collective consciousness, and law plays one of the key roles in this process. Conclusions. It was found that law today can only be considered in a broad, philosophical sense as a phenomenon, since in the modern consumer society it plays the role of a significant social regulator, and also reflects the most important and significant values and social changes occurring at the level of society p y y p Conclusions. It was found that law today can only be considered in a broad, philosophical sense as a phenomenon, since in the modern consumer society it plays the role of a significant social regulator, and also reflects the most important and significant values and social changes occurring at the level of society. Keywords: phenomenon of law, society, state, consumption, ontology, epistemology. Вступ. Актуальність цієї теми полягає в тому, що людина завжди виступає акто ром значних соціальних перетворень і не може залишатися осторонь цих процесів. Її буття невіддільне від буття суспільства, відповідно зміни, які в ньому відбуваються, впливають на неї, її свідомість, життя, поведінку безпосередньо. Суспільство спожи вання створює нові наративи, які потребують дослідження. Право і суспільство завжди існують разом, тому питання їх взаємодії є ключовими в розумінні соціального буття людини. Правові сенси в суспільстві сьогодні корелюють з уявленнями про складну природу права, його природу, людські виміри пізнання суспільства. Сучасне суспіль ство споживання потребує такого права, яке б було йому релевантним, відповідало сучасним викликам і водночас не втратило своєї природи. Проблема феномену права 20 ІSSN 2617-1929 (print) · Studies in History and Philosophy of Science and Technology · Vol 32 (2) · 2023 в суспільстві споживання є недостатньо дослідженою, а тому виникає багато питань щодо природи права сьогодні і його особливостей. р р р Питання права як елементу раціонального у свідомості людини розглядав Р. Алексі, який виділив основні проблеми сучасного права [1]. THE PHENOMENON OF LAW IN MODERN CONSUMER SOCIETY В праці [15] розглядаються проб леми природного права та його інтерпретацій. Впливу емоцій на процес споживання та прийняття економічних рішень присвячено працю [12]. Автори праці [7] розро бляли питання правової держави і різниці між правом і законом. Вони розглядають ці феномени в історичному, філософському, соціальному аспектах та намагаються визначити їх загальні й відмінні риси, а також можливі варіанти співіснування. Є. Ха ритонов і О. Харитонова приділяють увагу у своїх дослідженнях складним питанням правовідносин, зокрема у приватноправовій і публічноправовій сферах [9]. В праці [13] досліджуються умови соціалізації споживання коміксів на мікросоціальному рівні в контексті визначення особливостей формування та розвитку різноманітних поведінкових моделей попиту на цей продукт культури. Видатний філософ Е. Фромм у своїй праці «Мати або бути» [3] розглядав ключові проблеми людини як споживача, але водночас складність цього феномену ставить все більше питань, які потребують відповіді. Сучасний науковий контекст вимагає від досліджень широкого підходу до проб леми міждисциплінарності. Складність феномену права полягає перш за все в тому, що філософські, метафізичні уявлення про нього з необхідністю повинні поєднуватися з практикою права. Оскільки право може бути реалізованим сьогодні лише як право людини, тобто реальне, реалізоване право, то філософські питання права, а також питання його теорії і практики розглядаються поряд з економічними питаннями й актуальною соціальною дійсністю. Це є дуже доречним і в плані особливостей права в сучасному суспільстві споживання, дослідження яких може відбуватися лише в ши рокому світоглядному сенсі. р у у Мета дослідження: виявити специфічні риси феномену права в сучасному су спільстві, де споживання виступає ключовою характеристикою світогляду і буття людини. По-перше, визначити ті поняття, проблеми, які сприймаються людиною як раціональне в праві. По-друге, розглянути онтологічну природу права через різні форми його існування, що важливо з точки зору буття та сутності права. По-третє, проаналізувати аксіологію права, де воно розглядається як цінність людського буття. По-четверте, дослідити в культурному плані ключові риси сучасного суспільства і визначити його ключові вектори. По-п’яте, показати особливості права в сучасному суспільстві споживання, дослідити, що ж воно являє собою сьогодні. Предмет до слідження: онтологічні та аксіологічні виміри феномену права в сучасному соціумі. Розглядаючи феномен права, не можна залишити поза увагою дослідження пи тання про його раціональність. Проблемами раціональності і нормативності права, зокрема, займається відомий сучасний філософ права Р. Алексі. Він виділяє три основні проблеми природи права – це проблема природи норми, дійсності і правильності права [1]. Таким чином, ми бачимо, що проблематика раціональності права тісно пов’язана з філософією і розглядати її необхідно саме у філософському вимірі. THE PHENOMENON OF LAW IN MODERN CONSUMER SOCIETY Онтологічний підхід до права якраз дає змогу дослідити проблеми, пов’язані з природою, сутністю й існуванням права. Феномен права в цьому випадку розглядається через свою від мінність від інших феноменів і це дає змогу розкривати правову реальність як дещо особливе, специфічне, як те, що не може бути зведене до іншого і тим більше злива тися з ним. Раціональність права в цьому аспекті виражається перш за все в тому, що воно являє собою обов’язок, належність. Право раціональне тому, що містить у собі універсальний обов’язок. Водночас право метафізичне, ідеальне, являє собою як взі рець, до якого слід прагнути. І це потребує пояснень. Річ у тому, що у філософії права заведено ділити цей феномен на два різновиди: природне право і позитивне право. Щодо природного права існує два підходи до розуміння: перший визначає його як вище право, з позиції якого надається оцінка чинному законодавству, другий включає його в чинне законодавство як невіддільну складову частину і не бачить його існування поза 21 ІSSN 2617-1929 (print) · Дослідження з історії і філософії науки і техніки · Том 32 · № 2 · 2023 межами закону. А позитивне право розглядається як чинне законодавство в конкрет ній державі в конкретний час. І коли ми говоримо про універсальність і метафізику права, то маємо на увазі перш за все природне право [15]. В сучасному суспільстві споживання взагалі є тенденція не ускладнювати споживачеві його вибір складними пошуками істини, сенсу, причин та наслідків. Тому часто розуміння права як чинного законодавства є достатнім у такому суспільстві, оскільки саме право сприймається як річ, інструмент, яким можна користуватися лише з прагматичною метою. Пізнання ж права як складного феномену здійснюється лише в межах філософії. Онтологія права – це завжди стосовно його буття. Виникає питання про те, в яких формах право може існувати. У правовій реальності право існує як правосвідомість, правові норми та правовідносини. В сучасному суспільстві споживання ця структура зберігається, хоча змінюється сама правова реальність у її проявах. Філософські ас пекти права дуже ретельно висвітлюються саме в розробці категорії правосвідомість, оскільки вона містить уявлення людини щодо права, а це дуже комплексне явище, яке включає правові почуття, погляди, ідеї та інше, що відображує ставлення людини до всіх правових явищ. І ґрунтується правова свідомість завжди на тій впевненості, що право є необхідним у суспільстві і здатним виконувати свої завдання. Звичайно, що важливу роль в цьому випадку відіграє і правова культура особистості, і її вольові якості, і відчуття відповідальності, але необхідність і ефективність права – це основне. THE PHENOMENON OF LAW IN MODERN CONSUMER SOCIETY Воля сторін може бути реалізованою лише в соціальному плані, вона із суб’єктивного 22 ІSSN 2617-1929 (print) · Studies in History and Philosophy of Science and Technology · Vol 32 (2) · 2023 2023 волевиявлення виходить у план об’єктивності. Також важливо, що правовідносини мають цілеспрямований характер, чітко визначену мету, а її реалізація підкріплена юридичними обов’язками сторін. І раціональність правовідносин, як і права, можна визначити як їх цілеспрямованість і забезпеченість. Влучно Є. О. Харитонов і О. І. Ха ритонова звертають увагу на діалектику приватного та публічного в регулятивних пра вовідносинах [9]. Лише в такому аспекті проблема правовідносин може бути вписана в загальний контекст суспільного регулювання, де враховані інтереси як загального, так і індивідуального, як державного, так і приватного. Щодо охоронних правовідно син, то вони перш за все стосуються юридичної відповідальності, і в цьому випадку основне питання – це питання законності її підстав. В плані філософського розгляду важливим є те, що право – це певний вид соціальної комунікації і семіотики. І звичайно, феноменологічний метод дозволяє провести подібні дослідження. Як комунікація право є важливим елементом соціального спілкування й передачі інформації, яка транслюється як у часі, так і у просторі. Правова комунікація виступає як елемент культурної комунікації. Тому так важливо, щоб ця комунікація протікала не лише в межах правомірності, а й культурно сприйнятої в цьому суспіль стві раціональності. Це коли вона є зрозумілою всім учасникам правовідносин. Щодо семіотики права, то вона теж містить універсальну раціональність, завдяки якій може транслювати зрозумілу соціальну інформацію у вигляді знаків і символів. Це і правила, і оцінки, які на рівні колективної свідомості фіксуються, закріплюються і передаються. р ф у р р Філософська рефлексія завжди передбачає критичність. Щодо права ця критич ність є актуальною з точки зору оцінювання правильності й неправильності людської дії. Як показує логічний та системно-структурний аналіз, це є актуальним не лише щодо права, а й щодо моралі. Причому йдеться не лише про оцінку поведінкових ак тів конкретних суб’єктів права, а й про оцінку об’єктивності норм, їх релевантності, відповідності певним взірцям. Важливо визначитися з тим, про що йде мова – про філософську рефлексію чи про юридичну нормативність. З огляду на те що подібні оцінки даються з позиції аксіології, де ключовою категорією є цінності, можемо зро бити висновок, що йдеться про філософську рефлексію, яка зазвичай опосередкована специфічністю об’єкта дослідження – права. Водночас сама по собі філософська аксіо логія має природу нормативності, оскільки встановлює універсальні норми поведінки. І в плані загальної раціональності та нормативності аксіологія і правознавство схожі. THE PHENOMENON OF LAW IN MODERN CONSUMER SOCIETY Його раціональність у цьому плані полягає в його ефективності. р у у ф Правосвідомість відображає правову дійсність, надає їй оцінку. Такі оцінки мо жуть викликати певні емоції – позитивні або негативні, які спрямовані на феномен права і його реалізацію. В суспільстві споживання емоційний складник особливо виражений, тому що саме на емоції розраховують маркетологи, виробники, продавці товарів і послуг. Емоція є не лише психічною реакцією людини на події і явища, а й результатом, якого прагне людина. Наприклад, емоція радості від купленого товару, насолоди від оцінювання покупки іншими, задоволення від процесу вибору товару, його купівлі й користування ним. Правові прогнози й варіативність правових дій теж належить до правосвідомості. Право є певним взірцем поведінки, на яку орієнтуєть ся людина, а цінності містяться в її свідомості. Звичайно, не можна ототожнювати закон і право. Оскільки закон може не бути нормою, може не відображати суспільні відносини й суспільну поведінку [7]. Тому, коли мова йде про норму права, як елемент правової реальності завжди мається на увазі водночас її конкретність і універсальність, здатність до регулювання, актуальність, відповідність природному праву. В ній орга нічно поєднуються свобода і обов’язок, який не сприймається як обмеження свободи, а скоріше як необхідність, воля автономної особистості, виражена в універсальній формі у вигляді припису, правила, обов’язкового до виконання. Важливим критерієм раціональності норми є її зрозумілість. Сенс норми повинен бути переданий відповідно до того культурного контексту, в якому перебуває суспільство в цей час. Вимоги норми мають бути такими, щоб їх фактично можна було виконати. Тобто щоб норма не була пустою, декларативною, а щоб вона мала фактичну змогу бути реалізованою на прак тиці в реальних соціальних умовах, які склалися. Також норма повинна поєднувати в собі статичність і динамічність. З одного боку, вона має бути здатною, певний час не змінюючись, регулювати правовідносини, з іншого боку, перебуваючи в динаміч ному соціальному середовищі, змінюватися відповідно до його вимог. Особливо це актуально в сучасному суспільстві, в якому зміни відбуваються набагато динамічніше ніж коли-небудь. у На основі норм права виникають правовідносини, які являють собою третій елемент правової реальності. Як показує соціально-філософський аналіз, по суті норма права може бути реалізованою лише в процесі правовідносин, де всі сторони виступають як суб’єкти, мають свої права та обов’язки, виявляють свою волю і діють, керуючись власною волею. Характерною рисою правовідносин є й те, що в них беруть участь як мінімум дві сторони, і це свідчить про узгодження декількох волевиявлень. THE PHENOMENON OF LAW IN MODERN CONSUMER SOCIETY Створюється все більше нових товарів, які необхідно про дати – і цей феномен став ключовою стратегією сучасного суспільства. Це викликає певні різнопланові проблеми, до яких можна віднести звуження свідомості людини до рівня простого споживача, поглиблення соціальної нерівності, погіршення екології, енергоефективність та інші [2; 4]. Практичним аспектом філософії права є питання про реалізацію прав людини. Тобто не про право як загальну ідеальну категорію, а про конкретні права конкретної людини, які вона може реалізувати в певному суспільстві. І питання це виходить далеко за межі юриспруденції. Право набуває свого змісту лише як реально і практично можлива реалізація права людини. Лише в цьому випадку пра во стає елементом практичного соціального життя, що впорядковує і наповнює його, створює комунікацію і реалізує особистісну автономію, де кожна людина відчуває себе, з одного боку, причетною до соціально-культурного буття, а з іншого – самостійною особистістю. Але права людини мають не лише практичну цінність і самі по собі є цінністю, вони ще й являють собою загальну оцінку, певний стандарт, відповідно до якого повинні відбуватися певні соціальні процеси. Такий підхід відповідає принци пам громадянського суспільства, де загальний інтерес корелюється з індивідуальними інтересами громадян [14]. р р Яким може бути право в сучасному суспільстві споживання? Річ у тому, що соціальні норми в плані регулювання відображають ті економічні реалії, які існують у суспільстві [12]. Право як культурний феномен показує прийняті в суспільстві моделі споживання [13]. Сучасна людина як суб’єкт права також відображає образ споживача продуктів культури, який має свої специфічні риси й особливості культурного буття, які право повинне враховувати [5; 6; 11]. Оскільки для сучасного суспільства рівень споживання товарів і послуг є достат ньо високим, позитивне право повинне перш за все регулювати правовідносини між виробниками і споживачами, а також продавцями товарів. На перший план виходять питання захисту прав споживачів, які повинні реалізувати право на правдиву, достовірну й чітку інформацію щодо товарів та послуг, право на безпеку товарів, їх якість і право на звернення. Покупець повинен мати гарантії, які б забезпечували допомогу у випад ку порушення його прав, коли мова йде про шахрайство, недобросовісну торгівлю, зокрема постачання неякісних товарів і послуг. До основних питань, які є об’єктом правового регулювання в цьому сенсі, можна віднести ціноутворення, рекламу, якість товарів і послуг, договірні відносини та ін. Також актуальними сьогодні є врегулюван ня питань онлайн‑торгівлі та електронної комерції, розвиток яких відбувається дуже швидко. Зокрема, це проведення електронних транзакцій, електронний підпис, захист даних, регулювання додатків і онлайн-платформ. THE PHENOMENON OF LAW IN MODERN CONSUMER SOCIETY Але є звичайно й відмінності, оскільки філософська аксіологія оперує не лише кате горіями соціального світу як правознавство, а й категоріями метафізики. Аксіологічна раціональність і нормативність опосередкована цією метафізичністю. Метафізика як метод також використовується і у філософії права, яка застосо вує філософські методи до вивчення феномену права, а от правознавство має іншу, специфічну юридичну методологію. Таким чином, аксіологія містить метафізичну раціональність, а правознавство – соціальну раціональність. Чи можна їх поєднати? Цими проблемами займається філософія права. Зокрема, вона звертається до змісту природи норми права, проблем її онтології і гносеології, що надає цілісність і набагато ширші горизонти бачення проблеми нормативності. Аксіологія права займається питаннями цінностей у праві, а також розглядає право як цінність. Оскільки цінності в сучасній культурі розглядаються як все те, що може задовольнити не лише потреби людини, а й її бажання, важливим є питання про те, які цінності і як можуть регулюватися правом. Мало того, сучасна культура споживання все більше натякає нам на те, що цінність сьогодні – це все те, що приносить людині задоволення. Цінність необхідно набути або створити, а потім ще й зберегти, а бажано і збільшити. І в цьому плані вона являє собою мету, до якої прагне людина. Цінності можна розглядати як те, що задає сенс людському буттю, і як те, що забезпечує саме людське життя. Перші цінності в сучасній філософії вважаються вельми умовними, розмитими. Людина сама визначає, заради чого вона живе, або взагалі цього не визначає. А от інші цінності – ті, що супроводжують людину протягом її життя і задовольняють її потреби, в тому числі і матеріальні, в сучасному суспільстві споживання виходять 23 SSN 2617-1929 (print) · Дослідження з історії і філософії науки і техніки · Том 32 · № 2 · 2023 на перший план. Тобто йдеться більше не про філософські ідеали, а про предметно втілені цінності, такі як матеріальні речі, які не лише слугують практичним цілям, а й викликають задоволення від їх придбання та використання. р р Звичайно, сучасне суспільство споживання є результатом його поступового роз витку і певних культурних процесів, що відбувалися в ньому [8]. Споживання сьогодні є одною з основних характеристик культури [3]. Воно, по суті, визначає життя сучасної людини в тому плані, що стало його повсякденною невіддільною практикою. Люди все більше співвідносять себе зі споживанням, товарами, брендами, а також емоція ми, які виникають у зв’язку з процесом купівлі і споживанням [11]. Звичайно, цьому сприяє багато факторів – міфодизайн, що використовується в рекламі, маркетингу, зростання виробництва. «REFERENCES» 1. Alexy, R. (2010). The Argument from injustice: a reply to legal positivism. New York: Oxford University Press, 160 p. 1. Alexy, R. (2010). The Argument from injustice: a reply to legal positivism. New York: Oxford University Press, 160 p. 2. Andrade, S. L, & De Lima, G. E (2018). Reverse logistics and the facing of the phenomenon of programmed obsolescence. Revista de Direito da Cidade, 10(2). doi:10.12957/rdc.2018.30605 2. Andrade, S. L, & De Lima, G. E (2018). Reverse logistics and the facing of the phenomenon of programmed obsolescence. Revista de Direito da Cidade, 10(2). doi:10.12957/rdc.2018.30605 3. Fromm, E. (2015). To have or to be? (Reprint). London: Bloomsbury Academic. 4. Holanda, F. C. C., & Lima, K. L. C. R. (2022). The vulnerable consumer and the greenwashing on the fashion industry. Veredas Do Direito, 19(44), 33–55. doi:10.18623/rvd.v19i44.1863 4. Holanda, F. C. C., & Lima, K. L. C. R. (2022). The vulnerable consumer and the greenwashing on the fashion industry. Veredas Do Direito, 19(44), 33–55. doi:10.18623/rvd.v19i44.1863 5. Hudoshnyk, O. V., & Krupskyi, O. P. (2023). Media possibilities of comics: modern tools for the formation and presentation of organizational culture. European Journal of Management Issues, 31(1), 40–49. doi:10.15421/192304 (in Ukrainian). 6. Hudoshnyk, O., & Krupskyi, O. P. (2022). Science and comics: from popularization to the discipline of Comics Studies. History of Science and Technology, 12(2), 210–230. doi:10.32703/2415- 7422-2022-12-2-210-230 7. Ivanii, O. M., Aparov, A. M., & Kurova, A. A. et al. (2020). Pravova derzhava: realii ta perspektyvy rozbudovy v umovakh hlobalizatsii [The rule of law: realities and prospects of development in the context of globalization]. Sumy: SumDPU imeni A. S. Makarenka, 117 p. (in Ukrainian). 8. Jonker, J., Milo, M., & Vannerom, J. (2017). From hapless victims of desire to responsibly choosing citizens. BMGN-Low Countries Historical Review, 132(3), 115–138. doi:10.18352/ bmgn-lchr.10401 9. Kharytonov, Ye. O., & Kharytonova, O. I. (2018). Rehuliatyvni pravovidnosyny pryvatno- pravovyi ta publichno-pravovyi vymiry [Regulatory legal relations: private law and public law dimensions]. Odessa: Helvetica, 404 p. (in Ukrainian). 10. Mitchell, S. (2018). Narratives of Resistance and Repair in Consumer Society. Third Text, 32(1), 55–67. doi:10.1080/09528822.2018.1459110 11. Pavlov, R. A., & Pavlova, T. S. (2023). Obraz postmodernistskoho spozhyvacha produktiv kultury [The image of the postmodernist consumer of culture products]. Іn Pidpryiemnytstvo: suchasni vyklyky, trendy ta transformatsii [Entrepreneurship: modern challenges, trends and transformations]. Dnipro: Bila K. O., 310–333 (in Ukrainian). 12. Pavlov, R. A., Pavlova, T. S., & Levkovich, O. THE PHENOMENON OF LAW IN MODERN CONSUMER SOCIETY Розвиток права повинен відбуватися за цими напрямами, постійно розвиваючись і вдосконалюючись, а також адаптуючись під нові виклики сучасного суспільства споживання. у у Висновки. Показано, що право як складний філософський феномен одночасно має раціональну і метафізичну природу. Раціональність права, як показує соціально- філософський аналіз, об’єктивована перш за все в його обов’язковості, ефективності, зрозумілості, забезпеченості, які проявляються на рівні соціального життя як реаль ність права. Діалектика та онтологія права розкриває форми буття права в суспіль стві. Зокрема, це правосвідомість, яка відображає різні аспекти ставлення людини 24 ІSSN 2617-1929 (print) · Studies in History and Philosophy of Science and Technology · Vol 32 (2) · 2023 до права, його усвідомлення; норма права, що виступає як первинний елемент права і містить правило поведінки; правовідносини, які є формою реалізації норм права і, по суті, процесом їх здійснення. Право також є в суспільстві засобом комунікації, де інформація передається як усередині певної культури в певний час, так і між різними культурами й епохами. Воно також містить культурні знаки, які зрозумілі з точки зору того суспільства, до якого вони належать. І в цьому сенсі сьогодні право є символом сучасного суспільства, який містить актуальні наративи. Аксіологія права визначає ціннісну його природу, що розкриває право як взірець, з яким порівнюють людську поведінку. Цінності в суспільстві змінюються і відповідно до них змінюється право. Логічний та системно-структурний аналіз показує, що сучасне суспільство спожи вання постає як певний культурний феномен, що має свою специфіку й особливості правового регулювання. І не лише в сенсі реалізації позитивного права, а й у контексті самого розуміння права. Споживання стало важливою рисою людини та її повсякден ною життєвою практикою і звичайно вимагає правового забезпечення з урахуванням того моменту, що відбуваються постійні зміни, на які праву слід вчасно реагувати. Ці реакції повинні бути нормативними, релевантними, відповідати суспільним запитам, регулювати нові правові відносини, що виникають, бути універсальними й містити в собі дух закону. До основних сфер правового регулювання сучасного суспільства споживання можна віднести захист прав споживачів товарів і послуг та пов’язані з цим правовідносини. Також усе більший вплив на суспільство мають новітні цифрові технології, блокчейн, децентралізовані способи здійснення купівлі-продажу товарів і послуг, що має бути врегульовано законодавчо на національному та міжнародному рівнях з урахуванням природи права. 13. Pavlov, R. A., Pavlova, T. S., & Levkovich, O. V. (2019). Modeli spozhyvannia komiksiv yak variant postmodernistskoi sotsialno-ekonomichnoi komunikatsii [The models of comics consumption as an option of postmodern and social-economic communication]. Іn Upravlinnia rozvytkom subiektiv pidpryiemnytstva v umovakh vyklykiv XXI stolittia [Managing the development of business entities in the context of the challenges of the XXI century]. Dnipro: Bila K. O., 313–324 (in Ukrainian). 15. Pavlova, T., Zarutska, E., Pavlov, R., & Kolomoichenko, O. (2019). Ethics and law in Kant’s views: the principle of complementarity. International Journal of Ethics and Systems, 35(4), 651–664. doi:10.1108/ijoes-04-2019-0080. pidpryiemnytstva v umovakh vyklykiv XXI stolittia [Managing the development of business entities in the context of the challenges of the XXI century]. Dnipro: Bila K. O., 313–324 (in Ukrainian). 14. Pavlova, T. (2012). Filosofiia Hehelia ta hromadianske suspilstvo sohodni [Hegel’s philosophy and civil society today]. Kultura narodov Prichernomorya, 233, 149–152 (in Ukrainian). pidpryiemnytstva: mekhanizmy, realii, perspektyvy [Management of entrepreneurship development: mechanisms, realities, perspectives]. Dnipro: Bila K. O., 338–357 (in Russian). g y] p , ( ) 14. Pavlova, T. (2012). Filosofiia Hehelia ta hromadianske suspilstvo sohodni [Hegel’s philosophy and civil society today]. Kultura narodov Prichernomorya, 233, 149–152 (in Ukrainian). ІSSN 2617-1929 (print) · Дослідження з історії і філософії науки і техніки · Том 32 · № 2 · 2023 «REFERENCES» V. (2018). Integracija social’nyh norm v modelirovanie processa prinjatija finansovo-jekonomicheskih reshenij [Integration of social norms in modeling of the process of making financial-economic decision]. Іn Upravlinnia rozvytkom subiektiv 25 ІSSN 2617-1929 (print) · Дослідження з історії і філософії науки і техніки · Том 32 · № 2 · 2023 pidpryiemnytstva: mekhanizmy, realii, perspektyvy [Management of entrepreneurship development mechanisms, realities, perspectives]. Dnipro: Bila K. O., 338–357 (in Russian). 13. Pavlov, R. A., Pavlova, T. S., & Levkovich, O. V. (2019). Modeli spozhyvannia komiksiv yak variant postmodernistskoi sotsialno-ekonomichnoi komunikatsii [The models of comics consumption as an option of postmodern and social-economic communication]. Іn Upravlinnia rozvytkom subiektiv pidpryiemnytstva v umovakh vyklykiv XXI stolittia [Managing the development of business entities in the context of the challenges of the XXI century]. Dnipro: Bila K. O., 313–324 (in Ukrainian).i 14. Pavlova, T. (2012). Filosofiia Hehelia ta hromadianske suspilstvo sohodni [Hegel’s philosophy and civil society today]. Kultura narodov Prichernomorya, 233, 149–152 (in Ukrainian). 15. Pavlova, T., Zarutska, E., Pavlov, R., & Kolomoichenko, O. (2019). Ethics and law in Kant’s views: the principle of complementarity. International Journal of Ethics and Systems, 35(4), 651–664. doi:10.1108/ijoes-04-2019-0080. Received 23.07.2023 Received in revised form 13.09.2023 Accepted 15.09.2023 Received 23.07.2023 Received in revised form 13.09.2023 Accepted 15.09.2023 Received 23.07.2023 Received in revised form 13.09.2023 Accepted 15.09.2023 26 26
https://openalex.org/W4362549778	https://zenodo.org/records/7797004/files/Tafakkur%20Manzili-2023-APREL-1-qism-36-39.pdf	Russian	null	ПOВЫШЕНИЕ КAЧЕСТВO ВЫСOКOПРOЧНOГO МЕЛКOЗЕРНИСТOГO БЕТOНA	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	810	2023 APREL 2023 APREL ПOВЫШЕНИЕ КAЧЕСТВO ВЫСOКOПРOЧНOГO МЕЛКOЗЕРНИСТOГO БЕТOНA Нoрбoев Aзизбек Aкрoмжoн угли Нoрбoев Aзизбек Aкрoмжoн угли Мaгистрaнт QMBr-21 Тaшкентскoгo aрхитектурнo-стрoительнoгo университетa Мaгистрaнт QMBr-21 Тaшкентскoгo aрхитектурнo-стрoительнoгo университетa Aннoтaция: Пoвышение физикo-технических хaрaктеристик бетoнa дoстигaется вследствие нaпрaвленнoгo кoмплекснoгo вoздействия рaзрaбoтaннoгo нaнoимпрегнaтa нa фoрмирoвaние микрoструктуры цементнoгo кaмня в зoне кoнтaктa с зaпoлнителем зa счет дoпoлнительнoгo oбрaзoвaния уплoтняющих и упрoчняющих нoвooбрaзoвaний, идентичных гидрoaлюминaтaм и гидрoсиликaтaм кaльция, перерaспределения пoристoсти цементнoй мaтрицы в стoрoну увеличения кoличествa мезoпoр диaметрoм в интервaле oт 3 дo 33 нм при снижении oбщегo oбъемa пoр в 1,5 рaзa. Ключeвыe cлoвa: высoкoпрoчный мелкoзернистый бетoн, физикo- технические хaрaктеристики, мультикoмпoнентный нaнoимпрегнaт, кaвитaциoннoе суспензирoвaние. ВВEДEНИE Применение высoкoпрoчных мелкoзернистых бетoнoв (МЗБ) клaссa прoчнoсти нa сжaтие В60 и выше сooтветствует зaдaчaм стрaтегии рaзвития прoмышленнoсти стрoительных мaтериaлoв нa периoд дo 2020 гoдa и дaльнейшую перспективу дo 2030 гoдa пo рaсширению oтечественных, энергoи ресурсoсберегaющих технoлoгий и спoсoбoв пoвышения кaчествa и дoлгoвечнoсти железoбетoнных изделий и кoнструкций, в тoм числе при стрoительстве быстрoвoзвoдимых мoнoлитных и сбoрнo-мoнoлитных здaний и сooружений, a тaкже при выпoлнении aвaрийных и ремoнтнo-вoсстaнoвительных рaбoт. 2023 APREL Oблaдaя oднoрoднoстью и мелкoзернистoстью мaкрoструктуры, мелкoзернистый бетoн, пo срaвнению с трaдициoнным крупнoзернистым бетoнoм, хaрaктеризуется следующими техникo-экoнoмическими преимуществaми: вoзмoжнoстью сoздaния высoкoкaчественнoй микрo- и нaнoструктуры; пoвышеннoй тиксoтрoпией и спoсoбнoстью к эффективнoй мoдификaции микрo- и нaнoдисперсными дoбaвкaми; высoкoй технoлoгичнoстью (фoрмуемoстью, уплoтняемoстью рaзличными метoдaми: литья, экструзии, прессoвaния, штaмпoвaния, нaбрызгa и др.); легкoй трaнспoртируемoстью, в тoм числе пo трубoпрoвoдaм; вoзмoжнoстью пoлучения нoвых aрхитектурнo-кoнструкциoнных решений (тoнкoстенные и слoистые кoнструкции, изделия переменнoй плoтнoсти, гибридные кoнструкции) и применения местных сырьевых мaтериaлoв прирoднoгo и технoгеннoгo прoисхoждения; бoлее низкoй себестoимoстью. МAТEРИAЛЫ И МEТOДЫ МAТEРИAЛЫ И МEТOДЫ 36 2023 APREL Aлюмoсиликaтным кoмпoнентoм служил метaкaoлин (Al2O3·2SiO2) ‒ дисперсный мaтериaл, сoдержaщий aмoрфные мoдификaции oксидa aлюминия и oксидa кремния, пoлучaемый пoсле специaльнoй термическoй oбрaбoтки и пoмoлa кaoлинa, следующегo химическoгo сoстaвa (% пo мaссе): SiO2 – 42,83; Al2O3 – 50,61; Fe2O3 – 1,89; CaO – 0,15; (Na2O + К2O) – 0,72; MgO – 0,96; SO3 – 0,32; Cl – 0,04; прoчее – 2,48 [3]. Мoрoзoстoйкoсть oбрaзцoв oценивaлaсь пo изменению прoчнoсти нa сжaтие пoсле мнoгoкрaтнoгo зaмoрaживaния и oттaивaния ускoренным (втoрым) метoдoм (средa нaсыщения – 5 % вoдный рaствoр NaCl; средa и темперaтурa зaмoрaживaния – вoздушнaя, минус (18 ± 2) °С; средa и темперaтурa oттaивaния – 5 % вoдный рaствoр NaCl, (20 ± 2) °C. Результaты электрoннoй микрoскoпии пoкaзaли, чтo кaвитaциoннoе суспензирoвaние метaкaoлинa спoсoбствует эрoзии егo микрoчaстиц и aгрегaтoв вплoть дo нaнoдиaпaзoнa, a тaкже рaсщеплению нaнoтoлщинных гексaгoнaльных плaстинoк, сoдержaщихся в стoлбчaтых кoнглoмерaтaх (рис. 1). Рис. 1. Мoрфoлoгия чaстиц метaкaoлинa пoсле кaвитaциoннoгo суспензирoвaния Рис. 1. Мoрфoлoгия чaстиц метaкaoлинa пoсле кaвитaциoннoгo суспензирoвaния РEЗУЛЬТAТЫ И OБCУЖДEНИE Среди сoвременных эффективных спoсoбoв мoдифицирoвaния цементных бетoнoв, в тoм числе МЗБ, бoльшoй нaучнo-прaктический интерес предстaвляет импрегнирoвaние (прoпитывaние) их пoрoвoй структуры рaзличными прoпитывaющими [1] сoстaвaми (упрoчняющими, вoдooттaлкивaющими, oбеспыливaющими, oкрaшивaющими). В исследoвaниях применялись следующие метoды: рентгенoфлуoресцентнaя спектрoскoпия нa спектрoметре ARL 9900 XP, лaзернaя грaнулoметрия нa aнaлизaтoре Analysette 22 NanoTec plus (химический и грaнулoметрический сoстaв aлюмoсиликaтнoгo кoмпoнентa нaнoимпрегнaтa); фoтoннo-кoрреляциoннaя спектрoскoпия, электрoфoретическoе светoрaссеяние нa aнaлизaтoре ZetaPlus с системoй 90Plus/Bi-MAS (пoкaзaтели рaзмернoсти и aгрегaтивнoй устoйчивoсти чaстиц нaнoимпрегнaтa); электрoннaя микрoскoпия нa рaстрoвoм микрoскoпе TESCAN MIRA 3 LMU (мoрфoлoгия чaстиц aлюмoсиликaтнoгo кoмпoнентa и нaнoимпрегнaтa нa егo oснoве, микрoструктурa цементнoгo кaмня в кoнтaктнoй зoне с зaпoлнителем); aзoтнaя пoрoметрия нa прибoре Sorbi-M (рaспределение пoр пo рaзмерaм в цементнoм кaмне oтнoсительнo их oбщегo oбъемa) [2]. 37 37 2023 APREL быстрoвoзвoдимых здaний и сooружений, aвaрийных и ремoнтнo- вoсстaнoвительных рaбoт зa счет введения мультикoмпoнентнoгo нaнoимпрегнaтa aлюмoсиликaтнoгo сoстaвa в виде aгрегaтивнo-устoйчивoй суспензии с сoдержaнием чaстиц средним диaметрoм oкoлo 50 нм и дзетaпoтенциaлoм минус 67 мВ, рaзрaбoтaннoгo метoдoм кaвитaциoннoгo суспензирoвaния и aппретирoвaния метaкaoлинa плaстифицирующевoдoредуцирующим и гидрoфoбизирующим пoверхнoстнo- aктивными веществaми. ЛИТEРAТУРЫ быстрoвoзвoдимых здaний и сooружений, aвaрийных и ремoнтнo- вoсстaнoвительных рaбoт зa счет введения мультикoмпoнентнoгo нaнoимпрегнaтa aлюмoсиликaтнoгo сoстaвa в виде aгрегaтивнo-устoйчивoй суспензии с сoдержaнием чaстиц средним диaметрoм oкoлo 50 нм и дзетaпoтенциaлoм минус 67 мВ, рaзрaбoтaннoгo метoдoм кaвитaциoннoгo суспензирoвaния и aппретирoвaния метaкaoлинa плaстифицирующевoдoредуцирующим и гидрoфoбизирующим пoверхнoстнo- aктивными веществaми. ЛИТEРAТУРЫ ЗAКЛЮЧEНИE В хoде прoведения исследoвaний oбoснoвaнa вoзмoжнoсть пoвышения физикo- технических хaрaктеристик высoкoпрoчнoгo мелкoзернистoгo бетoнa для ЛИТEРAТУРЫ 1. Двoркин Л.И., Житкoвский В.В. Высoкo- прoчные мелкoзернистые бетoны с испoльзoвa- нием грaнитных oтсевoв // Технoлoгии бетoнoв. 2017. № 5-6 (130131). С. 21–25. 2. Лaрсен O.A., Дятлoв A.К. Пoвышение эф- фективнoсти мелкoзернистых бетoнoв дoбaвкaми пoликaрбoксилaтных плaстификaтoрoв для мo- нoлитнoгo дoмoстрoения // Технoлoгии бетoнoв. 2013. № 10 (87). С. 14–15. 3. Щепoчкинa Ю.A., Кaрaкoтенкo-Любимoв A.И. Мелкoзернистый бетoн с включением дo- бaвки пoлиaкрилaтa нaтрия // Инфoрмaциoннaя средa вузa. 2017. № 1 (1). С. 416–418. 4. Федoсoв С.В., Aкулoвa М.В., Слизневa Т.Е. Изучение зaкoнoмернoстей структурooбрa- зoвaния в цементнoм кaмне нa мехaнo-мaгнитo- aктивирoвaннoй вoде с дoбaвкoй ПВA // Academia. Aрхитектурa и стрoительствo. 2017. № 2. С. 117–122. 4. Федoсoв С.В., Aкулoвa М.В., Слизневa Т.Е. Изучение зaкoнoмернoстей структурooбрa- зoвaния в цементнoм кaмне нa мехaнo-мaгнитo- aктивирoвaннoй вoде с дoбaвкoй ПВA // Academia. Aрхитектурa и стрoительствo. 2017. № 2. С. 117–122. 4. Федoсoв С.В., Aкулoвa М.В., Слизневa Т.Е. Изучение зaкoнoмернoстей структурooбрa- зoвaния в цементнoм кaмне нa мехaнo-мaгнитo- aктивирoвaннoй вoде с дoбaвкoй ПВA // Academia. Aрхитектурa и стрoительствo. 2017. № 2. С. 117–122. 39 39
https://openalex.org/W2979894283	https://nottingham-repository.worktribe.com/preview/1478499/Aron	English	null	Raymond Aron’s “Machiavellian” Liberalism	Journal of the history of ideas	2,019	cc-by	13,842	2 This Aron is obviously linked to the Aron theorist of international relations, he who had the ear of Henry Kissinger. Stanley Hoffmann, “Raymond Aron and the Theory of International Relations,” International Studies Quarterly 29, no. 1 (1985): 13-27. 1 1 1 Stuart Campbell, “Raymond Aron: The Making of a Cold Warrior,” Historian 51, no. 4 (1989): 551– 573; Brian Anderson, ed., Raymond Aron: The Recovery of the Political (New Brunswick: Transaction Publishers, 1997), 1-18; Jan-Werner Mueller, “Fear and Freedom: On ‘Cold War Liberalism,’” European Journal of Political Theory 7, no. 1 (2008): 45–64; Aurelian Craiutu, Faces of Moderation: The Art of Balance in an Age of Extremes (Philadelphia: University of Pennsylvania Press, 2017), 60; Or Rosenboim, The Emergence of Globalism: Visions of World Order in Britain and the United States, 1939-1950 (Princeton: Princeton University Press, 2017), 27; Daniel Steinmetz-Jenkins, The Other Intellectuals: Raymond Aron and the United States (New York: Columbia University Press, forthcoming). 2 3 Daniel Mahoney, The Liberal Political Science of Raymond Aron (Lanham: Rowman and Littlefield, 1992); Jeremy Jennings, “Raymond Aron and the Fate of French Liberalism,” European Journal of Political Theory 2, no. 4 (2003): 365-371; Michael Behrent, “Liberal dispositions: recent scholarship on French liberalism,” Modern Intellectual History 13, no. 2 (2016): 447–477. 4 , y , ( ) 4 Stanley Hoffman, “Aron et Tocqueville,” Commentaire 8, no. 28-29 (1985): 200-212; and Stuart Campbell, “The Tocquevillian Liberalism and Political Sociology of Raymond Aron,” The Historian 53, no. 2 (1991): 303-316. Many thanks to Joshua Cherniss, Gregory Conti, Stefanos Geroulanos, William Selinger, Daniel Steinmetz-Jenkins, Iain Stewart, Or Rosenboim and two anonymous reviewers of the JHI for helpful comments to the drafts. Stuart Campbell, “Raymond Aron: The Making of a Cold Warrior,” Historian 51, no. 4 (1989): 551– 73; Brian Anderson, ed., Raymond Aron: The Recovery of the Political (New Brunswick: Transaction Publishers, 1997), 1-18; Jan-Werner Mueller, “Fear and Freedom: On ‘Cold War Liberalism,’” Many thanks to Joshua Cherniss, Gregory Conti, Stefanos Geroulanos, William Selinger, Daniel Steinmetz-Jenkins, Iain Stewart, Or Rosenboim and two anonymous reviewers of the JHI for helpful comments to the drafts. 1 Stuart Campbell, “Raymond Aron: The Making of a Cold Warrior,” Historian 51, no. 4 (1989): 551– 573; Brian Anderson, ed., Raymond Aron: The Recovery of the Political (New Brunswick: Transaction Publishers, 1997), 1-18; Jan-Werner Mueller, “Fear and Freedom: On ‘Cold War Liberalism,’” European Journal of Political Theory 7, no. 1 (2008): 45–64; Aurelian Craiutu, Faces of Moderation: The Art of Balance in an Age of Extremes (Philadelphia: University of Pennsylvania Press, 2017), 60; Or Rosenboim, The Emergence of Globalism: Visions of World Order in Britain and the United States, 1939-1950 (Princeton: Princeton University Press, 2017), 27; Daniel Steinmetz-Jenkins, The Other Intellectuals: Raymond Aron and the United States (New York: Columbia University Press, forthcoming). 2 This Aron is obviously linked to the Aron theorist of international relations, he who had the ear of Henry Kissinger. Stanley Hoffmann, “Raymond Aron and the Theory of International Relations,” International Studies Quarterly 29, no. 1 (1985): 13-27. 3 Daniel Mahoney, The Liberal Political Science of Raymond Aron (Lanham: Rowman and Littlefield, 1992); Jeremy Jennings, “Raymond Aron and the Fate of French Liberalism,” European Journal of Political Theory 2, no. 4 (2003): 365-371; Michael Behrent, “Liberal dispositions: recent scholarship on French liberalism,” Modern Intellectual History 13, no. 2 (2016): 447–477. 4 Stanley Hoffman, “Aron et Tocqueville,” Commentaire 8, no. 28-29 (1985): 200-212; and Stuart Campbell, “The Tocquevillian Liberalism and Political Sociology of Raymond Aron,” The Historian 53, no. 2 (1991): 303-316. Raymond Aron’s “Machiavellian” Liberalism Recent interest in Raymond Aron in Anglophone scholarship has centered on his “Cold War Liberalism.”1 There Aron, often paired with Isaiah Berlin and Karl Popper, is presented as an anti-Marxist and anti-Communist thinker who defended a “negative” or “minimum” version of liberalism, one sometimes associated with what Judith Shklar identified as the “liberalism of fear”: what needed to be avoided first and foremost was cruelty.2 As such, rather than propose a positive or indeed coherent political theory, Aron instead defended certain values (pluralism, tolerance) drawn, like all good liberals, from an idealized vision of England, and advocated an attitude or sensibility (prudence, moderation) in the face of the perils of nuclear Cold War politics. That characterization of Aron is undeniably true, but it nonetheless leaves open a large space within which to place him. Is he, as he has often been depicted, a Tocquevillian “liberal”?3 That association has been the longest and strongest,4 and 2 seems to have been publicly avowed in Aron’s introduction to his classic sociological study Les étapes de la pensée sociologique (1967), where he declared his admiration for the “limpide et triste” (clear and sad) prose of Tocqueville’s Democracy in America, leading to the sobriquet of labelling Aron himself a “liberal triste.”5 Yet if Aron’s long reflections on democracy naturally ties him to Tocqueville, that is not the full story. In fact, in the introduction to Les étapes in question, Aron places his general reflections under the banner of the “opposition Tocqueville-Marx.” He confesses that whilst he has been reading Marx for the past thirty-five years, he only recently turned to Tocqueville, in the last ten years, as a way of criticizing Marx. Ultimately, however, he would never hesitate between Capital, whose “mysteries” never ceased to intrigue him, and Democracy in America: if his conclusions belong to the “English School” of Tocqueville, then his “training” was in the “German School” of Marx.6 Bringing Marx – and in particular critics of Marx – back into the fold (Aron was to continue to write on Marx long after Les étapes),7 links the anti-Marxist and anti-Communist “Cold War Liberal” Aron to the Tocquevillian Aron thinker of modern democracy. This article will argue that what ties the anti-Communist to the Tocquevevillian Aron are the early twentieth century elite theorists of democracy whom he dubbed the “Machiavellians”: Gaetano Mosca, Vilfredo Pareto and Robert Michels. p p g q 7 Raymond Aron, D'une sainte famille à l'autre. Essai sur le marxisme imaginaire (Paris: Gallimard, 1969) and Le Marxisme de Marx (Paris: Éditions de Fallois, 2002). For Aron’s critique of Marx, see Daniel Mahoney, “Aron, Marx, and Marxism,” European Journal of Political Theory 2, no. 4 (2003): 415-427. y p p ( g y , ) 6 “Mes conclusions appartiennent à l’école anglaise, ma formation vient surtout de l’école allemande.” Aron, Les étapes de la pensée sociologique, 21. 7 5 “Je continue, presque malgré moi, à prendre plus d’intérêt aux mystères du Capital qu’à la prose limpide et triste de La Démocratie en Amérique.” Raymond Aron, Les étapes de la pensée sociologique (Paris: Gallimard, 1967), 21. This was translated by Richard Howard and Helen Weaver as Main Currents of Sociological Thought (New Brunswick: Transaction Publishers, 1998). I cite the French editions and all translations are my own. Giulio De Ligio, La Tristezza del pensatore politico: R d A il i t d líti (B l B i U i it P 2007) y g , zz p p Raymond Aron e il primato des político (Bologna: Bononia University Press, 2007). Paris: Gallimard, 1967), 21. This was translated by Richard Howard and Helen Weaver as Main Currents of Sociological Thought (New Brunswick: Transaction Publishers, 1998). I cite the French editions and all translations are my own. Giulio De Ligio, La Tristezza del pensatore politico: “Je continue, presque malgré moi, à prendre plus d’intérêt aux mystères du Capital qu’à la prose impide et triste de La Démocratie en Amérique.” Raymond Aron, Les étapes de la pensée sociologique Paris: Gallimard, 1967), 21. This was translated by Richard Howard and Helen Weaver as Main y g p p Raymond Aron e il primato des político (Bologna: Bononia University Press, 2007). 6 “M l i ti t à l’é l l i f ti i t t t d l’é ( ) 9 Serge Audier, Raymond Aron: La démocratie conflictuelle (Paris: Michalon, 2004), 53-5. 8 Stuart Campbell, “The Four Paretos of Raymond Aron,” Journal of the History of Ideas 47, no. 2 (1986): 287-298. p (1986): 287-298. Raymond Aron’s “Machiavellian” Liberalism It is through his engagement with these thinkers that Aron was able to 3 articulate, on the one hand, his anti-Marxist critique of totalitarianism during World War II and the Cold War, and, on the other, develop his theory of democracy, which took as its basis the anti-Marxist “fact of oligarchy,” that these authors had, on his account, first demonstrated. The importance Mosca, Michels and in particular Pareto played in the development of Aron’s thinking has been highlighted before, not least by Stuart Campbell’s study of “The Four Paretos of Raymond Aron,” and Serge Audier’s more recent Raymond Aron: La démocratie conflictuelle.8 The latter even develops what he calls a “Tocquevillian-Machiavellian” paradigm to interpret Aron’s democratic theory.9 Recognizing the role the Machiavellians play in identifying the hierarchical nature of modern society in Aron’s thought, Audier adds a Tocquevillian dimension to underline how Tocqueville had identified a specific egalitarian dynamic to modern life. Whilst Audier is undoubtedly correct in underlining this Tocquevillian dynamic, he is mistaken to think that the Machiavellians did not see the disappearance of old aristocracies: quite the opposite, their whole point was to show that even in modern egalitarian democracies that had overthrown their aristocratic class, elites stilled ruled, either through their theories of the “ruling class” (Mosca), “circulation of elites” (Pareto), or the “iron law of oligarchy” (Michels). Moreover, these theories were developed in explicit contradistinction to the Marxist notion that once the proletarian revolution accomplished, all hierarchies would melt away – that the “government of people,” as Engels, borrowing from Saint-Simon, put it, would leave way to the “administration of things” – which is why Aron, in his desire to criticize Marxism, was so taken by them. 4 Placing the Machiavellians back into the heart of Aron’s thinking allows us to see that Aron’s liberalism was not simply a negative or minimalist one. Rather, in articulating a theory of democracy based on the “fact of oligarchy,” Aron, notably in his seminal Démocratie et totalitarisme (1965), was able to elaborate a positive theory of democracy (a “Constitutional-Pluralist” regime), one which he actively defended against totalitarianism (a “Party Monopolistic” regime).10 The Machiavellian basis of his thought also provides a coherence to his political theory, from elaborating his critique of totalitarianism on the international sphere, to developing his sociological theory of hierarchical modern democratic society on the domestic. y ( g , ) 11 Iain Stewart, “Existentialist manifesto or conservative political science? Problems in interpreting Raymond Aron's Introduction à la philosophie de l'histoire,” European Review of History: Revue européenne d'histoire 16, no. 2 (2009): 217-233. 12 q ( ) 13 Perry Anderson, “Dégringolade: The Fall of France,” London Review of Books (September 2, 2004) and “Union Sucrée: The Normalizing of France,” London Review of Books (September 23, 2004). p ( ) 12 “Tâchons de dégager la loi de la nécessité industrielle à l’œuvre dans le drame des guerres et des empires, l’action de quelques-uns donnant forme et figure au procès d’industrialisation.” Raymond Aron, Dimensions de la conscience historique (Paris: Les Belles Lettres, 2011), 238. 10 “Les régimes constitutionnels-pluralistes” and “Un régime de parti monopolistique.” Raymond Aron, Démocratie et totalitarisme (Paris: Gallimard, 1965) This was translated by Valence Ionescu as Démocratie et totalitarisme (Paris: Gallimard, 1965) This was Democracy and Totalitarianism (New York: Praeger, 1969). 11 y ( g , ) 11 Iain Stewart, “Existentialist manifesto or conservative political science? Problems in interpreting Raymond Aron's Introduction à la philosophie de l'histoire ” European Review of History: Revue 10 “Les régimes constitutionnels-pluralistes” and “Un régime de parti monopolistique.” Raymond Aron, Démocratie et totalitarisme (Paris: Gallimard, 1965) This was translated by Valence Ionescu as Democracy and Totalitarianism (New York: Praeger, 1969). 11 Iain Stewart, “Existentialist manifesto or conservative political science? Problems in interpreting Raymond Aron's Introduction à la philosophie de l'histoire,” European Review of History: Revue européenne d'histoire 16, no. 2 (2009): 217-233. 12 “Tâchons de dégager la loi de la nécessité industrielle à l’œuvre dans le drame des guerres et des empires, l’action de quelques-uns donnant forme et figure au procès d’industrialisation.” Raymond Aron, Dimensions de la conscience historique (Paris: Les Belles Lettres, 2011), 238. 13 Perry Anderson, “Dégringolade: The Fall of France,” London Review of Books (September 2, 2004) and “Union Sucrée: The Normalizing of France,” London Review of Books (September 23, 2004). f gy , ( ) 16 Serge Audier, “The French Reception of American Neoliberalism in the late 1970s” in Stephen Sawyer and Iain Stewart ed., In Search of the Liberal Moment: Democracy, Anti-totalitarianism, and Intellectual Politics in France since 1950 (Basingstoke: Palgrave Macmillan, 2016), 167-8. 17 Daniel Stedman Jones, Masters of the Universe: Hayek, Friedman, and the Birth of Neoliberal Politics (Princeton: Princeton University Press, 2014); Angus Burgin, The Great Persuasion: Reinventing Free Markets since the Depression (Cambridge [MA]: Harvard University Press, 2015). France: Raymond Aron in Context,” History of European Ideas 39, no. 1 (2013): 35-50. 15 Nicholas Gane, “In and out of neoliberalism: Reconsidering the sociology of Raymond Aron,” Journal of Classical Sociology 16, no. 3 (2016): 261-279. 14 H. S. Jones and Iain Stewart, “Positive Political Science and the Uses of Political Theory in Post-War 14 H. S. Jones and Iain Stewart, “Positive Political Science and the Uses of Political Theory in Post-War France: Raymond Aron in Context,” History of European Ideas 39, no. 1 (2013): 35-50. 15 Nicholas Gane, “In and out of neoliberalism: Reconsidering the sociology of Raymond Aron,” Journal of Classical Sociology 16, no. 3 (2016): 261-279. f gy , ( ) 16 Serge Audier, “The French Reception of American Neoliberalism in the late 1970s” in Stephen Sawyer and Iain Stewart ed., In Search of the Liberal Moment: Democracy, Anti-totalitarianism, and Intellectual Politics in France since 1950 (Basingstoke: Palgrave Macmillan, 2016), 167-8. 17 14 H. S. Jones and Iain Stewart, “Positive Political Science and the Uses of Political Theory in Post-War France: Raymond Aron in Context ” History of European Ideas 39 no 1 (2013): 35-50 Politics (Princeton: Princeton University Press, 2014); Angus Burgin, The Great Persuasion: Reinventing Free Markets since the Depression (Cambridge [MA]: Harvard University Press, 2015). Raymond Aron’s “Machiavellian” Liberalism Moreover, beyond political theory, international relations and sociology, there is reason to believe that these elitist notions underpinned his work in the philosophy of history too:11 in Dimensions de la conscience historique (1961), Aron explains that history is the interplay between two central notions, drama and process. If process attempts to account for the necessary transformation of society, notably in this case the development of industrial society, drama captures the contingent action of men within this longer history. So if process is concerned with structural factors, drama is the fact of a small number of individuals; or, in other words, of an elite.12 This “Machiavellian” dimension to Aron’s thought undermines recent attempts to classify him as a “neo-liberal”.13 Aron was no doubt a “new” type of 5 liberal for the twentieth century,14 much like Tocqueville had been the for nineteenth, wiling to think politics in the “gros temps” of the Cold War, and he certainly attended the now infamous Colloque Walter Lippmann in Paris 1938, where the term was first coined.15 But the epithet “neo”, in particular in terms of what it has come to mean today, seems not to capture him well. For one, many of the participants in the Colloque – Ludwig von Mises, Friedrich von Hayek, Wilhelm Röpke, Aron himself – rejected the term or did not use it.16 What we now identify as neoliberalism developed later, in the 1970s, and is associated with the rising influence of Milton Friedman, Gary Becker’s Chicago School, and the Virginia school of public choice theorists James Buchanan and Gordon Tullock.17 But that type of neoliberalism, which wished, in particular in its Becker Chicago School incarnation, to extend economic logic to all aspects of life, is far removed from the type of political liberalism Aron wanted to defend, which formally drew from a group of Francophone liberal thinkers (Montesquieu, Tocqueville, Constant, Guizot) utterly foreign to the economic thinking of the Chicago School: a political liberalism premised on a clear separation between economic and political spheres. Nor can Aron’s thought be subsumed under the banner of Hayek’s libertarianism. 18 Mueller, “Fear and Freedom,” 56. See Aron’s critique of Hayek “La définition libérale de la liberté,” in Raymond Aron, Études Politiques (Paris: Gallimard, 1972) , 195-215 ; his Essai sur les libertés (Paris: Pluriel, 2014), and his final cours at the Collège de France, Liberté et égalité (Paris: Editions de l’EHESS, 2013). p p q , ( ) 21 Michael Behrent, “Foucault and France’s Liberal Moment” in Sawyer and Stewart, In Search of the Liberal Moment, 156. f 20 Gwendal Châton, “Libéralisme ou démocratie ? Raymond Aron lecteur de Friedrich Hayek,” Revue de philosophie économique 17, no. 1 (2016): 103-134. 21 , 22 Audier, Raymond Aron: La démocratie conflictuelle, 61-88. , ) 19 Craiutu, Faces of Moderation, 62-5. 20 , 22 Audier, Raymond Aron: La démocratie conflictuelle, 61-88. y y ( y ) 25 Quentin Skinner, Liberty Before Liberalism (Cambridge: Cambridge University Press, 1998); an Philip Pettit, Republicanism: A Theory of Freedom and Government (Oxford: Clarendon Press, 1997). 26 23 John Pocock, The Machiavellian Moment: Florentine Political Thought and the Atlantic Republican Tradition (Princeton: Princeton University Press, 2016). 24 ( y , ) 24 Serge Audier, Machiavel, conflit et liberté (Paris: Vrin, 2005); Warren Breckman, Adventure of th Symbolic: Post-Marxism and Radical Democracy (New York: Columbia University Press, 2013). 23 John Pocock, The Machiavellian Moment: Florentine Political Thought and the Atlantic Republican Tradition (Princeton: Princeton University Press 2016) p , p y f ( , ) 26 Audier, Machiavel, conflit et liberté, 28. Raymond Aron’s “Machiavellian” Liberalism As Jan-Werner Mueller succinctly puts it: “Aron explicitly criticized Hayek’s notion of liberty for being one-dimensional and ahistorical, and argued that the advanced industrial societies of the West had managed to find a synthèse 6 démocratico-libérale which had absorbed the socialist critique of a purely negative understanding of liberty.”18 Moreover, Aron was willing to entertain a degree of economic planning and welfare redistribution, something anathema to Hayek, and which led, on the latter’s account, onto The Road to Serfdom (1944).19 For Aron it was democracy, that is to say politics, that came first, with the market a tool to help foster political liberties, whereas for Hayek it was the market that needed to be defended from the encroachments of democratic politics.20 As Michael Behrent, channeling Churchill, has written in another context: if Aron was “in” the neoliberal moment, he was not “of” it.21 This openness on Aron’s part to entertain market planning and social redistribution – whether for conservative reasons or not, or indeed whether within different historical circumstances he would have defended the same ideas – has led Audier to argue that Aron is best understood as a “social-liberal,” one willing to try to reconcile socialism (Aron identified his own intellectual roots as coming from the left) with liberalism.22 In attempting to combine liberty and equality, this, of course, links Aron back to Tocqueville, but also, this article contends, to the Machiavellians. Building on Campbell and Audier’s work, this article will at first deepen Aron’s engagement with the Machiavellians, by tracing his intellectual dialogue with Pareto and how that provided him with important intellectual tools to critique totalitarian regimes on the one hand and develop a positive theory of democracy on the other. It will be particularly attentive to the shift in Aron’s appreciation of Pareto, 7 from seeing him in his early days as an apologist of Fascism to a fellow-in-arms critic of totalitarianism and defender of democracy. The role the ex-Trotskyist James Burnham’s now forgotten book, The Machiavellians: Defenders of Liberty (1943), played in this change of heart will be key, and it is he who will give the elite theorists the Machiavellian appellation Aron will subsequently make his own. p , p y f 26 Audier, Machiavel, conflit et liberté, 28. ip Pettit, Republicanism: A Theory of Freedom and Government (Oxford: Clarendon Press, 1997). udier, Machiavel, conflit et liberté, 28. Raymond Aron’s “Machiavellian” Liberalism The second section will turn its attention to Aron’s often overlooked sociological writings of the 1950s and 1960s, where he developed, through his engagement with the Machiavellian thinkers, his concept of a “divided” (“divisée”) and “unified” (“unifiée”) elite, which was to serve as the basis for distinguishing liberal-democratic from non-democratic regimes. How Aron articulates the passage from political sociology to political philosophy, notably in Les étapes and Démocratie et totalitarisme, will be of particular interest. from seeing him in his early days as an apologist of Fascism to a fellow-in-arms critic of totalitarianism and defender of democracy. The role the ex-Trotskyist James Burnham’s now forgotten book, The Machiavellians: Defenders of Liberty (1943), played in this change of heart will be key, and it is he who will give the elite theorists the Machiavellian appellation Aron will subsequently make his own. The second section will turn its attention to Aron’s often overlooked sociological writings of the 1950s and 1960s, where he developed, through his engagement with the Machiavellian thinkers, his concept of a “divided” (“divisée”) and “unified” (“unifiée”) elite, which was to serve as the basis for distinguishing liberal-democratic from non-democratic regimes. How Aron articulates the passage from political sociology to political philosophy, notably in Les étapes and Démocratie et totalitarisme, will be of particular interest. from seeing him in his early days as an apologist of Fascism to a fellow-in-arms critic of totalitarianism and defender of democracy. The role the ex-Trotskyist James Burnham’s now forgotten book, The Machiavellians: Defenders of Liberty (1943), Aron’s “Machiavellianism” has led Audier, building on John Pocock’s The Machiavellian Moment,23 to posit a French post-war “Machiavellian moment” encompassing Aron, Maurice Merlau-Ponty and Claude Lefort.24 And although his concerns about the corruption of political regimes ties in well with the themes of the original Florentine “Machiavellian Moment”, Aron did not develop the type of “non- domination” republicanism that characterizes Quentin Skinner and Philip Pettit’s work.25 Instead, as Audier has argued, Aron’s “Machiavellianism” centers on a “conflictual pluralism”26 that sees liberty as emerging from within the space opened up by competing parties, interests and groups. And that conception of liberty, this 8 article submits, emerged through his engagement with his own Machiavellians – Pareto, Mosca and Michels – instead of Machiavelli as such. The French moment did not die, however, with Aron. Raymond Aron’s “Machiavellian” Liberalism The theme of “conflictual pluralism” is present throughout the work of many of the members of the Centre Raymond Aron that was founded in his name: not solely in Lefort’s work, but also in the work of Pierre Manent, Bernard Manin and Pierre Rosanvallon, to name but three. Indeed, Rosanvallon’s dialogue with Michels and Moisie Ostrogorski – a figure almost entirely forgotten today – dates back at least to his time as a young auto- gestionnaire. The third part will thus explore the legacy of Aron’s Machiavellianism and how its figures were used to address new questions, notably that of representation. In conclusion the article will ask whether such an account of democracy still has anything to offer us today. 27 Raymond Aron, “La sociologie de Pareto,” Zeitschrift für Sozialforschung 6, no. 3 (1937): 489-521. ( g , ), ; y “Introduction: Raymond Aron and the Persistence of the Political,” Perspectives on Political Science 35, no. 2 (2006): 73-74. 29 29 On Aron and “convergence theory” see Daniel Mahoney, “The Totalitarian Negation of Man: Raymond Aron on Ideology and Totalitarianism” in The Companion to Raymond Aron, ed. Colen and Dutartre-Michaut, 137-148. 30 Giulio De Ligio, The Question of Political Regime and the Problems of Democracy: Aron and the Alternative of Tocqueville” in The Companion to Raymond Aron, ed. José Colen and Elisabeth Dutartre-Michaut (New York: Palgrace Macmillan, 2015), 119-135; and Daniel Mahoney I: Aron, Pareto and Burnham Aron engaged with Pareto from early on, and that engagement was to continue throughout his productive career. His first published piece appeared in 1937, whilst he was still finishing his PhD. It was entitled “La sociologie de Pareto,” and it was published in Horkheimer’s Zeitschrift für Sozialforschung.27 In it Aron was critical of Pareto, presenting him as a proto-Fascist thinker: by rejecting the Marxist idea of a forthcoming proletarian revolution that would do away with class inequality by affirming the historical, social and political persistence of elites, Aron saw in Pareto’s sociology a theory reactionary bourgeois could seize upon to fight a rear-guard action 9 against revolutionary forces. Yet Aron also drew three key insights from his study of Pareto, which he retained throughout his life. First, that the sphere of politics was autonomous from the economic and social spheres – a highly significant move in a French context dominated by Marxist accounts of the primacy of economics, or Durkheimian views on the pre-eminence of the social.28 Aron’s view that it was politics that came first and foremost is crucial to understanding the fact that whilst the modern world is characterized by being an industrial society, the type of political regime that goes with it – democratic or not – is ultimately a political question, and it is that question that will mark the trilogy of lectures Aron will give at the Sorbonne in the 1950s and 1960s: Dix-huit leçons sur la société industrielle (1963), La Lutte des classes (1964), and Démocratie et totalitarisme (1965).29 Second, Aron drew from Pareto’s sociology a theory of fascist leadership: a hypocritical demagogue, willing to use any type of myth to excite the crowds, but who defends the interests of the elites, whom he ultimately rules in favor of.30 This more analytical Pareto was one Aron grew closer to over the next decades. Indeed, Pareto the analyst of Fascism, rather than its spokesman, was to become the dominant interpretation of Pareto that Aron would develop over time, most notably in his later Les étapes. , 30 Aron, “La sociologie,” 516-9. g 31 Aron, “La sociologie,” 518-9. y gy Dutartre-Michaut, 137-148. 28 Giulio De Ligio, “The Question of Political Regime and the Problems of Democracy: Aron and the 28 Giulio De Ligio, “The Question of Political Regime and the Problems of Democracy: Aron and the Alternative of Tocqueville” in The Companion to Raymond Aron, ed. José Colen and Elisabeth D t t Mi h t (N Y k P l M ill 2015) 119 135 d D i l M h g , Q g y Alternative of Tocqueville” in The Companion to Raymond Aron, ed. José Colen and Elisabeth Dutartre-Michaut (New York: Palgrace Macmillan, 2015), 119-135; and Daniel Mahoney “Introduction: Raymond Aron and the Persistence of the Political,” Perspectives on Political Science , ), 35 Raymond Aron, “Le machiavélisme, doctrine des tyrannies modernes” in Raymond Aron, Penser la liberté, penser la démocratie (Gallimard: Paris, 2005), 115-124. See also “L’Homme contre les tyrants” in Penser la liberté, 107-384 and in Aron, Machiavel et les tyrannies modernes. p , , 34 Raymond Aron, “Lectures de Pareto” in Machiavel et les tyrannies modernes (Paris: Editions de Fallois, 1993), 263-7. 3 32 Raymond Aron, “La sociologie de Pareto,” Revue européenne des science sociales 16, no. 43 (1978) 5-33. 33 C b ll “F P t ” 287 I: Aron, Pareto and Burnham Aron imparts blame for the rise of the fascist leader to the liberal bourgeoisie, who, losing their nerve in face of communist agitation, were willing to throw in their lot with a violent elite.31 This is quite perceptive in terms of explaining the rise of both Mussolini and Hitler, who relied at first on traditional conservative Second, Aron drew from Pareto’s sociology a theory of fascist leadership: a hypocritical demagogue, willing to use any type of myth to excite the crowds, but who defends the interests of the elites, whom he ultimately rules in favor of.30 This more analytical Pareto was one Aron grew closer to over the next decades. Indeed, 10 elites to cement their power before disposing of them, and it also offers Aron his third insight derived from Pareto: that liberalism has to be defended, sometimes even with force. When the article was reprinted some forty years later in 1978 in the Revue européenne des sciences sociales, Aron explicitly distanced himself from his early piece, explaining in a brief preamble that the views expressed there no longer represented his current views.32 What had changed? 33 Campbell, “Four Paretos,” 287. 33 Campbell, “Four Paretos,” 287. 34 p , , 40 Aron, “La sociologie,” 519-520; Aron, Les étapes, 20; Aron, “Lectures,” 263. On Aron’s cynicism, see Campbell, “Four Paretos,” 297-8. 37 Nicolas Guilhot, After the Enlightenment: Political Realism and International Relations in the Mid Twentieth Century (Cambridge: Cambridge University Press, 2017). 38 C b ll “ ” 289 38 Campbell, “Four Paretos,” 289. University Press, forthcoming). 37 Nicolas Guilhot, After the Enlightenment: Political Realism and International Relations in the Mid Twentieth Century (Cambridge: Cambridge University Press, 2017). 38 Campbell, “Four Paretos,” 289. 39 Campbell, “Four Paretos," 287. 40 I: Aron, Pareto and Burnham In a seminal article published in 1986, based on an interview with Aron conducted in 1982, one year before his death, Stuart Campbell analyzed the “four Paretos” Aron claimed to have been in existence in his work.33 These four Paretos, which Aron himself identified in his 1973 text “Lectures de Pareto,” were: the fascist Pareto, the authoritarian Machiavellian Pareto, the liberal Machiavellian Pareto, and the Pareto the cynic.34 There is much to be said about reading Aron’s Paretos in this way: we have already explored the fascist Pareto, and it is true that during his wartime journalist writings Aron started to use the more analytical Pareto as a way of making sense of rising totalitarianism in Europe, whether Fascist, National-Socialist, or Communist – what Campbell identifies as the “authoritarian Machiavellian Pareto.” Indeed, the first piece he wrote for La France libre, which he was editing from London as part of the wartime effort under de Gaulle, with whom he had an oftentimes fractious relationship, was entitled “Le machiavélianisme, doctrine des tyrannies modernes” (1940).35 Inspired by his mentor “Lectures de Pareto,” were: the fascist Pareto, the authoritarian Machiavellian Pareto, the liberal Machiavellian Pareto, and the Pareto the cynic.34 There is much to be said about reading Aron’s Paretos in this way: we have already explored the fascist Pareto, and it is true that during his wartime journalist writings Aron started to use the more analytical Pareto as a way of making sense of rising totalitarianism in Europe, whether Fascist, National-Socialist, or Communist – what Campbell identifies as the “authoritarian Machiavellian Pareto.” Indeed, the first piece he wrote for La France libre, which he was editing from London as part of the wartime effort under de Gaulle, with whom he had an oftentimes fractious relationship, was entitled “Le machiavélianisme, doctrine des tyrannies modernes” (1940).35 Inspired by his mentor 11 Elie Halevy’s L’Ere des tyrannies (1938),36 Aron set out to analyze both the rise of Fascism and Communism through the lenses of “Machiavellianism.”37 Fascism on this account, as we saw above, adopted a Machiavellian/Paretean philosophy, whilst Communism adopted Machiavellian tactics.38 Whilst these four Paretos offer undeniably helpful prisms through which to interpret Aron’s work, it would be a mistake, however, to think of them as somehow temporal and sequential, mapping themselves back onto Aron’s own development. 36 Iain Stewart, Raymond Aron and the History of Liberal Thought, 1926-1983 (Cambridge: Cambridge University Press, forthcoming). 36 Iain Stewart, Raymond Aron and the History of Liberal Thought, 1926-1983 (Cambridge: Cambridge U i it P f th i ) Twentieth Century (Cambridge: Cambridge University Press, 2017). 38 p 39 Campbell, “Four Paretos," 287. 36 Iain Stewart, Raymond Aron and the History of Liberal Thought, 1926-1983 (Cambridge: Cambridge University Press, forthcoming). 37 Nicolas Guilhot, After the Enlightenment: Political Realism and International Relations in the Mid- T i h C (C b id C b id U i i P 2017) I: Aron, Pareto and Burnham Whilst these four Paretos offer undeniably helpful prisms through which to interpret Aron’s work, it would be a mistake, however, to think of them as somehow temporal and sequential, mapping themselves back onto Aron’s own development. Rather than these four different moments, what seems of most importance is the shift from Aron’s early view of Pareto as a proto-Fascist to him having a more positive view of Pareto – a Pareto who ultimately served the cause of liberty – as Aron himself acknowledged in the 1982 interview.39 And that shift came about as a result of his discovery of Burnham in the 1940s. Indeed, we have already explored the evolution of the “Fascist Pareto,” from apologist of Fascism in the pre-War writings to analyst of Fascism post-War. Pareto the cynic, which we haven’t much discussed, is present throughout Aron’s writing on Pareto, already in his early 1937 piece on “La Sociologie,” to his latter 1967 Les étapes, and through to his “Lectures de Pareto” of 1974 – although that Aron’s cynicism would increase in his later life is a point well made.40 Finally, as we shall now turn to, it seems difficult to disentangle the so-called “authoritarian” from the “liberal” Machiavellian Pareto of the 1940s/50s to 1960s, the Pareto used both to critique totalitarianism and also to develop a theory of democracy, an inseparable task in the context of the Cold War. Rather than these four different moments, what seems of most importance is the shift from Aron’s early view of Pareto as a proto-Fascist to him having a more positive view of Pareto – a Pareto who ultimately served the cause of liberty – as Aron himself acknowledged in the 1982 interview.39 And that shift came about as a result of his discovery of Burnham in the 1940s. Indeed, we have already explored the evolution of the “Fascist Pareto,” from apologist of Fascism in the pre-War writings to analyst of Fascism post-War. p , , 39 Campbell, “Four Paretos," 287. Campbell, Four Paretos, 289. 39 Campbell, “Four Paretos," 287. 40 Aron “La sociologie ” 519 520; Aron Les étapes 20; Aron “Lectures ” 263 On Aron’s cynicism University Press, forthcoming). 37 Nicolas Guilhot, After the Enlightenment: Political Realism and International Relations in the Mid- Twentieth Century (Cambridge: Cambridge University Press, 2017). 38 C b ll “F P ” 289 I: Aron, Pareto and Burnham Pareto the cynic, which we haven’t much discussed, is present throughout Aron’s writing on Pareto, already in his early 1937 piece on “La Sociologie,” to his latter 1967 Les étapes, and through to his “Lectures de Pareto” of 1974 – although that Aron’s cynicism would increase in his later life is a point well made.40 Finally, as we shall now turn to, it seems difficult to disentangle the so-called “authoritarian” from the “liberal” Machiavellian Pareto of the 1940s/50s to 1960s, the Pareto used both to critique totalitarianism and also to develop a theory of democracy, an inseparable task in the context of the Cold War. 12 James Burnham, a disappointed Trotskyist turned reactionary critic of bureaucracy, is best remembered for his 1941 book The Managerial Revolution. That book, in the era of Trump, is going through a mini-renaissance,41 but his 1943 follow- up The Machiavellians: Defenders of Liberty is now almost completely forgotten. Aron however, who met Burnham, was very taken by it, and personally arranged for it to be published in his “Liberté de l’esprit” series he was directing at Calmann-Lévy in 1949.42 In an 1949 article “Histoire et politique,” Aron registered his debt to Burnham, explaining that his reading of Pareto and the other Machiavellians, who proposed a more “realistic” or “pessimistic” account of power, in which power was needed to check power, served as a critique of Communist millenarianism: that regimes that aim for the highest level of perfection are in fact the ones most likely to use oppressive and totalitarian means to achieve it.43 What was needed instead was a divided elite (“divisées”) that aimed not for a perfect society, but made best do with the imperfect societies in which they lived.44 James Burnham, a disappointed Trotskyist turned reactionary critic of bureaucracy, is best remembered for his 1941 book The Managerial Revolution. That book, in the era of Trump, is going through a mini-renaissance,41 but his 1943 follow- up The Machiavellians: Defenders of Liberty is now almost completely forgotten. 44 “les élites les plus supportables son celles qui sont divisées…Il n’y a pas de société parfait, mais il y a des degrés dans l’imperfection.” Aron, “Histoire et politique," 533. American Affairs was launched in 2017 to “help explain Trumpism”. See further Alan Wald, “From Trotsky to Buckley,” Jacobin, 15/09/2017. y y, , 42 James Burnham, Les Machiavéliens: Défenseurs de la liberté (Paris: Calmann-Lévy, 1949). 43 43 “Souvent les prophètes de la société parfaite sont précisément ceux qui édifient la société la plus oppressive.” Raymond Aron, “Histoire et politique” in Aron, Penser la liberté, penser la démocratie, 533. 41 Julius Krein, “James Burnham’s Managerial Elite,” American Affairs 1, no. 1 (2017): 126-51. 41 Julius Krein, “James Burnham’s Managerial Elite,” American Affairs 1, no. 1 (2017): 126-51. American Affairs was launched in 2017 to “help explain Trumpism”. See further Alan Wald, “From Trotsky to Buckley,” Jacobin, 15/09/2017. American Affairs was launched in 2017 to help explain Trumpism . See furth Trotsky to Buckley,” Jacobin, 15/09/2017. , , 48 Raymond Aron, “Catégories dirigeantes ou classe dirigeante?” in Raymond Aron, Études Sociologiques (Paris: PUF, 1988), 88. I: Aron, Pareto and Burnham Aron however, who met Burnham, was very taken by it, and personally arranged for it to be published in his “Liberté de l’esprit” series he was directing at Calmann-Lévy in 1949.42 In an 1949 article “Histoire et politique,” Aron registered his debt to Burnham, explaining that his reading of Pareto and the other Machiavellians, who proposed a more “realistic” or “pessimistic” account of power, in which power was needed to check power, served as a critique of Communist millenarianism: that regimes that aim for the highest level of perfection are in fact the ones most likely to use oppressive and totalitarian means to achieve it.43 What was needed instead was a divided elite (“divisées”) that aimed not for a perfect society, but made best do with the imperfect societies in which they lived.44 This critique of millenarian Marxism, married to a more positive formulation of what a society that wishes to uphold liberty should look like, means that the Machiavellian authoritarian and the Machiavellian liberal Pareto go hand-in-hand. It also underlines how central Burnham’s reading of Pareto and his Machiavellian colleagues were to Aron’s understanding of them, and particularly in his more positive, post-War, reappraisal of them. Indeed, by emphasizing how power needs to be checked and not given unlimited reign – that it is counter-powers that are the best 13 guarantors of freedom – Pareto and the Machiavellians, on Aron’s account, fundamentally furthered the cause of modern liberty, as the subtitle to Burnham’s book intimated.45 As he will come to fully theorize in both his sociological and political writings of the 1950s and 1960s, liberty was to be found, for Aron, from the fact that different political, social and economic elites all compete for power. It is within the space opened up between these opposing forces that liberty can flourish. guarantors of freedom – Pareto and the Machiavellians, on Aron’s account, guarantors of freedom – Pareto and the Machiavellians, on Aron’s account, fundamentally furthered the cause of modern liberty, as the subtitle to Burnham’s book intimated.45 As he will come to fully theorize in both his sociological and political writings of the 1950s and 1960s, liberty was to be found, for Aron, from the fact that different political, social and economic elites all compete for power. It is within the space opened up between these opposing forces that liberty can flourish. 45 Aron, of course, might have gleaned counter-powers from Montesquieu, but the latter does not offe the reflections on elites within a modern industrial society that the Machiavellians do. 46 “De la philosophie à la sociologie politique ” Aron Démocratie 38 p p g p q , , 47 Aron, Démocratie, 38-41. 48 46 “De la philosophie à la sociologie politique.” Aron, Démocratie, 38. 47 Aron Démocratie 38-41 46 “De la philosophie à la sociologie politique.” Aron, Démocratie, 38. 47 Aron, Démocratie, 38-41. 47 Aron, Démocratie, 38-41. 48 II: Divided and unified elites In the second chapter of Démocratie et totalitarisme, entitled “From philosophy to political sociology,” Aron questions the relation political philosophy, which he defines as the exercise of judging political regimes, entertains with sociology, which comprises a factual study of different regimes.46 He starts with Aristotle, whose Politics combined both political sociology, in its classification of regimes into monarchies, aristocracies and polities – alongside their corrupted versions tyranny, oligarchy and democracy – and political philosophy, in that it judged these regimes according to a human telos.47 In a contemporary sociological text, one we’ll have occasion to return to, Aron points out that when Aristotle comes to the detailed description of the ancient Greek cities, he leaves aside his abstract classification and posits instead a perennial conflict between oligarchy and democracy, between the rich and the poor, between the rulers and the ruled.48 14 Nevertheless, what follows in Démocratie is a potted history of ideas, where Aron discusses the relation between sociology and politics in figures such as Montesquieu, Hobbes, Marx and Popper. Montesquieu’s new classification of regimes into republic, monarchy and despotism will in fact serve as the opening for the later Les étapes, where Aron examines the passage from political theory to sociology by exploring how Montesquieu, after elaborating his new conceptual schema, will turn to studying the political sociology of these regimes, by analyzing both their material (climate, geography) and social (religion, commerce) causes.49 But the notion of the conflict between ruler and ruled will return. Aron locates the birth of modern political sociology in the nineteenth century, notably with the work of Comte and Marx. This modern sociology teaches us two things: that all regimes are essentially defined by the struggle for power, and the fact that it is always the few who rule.50 These two new “savoirs” Aron attributes to the Machiavellians, and to Pareto in particular: whilst Marx was right to identify the conflictual nature of politics, he was mistaken to think that the class struggle would come to an end after the proletarian revolution, and that the “rule of the (few) men” could be replaced with the “administration of things.” Pareto’s answer to Marx was that conflict would continue in the future, and the question of politics would continue to be “who rules?”.51 But the notion of the conflict between ruler and ruled will return. Aron, Démocratie, 51. 51 “Le vrai problème est de savoir qui gouverne…le fait qu’un petit nombre d’hommes exercent le pouvoir.” Aron, Démocratie, 49-50. 49 Aron, Les étapes, 27-52. 50 50 Aron, Démocratie, 51. 51 50 Aron, Démocratie, 51. 51 Aron, Les étapes, 27-52. 50 Aron, Démocratie, 51. 51 50 Aron, Démocratie, 51. 51 50 Aron, Démocratie, 51. 51 “Le vrai problème est de savoir qui gouverne le fait qu’un petit nombre d’hommes exercent le 52 Raymond Aron, Introduction à la philosophie politique: Démocratie et révolution (Paris: Éditions du Fallois, 1997), 55-8. 53 , , 54 “la recherché du pouvoir légitime…du régime le meilleur.” Aron, Démocratie, 51-3. Aron, Démocratie, 51-3. 4 “la recherché du pouvoir légitime…du régime le meilleur.” Aron, Démocratie, 51-3. , ), 53 Aron, Démocratie, 51-3. 54 , ), 53 Aron, Démocratie, 51-3. Fallois, 1997), 55 8. 53 Aron, Démocratie, 51-3. 54 II: Divided and unified elites Aron locates the birth of modern political sociology in the nineteenth century, notably with the work of Comte and Marx. This modern sociology teaches us two things: that all regimes are essentially defined by the struggle for power, and the fact that it is always the few who rule.50 These two new “savoirs” Aron attributes to the Machiavellians, and to Pareto in particular: whilst Marx was right to identify the conflictual nature of politics, he was mistaken to think that the class struggle would come to an end after the proletarian revolution, and that the “rule of the (few) men” could be replaced with the “administration of things.” Pareto’s answer to Marx was that conflict would continue in the future, and the question of politics would continue to be “who rules?”.51 This “Machiavellian” critique of democracy – that all regimes are in fact oligarchic, that the few always rule – Aron had already developed in his 1950s lectures Introduction à la philosophie politique at the École nationale d'administration (ENA), set-up by de Gaulle after the war to train the future high civil 15 service in charge of reconstructing the country, and in reality continues to furnish France with a large portion of its political class even today, notably Emmanuel Macron. There he explicitly cites Pareto, Mosca and Burnham as being the originators of this theory, but he does not leave it at that, arguing that once the oligarchic nature of democracy had been stated, then the question of how that oligarchy is constituted, and what its relation to the masses is, become the key political questions.52 In Démocratie Aron goes further still, criticizing the “Machiavellian” conception as being too “cynical” – a throwback to our discussion of Pareto above – as it concentrates solely on the struggle for power, but overlooks the fact that one can still judge between regimes to see which one is best.53 service in charge of reconstructing the country, and in reality continues to furnish France with a large portion of its political class even today, notably Emmanuel Macron. II: Divided and unified elites There he explicitly cites Pareto, Mosca and Burnham as being the originators of this theory, but he does not leave it at that, arguing that once the oligarchic nature of democracy had been stated, then the question of how that oligarchy is constituted, and what its relation to the masses is, become the key political questions.52 In Démocratie Aron goes further still, criticizing the “Machiavellian” conception as being too “cynical” – a throwback to our discussion of Pareto above – as it concentrates solely on the struggle for power, but overlooks the fact that one can still judge between regimes to see which one is best.53 service in charge of reconstructing the country, and in reality continues to furnish France with a large portion of its political class even today, notably Emmanuel Macron. There he explicitly cites Pareto, Mosca and Burnham as being the originators of this theory, but he does not leave it at that, arguing that once the oligarchic nature of democracy had been stated, then the question of how that oligarchy is constituted, and what its relation to the masses is, become the key political questions.52 In The type of political sociology, then, that Aron wishes to practice is one that does not simply affirm the Machiavellian struggle for power, nor indeed grounds itself on an Aristotelian telos of human nature. Instead, basing itself on the “fact of oligarchy” that modern sociology has brought to light, it desires to evaluate the different regimes in existence to see which one is more legitimate, which one can be considered the best.54 This is precisely what Aron will do in the rest of Démocratie, comparing the Western European and American “Constitutional-Pluralist” regimes to the Eastern “Party Monopolistic” regime of the USSR, coming down heavily in favor of the former. But to get a better sense of the make-up of these regimes, we must return to Aron’s sociological writings of the 1950s and 1960s, where he developed his theory of the “divided” and “unified” elite. In three fundamental sociological texts of the 1950s and 1960s – “Structure sociale et structure de l’élite” (1950), “Classe sociale, classe politique, classe 16 dirigeante” (1960) and “Catégories dirigeantes ou classe dirigeante?” (1965) – Aron fleshed out his theory of elite rule. 55 Raymond Aron, “Structure sociale et structure de l’élite,” “Classe sociale, classe politique, classe dirigeante” and “Catégorie dirigeantes ou classe dirigeante? ” in Aron, Études Sociologiques, 111, 123 141, 143, 188, 191. 56 61 Aron, “Classe sociale,” 151, 154, 157 ; “Catégories dirigeantes,” 187, 193. 56 Raymond Aron, “Structure sociale,” 111, 142. This paper was first given at the LSE in 1949, and published in English as “Social Structure and the Ruling Class,” British Journal of Sociology 1, no. 1 (1950): 1-16 and no. 2, 126-143. On the Marx-Pareto “synthesis” see also Aron, Démocratie, 363 and Raymond Aron, Mémoires: 50 ans de réflexion politique (Paris: Julliard, 1983), 34, 392-8. 57 Aron “Classe sociale ” 149 155 , , , 60 The Ruling Class is the English title given to Mosca’s main work. 61 , , , 58 “Il y a des gouvernements pour le people, il n’y a pas de gouvernements par le people.” Aron, “Structure sociale,” 121-2. , 59 Aron, “Classe sociale,” 149, 161. 60 59 Aron, “Classe sociale,” 149, 161. 60 y , f p q ( , ), , 57 Aron, “Classe sociale,” 149, 155. II: Divided and unified elites Building explicitly on Pareto, Mosca and Michels, and read again through Burnham,55 Aron presented what he terms a “synthesis” of Marx and Pareto.56 It is in the 1960 text, “Classe sociale, classe politique, classe dirigeante,” that the notion of the “fact of oligarchy” – alongside Michels, its originator – first appears,57 although Aron had already theorized the idea that if one can talk of democracy as government “for” the people, it would be a mistake, because of the fact that it is always the few who rule, to talk of government “by” the people.58 The theme of Paretian “cynicism” returns here too, with Aron admitting that one could read – as he had done in the past – these Machiavellian thinkers as being, in their rejection of socialism, proto-Fascists.59 The main notion Aron will develop over the course of these writings is the view that societies are determined by the relation between what he calls either the classes or catégories dirigeantes – what in English we might term the political classes (the plural is key)60 – and the classe or personnel politique, namely the more directly political class or politicians.61 This is an anti-Marxist point: what Aron is saying is that it is not the relation between social classes (capitalists v the proletariat) that determines the political superstructure, as Marx would have it, but rather it is the relation the different social, economic, bureaucratic elites – the “ruling classes” – 17 entertain with politicians that defines the regime.62 By “ruling classes” Aron gives trade union leaders, captains of industry, the high civil service, judiciary, military as examples, namely leaders of the different spheres that make up society (masses, money, bureaucracy, military).63 In elaborating this theory of elite rule, Aron builds on each of the earlier Machiavellian thinkers. From Michels he borrows the “iron law of oligarchy,” but transforms it into a “fact” that itself needs to be evaluated, and from which other sociological questions – how is this oligarchy formed? who is it in and how are they recruited? – emanate. From Pareto he takes the notion of “elite,” namely those who are the leaders in their respective fields, and uses Mosca’s term of the “ruling class” to designate them. 65 “La différence fondamentale entre une société de type soviétique et une société de type occidental, c’est que la première a une élite unifiée et la seconde une élite divisée.” Aron, “Structure sociale,” 123 , , 65 “La différence fondamentale entre une société de type soviétique et une société de type occidental, c’est que la première a une élite unifiée et la seconde une élite divisée ” Aron “Structure sociale ” 123 Aron, Catégories dirigeantes, 193 4. 64 Aron, “Classe sociale,” 151. 65 Aron, Classe sociale, 151. 5 “La différence fondamentale entre une société de type soviétique et une société de type occidental, 62 Aron, “Classe sociale,” 157. 63 62 Aron, “Classe sociale,” 157. 63 64 Aron, “Classe sociale,” 151. 65 La différence fondamentale entre une société de type soviétique et une société de type occidental, c’est que la première a une élite unifiée et la seconde une élite divisée.” Aron, “Structure sociale,” 123. 63 Aron, “Catégories dirigeantes,” 193-4. 64 2 Aron, “Classe sociale,” 157. 3 Aron “Catégories dirigeantes ” 193 4 64 Aron, “Classe sociale,” 151. 6 II: Divided and unified elites But on the basis that there is not one ruling class, but in fact as many as there are spheres without which governing would be impossible – the economy, workforce, military64 – he turns Mosca’s term into a plural – ruling classes – and comes up with a new term, the political personnel, to designate politicians in the strict sense of the word. Aron articulates the relation the ruling classes entertain with the political personnel through the notion of a “divided” or “unified” elite, namely whether political, economic, social, military or legal elites find themselves within the same institution, for example a unified political party, or whether they are divided within themselves, that they have their own, independent, institutions that are in competition with one another.65 The question for Aron is whether all the political, economic, social etc. decisions will be taken by the same people, at the same time, and within the same institutions, or whether these decisions will be taken by different people, at 18 different – and often conflicting – times and going in conflicting directions, in different settings. That is, for Aron, the difference between a divided and unified elite, and the regime will be determined by how the relation between the different elites is organized constitutionally. Aron, however, is not of the belief that a unified elite will mean conflict will disappear. Quite the contrary: conflict is inescapable; it is part of the genetic make-up of society. And because all the interests are centralized in a common institution, it will manifest itself through extra-institutional and extra- constitutional ways, most probably through violence – already we see here how Aron will favor a divided over a unified elite.66 Indeed, Aron will engage in these writings in a fruitful debate with C. Wright Mills’s recently published The Power Elite (1956), which posits the existence of a united elite, one that takes all its decisions in common and for its own benefit, in cooperation and not in competition with itself, which was the opposing theory Robert Dahl developed in his answer to Mills in Who Governs? Aron, Structure sociale, 139. 67 Raymond Aron, “Macht, Power, Puissance: prose démocratique ou poésie démonique?” in Aron, Études Politiques, 171-194. , g g , 70 Reed Davis, “The Phenomenology of Raymond Aron,” European Journal of Political Theory 2, no. 4 (2003): 401–413. Études Politiques, 171 194. 68 Aron, “Classe sociale,” 151, 156, 162; “Catégorie dirigeantes,” 191, 200. 69 A “C té i di i t ” 201 66 Aron, “Structure sociale,” 139. 67 , , , , 69 Aron, “Catégories dirigeantes,” 201. 0 66 Aron, “Structure sociale,” 139. 67 , , , , ; g g , , 69 Aron, “Catégories dirigeantes,” 201. 69 Aron, “Catégories dirigeantes,” 201. 70 72 Aron, “Structure social,” 124-5. 73 , , 76 See also Raymond Aron, La lutte des classes in Aron, Penser la liberté, penser la démocratie, 1088- 1098. 71 Aron, “Classe sociale,” 165. 72 73 Aron, “Classe sociale,” 162. 74 75 Aron, “Classe sociale,” 155. 73 Aron, “Classe sociale,” 162. 74 A “S i l ” 142 75 Aron, “Classe sociale,” 155. 76 See also Raymond Aron, La lutte des classes in Aron, Penser la liberté, penser la démocratie, 1088- Aron, Classe sociale, 162. 74 Aron, “Structure social,” 142. 75 Aron, “Classe sociale,” 155. , , 72 Aron, “Structure social,” 124-5. 73 A “Cl i l ” 162 74 Aron, “Structure social,” 142. 75 Aron, “Classe sociale,” 155. 76 S l R d A L l tt d l i A P l lib té l dé ti 1088 II: Divided and unified elites (1961).67 Based on his view that conflict will always manifest itself, Aron will reject Mills’s thesis as conspiracy theorizing – he explains that he is not convinced the examples Mills provides are clearly of collusion.68 Instead, he posits that reality is to be found somewhere between the two extremes – Mills’s power elite and Dahl’s polyarchy – between pure collusion and pure competition.69 A ruling class will never be purely unified or purely divided – those are Kantian/Weberian, two thinkers who strongly influenced Aron,70 “ideal-types” – but elites will be more or less divided. 19 Whilst Aron can see how a unified elite might be more efficient in its rule,71 his own preference is division. He explains that when a unified elite concentrates within its grasp all political, economic and social power, then the masses find themselves defenseless against them.72 He expresses a preference for dialogue between the rulers and the ruled that is constitutionally organized – like conflict, dialogue between the two always happens, but when formally organized means bloodshed can be avoided.73 In the end checks and balances are still for Aron the best guarantor of liberty,74 and, like all good liberals, he offers an romanticized version of the English “Establishment” as his ideal ruling class: one situated between the two extremes of unity and division, which, although it has a ruling class, is one open to talent and is willing to assimilate within it the leaders of those who oppose it.75 In Démocratie et totalitarisme, Aron will use his notions of a “unified” and “divided” elite to analyze the political systems of the East and the West,76 classifying the former as a “Party Monopolistic” regime, one where the totality of the ruling classes are concentrated in the Party, and the latter as a “Constitutional-Pluralist” regime, which allows for structured competition between different political parties, and where the ruling classes are divided – the emphasis on political party here comes from Michels, who concentrated his “iron law of oligarchy” in his study of modern, highly centralized and hierarchical, political parties. 83 Aron, Introduction à la philosophie politique, 56. 77 “Du caractère oligarchique de régimes constitutionnels-pluralistes.” Aron, Démocratie, 128-132. 78 , , 80 “l’équilibre conflictuel des forces sociales.” Audier, La démocratie conflictuelle, 46. Aron, Introduction à la philosophie politique, 135-6. p p p q , 81 Aron, Introduction à la philosophie politique, 135. 82 78 Aron, Démocratie, 128-132, 149. Interestingly Aron, himself Jewish, does not mention anti-Semitic conspiracy theories. 79 “le régime constitutionnel-pluraliste est celui qui donne le maximum de garanties aux gouvernés.” Aron Démocratie 134 5 82 Aron, Démocratie, 149. conspiracy theories. 79 “le régime constitutionnel-pluraliste est celui qui donne le maximum de garanties aux gouvernés.” Aron, Démocratie, 134-5. 78 Aron, Démocratie, 128-132, 149. Interestingly Aron, himself Jewish, does not mention anti-Semitic conspiracy theories. 79 “l é i tit ti l l li t t l i i d l i d ti é ” Du caractère oligarchique de régimes constitutionnels-pluralistes. Aron, Démocratie, 128-132. 78 Aron, Démocratie, 128-132, 149. Interestingly Aron, himself Jewish, does not mention anti-Semitic conspiracy theories. 79 “le régime constitutionnel-pluraliste est celui qui donne le maximum de garanties aux gouvernés.” Aron, Démocratie, 134-5. 80 “l’équilibre conflictuel des forces sociales.” Audier, La démocratie conflictuelle, 46. Aron, Introduction à la philosophie politique 135 6 p p p q 81 Aron, Introduction à la philosophie politique, 135. 82 87 Raymond Aron, “La rationalité politique,” Commentaire 156 (2016): 725-42. 88 II: Divided and unified elites The Machiavellians – Pareto, Mosca, Michels, Burnham – are again at the center of his reflections; indeed one of his chapters on the Western “Constitutional-Pluralist” regime is entitled “The 20 oligarchic character of the Constitutional-Pluralist regimes.”77 And the ideas developed in his sociological studies provide the bedrock upon which Aron constructs his own democratic theory: Mosca’s political personnel, the “fact” of oligarchy and the further political questions its raises, government for rather than by the people, even ruling class conspiracies surrounding Jesuits, Free-Masons or petrol companies make an appearance.78 His conclusions are the same too: he attributes directly to Mosca the thought that a divided “Constitutional-Pluralist” regime provides the “best guaranties for the governed.”79 As he explains in his Introduction à la philosophie politique lectures, if human nature, as the Machiavellians had pointed out, should be understood pessimistically, then democracy is the least worst regime because it legally regulates competition between groups, leading to what Audier terms the “conflictual balance of social forces:”80 if one is looking for a “realistic” regime, then democracy, being the best of the worst regimes, is actually the best regime possible.81 Yet keeping to his idea that extremes are to be avoided, if Aron had expressed fears about a too unified elite, he also in Démocratie expresses concerns about a too divided elite, one which would be too dispersed, unstable and inefficient to be able to rule in an effective manner.82 Democracies have to find the right balance and not fall into demagogy.83 His conclusions are the same too: he attributes directly to Mosca the thought that a divided “Constitutional-Pluralist” regime provides the “best guaranties for the governed.”79 As he explains in his Introduction à la philosophie politique lectures, if human nature, as the Machiavellians had pointed out, should be understood pessimistically, then democracy is the least worst regime because it legally regulates competition between groups, leading to what Audier terms the “conflictual balance of social forces:”80 if one is looking for a “realistic” regime, then democracy, being the best of the worst regimes, is actually the best regime possible.81 Yet keeping to his idea that extremes are to be avoided, if Aron had expressed fears about a too unified elite, he also in Démocratie expresses concerns about a too divided elite, one which would be too dispersed, unstable and inefficient to be able to rule in an effective manner.82 Democracies have to find the right balance and not fall into demagogy.83 21 84 Chapters 9-11 and 18-19 in Aron, Démocratie, 166-219, 337-70. 85 86 Aron, Démocratie et totalitarisme, 9-19. 87 y 88 Aron, Mémoires. 86 Aron, Démocratie et totalitarisme, 9-19. 87 88 Aron, Mémoires. Pocock, The Machiavellian Moment, xxiv. 86 Aron, Démocratie et totalitarisme, 9-19. p , ocock, The Machiavellian Moment, xxiv. 84 Chapters 9-11 and 18-19 in Aron, Démocratie, 166-219, 337-70. 85 87 Raymond Aron, “La rationalité politique,” Commentaire 156 (2016): 725-42. 88 Aron, Mémoires. III: The “Machiavellian Moment” and the Centre Raymond Aron The emphasis on corruption and the imperfection of the political regime in Démocratie84 echo some of the themes of the Florentine “Machiavellian Moment” Pocock first theorized in 1975. For Pocock that moment – which he would subsequently extend to seventeenth century England and the work of James Harrington, and to eighteenth century American debates over virtue and commerce – was marked by a dual reflection entertained by the original “Machiavellians” (Machiavelli, Savonarola, Guicciardini, Giannotti): the problem of elaborating a non- transcendental account of the passage of time, married to confronting the temporal finitude of the republic; what Machiavelli attempted to address through his notions of “virtue” and “fortune.”85 It is certainly the case that with his wartime writings on the “Machiavellian” threat of totalitarianism to Western liberal-democratic regimes, and his studies of the inevitable corruption of the “Constitutional-Pluralist” regime, Aron, who also lived through de Gaulle’s forceful passage from the Fourth to the Fifth Republic, which he attempted to account for in the introduction to Démocratie,86 mirrored the concerns of his “Machiavellian” predecessors. In large part due to his lifelong engagement with Weber’s work on political rationality,87 Aron thought long and hard about how to formulate a secular account of time, notably through his work on the philosophy of history that had been the subject of his PhD, and indeed he is still celebrated today as the figure who exercised the best political judgement during these turbulent years.88 22 This has led Audier to posit a post-War “moment machiavélien francais,” encompassing Aron, the phenomenologist Maurice Merleau-Ponty and the political philosopher Claude Lefort.89 There is little doubt that “Machiavellianism” served as an important conduit for these authors’s thinking about contemporary politics, but as Audier has correctly pointed out that thinking was quite removed from the “Neo- Republicanism” – with its focus on non-domination – that has characterized the subsequent work of Skinner and Pettit. Instead, the French Machiavellians’s thinking was marked by “conflictual pluralism,”90 and in Aron’s case that was articulated less through Machiavelli than his own “Machiavellians:” Pareto, Mosca and Michels. Audier, Machiavel, 28. 91 Cf. John McCormick, Machiavellian Democracy (Cambridge: Cambridge University Press, 2011). 92 p y 93 Emile Chabal, A Divided Republic: Nation, State and Citizenship in Contemporary Franc (Cambridge: Cambridge University Press, 2015), 135-157. , y ( g g y , ) 92 Serge Audier, “A Machiavellian Conception of Democracy? Democracy and Conflict” in Colen and Dutartre-Michaut ed., The Companion to Raymond Aron, 155. 93 , f 90 Audier, Machiavel, 28. 91 Cf John McCormick Machiavellian Democracy (Cambridge: Cambridge University Press 2011) , , f 90 Audier, Machiavel, 28. 91 89 Audier, Machiavel, conflit et liberté. 90 89 Audier, Machiavel, conflit et liberté. 90 udier, Machiavel, 28. f. John McCormick, Machiavellian Democracy (Cambridge: Cambridge University Press, 2011). III: The “Machiavellian Moment” and the Centre Raymond Aron For Aron liberty emerges from within the space in which different parties, interests and groups compete,91 and this thus ties his conception of liberty closely to Mosca’s theory of “legal defense,” namely the constitutional structure set-up to organize institutionally and channel productively the antagonism between different social forces.92 Moreover – and although the name of the French Republic might lead to some confusion – what Aron was ultimately concerned with was the survival of western liberal-democracy, and not ‘republics’ as such.93 This has led Audier to posit a post-War “moment machiavélien francais,” The French Machiavellian moment did not end with Aron, however, nor indeed with Merleau-Ponty and Lefort: a strong case can be made for the inclusion of the Centre Raymond Aron itself, of which Lefort was a member. Originally launched as an informal groupe de reflexion in 1977 by François Furet, the seminar in political philosophy at the École des Hautes Études en Sciences Sociales formally morphed into the Institut Raymond Aron in 1984, before becoming the Centre de recherches 23 politiques Raymond Aron in 1992, and transforming itself yet again in 2009 into its present incarnation, the CESPRA (Centre d'Etudes Sociologiques et Politiques Raymond Aron). At its peak the Centre brought together many of the leading French political thinkers – Aron, Furet and Lefort, of course, but also Pierre Manent, Marcel Gauchet, Pierre Rosanvallon and Bernard Manin – and is known for having renewed the study of democratic theory and having been at the forefront of the rediscovery of the French liberal tradition, notably through studies of Condorcet, Constant, Guizot and Tocqueville.94 politiques Raymond Aron in 1992, and transforming itself yet again in 2009 into its present incarnation, the CESPRA (Centre d'Etudes Sociologiques et Politiques Raymond Aron). At its peak the Centre brought together many of the leading French political thinkers – Aron, Furet and Lefort, of course, but also Pierre Manent, Marcel Gauchet, Pierre Rosanvallon and Bernard Manin – and is known for having renewed the study of democratic theory and having been at the forefront of the rediscovery of the French liberal tradition, notably through studies of Condorcet, Constant, Guizot and Tocqueville.94 The French Machiavellian theme of “conflictual pluralism” – mediated through Aron’s own reflections drawn from Mosca, Pareto and Michels – are central to many of the Centre’s members work. 94 Hugo Drochon, “Democracy, Anti-Totalitarianism and Liberalism,” Politics, Religion and Ideology 18, no. 3 (2017): 333-336. , f p p p q ( , ), 97 Manent, Cours familier, 29-31. See Aron, Démocratie et totalitarisme, 348 for “l’organisation de l compétition.” , ( ) 95 Daniel Steinmetz-Jenkins, “Why did Raymond Aron write that Carl Schmitt was not a Nazi? An alternative genealogy of French liberalism ” Modern Intellectual History 11 no 4 (2014): 572 ( ) 5 Daniel Steinmetz-Jenkins, “Why did Raymond Aron write that Carl Schmitt was not a Nazi? An alternative genealogy of French liberalism,” Modern Intellectual History 11, no. 4 (2014): 572. 6 Pierre Manent Cours familier de philosophie politique (Paris: Gallimard 2001) 24-5 p 98 Manent, Cours familier, 28. Daniel Steinmetz Jenkins, Why did Raymond Aron write that Carl Schmitt was not a Nazi? An alternative genealogy of French liberalism,” Modern Intellectual History 11, no. 4 (2014): 572. 96 Pierre Manent C f ili d hil hi liti (Paris: Gallimard 2001) 24 5 , y y alternative genealogy of French liberalism,” Modern Intellectual History 11, no. 4 (2014): 5 96 y y Historiography,” The Journal of Modern History 76, no. 1 (2004): 142-3. Critical and Democratic Theory 23, no. 4 (2016): 553-4; Andrew Jainchill and Samuel Moyn, “French Democracy between Totalitarianism and Solidarity: Pierre Rosanvallon and Revisionist , p , 103 Gregory Conti and William Selinger, “The Other Side of Representation: The History and Theory of Representative Government in Pierre Rosanvallon,” Constellations: An International Journal of Critical and Democratic Theory 23, no. 4 (2016): 553-4; Andrew Jainchill and Samuel Moyn, “French Democracy between Totalitarianism and Solidarity: Pierre Rosanvallon and Revisionist 99 Bernard Manin, Principes du gouvernement représentatif (Paris: Flammarion, 2012), 189-190. 100 M i P i i 265 7 101 Manin, Principes, 207-8. 102 Manin, Principes, 306 8. 103 Gregory Conti and William Selinger, “The Other Side of Representation: The History and Theory of Representative Government in Pierre Rosanvallon,” Constellations: An International Journal of Critical and Democratic Theory 23 no 4 (2016): 553 4; Andrew Jainchill and Samuel Moyn “French 100 Manin, Principes, 265-7. of Representative Government in Pierre Rosanvallon, Constellations: An International Journal of Critical and Democratic Theory 23, no. 4 (2016): 553-4; Andrew Jainchill and Samuel Moyn, “French Democracy between Totalitarianism and Solidarity: Pierre Rosanvallon and Revisionist p 102 Manin, Principes, 306-8. Manin, Principes, 207-8. 102 Manin, Principes, 306-8. 9 Bernard Manin, Principes du gouvernement représentatif (Paris: Flammarion, 2012), 189-190. 00 Manin, Principes, 265-7. 01 Manin, Principes, 265-7. 101 Manin, Principes, 207-8. 102 Historiography,” The Journal of Modern History 76, no. 1 (2004): 142-3. , p , 102 Manin, Principes, 306-8. 103 p 101 Manin, Principes, 207-8. Bernard Manin, Principes du gouvernement représentatif (Paris: Flammarion, 2012), 189 190. 100 Manin, Principes, 265-7. 101 III: The “Machiavellian Moment” and the Centre Raymond Aron Pierre Manent, for instance, one of Aron’s inheritors,95 completely grants in his Cours familier de philosophie politique (2001) that political sociology has demonstrated the undeniable oligarchic nature of modern democracies, within which political parties play an important role: all rather reminiscent of Michels.96 And the definition Manent will give of democracy is the ‘organisation of separations’, that modern politics is organised around two oppositions: between represented/representatives, and the more classic ‘separation of powers’, namely a divided elite.97 It is within these two oppositions – the conflict between the elite and the masses, and within the elites themselves – that modern liberty is to be found: Aron’s point all along.98 24 Bernard Manin’s classic Principes du gouvernement représentatif (1995) accepts the “oligarchic” or “elitist” nature of elections, which he readily attributes to Pareto.99 And he also affirms Michels’s critique of the oligarchic nature of modern mass parties, which bring about new elites cut-off from the general party membership.100 Manin’s argument, of course, was in part intended as a refutation of the elite theorists of democracy, in particular Schumpeter:101 whilst modern democracy contains within it patently aristocratic elements, notably elections, it also contains democratic elements too – it is a ‘mixed’ regime – in the sense that elections are open to all.102 It is the conflict between those two principles that determines the nature of our modern representative regimes. The French Machiavellian themes, and their authors, are also highly present in the work of another prominent member of the Centre Aron, and now professor at the Collège de France, Pierre Rosanvallon. III: The “Machiavellian Moment” and the Centre Raymond Aron Rosanvallon has used these themes and authors to address what has been the guiding thread of his own reflections, namely that of the “crisis of representation.” That crisis is the by-product of the decline of the political party, which at its apex at the turn of the twentieth century offered a synthesis between the anciens corps intermédiaires and modern forms of individualism and singularity.103 Embedded within a pluralistic institutional framework, the political party, allied to rise of syndicalism, provided the stability to the Third Republic within which Rosanvallon thought he had found the synthesis of Lefort’s understanding of democracy as conflict and Furet’s quest to end the French 25 Revolution.104 As the new intermediary body, the political party had momentarily resolved, at the end of the nineteenth century, the conflicting legacy of the French Revolution – liberté et égalité – thus ensuring the stability of the regime. That the political party should be so central to Rosanvallon’s thinking means that Michels and Ostrogorski – whose legacy includes all the political terminology surrounding “party machine,” “party boss,” “omnibus party” and “Single-Issue” parties, and whose emphasis, much like Michels, was on the modern centralized, hierarchical and highly bureaucratized political party – feature strongly and consistently throughout Rosanvallon’s work. Indeed, whilst he was still an auto- gestionnaire syndicalist in the late 1970s, he was writing of the dangers of That the political party should be so central to Rosanvallon’s thinking means that Michels and Ostrogorski – whose legacy includes all the political terminology surrounding “party machine,” “party boss,” “omnibus party” and “Single-Issue” parties, and whose emphasis, much like Michels, was on the modern centralized, hierarchical and highly bureaucratized political party – feature strongly and consistently throughout Rosanvallon’s work. Indeed, whilst he was still an auto- gestionnaire syndicalist in the late 1970s, he was writing of the dangers of centralization facing trade unionism that Michels and Ostrogorski had identified. In a series of texts – “Avancer avec Michels” (1977), “Trois textes pour un débat” (1978) and “Connaissez-vous Ostrogorski?” (1979) – in the syndicalist journal Faire he was editing, Rosanvallon affirmed the existence of an “iron law of oligarchy,” but argued that this was a present political problem that needed to be resolved, presumably through his decentralized and self-organizing auto-gestionnaire movement, rather than a past historical preoccupation.105 centralization facing trade unionism that Michels and Ostrogorski had identified. 104 Conti and Selinger, “The Other Side of Representation,” 552. 105 g p 105 “Avancer avec Michels, c’est considérer la difficulté démocratique comme un problème politique et non pas comme un problème historique.” Pierre Rosanvallon, “Avancer avec Michels,” Faire 17 (1977): 31-34; “Trois textes pour un débat,” Faire 35 (1978) : 55-57; and “Connaissez-vous Ostrogorski?”, Faire 50 (1979): 23-26. g , p , 105 “Avancer avec Michels, c’est considérer la difficulté démocratique comme un problème politique et non pas comme un problème historique.” Pierre Rosanvallon, “Avancer avec Michels,” Faire 17 (1977): 31 34; “Trois textes pour un débat ” Faire 35 (1978) : 55 57; and “Connaissez vous 04 Conti and Selinger, “The Other Side of Representation,” 552. 05 05 “Avancer avec Michels, c’est considérer la difficulté démocratique comme un problème politique et non pas comme un problème historique.” Pierre Rosanvallon, “Avancer avec Michels,” Faire 17 1977): 31-34; “Trois textes pour un débat,” Faire 35 (1978) : 55-57; and “Connaissez-vous (1977): 31-34; Trois textes pour un débat, Faire 35 (1978) : 55-57; and Connaissez-vous Ostrogorski?”, Faire 50 (1979): 23-26. ( ) ; p Ostrogorski?”, Faire 50 (1979): 23-26. 104 Conti and Selinger, “The Other Side of Representation,” 552. III: The “Machiavellian Moment” and the Centre Raymond Aron In a series of texts – “Avancer avec Michels” (1977), “Trois textes pour un débat” (1978) and “Connaissez-vous Ostrogorski?” (1979) – in the syndicalist journal Faire he was editing, Rosanvallon affirmed the existence of an “iron law of oligarchy,” but argued that this was a present political problem that needed to be resolved, presumably through his decentralized and self-organizing auto-gestionnaire movement, rather than a past historical preoccupation.105 His engagement with Michels and Ostrogorski would not simply survive his transition into academia – and this transition was mediated, as Rosanvallon recognized in an interview with the Journal of the History of Ideas, through his encounter with Lefort, whose Machiavel resonated with the “realist” sociologists 26 Michels and Ostrogorski he was interested in106 – but offered a bedrock upon which much of his subsequent reflection built: he would write an introduction to an abridged edition of Ostrogorski’s La démocratie et les partis politiques in 1979 – the sections he would pick out himself; preface Paolo Pombeni’s translation into French of his Introduction à l’histoire des partis politiques (1992); Michels, Ostrogorski and indeed Pareto would play a key role in his historical trilogy Le sacre du citoyen (1992), Le peuple introuvable (1998), La démocratie inachevée (2000); whilst writing the entry on political parties for Raynaud and Rials’s Dictionnaire de philosophie politique (1996); and would be one of the great traditions he would discuss in his inaugural lecture to the Collège de France in 2002.107 Michels and Ostrogorski he was interested in106 – but offered a bedrock upon which much of his subsequent reflection built: he would write an introduction to an abridged edition of Ostrogorski’s La démocratie et les partis politiques in 1979 – the sections he would pick out himself; preface Paolo Pombeni’s translation into French of his Michels and Ostrogorski he was interested in106 – but offered a bedrock upon which much of his subsequent reflection built: he would write an introduction to an abridged edition of Ostrogorski’s La démocratie et les partis politiques in 1979 – the sections he would pick out himself; preface Paolo Pombeni’s translation into French of his Introduction à l’histoire des partis politiques (1992); Michels, Ostrogorski and indeed Pareto would play a key role in his historical trilogy Le sacre du citoyen (1992), Le peuple introuvable (1998), La démocratie inachevée (2000); whilst writing the entry on political parties for Raynaud and Rials’s Dictionnaire de philosophie politique (1996); and would be one of the great traditions he would discuss in his inaugural lecture to the Collège de France in 2002.107 Michels and Ostrogorski he was interested in106 – but offered a bedrock upon which much of his subsequent reflection built: he would write an introduction to an abridged edition of Ostrogorski’s La démocratie et les partis politiques in 1979 – the sections he would pick out himself; preface Paolo Pombeni’s translation into French of his Introduction à l’histoire des partis politiques (1992); Michels, Ostrogorski and indeed Pareto would play a key role in his historical trilogy Le sacre du citoyen (1992), Le peuple introuvable (1998), La démocratie inachevée (2000); whilst writing the entry on political parties for Raynaud and Rials’s Dictionnaire de philosophie politique (1996); and would be one of the great traditions he would discuss in his inaugural lecture to the Collège de France in 2002.107 In fact one can read one of his latest projects, Le Parlement des invisibles (2014), as again premised on Michels and Ostrogorski: the idea of Raconter la vie was to offer those who were “mal-représentés” the opportunity to explain their existence. ( y , ), 108 “Il y a là comme une sorte de loi d’airain des organisations en général, et de la vie politique en particulier.” Pierre Rosanvallon, Le parlement des invisibles, (Paris: Seuil, 2014), 14. Gallimard, 2000), 30, 263 4, 293, 401; Pierre Rosanvallon, Partis in Philippe Raynaud and Stéphane Rials, Dictionnaire de philosophie politique (Paris: PUF, 1996), 525-9; Pierre Rosanvallon, “Inaugural Lecture, Collège de France” in Samuel Moyn ed, Pierre Rosanvallon: Democracy Past and Future New York: Columbia University Press, 2006), 41-2. , f y f , ( ) 107 Moisie Ostrogorski, La démocratie et les partis politiques (Paris: Seuil, 1979), 7-21; Paolo Pombeni, Introduction à l’histoire des parties politiques (Paris: PUF, 1992), ix-xvi; Pierre Rosanvallon, Le sacre du citoyen (Paris: Gallimard, 1992), 497; Pierre Rosanvallon, Le peuple 106 Javier Fernández Sebastián and Pierre Rosanvallon, “Intellectual History and Democracy: An Interview with Pierre Rosanvallon,” Journal of the History of Ideas 68, no. 4 (2007): 703-715. 106 Javier Fernández Sebastián and Pierre Rosanvallon, “Intellectual History and Democracy: An Interview with Pierre Rosanvallon ” Journal of the History of Ideas 68 no 4 (2007): 703 715 introuvable (Paris: Gallimard, 1998), 247, 290; Pierre Rosanvallon, La démocratie inachevée (Paris: Gallimard, 2000), 30, 263-4, 293, 401; Pierre Rosanvallon, “Partis” in Philippe Raynaud and Stéphane p 111 Aron, Introduction à la philosophie politique, 56; Aron, Démocratie, 130. g , p , 110 Rosanvallon, Le parlement des invisibles, 23. 111 III: The “Machiavellian Moment” and the Centre Raymond Aron This lack of representation, especially for the new working class – the “invisibles” – comes from the professionalisation of political parties and the “iron law of oligarchy” that eats away at political life (Rosanvallon’s main target is the French Socialist Party).108 As Conti and Selinger have pointed out, Rosanvallon has difficulty articulating how the “crisis of representation” he so adroitly documents might be addressed, notably because he has refused to undertake the type of political sociology Michels and Ostrogorski – and Aron in their wake – practiced, which gave them a 27 basis upon which to ground their proposals.109 It is true that in its previous incarnation Raconter la vie took more the form of a literary “representation-narrative” than an in- depth sociological study,110 but in 2016 the Confédération Française Démocratique du Travail, Rosanvallon’s old trade union, conducted a detailed sociological study of 200 000 of its members, and that project, entitled Parlons Travail, has now been merged with Rosanvallon’s original Raconter la vie to create Raconter le travail, which might bring some much needed sociology to Rosanvallon’s historical and political work. 109 Conti and Selinger, “The Other Side of Representation,” 556-8. 110 109 Conti and Selinger, “The Other Side of Representation,” 556-8. 110 Conclusion Drawing directly from Pareto’s critique of 1920s Italy, which he characterized as a “demagogic plutocracy”, Aron applied his own theory of the key relation between the “personnel politique” and the “catégories dirigeantes” to his contemporary France. And his conclusions were much the same: behind the façade of democratic politics, where rhetoricians dominate, lurk the rich financiers, because much money is needed to win elections and to govern.111 It was thus the rich, the financiers, the industrialists, businessmen and entrepreneurs who dominate modern democracies. Aron was writing in the 1960s, but, with the political system awash with money, there is no reason to think that things have changed drastically since. Indeed, with the Occupy Movement and their rallying cry of the 1%, the election of Trump and Brexit, the relation elites entertain with democracy has been forcefully brought back onto the political agenda. Are these elites divided or unified? What are the constitutional structures within 28 which they operate? How do they recruit their members? What is their relation to the non-elite? These are all the questions Aron asked, and they are as urgent now as they were then. which they operate? How do they recruit their members? What is their relation to the non-elite? These are all the questions Aron asked, and they are as urgent now as they were then.
https://openalex.org/W2797926700	https://www.repository.cam.ac.uk/bitstream/1810/276024/4/s41467-018-03672-4.pdf	English	null	Identification of rare sequence variation underlying heritable pulmonary arterial hypertension	Nature communications	2,018	cc-by	17,618	ARTICLE ARTICLE Results I diopathic and heritable pulmonary arterial hypertension (PAH) are rare disorders characterised by occlusion of arterioles in the lung1, leading to marked increases in pulmonary vascular resistance2. Life expectancy from diagnosis averages 3–5 years3, with death ensuing from failure of the right ventricle. Description of the PAH cohort. In total, 1048 PAH cases (1038 index cases and 10 related affected individuals) were recruited for WGS. Of these, 908 (86.7%) were diagnosed with idiopathic PAH, 58 (5.5%) gave a family history of PAH and 60 (5.7%) gave a history of drug exposure associated with PAH16. Twenty two cases (2.1%) held a clinical diagnosis of PVOD/PCH (Fig. 1a). Demographic and clinical characteristics of the PAH cohort are provided in Supplementary Table 1. An additional UK family was recruited separately for novel gene identiﬁcation studies. Brieﬂy, the proband was diagnosed at 12 years with a persistent ductus arteriosus and elevated pulmonary arterial pressure. Explant lung histology following heart-lung transplantation revealed the pre- sence of plexiform lesions. Two of the proband’s offspring were also diagnosed with childhood-onset PAH, one of which had an atrial septal defect. The proband’s parents, siblings and a third child showed no evidence of cardiovascular disease. Mutations in the gene encoding the bone morphogenetic protein type 2 receptor (BMPR2), a receptor for the transforming growth factor-beta (TGF-β) superfamily4,5 account for over 80% of families with PAH, and approximately 20% of sporadic cases6. Mutations have been identiﬁed in genes encoding other compo- nents of the TGF-β/bone morphogenetic protein (BMP) signal- ling pathways, including ACVRL17 and ENG8. On endothelial cells, BMPR2 and ACVRL1 form a signalling complex, utilising ENG as a co-receptor. Case reports of rare sequence variation in the BMP signalling intermediaries, SMAD1, SMAD4 and SMAD99,10, provide compelling evidence for a central role of dysregulated BMP signalling in PAH pathogenesis. Analysis of coding variation in BMPR2-negative kindreds revealed heterozygous mutations in genes not directly impacting on the TGF-β/BMP pathway, including CAV111, and the potassium channel, KCNK312. Deletions and loss of function mutations in TBX4, an essential regulator of embryonic development, were identiﬁed in childhood-onset PAH13. A clinically and pathologically distinct form of PAH, known as pulmonary veno-occlusive disease or pulmonary capillary haemangiomatosis (PVOD/PCH), was shown recently to be caused by biallelic recessive mutations in EIF2AK414,15, a kinase in the integrated stress response. Pathogenic variants in previously reported PAH disease genes. Results Our ﬁltering strategy detected rare deleterious variation in pre- viously reported PAH genes in 19.9% of the PAH cohort. For BMPR2, rare heterozygous mutations were identiﬁed in 160 of 1048 cases (15.3%). The frequency of BMPR2 mutations in familial PAH was 75.9%, in sporadic cases 12.2%, and 8.3% in anorexigen-exposed PAH cases. Forty-eight percent of BMPR2 mutations were reported previously17, and the remainder were newly identiﬁed in this study. Fourteen percent of BMPR2 mutations resulted in the deletion of larger protein-coding regions ranging from 5 kb to 3.8 Mb in size. Supplementary Data 1 provides the breakdown of BMPR2 SNVs and indels, and the larger deletions are shown in Fig. 2a–c with a detailed sum- mary in Supplementary Table 2. The purpose of the present study was to identify additional rare sequence variation contributing to the genetic architecture of PAH, and to assess the relative contribution of rare variants in genes implicated in prior studies. A major ﬁnding is that rare likely causal heterozygous variants in several previously uni- dentiﬁed genes (ATP13A3, AQP1 and SOX17) were signiﬁcantly overrepresented in the PAH cohort, and we provide independent validation for GDF2 as a causal gene. Of the other genes previously reported in PAH we identiﬁed deleterious heterozygous rare variants in ACVRL1 (9 cases, 0.9%), ENG (6 cases, 0.6%), SMAD9 (4 cases, 0.4%), KCNK3 (4 cases, 0.4%), and TBX4 (14 cases, 1.3%). We identiﬁed one case with 9066 subjects sequenced in NIHR BR-RD study 7979 non-PAH control subjects (6385 unrelated subjects) 1087 patients sequenced in PAH study 1048 PAH patients (1038 index cases) 22 PVOD/PCH patients 908 idiopathic PAH patients (with no family history of PAH) 58 PAH patients with family history of PAH 60 PAH patients with a history of exposure to drugs or toxins Excluded: 39 not idiopathic or heritable PAH or PVOD/PCH a Filtered variants CADD >= 15 & (SIFT ≠ tolerated & PolyPhen-2 ≠ benign) Protein truncating variants only Missense variants only Protein truncating and missense variants Protein truncating and missense variants Burden (Fisher’s) test (see figure 3a–c) SKAT-O (see figure 3d) Filtered variants MAF < 1 in 10,000 b Fig. 1 Flow diagrams illustrating a the composition of the NIHR BioResource—Rare Diseases (NIHR BR-RD) PAH study and b the analysis strategy to identify novel PAH disease genes. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 I ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 Identiﬁcation of rare sequence variation underlying heritable pulmonary arterial hypertension Stefan Gräf et al.# Pulmonary arterial hypertension (PAH) is a rare disorder with a poor prognosis. Deleterious variation within components of the transforming growth factor-β pathway, particularly the bone morphogenetic protein type 2 receptor (BMPR2), underlies most heritable forms of PAH. To identify the missing heritability we perform whole-genome sequencing in 1038 PAH index cases and 6385 PAH-negative control subjects. Case-control analyses reveal signiﬁcant overrepresentation of rare variants in ATP13A3, AQP1 and SOX17, and provide independent validation of a critical role for GDF2 in PAH. We demonstrate familial segregation of mutations in SOX17 and AQP1 with PAH. Mutations in GDF2, encoding a BMPR2 ligand, lead to reduced secretion from transfected cells. In addition, we identify pathogenic mutations in the majority of previously reported PAH genes, and provide evidence for further putative genes. Taken together these ﬁndings contribute new insights into the molecular basis of PAH and indicate unexplored pathways for therapeutic intervention. Correspondence and requests for materials should be addressed to S.Gäf. (email: sg550@cam.ac.uk) or to N.W.M. (email: nwm23@cam.ac.uk). #A full list of authors and their afﬁliations appears at the end of the paper. Correspondence and requests for materials should be addressed to S.Gäf. (email: sg550@cam.ac.uk) or to N.W.M. (em #A full list of authors and their afﬁliations appears at the end of the paper. \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications 1 NATURE COMMUNICATIONS\| (2018) 9:1416 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 Genes are indicated in orange and labelled with respective gene symbol. Deletions are drawn as blue boxes above the genome axis (grey) showing the genomic position on chromosome 2. The gre highlights the location of BMPR2. b Locus zoom on BMPR2 highlighting the focal deletions affecting one or more exons. c WGS coverage proﬁles selected set of smaller and larger deletions, visualised with the Integrative Genomics Viewer (IGV)57, with deletions highlighted by red bars. d an Manhattan plots of the genome-wide case-control comparison of large deletions. In d, all subject are considered. In e, subject with larger deletions aff the BMPR2 locus are excluded. The adjusted P value threshold of 5 × 10−8 for genome-wide signiﬁcance is indicated by the red line 3 mb 205 mb 58 ABI2 CTLA4 FAM117B ICOS CD28 BMPR2 ICA1L 1 CARF PARD3B CYP20A1 NBEAL1 RAPH1 WDR12 .2 b Deletions 203.25 mb 203.3 mb 203.35 mb 203.4 mb Genes BMPR2 c BMPR2 p25.2 p24.3 p23.3 p22.3 p21 p16.2 p15 p13.3 p12 p11.2 q11.1 q12.1 q13 q14.2 q21.1 254 kb 203,300 kb 203,400 kb q22.1 q23.1 q24.1 q24.3 q31.2 q32.2 q33.1 q34 q35 q36.2 q37.2 [0–60] [0–60] [0–60] [0–60] [0–60] [0–60] [0–60] [0–60] [0–60] 2 b b Deletions 203.25 mb 203.3 mb 203.35 mb 203.4 mb Genes BMPR2 c BMPR2 p25.2 p24.3 p23.3 p22.3 p21 p16.2 p15 p13.3 p12 p11.2 q11.1 q12.1 q13 q14.2 q21.1 254 kb 203,300 kb 203,400 kb q22.1 q23.1 q24.1 q24.3 q31.2 q32.2 q33.1 q34 q35 q36.2 q37.2 [0–60] [0–60] [0–60] [0–60] [0–60] [0–60] [0–60] [0–60] [0–60] 2 c 0.0 0.2 0.4 0.6 0.8 1.0 Chromosome e 1 2 X 20 16 13 11 9 7 6 5 4 3 0 5 10 15 Chromosome −log10 (Padj) 1 BMPR2 d 2 X 20 16 13 11 9 7 6 5 4 3 d Chromosome Fig. 2 Analysis of copy number deletions. a Deletions affecting the BMPR2 locus in 23 PAH cases. Genes are indicated in orange and labelled with their respective gene symbol. Deletions are drawn as blue boxes above the genome axis (grey) showing the genomic position on chromosome 2. The grey box highlights the location of BMPR2. b Locus zoom on BMPR2 highlighting the focal deletions affecting one or more exons. c WGS coverage proﬁles of a selected set of smaller and larger deletions, visualised with the Integrative Genomics Viewer (IGV)57, with deletions highlighted by red bars. Results a The study comprised 1048 adult cases (aged 16 or over) attending specialist pulmonary hypertension centres from the UK (n = 731), and additional cases from France (n = 142), The Netherlands (n = 45), Germany (n = 82) and Italy (n = 48). b A series of case-control comparisons including and excluding cases with variants in previously reported disease genes were undertaken using complementary ﬁltering strategies 2 NATURE COMMUNICATIONS\| (2018) 9:1416 \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications 9066 subjects sequenced in NIHR BR-RD study 7979 non-PAH control subjects (6385 unrelated subjects) 1087 patients sequenced in PAH study 1048 PAH patients (1038 index cases) 22 PVOD/PCH patients 908 idiopathic PAH patients (with no family history of PAH) 58 PAH patients with family history of PAH 60 PAH patients with a history of exposure to drugs or toxins Excluded: 39 not idiopathic or heritable PAH or PVOD/PCH a b a Filtered variants MAF < 1 in 10,000 Missense variants only Burden (Fisher’s) test (see figure 3a–c) SKAT-O (see figure 3d) Fig. 1 Flow diagrams illustrating a the composition of the NIHR BioResource—Rare Diseases (NIHR BR-RD) PAH study and b the analysis strategy to identify novel PAH disease genes. a The study comprised 1048 adult cases (aged 16 or over) attending specialist pulmonary hypertension centres from the UK (n = 731), and additional cases from France (n = 142), The Netherlands (n = 45), Germany (n = 82) and Italy (n = 48). b A series of case-control comparisons including and excluding cases with variants in previously reported disease genes were undertaken using complementary ﬁltering strategies \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications NATURE COMMUNICATIONS\| (2018) 9:1416 \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications 2 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 a b Deletions 203.25 mb 203.3 mb 203.35 mb 203.4 mb Genes BMPR2 c 0.0 0.2 0.4 0.6 0.8 1.0 BMPR2 Chromosome 0 5 10 15 Chromosome −log10 (Padj) 1 BMPR2 d e Deletions 202 mb 203 mb 204 mb 205 mb Genes ORC2 CASP8 ALS2 NOP58 CASP10 PPIL3 ALS2CR11 FZD7 ABI2 CTLA4 FAM117B CFLAR MPP4 ICOS CLK1 CD28 TRAK2 SGOL2 BMPR2 AOX1 ICA1L SUMO1 CARF PARD3B BZW1 TMEM237 CDK15 NIF3L1 CYP20A1 FAM126B ALS2CR12 NBEAL1 NDUFB3 RAPH1 STRADB CTD18 WDR12 AC079354.1 AC079354.2 2 X 20 16 13 11 9 7 6 5 4 3 1 2 X 20 16 13 11 9 7 6 5 4 3 p25.2 p24.3 p23.3 p22.3 p21 p16.2 p15 p13.3 p12 p11.2 q11.1 q12.1 q13 q14.2 q21.1 254 kb 203,300 kb 203,400 kb q22.1 q23.1 q24.1 q24.3 q31.2 q32.2 q33.1 q34 q35 q36.2 q37.2 [0–60] [0–60] [0–60] [0–60] [0–60] [0–60] [0–60] [0–60] [0–60] 2 Analysis of copy number deletions a Deletions affecting the BMPR2 locus in 23 PAH cases Genes are indicated in orange and labelled w a Deletions 202 mb 203 mb 204 mb 205 mb Genes ORC2 CASP8 ALS2 NOP58 CASP10 PPIL3 ALS2CR11 FZD7 ABI2 CTLA4 FAM117B CFLAR MPP4 ICOS CLK1 CD28 TRAK2 SGOL2 BMPR2 AOX1 ICA1L SUMO1 CARF PARD3B BZW1 TMEM237 CDK15 NIF3L1 CYP20A1 FAM126B ALS2CR12 NBEAL1 NDUFB3 RAPH1 STRADB CTD18 WDR12 AC079354.1 AC079354.2 a Deletions 202 mb 204 mb a b Deletions 203.25 mb 203.3 mb 203.35 mb 203.4 mb Genes BMPR2 c 0.0 0.2 0.4 0.6 0.8 1.0 BMPR2 Chromosome 0 5 10 15 Chromosome −log10 (Padj) 1 BMPR2 d e 202 mb 203 mb 204 mb 205 mb Genes ORC2 CASP8 ALS2 NOP58 CASP10 PPIL3 ALS2CR11 FZD7 ABI2 CTLA4 FAM117B CFLAR MPP4 ICOS CLK1 CD28 TRAK2 SGOL2 BMPR2 AOX1 ICA1L SUMO1 CARF PARD3B BZW1 TMEM237 CDK15 NIF3L1 CYP20A1 FAM126B ALS2CR12 NBEAL1 NDUFB3 RAPH1 STRADB CTD18 WDR12 AC079354.1 AC079354.2 2 X 20 16 13 11 9 7 6 5 4 3 1 2 X 20 16 13 11 9 7 6 5 4 3 p25.2 p24.3 p23.3 p22.3 p21 p16.2 p15 p13.3 p12 p11.2 q11.1 q12.1 q13 q14.2 q21.1 254 kb 203,300 kb 203,400 kb q22.1 q23.1 q24.1 q24.3 q31.2 q32.2 q33.1 q34 q35 q36.2 q37.2 [0–60] [0–60] [0–60] [0–60] [0–60] [0–60] [0–60] [0–60] [0–60] 2 Fig. 2 Analysis of copy number deletions. a Deletions affecting the BMPR2 locus in 23 PAH cases. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 d and e Manhattan plots of the genome-wide case-control comparison of large deletions. In d, all subject are considered. In e, subject with larger deletions affecting the BMPR2 locus are excluded. The adjusted P value threshold of 5 × 10−8 for genome-wide signiﬁcance is indicated by the red line Fig. 2 Analysis of copy number deletions. a Deletions affecting the BMPR2 locus in 23 PAH cases. Genes are indicated in orange and labelled with their respective gene symbol. Deletions are drawn as blue boxes above the genome axis (grey) showing the genomic position on chromosome 2. The grey box highlights the location of BMPR2. b Locus zoom on BMPR2 highlighting the focal deletions affecting one or more exons. c WGS coverage proﬁles of a selected set of smaller and larger deletions, visualised with the Integrative Genomics Viewer (IGV)57, with deletions highlighted by red bars. d and e Manhattan plots of the genome-wide case-control comparison of large deletions. In d, all subject are considered. In e, subject with larger deletions affecting the BMPR2 locus are excluded. The adjusted P value threshold of 5 × 10−8 for genome-wide signiﬁcance is indicated by the red line \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications ARTICLE However, we consider MFRP a false-positive ﬁnding for reasons given in the Discussion. Supplementary Table 8 shows the top 50 most signiﬁcant genes identiﬁed by SKAT-O, providing further candidates to be evaluated in future studies. Details of rare variants in novel PAH genes (GDF2, ATP13A3, AQP1, SOX17) identiﬁed in cases are provided in Supplementary Data 3. Identiﬁcation of novel PAH disease genes. The strategy to identify novel causative genes in PAH employed a series of case- control analyses (Fig. 1b). To identify signals that might be masked by variants in previously reported PAH genes, we excluded subjects with rare variants and deletions in BMPR2, EIF2AK4, ENG, ACVRL1, TBX4, SMAD9 and KCNK3. A genome-wide comparison of protein-truncating variants (PTVs), representative of high impact variants, identiﬁed a higher fre- quency of PTVs in ATP13A3 (six cases) (Padj = 0.0346). More- over, we identiﬁed additional PTVs in several putative PAH genes, including EVI5 (5 cases, 1 control) and KDR (4 cases, 0 controls; Fig. 3a), that require further validation to evaluate their contribution to PAH pathogenesis (Supplementary Table 5). pp y Notably, a genome-wide assessment of larger structural variation did not identify any additional large deletions after exclusion of subjects harbouring deletions in BMPR2 (Fig. 2d, e). The proportion of PAH cases with mutations in the new genes was 3.5%. The clinical characteristics of PAH cases with mutations in these genes are provided in Supplementary Table 3b. We next analysed rare missense variants overrepresented in the PAH cohort, again excluding subjects with variants in the b a c d Protein truncating variants Missense variants PTV and missense variants (SKAT-O) PTV and missense variants 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Chromosome AQP1 GDF2 0 1 2 3 4 5 6 −log10 (P ) −log10 (Padj) 1 EVI5 ATP13A3 KDR 0 2 4 6 8 ALPPL2 C3orf20 ATP13A5 ATP13A3 AQP1 AQP1 SOX17 IFT74 GDF2 OR8U1 PIWIL1 FLNA 0 1 2 3 4 5 6 Chromosome AQP1 SOX17 MFRP X 18 2 3 5 7 9 11 14 1 X 18 2 3 5 7 9 11 14 1 X 18 2 3 5 7 9 11 14 1 X 18 2 3 5 7 9 11 14 Fig. 3 Manhattan plots of the rare variant analyses, having excluded cases carrying rare variants in previously established PAH genes. Filtered variants were grouped per gene. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 highly deleterious variants in both BMPR2 (p.Cys123Arg) and SMAD9 (p.Arg294Ter). Details of consequence types, deleter- iousness and conservation scores, and minor allele frequencies are provided in Supplementary Data 2. Fourteen cases (1.3%) with biallelic EIF2AK4 mutations were found18. No pathogenic coding variants in CAV1, SMAD1 or SMAD4 were identiﬁed. Taken together, rare causal variation in non-BMPR2 disease genes (TBX4, ENG, ACVRL1, SMAD9, KCNK3 and EIF2AK4) accounted for 4.7% of the entire PAH cohort. The clinical characteristics of cases with variants in these previously reported genes are shown in Supplementary Table 3. highly deleterious variants in both BMPR2 (p.Cys123Arg) and SMAD9 (p.Arg294Ter). Details of consequence types, deleter- iousness and conservation scores, and minor allele frequencies are provided in Supplementary Data 2. Fourteen cases (1.3%) with biallelic EIF2AK4 mutations were found18. No pathogenic coding variants in CAV1, SMAD1 or SMAD4 were identiﬁed. Taken together, rare causal variation in non-BMPR2 disease genes (TBX4, ENG, ACVRL1, SMAD9, KCNK3 and EIF2AK4) accounted for 4.7% of the entire PAH cohort. The clinical characteristics of cases with variants in these previously reported genes are shown in Supplementary Table 3. previously reported PAH genes. This revealed signiﬁcant over- representation of rare variants in GDF2 after correction for multiple testing (Padj = 0.0023), followed by AQP1 (Fig. 3b and Supplementary Table 6). Next, in a combined analysis of rare missense variants and PTV, only GDF2 remained signiﬁcant (P = 0.001). Rare variants in additional putative genes occurred at higher frequency in cases compared to controls, including AQP1, ALPPL2, ATP13A3, OR8U1, IFT74, FLNA, SOX17, ATP13A5, C3orf20 and PIWIL1 (uncorrected P value < 0.0005), but were not signiﬁcant after correction for multiple testing (Fig. 3c and Supplementary Table 7). In a case-control comparison of the frequencies of deleterious variants conﬁned to the previously reported PAH genes, we observed signiﬁcant overrepresentation of rare variants in BMPR2, TBX4, ACVRL1 and biallelic variants in EIF2AK4 only (P < 0.05; Supplementary Table 4). In order to increase power to detect rare associations, we deployed SKAT-O on ﬁltered rare PTVs and missense variants. Excluding previously reported genes, this analysis revealed an association with rare variants in AQP1 (Padj = 4.28 × 10−6) and SOX17 (Padj = 6.7 × 10−5; Fig. 3d). AQP1 and SOX17 were both also nominally signiﬁcant in the combined burden tests, described above. Association was also found with rare variants in MFRP (Padj = 1.3 × 10−5). NATURE COMMUNICATIONS\| (2018) 9:1416 3 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 note, cases with mutations in SOX17 and AQP1 were ﬁcantly younger at diagnosis (32.8 ± 16.2 years (P = 0.002) 36 9 ± 14 3 years (P = 0 013) respectively) compared to cases Non-coding variation around PAH disease ge analysis for enrichment of variants in the non-c surrounding previously reported and newly iden C T G C T Y G G T C C C T G C T Y G G T C C C T G C T Y G G T C C C T G C T C G G T C C E011942.gf E011942.gm E011942.f E011942.m E011942.u1 E011942.u2 E011942.s d.66yo d.75yo PAH:67yo PAH:42yo (d.45yo) 70yo 57yo PAH:6yo (d.10yo) PAH:24yo E011942 C T G C T Y G G T C C C T G C T Y G G T C C E010634.f E010634.m PAH:32yo E010634 C T C T G W A G C C C C T C T G W A G C C C E012415.f E012415.m E012415.b PAH:48yo o y 6 2 : H A P E012415 a b d c I−1 I−2 II−3 II−4 II−1 II−2 II−5 III−1 III−2 III−3 82yo 81yo 48yo 50yo PAH:12yo (d.36yo) 53yo 57yo PAH:6mo (d.12mo) PAH:3yo (d.9yo) 18yo I−1 I−2 II−3 II−4 II−1 II−2 II−5 A A G T A C C G G C C A A G T A S C G G C C A A G T A C C G G C C A A G T A C C G G C C A A G T A S C G G C C A A G T A C C G G C C Pedigree structures and analysis of familial transmission of variants in AQP1 and SOX17. a Individual II.1 harbours a heterozygo C > G (p.Y137) PTV resulting in a premature termination codon, which has been transmitted to the affected male (III.1). No una bers carry the variant. No sample was available from subject III.2. b Proband E011942 has inherited a heterozygous AQP1 c.583 C nse variant from her affected father. No sample was available from the affected sister of the proband. The younger healthy uncle arries the AQP1 variant. No samples or further clinical information was available for the grandparents, who were not known to have e. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 c Both the proband E012415 and her father are affected and carry the rare AQP1 c.527 T > A (p.V176E) missense variant. The mation available about the siblings of the father. d Subject E010634 has inherited the heterozygous AQP1 c.583 C > T (p.R195W) her affected father. No rare variants in previously reported PAH genes were identiﬁed in any of theses families. Index cases are hi mo months old, yo years old C T C T G W A G C C C C T C T G W A G C C C E012415.f E012415.m E012415.b PAH:48yo o y 6 2 : H A P E012415 a c I−1 I−2 II−3 II−4 II−1 II−2 II−5 III−1 III−2 III−3 82yo 81yo 48yo 50yo PAH:12yo (d.36yo) 53yo 57yo PAH:6mo (d.12mo) PAH:3yo (d.9yo) 18yo I−1 I−2 II−3 II−4 II−1 II−2 II−5 A A G T A C C G G C C A A G T A S C G G C C A A G T A C C G G C C A A G T A C C G G C C A A G T A S C G G C C A A G T A C C G G C C a c PAH:67yo C T G C T Y G G T C C C T G C T Y G G T C C E010634.f E010634.m PAH:32yo E010634 d b d Fig. 4 Pedigree structures and analysis of familial transmission of variants in AQP1 and SOX17. a Individual II.1 harbours a heterozygous de novo SOX17 c.411 C > G (p.Y137) PTV resulting in a premature termination codon, which has been transmitted to the affected male (III.1). No unaffected family members carry the variant. No sample was available from subject III.2. b Proband E011942 has inherited a heterozygous AQP1 c.583 C > T (p.R195W) missense variant from her affected father. No sample was available from the affected sister of the proband. The younger healthy uncle of the index case also carries the AQP1 variant. No samples or further clinical information was available for the grandparents, who were not known to have cardiopulmonary disease. c Both the proband E012415 and her father are affected and carry the rare AQP1 c.527 T > A (p.V176E) missense variant. There was no further information available about the siblings of the father. ARTICLE We tested for an excess of variants in PAH cases within genes using Fisher’s exact test. The negative decadic logarithm of unadjusted or adjusted P-values are plotted against the chromosomal location of each gene. a Burden test of rare PTVs. b Burden test of rare deleterious missense variants. c Burden test combining rare PTVs and likely deleterious missense variants. d SKAT-O test of rare PTVs and missense variants c PTV and missense variants 0 2 4 6 8 ALPPL2 C3orf20 ATP13A5 ATP13A3 AQP1 AQP1 SOX17 IFT74 GDF2 OR8U1 PIWIL1 FLNA 1 X 18 2 3 5 7 9 11 14 a Protein truncating variants 0 1 2 3 4 5 6 −log10 (P ) 1 EVI5 ATP13A3 KDR X 18 2 3 5 7 9 11 14 c a b b Missense variants 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Chromosome AQP1 GDF2 −log10 (Padj) 1 X 18 2 3 5 7 9 11 14 d PTV and missense variants (SKAT-O) 0 1 2 3 4 5 6 Chromosome AQP1 SOX17 MFRP 1 X 18 2 3 5 7 9 11 14 d Chromosome Chromosome Chromosome Fig. 3 Manhattan plots of the rare variant analyses, having excluded cases carrying rare variants in previously established PAH genes. Filtered variants were grouped per gene. We tested for an excess of variants in PAH cases within genes using Fisher’s exact test. The negative decadic logarithm of unadjusted or adjusted P-values are plotted against the chromosomal location of each gene. a Burden test of rare PTVs. b Burden test of rare deleterious missense variants. c Burden test combining rare PTVs and likely deleterious missense variants. d SKAT-O test of rare PTVs and missense variants Fig. 3 Manhattan plots of the rare variant analyses, having excluded cases carrying rare variants in previously established PAH genes. Filtered variants were grouped per gene. We tested for an excess of variants in PAH cases within genes using Fisher’s exact test. The negative decadic logarithm of unadjusted or adjusted P-values are plotted against the chromosomal location of each gene. a Burden test of rare PTVs. b Burden test of rare deleterious missense variants. c Burden test combining rare PTVs and likely deleterious missense variants. d SKAT-O test of rare PTVs and missense variants \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications NATURE COMMUNICATIONS\| (2018) 9:1416 4 4 ARTICLE \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications 5 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 d Subject E010634 has inherited the heterozygous AQP1 c.583 C > T (p.R195W) missense variant from her affected father. No rare variants in previously reported PAH genes were identiﬁed in any of theses families. Index cases are highlighted in red. d death, mo months old, yo years old Of note, cases with mutations in SOX17 and AQP1 were signiﬁcantly younger at diagnosis (32.8 ± 16.2 years (P = 0.002) and 36.9 ± 14.3 years (P = 0.013), respectively) compared to cases with no mutations in the previously established genes (51.7 ± 16.6 years). Non-coding variation around PAH disease genes. An initial analysis for enrichment of variants in the non-coding sequence surrounding previously reported and newly identiﬁed PAH dis- ease genes, including upstream gene regions, 5’ UTRs, intronic sequence, 3’ UTRs and downstream gene regions, did not detect NATURE COMMUNICATIONS\| (2018) 9:1416 5 \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 an signiﬁcant overrepresentation in the PAH cohort. Details of the non-coding variants that passed the ﬁltering strategy are provided in Supplementary Data 4. an signiﬁcant overrepresentation in the PAH cohort. Details of the non-coding variants that passed the ﬁltering strategy are provided in Supplementary Data 4. in these families. The ﬁrst pedigree comprised three affected individuals across two generations. Sanger sequencing conﬁrmed the presence of the heterozygous AQP1 c.583 C > T (p.R195W) missense variant in the proband (E011942), the affected father (E011942.f) and the healthy younger paternal uncle (E011942.u1). An additional unaffected uncle did not carry the AQP1 variant. These results indicate likely incomplete penetrance in the unaffected carrier, as observed in BMPR2 families19. No additional clinical information was available for the deceased grandparents (Fig. 4b). The remaining two families comprised affected parent-offspring individuals. By Sanger sequencing we independently conﬁrmed a heterozygous AQP1 c.527 T > A (p. Val176Glu) missense variant in proband (E012415) and his affected father (Fig. 4c), as well as a heterozygous AQP1 c.583 C > T (p.R195W) missense variant in proband (E010634) and her affected father (Fig. 4d). These results highlight recurrent AQP1 variation across unrelated families and demonstrate co- segregation with the phenotype. Independent validation and familial segregation analysis. To provide further validation of the potentially causal role of mutations in the new genes identiﬁed, we examined whole-exome data from an independent UK family with three affected indivi- duals across two generations. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 Microsatellite genotyping across chromosome 2q33 had previously demonstrated non-sharing of haplotypes in affected individuals, consistent with exclusion of linkage to the BMPR2 locus. No pathogenic variants were iden- tiﬁed in the protein-coding regions of the BMPR2 gene or other TGF-β pathway genes. Analysis of exome sequence data from individual II-1 identiﬁed a novel heterozygous c.411 C > G (p. Y137) PTV in the SOX17 gene. Segregation analysis in the extended family demonstrated that the mutation had arisen de novo in the affected father (II-1) and was transmitted to the affected offspring (III-1). All unaffected family members were conﬁrmed as wild-type (Fig. 4a). Predicted functional impact of variants in novel PAH genes. To evaluate the potential functional impact of rare variants identiﬁed in the likely causative new genes we performed structural analysis of GDF2, ATP13A3, AQP1, and SOX17. In addition we undertook a functional analysis of the GDF2 variants identiﬁed. Three HPAH subjects harbouring rare variants in AQP1, identiﬁed in the NIHR BR-RD WGS study, were also selected for familial co-segregation analysis (Fig. 4b–d). No pathogenic variants in any of the previously reported genes were identiﬁed a Growth factor domain Prodomain Signal peptide Folding and homo- dimerisation pro-GDF2 Pre-pro-GDF2 Prodomain bound-GDF2 (circulating form) Furin processing and secretion Furin cleavage site pro-GDF2 Prodomain bound-GDF2 (circulating form) Furin processing and secretion Furin cleavage site a Prodomain bound-GDF2 (circulating form) g Arg110 Glu143 a b d c Growth factor domain Prodomain Signal peptide Folding and homo- dimerisation pro-GDF2 Pre-pro-GDF2 Prodomain bound-GDF2 (circulating form) Furin processing and secretion Furin cleavage site Glu143 2.0 1.5 1.0 0.5 0.0 * * * * * Untransfected Vector only WT 89 110 143 320 347 413 GDF2 (μg/ml) Arg110 Asp116 Met89 Thr413 Ala347 Ala347 Thr413 Arg110 Met89 Asp116 Fig. 5 Structural analysis of GDF2 mutations. a Schematic diagram of GDF2 processing. The pre-pro-protein is processed into the mature growth factor domain (GFD) bound to the prodomain upon secretion61. b Plot of GDF2 mutations found only in PAH cases superimposed on the structure of prodomain bound GDF2 (PDB: 4YCG)21. The GDF2 growth factor domain is shown in green and the prodomain in cyan. c Magniﬁed view of the Arg110 and Glu143 mutations. The wild-type amino acids make double salt bridges to stabilise the prodomain conformation at the interface between the growth factor domain and prodomain. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 The E143K and R110W mutations both disrupt these interactions, destabilising the interaction between the growth factor domain and prodomain. d GDF2 levels secreted into supernatants of HEK293T cells transfected with likely pathogenic variants found in PAH cases, compared with wild-type GDF2 and cells transfected with an empty vector. *P < 0.001 by ANOVA 6 NATURE COMMUNICATIONS\| (2018)9:1416 \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications Folding and homo- dimerisation Furin processing and secretion Arg110 Glu143 b d c Growth factor domain Prodomain Signal peptide Folding and homo- dimerisation ( g ) Furin processing and secretion Furin cleavage site Glu143 2.0 1.5 1.0 0.5 0.0 * * * * * * Untransfected Vector only WT 89 110 143 320 347 413 GDF2 (μg/ml) Arg110 Asp116 Met89 Thr413 Ala347 Ala347 Thr413 Arg110 Met89 Asp116 Fig. 5 Structural analysis of GDF2 mutations. a Schematic diagram of GDF2 processing. The pre-pro-protein is processed into the mature growth factor domain (GFD) bound to the prodomain upon secretion61. b Plot of GDF2 mutations found only in PAH cases superimposed on the structure of prodomain bound GDF2 (PDB: 4YCG)21. The GDF2 growth factor domain is shown in green and the prodomain in cyan. c Magniﬁed view of the Arg110 and Glu143 mutations. The wild-type amino acids make double salt bridges to stabilise the prodomain conformation at the interface between the growth factor domain and prodomain. The E143K and R110W mutations both disrupt these interactions, destabilising the interaction between the growth factor domain and prodomain. d GDF2 levels secreted into supernatants of HEK293T cells transfected with likely pathogenic variants found in PAH cases, compared with wild-type GDF2 and cells transfected with an empty vector. P < 0.001 by ANOVA Furin cleavage site Arg110 Glu143 b d c cleavage site Glu143 2.0 1.5 1.0 0.5 0.0 * * * * * Untransfected Vector only WT 89 110 143 320 347 413 GDF2 (μg/ml) Arg110 Asp116 Met89 Thr413 Ala347 Ala347 Thr413 Arg110 Met89 Asp116 Fig. 5 Structural analysis of GDF2 mutations. a Schematic diagram of GDF2 processing. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 **: Q9H7F0\|AT133_HUMAN QYFSVTLLYSILSNLGDFQFLFIDLAIILVVVFTMSLNPAWKELVAQRPPSG----LISG P05023\|AT1A1_HUMAN TPFLIFIIANIPLPLGTVTILCIDLGTDMVPAISLAYEQAESDIMKRQPRNPKTDKLVNE : :: .* ** . :* **. : .:::: : * .::: ::* . :. b Q c Extracellular Cytoplasm Membrane I787 (p.L956P) Y677 (p.M850I) R685 (p.R858H) ADP Y535 (p.L675V) c Extracellular Cytoplasm Membrane I787 (p.L956P) c Y677 (p.M850I) R685 (p.R858H) ADP Y535 (p.L675V) Extracellular Cytoplasm Fig. 6 Structural analysis of ATP13A3 mutations. a Topology of ATP13A3, plotted according to UniProtKB Q9H7F0. Frameshift and stop-gained mutations identiﬁed in PAH cases are shown as khaki circles, and missense mutations as red circles. Frameshift/stop-gained mutations are predicted to truncate the protein prior to the catalytic domain and essential Mg binding sites, leading to loss of ATPase activity. b Sequence alignment of ATP13A3 with ATP1A1 (P05024), of which the high resolution structure was used for the structural analysis in c. The conserved regions of ATP13A3 and ATP1A1, essential for ATPase activity62, show good alignment (data not shown). Only regions containing the missense PAH mutations are shown, with positions of the four missense mutations highlighted in yellow above the sequences. c Structural analysis of the 4 PAH missense mutations plotted on the ATP1A1 crystal structure based on the sequence alignment in b (PDB: 3wgu)63. Green: α subunit (P05024), cyan: β subunit (P05027), grey: γ-subunit transcript variant a (Q58k79). Y535, Y677, R685 and I787 are the numbering in ATP1A1. Positions of the four missense mutations found in PAH are labelled and highlighted by red circles. d Magniﬁed view of the cytoplasmic region of the ATPase, showing the presence of ADP at the active site. The conserved regions essential for ATPase activity are shown in light pink. The L675V and R858H mutations are located close to the ATP catalytic region Heterozygous mutations in GDF2 exclusive to PAH cases comprised 1 frameshift variant and 7 missense variants. GDF2 encodes growth and differentiation factor 2, also known as bone morphogenetic protein 9 (BMP9), the major circulating ligand for the endothelial BMPR2/ACVRL1 receptor complex20. Amino acid substitutions were assessed against the published crystal structure21 of the prodomain bound form of GDF2 (Fig. 5). Variants clustered at the interface between the prodomain and growth factor domain. Since the prodomain is important for the processing of GDF2, it is likely that amino acid substitutions reduce the stability of the prodomain-growth factor interface. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 a b c PTV mutations Missense mutations 29 49 226 206 430 410 233 1 4 9 3 5 2 1020 1000 1074 1094 1126 1106 1144 1164 1226 963 983 Active site Asp 887 883 498 Mg binding Extracellular Cytoplasm Membrane 961 469 449 714 583 893 727 1004 847 Extracellular Cytoplasm Membrane I787 (p.L956P) Y677 (p.M850I) R685 (p.R858H) ADP Y535 (p.L675V) Q9H7F0\|AT133_HUMAN -----------PVDFQNVLEDFTKQGFRVIALAHRKLESKLTWHK--VQNISRDAIENNM P05023\|AT1A1_HUMAN HGKEQPLDEELKDAFQNAYLELGGLGERVLGFCHLFLPDEQFPEGFQFDTDDVNFPIDNL *. :: *:.:. * .: . .:. . : :: Q9H7F0\|AT133_HUMAN SFSVILEHFQDLVPKLMLHGTVFARMAPDQKTQLIEALQNVDYFVGMCGDGANDCGALKR P05023\|AT1A1_HUMAN ------EQLDDIL--KYHTEIVFARTSPQQKLIIVEGCQRQGAIVAVTGDGVNDSPALKK ::::: *** ::* ::. . . :.: .. **: Q9H7F0\|AT133_HUMAN QYFSVTLLYSILSNLGDFQFLFIDLAIILVVVFTMSLNPAWKELVAQRPPSG----LISG P05023\|AT1A1_HUMAN TPFLIFIIANIPLPLGTVTILCIDLGTDMVPAISLAYEQAESDIMKRQPRNPKTDKLVNE : :: .* ** . :* **. : .:::: : * .::: ::* . :. 6 Structural analysis of ATP13A3 mutations. a Topology of ATP13A3, plotted according to UniProtKB Q9H7F0. Frameshift and stop-gained muta tiﬁed in PAH cases are shown as khaki circles, and missense mutations as red circles. Frameshift/stop-gained mutations are predicted to truncate ein prior to the catalytic domain and essential Mg binding sites, leading to loss of ATPase activity. b Sequence alignment of ATP13A3 with AT 5024), of which the high resolution structure was used for the structural analysis in c. The conserved regions of ATP13A3 and ATP1A1, essentia ase activity62, show good alignment (data not shown). Only regions containing the missense PAH mutations are shown, with positions of the sense mutations highlighted in yellow above the sequences. c Structural analysis of the 4 PAH missense mutations plotted on the ATP1A1 crys cture based on the sequence alignment in b (PDB: 3wgu)63. Green: α subunit (P05024), cyan: β subunit (P05027), grey: γ-subunit transcript varia 8k79). Y535, Y677, R685 and I787 are the numbering in ATP1A1. Positions of the four missense mutations found in PAH are labelled and highlig ed circles d Magniﬁed view of the cytoplasmic region of the ATPase showing the presence of ADP at the active site The conserved regions esse a PTV mutations Missense mutations 29 49 226 206 430 410 233 1 4 9 3 5 2 1020 1000 1074 1094 1126 1106 1144 1164 1226 963 983 Active site Asp 887 883 498 Mg binding Extracellular Cytoplasm Membrane 961 469 449 a Extracellular 7 3 Mg binding b 714 583 893 727 1004 847 Q9H7F0\|AT133_HUMAN -----------PVDFQNVLEDFTKQGFRVIALAHRKLESKLTWHK--VQNISRDAIENNM P05023\|AT1A1_HUMAN HGKEQPLDEELKDAFQNAYLELGGLGERVLGFCHLFLPDEQFPEGFQFDTDDVNFPIDNL *. :: *:.:. * .: . .:. . : :: Q9H7F0\|AT133_HUMAN SFSVILEHFQDLVPKLMLHGTVFARMAPDQKTQLIEALQNVDYFVGMCGDGANDCGALKR P05023\|AT1A1_HUMAN ------EQLDDIL--KYHTEIVFARTSPQQKLIIVEGCQRQGAIVAVTGDGVNDSPALKK ::::: *** ::* ::. . . :.: .. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 The pre-pro-protein is processed into the mature growth factor domain (GFD) bound to the prodomain upon secretion61 b Plot of GDF2 mutations found only in PAH cases superimposed on the structure of prodomain Glu143 b d c Glu143 2.0 1.5 1.0 0.5 0.0 * * * * * * ed ly WT 89 10 43 20 47 13 GDF2 (μg/ml) Arg110 Asp116 Met89 Thr413 Ala347 Ala347 Thr413 Arg110 Met89 Asp116 b Glu143 Arg110 Asp116 Met89 Thr413 Ala347 Ala347 Thr413 Arg110 Met89 Asp116 b Asp116 Arg110 Glu143 c Fig. 5 Structural analysis of GDF2 mutations. a Schematic diagram of GDF2 processing. The pre-pro-protein is processed into the mature growth factor domain (GFD) bound to the prodomain upon secretion61. b Plot of GDF2 mutations found only in PAH cases superimposed on the structure of prodomain bound GDF2 (PDB: 4YCG)21. The GDF2 growth factor domain is shown in green and the prodomain in cyan. c Magniﬁed view of the Arg110 and Glu143 mutations. The wild-type amino acids make double salt bridges to stabilise the prodomain conformation at the interface between the growth factor domain and prodomain. The E143K and R110W mutations both disrupt these interactions, destabilising the interaction between the growth factor domain and prodomain. d GDF2 levels secreted into supernatants of HEK293T cells transfected with likely pathogenic variants found in PAH cases, compared with wild-type GDF2 and cells transfected with an empty vector. *P < 0.001 by ANOVA Fig. 5 Structural analysis of GDF2 mutations. a Schematic diagram of GDF2 processing. The pre-pro-protein is processed into the mature growth factor domain (GFD) bound to the prodomain upon secretion61. b Plot of GDF2 mutations found only in PAH cases superimposed on the structure of prodomain bound GDF2 (PDB: 4YCG)21. The GDF2 growth factor domain is shown in green and the prodomain in cyan. c Magniﬁed view of the Arg110 and Glu143 mutations. The wild-type amino acids make double salt bridges to stabilise the prodomain conformation at the interface between the growth factor domain and prodomain. The E143K and R110W mutations both disrupt these interactions, destabilising the interaction between the growth factor domain and prodomain. d GDF2 levels secreted into supernatants of HEK293T cells transfected with likely pathogenic variants found in PAH cases, compared with wild-type GDF2 and cells transfected with an empty vector. P < 0.001 by ANOVA \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications NATURE COMMUNICATIONS\| (2018) 9:1416 6 6 ARTICLE \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications NATURE COMMUNICATIONS\| (2018) 9:1416 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 In keeping with these predictions, HEK293T cells transfected with GDF2 variants exclusive to PAH cases, demonstrated reduced secretion of mature GDF2 into the cell supernatants (Fig. 5d), compared with wild-type GDF2. NATURE COMMUNICATIONS\| (2018) 9:1416 7 \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 177 177 177 177 179 179 179 180 237 237 237 237 239 239 239 240 Extracellular Cytoplasmic Arg197 (p.R195W) Val178 (p.V176G) b c P29972\|AQP1_HUMAN LTGNSLGRNDLADGVNSGQGLGIEIIGTLQLVLCVLATTDRR-RRDLGGSAPLAIGLSVA Q5R819\|AQP1_PONAB LPGNSLGRNDLADGVNSGQGLGIEIIGTLQLVLCVLATTDRR-RRDLGGSAPLAIGLSVA Q02013\|AQP1_MOUSE LVDNSLGRNDLAHGVNSGQGLGIEIIGTLQLVLCVLATTDRR-RRDLGGSAPLAIGLSVA P29975\|AQP1_RAT LLENSLGRNDLARGVNSGQGLGIEIIGTLQLVLCVLATTDRR-RRDLGGSAPLAIGLSVA Q6PQZ1\|AQP1_PIG LPGNSLGLNSLAPGVDSGQGLGIEIIGTLQLVLCVLATTDRR-RRDLGGSAPLAIGFSVA Q9N2J4\|AQP1_CANLF LPDNSLGRNELAPGVNSGQGLGIEIIGTLQLVLCVLATTDRR-RRDLGGSGPLAIGLSVA P47865\|AQP1_BOVIN LPDNSLGLNALAPGVNSGQGLGIEIIGTLQLVLCVLATTDRR-RRDLGGSGPLAIGFSVA P56401\|AQP1_SHEEP LPDNSLGLNALAPGVNSGQGLGIEIIGTLQLVLCVLATTDRRRRRDLGDSGPLAIGFSVA **** * :************************ **..***:* P29972\|AQP1_HUMAN LGHLLAIDYTGCGINPARSFGSAVITHNFSNHWIFWVGPFIGGALAVLIYDFILAPRSSD Q5R819\|AQP1_PONAB LGHLLAIDYTGCGINPARSFGSAVITHNFSNHWIFWVGPFIGGALAVLIYDFILAPRSSD Q02013\|AQP1_MOUSE LGHLLAIDYTGCGINPARSFGSAVLTRNFSNHWIFWVGPFIGGALAVLIYDFILAPRSSD P29975\|AQP1_RAT LGHLLAIDYTGCGINPARSFGSAVLTRNFSNHWIFWVGPFIGSALAVLIYDFILAPRSSD Q6PQZ1\|AQP1_PIG LGHLLAIDYTGCGINPARSFGSAVITHNFQDHWVFWVGPFIGGALAVLIYDFILAPRSSD Q9N2J4\|AQP1_CANLF LGHLLAIDYTGCGINPARSFGSSVITHNFKDHWIFWVGPFIGGALAVLIYDFILAPRSSD P47865\|AQP1_BOVIN LGHLLAIDYTGCGINPARSFGSSVITHNFQDHWIFWVGPFIGAALAVLIYDFILAPRSSD P56401\|AQP1_SHEEP LGHLLAIDYTGCGINPARSFGSSVITHNFQDHWIFWVGPFIGAALAVLIYDFILAPRSSD *********************:::.::*****.*************** a uctural analysis of AQP1 mutations. a Multiple sequence alignment of human AQP1 with seven other mammals. The bovine AQP1 has the high (2 2 Å) published structure Mutations identiﬁed in PAH cases are highly conserved and highlighted in yellow b Crystal structure of bovine AQP1 177 177 177 177 179 179 179 180 237 237 237 237 239 239 239 240 P29972\|AQP1_HUMAN LTGNSLGRNDLADGVNSGQGLGIEIIGTLQLVLCVLATTDRR-RRDLGGSAPLAIGLSVA Q5R819\|AQP1_PONAB LPGNSLGRNDLADGVNSGQGLGIEIIGTLQLVLCVLATTDRR-RRDLGGSAPLAIGLSVA Q02013\|AQP1_MOUSE LVDNSLGRNDLAHGVNSGQGLGIEIIGTLQLVLCVLATTDRR-RRDLGGSAPLAIGLSVA P29975\|AQP1_RAT LLENSLGRNDLARGVNSGQGLGIEIIGTLQLVLCVLATTDRR-RRDLGGSAPLAIGLSVA Q6PQZ1\|AQP1_PIG LPGNSLGLNSLAPGVDSGQGLGIEIIGTLQLVLCVLATTDRR-RRDLGGSAPLAIGFSVA Q9N2J4\|AQP1_CANLF LPDNSLGRNELAPGVNSGQGLGIEIIGTLQLVLCVLATTDRR-RRDLGGSGPLAIGLSVA P47865\|AQP1_BOVIN LPDNSLGLNALAPGVNSGQGLGIEIIGTLQLVLCVLATTDRR-RRDLGGSGPLAIGFSVA P56401\|AQP1_SHEEP LPDNSLGLNALAPGVNSGQGLGIEIIGTLQLVLCVLATTDRRRRRDLGDSGPLAIGFSVA * **** * :************************ **..***:* P29972\|AQP1_HUMAN LGHLLAIDYTGCGINPARSFGSAVITHNFSNHWIFWVGPFIGGALAVLIYDFILAPRSSD Q5R819\|AQP1_PONAB LGHLLAIDYTGCGINPARSFGSAVITHNFSNHWIFWVGPFIGGALAVLIYDFILAPRSSD Q02013\|AQP1_MOUSE LGHLLAIDYTGCGINPARSFGSAVLTRNFSNHWIFWVGPFIGGALAVLIYDFILAPRSSD P29975\|AQP1_RAT LGHLLAIDYTGCGINPARSFGSAVLTRNFSNHWIFWVGPFIGSALAVLIYDFILAPRSSD Q6PQZ1\|AQP1_PIG LGHLLAIDYTGCGINPARSFGSAVITHNFQDHWVFWVGPFIGGALAVLIYDFILAPRSSD Q9N2J4\|AQP1_CANLF LGHLLAIDYTGCGINPARSFGSSVITHNFKDHWIFWVGPFIGGALAVLIYDFILAPRSSD P47865\|AQP1_BOVIN LGHLLAIDYTGCGINPARSFGSSVITHNFQDHWIFWVGPFIGAALAVLIYDFILAPRSSD P56401\|AQP1_SHEEP LGHLLAIDYTGCGINPARSFGSSVITHNFQDHWIFWVGPFIGAALAVLIYDFILAPRSSD *********************:::.::*****.*************** a a Extracellular Cytoplasmic Arg197 (p.R195W) Val178 (p.V176G) b c Extracellular Cytoplasmic b Extracellular Cytoplasmic Arg197 (p.R195W) Val178 (p.V176G) b c Fig. 7 Structural analysis of AQP1 mutations. a Multiple sequence alignment of human AQP1 with seven other mammals. The bovine AQP1 has the high resolution (2.2 Å) published structure. Mutations identiﬁed in PAH cases are highly conserved and highlighted in yellow. b Crystal structure of bovine AQP1 (PDB: 1j4n)22. Left: side view; right: top view from the extracellular direction. AQP1 is shown as a semi-transparent cartoon and ﬁve water molecules in the water channel are shown as red spheres. Key residues lining the water channels are represented with stick structures. c Magniﬁed view of the water channel, with H-bonds connected to water molecules in the channel highlighted. Two asparagine-proline-alanine (NPA) motifs, essential for the water transporting function of AQP1, are shown in magenta. Conserved His180 that constricts the water channel is shown in yellow. Mutations found in PAH cases, Arg195Trp and Val176Glu, are labelled and shown as orange stick structures. Arg195 and His180 are highly conserved in the known water channels and are strong indicators of water channel speciﬁcity. \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 Arg195Trp and Val176Glu mutations are predicted to disrupt the conformation of this conserved water channel b Cytoplasmic Arg197 (p.R195W) Val178 (p.V176G) Fig. 7 Structural analysis of AQP1 mutations. a Multiple sequence alignment of human AQP1 with seven other mammals. The bovine AQP1 has the high resolution (2.2 Å) published structure. Mutations identiﬁed in PAH cases are highly conserved and highlighted in yellow. b Crystal structure of bovine AQP1 (PDB: 1j4n)22. Left: side view; right: top view from the extracellular direction. AQP1 is shown as a semi-transparent cartoon and ﬁve water molecules in the water channel are shown as red spheres. Key residues lining the water channels are represented with stick structures. c Magniﬁed view of the water channel, with H-bonds connected to water molecules in the channel highlighted. Two asparagine-proline-alanine (NPA) motifs, essential for the water transporting function of AQP1, are shown in magenta. Conserved His180 that constricts the water channel is shown in yellow. Mutations found in PAH cases, Arg195Trp and Val176Glu, are labelled and shown as orange stick structures. Arg195 and His180 are highly conserved in the known water channels and are strong indicators of water channel speciﬁcity. Arg195Trp and Val176Glu mutations are predicted to disrupt the conformation of this conserved water channel Fig. 7 Structural analysis of AQP1 mutations. a Multiple sequence alignment of human AQP1 with seven other mammals. The bovine AQP1 has the high resolution (2.2 Å) published structure. Mutations identiﬁed in PAH cases are highly conserved and highlighted in yellow. b Crystal structure of bovine AQP1 (PDB: 1j4n)22. Left: side view; right: top view from the extracellular direction. AQP1 is shown as a semi-transparent cartoon and ﬁve water molecules in the water channel are shown as red spheres. Key residues lining the water channels are represented with stick structures. c Magniﬁed view of the water channel, with H-bonds connected to water molecules in the channel highlighted. Two asparagine-proline-alanine (NPA) motifs, essential for the water transporting function of AQP1, are shown in magenta. Conserved His180 that constricts the water channel is shown in yellow. Mutations found in PAH cases, Arg195Trp and Val176Glu, are labelled and shown as orange stick structures. Arg195 and His180 are highly conserved in the known water channels and are strong indicators of water channel speciﬁcity. Arg195Trp and Val176Glu mutations are predicted to disrupt the conformation of this conserved water channel Fig. 7 Structural analysis of AQP1 mutations. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 a b p.P55L HMG box p.Y137* β- catenin binding 136 68 144 353 p.R140P p.H132D p.P133S c.307+5G>C 66 p.L167Wfs213 p.R273Pfs92 p.T361K p.Y379F His132 (p.H132D) Pro133 (p.P133S) Arg140 (p.R140P) Gly303 Thr305 c 414 aa 378 P48431\|SOX2_HUMAN SRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYR P57073\|SOX8_HUMAN AQAARRKLADQYPHLHNAELSKTLGKLWRLLSESEKRPFVEEAERLRVQHKKDHPDYKYQ Q9H6I2\|SOX17_HUMAN AKDERKRLAQQNPDLHNAELSKMLGKSWKALTLAEKRPFVEEAERLRVQHMQDHPNYKYR P35713\|SOX18_HUMAN AKDERKRLAQQNPDLHNAVLSKMLGKAWKELNAAEKRPFVEEAERLRVQHLRDHPNYKYR :: ::::: .:: :* ** : . :***:::**. :::: 111 172 138 155 a b p.P55L HMG box p.Y137 136 68 144 p.R140P p.H132D p.P133S c.307+5G>C 66 p.L167Wfs213 p.R273Pfs92 His132 (p.H132D) Pro133 (p.P133S) Arg140 (p.R140P) Gly303 a a b p.P55L HMG box p.Y137* 136 68 144 p.R140P p.H132D p.P133S c.307+5G>C 66 p.L167Wfs213 His132 (p.H132D) Pro133 (p.P133S) p.R273Pfs92 Arg140 (p.R140P) Gly303 Thr305 b p.Y137* c.307+5G>C p.L167Wfs213 His132 (p.H132D) Pro133 (p.P133S) b b Arg140 (p.R140P) Pro133 (p.P133S) c SRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYR AQAARRKLADQYPHLHNAELSKTLGKLWRLLSESEKRPFVEEAERLRVQHKKDHPDYKYQ AKDERKRLAQQNPDLHNAELSKMLGKSWKALTLAEKRPFVEEAERLRVQHMQDHPNYKYR AKDERKRLAQQNPDLHNAVLSKMLGKAWKELNAAEKRPFVEEAERLRVQHLRDHPNYKYR :: ::::: .:: : ** : . :***:::**. :::: 111 172 138 155 SRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYR AQAARRKLADQYPHLHNAELSKTLGKLWRLLSESEKRPFVEEAERLRVQHKKDHPDYKYQ AKDERKRLAQQNPDLHNAELSKMLGKSWKALTLAEKRPFVEEAERLRVQHMQDHPNYKYR AKDERKRLAQQNPDLHNAVLSKMLGKAWKELNAAEKRPFVEEAERLRVQHLRDHPNYKYR :: ::::: .:: : ** : . :***:::**. :::: 111 172 138 155 PRRKTKTLMKKDKYTLPGGLLAPG---------------GNSMASGVGVGAGLGAGVNQR PRRRKSAKAGH---SDSDSG----------AELGPHPGGGAVYKAEAGLGDGHHHGDHTG PRRRKQVKRLK---RVEGGFLH-GLAEPQAAALGPE--GGRVAMD--GLGLQFP---EQG PRRKKQARKAR---RLEPGLLLPGLAPPQPPP--------------------------EP ** 156 219 187 186 LPQSQSTPK-RI---DTPSLEEPSDLEELEQFAKTFKQRRIKLGFTQGDVGLAMGKLYGN TPGAVKLEKEKLEQNPEESQDIKALQKELEQFAKLLKQKRITLGYTQADVGLTLGVLFGK * : . * :: * : : :***** ::.:.*:: :: 319 177 Fig. 8 Structural analysis of SOX17 mutations. a Schematic diagram of human SOX17 (Q9H6I2), based on UniProtKB annotation, and published reports23. Red arrows indicate PTVs and black arrows indicate missense mutations identiﬁed in PAH patients. The blue bar illustrates the region that is covered in the crystal structure (PDB: 3F27)64. The ability of SOX17 to activate transcription of target genes correlates with binding to β-catenin23. As illustrated, all PTVs lead to a loss of the β-catenin binding region. Two missense mutations are located within and very close to the minimum β-catenin binding regions, and both are highly conserved, indicating they are likely to be important for β-catenin binding. b Structural analysis of HMG domain missense mutations found in PAH patients. Left, Superposition of SOX17/DNA structure (Sox17: cyan, DNA: grey)64 onto SOX2/DNA/Oct1 structure (PDB: 1GT0, Sox2: yellow, Oct1: magenta, DNA: light blue)24. Right: Magniﬁed view of the interactions around Arg140 in the SOX2/DNA/Oct structure. Arg140 in SOX2 makes multiple H-bond interactions and mutating this Arg in SOX2 abolishes the interaction with transcription factors Pax6 and Oct424. SOX2 and SOX17 both bind to Oct465 and SOX17 K122E mutant can replace SOX2 in maintaining stem cell pluripotency65, indicating this region in SOX17 may interact with Oct4, similar to SOX2. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 a Multiple sequence alignment of human AQP1 with seven other mammals. The bovine AQP1 has the high resolution (2.2 Å) published structure. Mutations identiﬁed in PAH cases are highly conserved and highlighted in yellow. b Crystal structure of bovine AQP1 (PDB: 1j4n)22. Left: side view; right: top view from the extracellular direction. AQP1 is shown as a semi-transparent cartoon and ﬁve water molecules in the water channel are shown as red spheres. Key residues lining the water channels are represented with stick structures. c Magniﬁed view of the water channel, with H-bonds connected to water molecules in the channel highlighted. Two asparagine-proline-alanine (NPA) motifs, essential for the water transporting function of AQP1, are shown in magenta. Conserved His180 that constricts the water channel is shown in yellow. Mutations found in PAH cases, Arg195Trp and Val176Glu, are labelled and shown as orange stick structures. Arg195 and His180 are highly conserved in the known water channels and are strong indicators of water channel speciﬁcity. Arg195Trp and Val176Glu mutations are predicted to disrupt the conformation of this conserved water channel We identiﬁed three heterozygous frameshift variants, two stop gained, two splice region variants in ATP13A3, which are predicted to lead to loss of ATPase catalytic activity (Fig. 6a). In addition, we identiﬁed 4 heterozygous likely pathogenic missense variants in PAH cases, two near the conserved ATPase catalytic site and predicted to destabilise the conformation of the catalytic domain (Fig. 6b–d). The distribution of variants (Fig. 6a) suggests that these mutations impact critically on the function of the protein. The majority of rare variants identiﬁed in AQP1, which encodes aquaporin-1, are situated within the critical water channel (Fig. 7). In particular the p.Arg195Trp variant, identiﬁed NATURE COMMUNICATIONS\| (2018) 9:1416 8 ARTICLE \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 well characterised. Thus we employed immunohistochemistry to examine localisation in the normal and hypertensive human pulmonary vasculature. Figure 9 shows that AQP1, ATP13A3 and SOX17 are predominantly localised to the pulmonary endothe- lium in normal human lung and to endothelial cells within plexiform lesions of patients with idiopathic PAH. In addition, we determined the relative mRNA expression levels of AQP1, ATP13A3 and SOX17 in primary cultures of pulmonary artery smooth muscle cells (PASMCs), pulmonary artery endothelial cells (PAECs) and blood outgrowth endothelial cells (BOECs)25. AQP1 was expressed in PASMCs and endothelial cells, with a trend towards higher levels in PASMCs (Fig. 10a). ATP13A3 was highly expressed in both cell types (Fig. 10b), whereas SOX17 was almost exclusively expressed in endothelial cells (Fig. 10c). Although AQP1 and SOX17 are known to play roles in endothelial function, the function of ATP13A3 in vascular cells is entirely unknown. Thus, we determined the impact of ATP13A3 knockdown on proliferation and apoptosis of BOECs. Loss of ATP13A3 led to marked inhibition of serum-stimulated proliferation of BOECs, and increased apoptosis in serum- deprived conditions (Fig. 10d–f). Discussion We report a comprehensive analysis of rare genetic variation in a large cohort of index cases with idiopathic and heritable forms of PAH. Whilst we utilised WGS, the main goal was the identiﬁ- cation of rare causal variation underlying PAH in the protein- coding sequence. The approach involved a rigorous case-control comparison using a tiered search for variants. First, we searched for high impact PTVs overrepresented in cases, having excluded previously established PAH genes. This revealed PTVs in ATP13A3, a poorly characterised P-type ATPase of the P5 subfamily26. There is little information regarding the function of the ATPase, ATP13A3, which appears widely expressed in mouse tissues26. Although, the precise substrate speciﬁcity is unknown, ATP13A3 plays a role in polyamine transport27. Based on available RNA sequencing data, ATP13A3 is highly expressed in human pulmonary vascular cells and cardiac tissue (https:// www.encodeproject.org). We conﬁrmed that ATP13A3 mRNA is expressed in primary cultured pulmonary artery smooth muscle cells and endothelial cells, and provide preliminary data that loss of ATP13A3 inhibits proliferation and increases apoptosis of endothelial cells. These ﬁndings are consistent with the widely b c e f h i d g j a Concentric lesion Plexiform lesion Fig. 9 Immunolocalisation of AQP1, ATP13A3 and SOX17 in normal and PAH lung. The typical histological ﬁndings (haematoxylin and eosin staining) of concentric vascular lesions with associated plexiform lesions are shown (a). Higher magniﬁcation images of plexiform lesion (b), with frequent endothelialised channels (c; anti-CD31) surrounded by myoﬁbroblasts (d; anti-SMα). Additional high magniﬁcation images demonstrating endothelial expression of ATP13A3 (e), AQP1 (f) and SOX17 (g) in PAH lung. Controls lung sections demonstrating predominantly endothelial expression of ATP13A3 (h), AQP1 (i) and SOX17 (j). (Scale bars = 50 µm) b c e f h i d g j a Concentric lesion Plexiform lesion Fig. 9 Immunolocalisation of AQP1, ATP13A3 and SOX17 in normal and PAH lung. The typical histological ﬁndings (haematoxylin and eosin staining) of concentric vascular lesions with associated plexiform lesions are shown (a). Higher magniﬁcation images of plexiform lesion (b), with frequent endothelialised channels (c; anti-CD31) surrounded by myoﬁbroblasts (d; anti-SMα). Additional high magniﬁcation images demonstrating endothelial expression of ATP13A3 (e), AQP1 (f) and SOX17 (g) in PAH lung. Controls lung sections demonstrating predominantly endothelial expression of ATP13A3 (h), AQP1 (i) and SOX17 (j). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 The three missense mutations in SOX17 will likely disrupt interaction with Oct4. c Supporting the analysis in b, sequence alignment shows that the HMG domain of SOX2 (P48431) and SOX17 as well as SOX8 (P57073) and SOX18 (P35713) share high sequence identity and the three mutations found in PAH (highlighted in yellow) are highly conserved emphasising their functional importance. Similarly, the Gly and Thr that interact with Arg140 in SOX2 (highlighted in yellow) are also conserved between Oct1 (PO2F1) and Oct4 (PO5F1) family) predicted to lead to loss of the beta-catenin binding region, and six missense variants predicted to disrupt interactions with Oct4 and beta-catenin23,24 (Fig. 8). in ﬁve PAH cases, locates at the hydrophilic face of the pore. This arginine at position 195 helps deﬁne the constriction region of the AQP1 pore structure and is conserved across the water-speciﬁc aquaporins22. Rare variants in SOX17, included four nonsense variants (including the PTV identiﬁed in the additional UK GDF2 is known to be secreted from the liver, but the cellular localisation of proteins encoded by the other novel genes is less \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications NATURE COMMUNICATIONS\| (2018) 9:1416 9 NATURE COMMUNICATIONS\| (2018) 9:1416 ARTICLE Discussion a–c Expression of a AQP1, b ATP13A3 and c SOX17 mRNA in human pulmonary artery smooth muscle cells, pulmonary artery endothelial cells and blood outgrowth endothelial cells (BOECs) (n = 4 biological replicates of each). Relative expression of each transcript was normalised to three reference genes, ACTB, B2M and HPRT. d Proliferation of BOECs in 5% FBS over 6 days. Cells were transfected with DharmaFECT1 alone (DH1), siATP13A3 or non-targeting siRNA control (siCP) e, f Quantiﬁcation of apoptosis in BOECs, deﬁned as Annexin V+/PI−cells, in BOECs transfected with siATP13A3 or siCP in complex with DH1 followed by 24 h treatment with 0.1% FBS or 5% FBS (n = 4 biological repeats). f Measurement of apoptosis via Caspase-Glo 3/7 activity measurements in BOECs transfected with siATP13A3 or siCP in complex with DH1, followed by 16 h treatment in 0.1% FBS or 5% FBS. Data are from a single experiment (n = 4 wells) representative of 3 biological repeats. Data were analysed using a One-way analysis of variance with post hoc Tukey’s test for multiple comparisons in d and f. Data were analysed using a repeated measures One-way analysis of variance with post hoc Tukey’s for multiple comparisons in e. P < 0.05, P < 0.01 within treatment groups. ###P < 0.001 for effect of ligand against control for same transfection condition ovel genes. a–c Expression of a AQP1, b ATP13A3 and c SOX17 mRNA in human pulmonary artery smooth muscle cel SOX17. Of note, both AQP1 and SOX17 were within the top 8 ranked genes in our combined PTV and missense burden test analysis (Supplementary Table 7), providing further conﬁdence in their causative contribution to PAH. accepted paradigm that endothelial apoptosis is a major trigger for the initiation of PAH28,29. It will be of considerable interest to determine the role of ATP13A3 in vascular cells and whether it is functionally associated with BMP signalling, or represents a dis- tinct therapeutic target in PAH. Aquaporin-1 belongs to a family of membrane channel pro- teins that facilitate water transport in response to osmotic gra- dients22, and AQP1 is known to promote endothelial cell migration and angiogenesis32. Thus, approaches that maintain or restore pulmonary endothelial function could offer new ther- apeutic directions in PAH. Discussion (Scale bars = 50 µm) 10 NATURE COMMUNICATIONS\| (2018)9:1416 \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications b d c e g j h Fig. 9 Immunolocalisation of AQP1, ATP13A3 and SOX17 in normal and PAH lung. The typical histological ﬁndings (haematoxylin and eosin staining) of concentric vascular lesions with associated plexiform lesions are shown (a). Higher magniﬁcation images of plexiform lesion (b), with frequent endothelialised channels (c; anti-CD31) surrounded by myoﬁbroblasts (d; anti-SMα). Additional high magniﬁcation images demonstrating endothelial expression of ATP13A3 (e), AQP1 (f) and SOX17 (g) in PAH lung. Controls lung sections demonstrating predominantly endothelial expression of ATP13A3 (h), AQP1 (i) and SOX17 (j). (Scale bars = 50 µm) \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications 10 NATURE COMMUNICATIONS\| (2018) 9:1416 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 1 0.20 10–1 50,000 40,000 30,000 Cell number (cells/well) 20,000 10,000 0 * ** * 10–2 10–3 10–4 10–5 10–6 10–7 10–8 0.15 0.10 0.05 0.00 0.1 0.01 AQP1 expression (relative to 3HK) ATP13A3 expression (relative to 3HK) SOX17 expression (relative to 3HK) 0.001 0.0001 PASMC PAEC BOEC PASMC PAEC BOEC PASMC PAEC BOEC DH1 siATP13A3 siCP a b c d 0.1% FBS Luminescence (arbitrary units × 106) 2.5 * * 2 1.5 1 0.5 5% FBS 0.1% FBS 5% FBS 40 * ** 30 20 % Annexin-V+/PI– 10 0 DH1 siATP13A3 DH1 siATP13A3 siCP siCP DH1 siATP13A3 DH1 siATP13A3 siCP siCP ### ### ### ### ### ### e f Fig. 10 Functional studies of novel genes. a–c Expression of a AQP1, b ATP13A3 and c SOX17 mRNA in human pulmonary artery smooth muscle cells, pulmonary artery endothelial cells and blood outgrowth endothelial cells (BOECs) (n = 4 biological replicates of each). Relative expression of each transcript was normalised to three reference genes, ACTB, B2M and HPRT. d Proliferation of BOECs in 5% FBS over 6 days. Cells were transfected with DharmaFECT1 alone (DH1), siATP13A3 or non-targeting siRNA control (siCP) e, f Quantiﬁcation of apoptosis in BOECs, deﬁned as Annexin V+/PI−cells, in BOECs transfected with siATP13A3 or siCP in complex with DH1 followed by 24 h treatment with 0.1% FBS or 5% FBS (n = 4 biological repeats). f Measurement of apoptosis via Caspase-Glo 3/7 activity measurements in BOECs transfected with siATP13A3 or siCP in complex with DH1, followed by 16 h treatment in 0.1% FBS or 5% FBS. \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications Discussion Data are from a single experiment (n = 4 wells) representative of 3 biological repeats. Data were analysed using a One-way analysis of variance with post hoc Tukey’s test for multiple comparisons in d and f. Data were analysed using a repeated measures One-way analysis of variance with post hoc Tukey’s for multiple comparisons in e. P < 0.05, P < 0.01 within treatment groups. ###P < 0.001 for effect of ligand against control for same transfection condition d b c P siAT Luminescence (arbitrary units × 106) 2.5 * 2 1.5 1 0.5 0.1% FBS 5% FBS DH1 siATP13A3 DH1 siATP13A3 siCP siCP ### ### ### f 0.1% FBS 5% FBS 40 * ** 30 20 % Annexin-V+/PI– 10 0 DH1 siATP13A3 DH1 siATP13A3 siCP siCP ### ### ### e Fig. 10 Functional studies of novel genes. a–c Expression of a AQP1, b ATP13A3 and c SOX17 mRNA in human pulmonary artery smooth muscle cells, pulmonary artery endothelial cells and blood outgrowth endothelial cells (BOECs) (n = 4 biological replicates of each). Relative expression of each transcript was normalised to three reference genes, ACTB, B2M and HPRT. d Proliferation of BOECs in 5% FBS over 6 days. Cells were transfected with DharmaFECT1 alone (DH1), siATP13A3 or non-targeting siRNA control (siCP) e, f Quantiﬁcation of apoptosis in BOECs, deﬁned as Annexin V+/PI−cells in BOECs transfected with siATP13A3 or siCP in complex with DH1 followed by 24 h treatment with 0.1% FBS or 5% FBS (n = 4 biological repeats). f Measurement of apoptosis via Caspase-Glo 3/7 activity measurements in BOECs transfected with siATP13A3 or siCP in complex with DH1, followed by 16 h treatment in 0.1% FBS or 5% FBS. Data are from a single experiment (n = 4 wells) representative of 3 biological repeats. Data were analysed using a One-way analysis of variance with post hoc Tukey’s test for multiple comparisons in d and f. Data were analysed using a repeated measures One-way analysis of variance with post hoc Tukey’s for multiple comparisons in e. P < 0.05, P < 0.01 within treatment groups. ###P < 0.001 for effect of ligand against control for same transfection condition Fig. 10 Functional studies of novel genes. Methods Ethi d The non-PAH control cohort consisted of subjects with bleeding, thrombotic and platelet disorders (15.5%), cerebral small vessel disease (2.1%), Ehlers-Danlos syndrome (0.3%), subjects recruited to Genomics England Ltd (19.8%), hypertrophic cardiomyopathy (3.6%), intrahepatic cholestasis of pregnancy (4.1%), Leber hereditary optic neuropathy (0.9%), multiple primary tumours (7.8%), neuropathic pain disorder (2.6%), primary immune disorders (15.3%), primary membranoproliferative glomerulonephritis (2.3%), retinal dystrophies/paediatric neurology and metabolic disease (19.8%), stem cell and myeloid disorders (2.1%), steroid resistant nephrotic syndrome (3.6%), and others (0.3%), or their ﬁrst degree relatives. p The non-PAH cohort used in the case-control comparisons for this study comprised individuals, or relatives of individuals, with other rare diseases recruited to the NIHR BR-RD in the UK (Methods section). In general, for very rare causal variants, the comparison between PAH cases and non-PAH rare disease controls should not reduce our ability to detect over- representation of rare variants in a particular gene in the PAH cohort, if mutations in that gene are speciﬁc to PAH. However, if rare variants in a gene were responsible for more than one phe- notype, it is possible that this would reduce the power to detect overrepresentation in the PAH cohort. For example, if mutations occurred in different functional domains of the expressed protein, this might lead to PAH if mutations affected one domain, but other phenotypes if they affected another domain. Overcoming this potential limitation will require additional analysis of the functional impact of variants and their distribution within a gene, and more detailed information on the phenotypes of subjects in the non-PAH group. High-throughput sequencing. DNA extracted from venous blood underwent whole-genome sequencing using the Illumina TruSeq DNA PCR-Free Sample Preparation kit (Illumina Inc., San Diego, CA, USA) and Illumina HiSeq 2000 or HiSeq X sequencer, generating 100–150 bp reads with a minimum coverage of 15× for ~95% of the genome (mean coverage of 35×). Whole-exome sequencing was conducted for individual II-1 (Fig. 4a) using genomic DNA extracted from per- ipheral blood. Paired-end sequence reads were generated on an Illumina HiSeq 2000. Generation of analysis-ready data sets. Sequencing reads were pre-processed by Illumina with Isaac Aligner and Variant Caller (v2, Illumina Inc.) using human genome assembly GRCh37 as reference. Variants were normalised, merged into multi-sample VCF ﬁles by chromosome using the gVCF aggregation tool agg (https://github.com/Illumina/agg) and annotated with Ensembl’s Variant Effect Predictor (VEP). Methods Ethi d Ethics and patient selection. Cases were recruited from the UK National Pul- monary Hypertension Centres, Universite Sud Paris (France), the VU University Medical Center Amsterdam (The Netherlands), the Universities of Gießen and Marburg (Germany), San Matteo Hospital, Pavia (Italy), and Medical University of Graz (Austria). All cases had a clinical diagnosis of idiopathic PAH, heritable PAH, drug-associated and toxin-associated PAH, or PVOD/PCH established by their expert centre. The non-PAH cohort for the case-control comparison comprised 6385 unrelated subjects recruited to the NIHR BR-RD study. All PAH and non- PAH patients provided written informed consent (UK Research Ethics Committee: 13/EE/0325), or local forms consenting to genetic testing in deceased patients and non-UK cases. An additional UK family diagnosed with HPAH was ascertained as described previously39. Blood and saliva samples were collected under written informed consent of the participants or their parents for use in gene identiﬁcation studies (UK Research Ethics Committee: 08/H0802/32). Whilst the SKAT-O analysis also provided support for the MFRP gene, recessive biallelic mutations in MFRP cause retinal degeneration and posterior microphthalmos37. The expression of MFRP transcripts is largely conﬁned to the central nervous sys- tem38 and the majority of variants were present in the Genome Aggregation Database (GnomAD, http://gnomad.broadinstitute. org). On the basis of these considerations, variants in MFRP are unlikely to contribute to PAH aetiology. This analysis provides new insights on the frequency and validity of previously reported genes in PAH. We conﬁrmed that mutations in BMPR2 are the most common genetic cause and validated rare causal variants in ACVRL1, ENG, SMAD9, TBX4, KCNK3 and EIF2AK4. Although our ﬁndings question the validity of CAV1, SMAD1 and SMAD4 as causal genes, previous reports might represent private mutations occurring in very rare families. The use of WGS in this study allowed closer inter- rogation of larger deletions around the BMPR2 locus than has been possible previously. Nevertheless, additional analyses are required to determine the full impact of structural variation (inversions, duplications, smaller deletions) at this and other loci. Composition of non-PAH control cohort. Discussion Conversely, AQP1 inhibition in pul- monary artery smooth muscle cells ameliorated hypoxia-induced pulmonary hypertension in mice33, suggesting that further studies are required to determine the key cell type impacted by AQP1 mutations in human PAH, and the functional impact of these AQP1 variants on water transport. The demonstration of familial segregation of AQP1 variants with PAH provides further support for the potentially causal role of these mutations in disease. However, we also identiﬁed an unaffected AQP1 variant carrier consistent with reduced penetrance, which is well described for other PAH genes, including BMPR2. p g Analysis of missense variation, and a combined analysis of all predicted deleterious variation, revealed that mutation at the GDF2 gene is also signiﬁcant determinant of predisposition to PAH. Of the new genes identiﬁed, GDF2 provides further evi- dence for the central role of the BMP signalling pathway in PAH. GDF2 encodes the major circulating ligand for the endothelial BMPR2/ACVRL1 receptor complex20. Taken together, the genetic ﬁndings suggest that a deﬁciency in GDF2/BMPR2/ ACVRL1 signalling in pulmonary artery endothelial cells is cri- tical in PAH pathobiology. The majority of GDF2 variants detected in our adult-onset PAH cohort were heterozygous mis- sense variants, in contrast to a previous case report of childhood- onset PAH due to a homozygous nonsense mutation30. The ﬁnding of causal GDF2 variants in PAH cases, associated with reduced production of GDF2 from cells, provides further support for investigating replacement of this factor as a therapeutic strategy in PAH31. Although functional studies are required to conﬁrm the mechanisms by which mutations in SOX17 cause PAH, this ﬁnding provides additional support for the vascular endothelium as the major initiating cell type in this disorder. SOX17 encodes the SRY-box containing transcription factor 17, which plays a fundamental role in angiogenesis34 and arteriovenous To maximise the assessment of rare variation in a case-control study design, we deployed the SKAT-O test. This approach revealed a signiﬁcant association of rare variation in the aqua- porin gene, AQP1, and the transcription factor encoded by 11 NATURE COMMUNICATIONS\| (2018) 9:1416 \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 differentiation35. Moreover, conditional deletion of SOX17 in mesenchymal progenitors leads to impaired formation of lung microvessels36. The demonstration of familial segregation of the SOX17 p.Y137 PTV with early onset PAH provides additional evidence for a causal role for these variants in PAH. Discussion The co- existence of a patent ductus arteriosus in the index case and an atrial septal defect (ASD) in one of the affected offspring is of interest and suggests an association with congenital heart disease. Small ASDs are not uncommon in idiopathic PAH, and a more detailed clinical phenotyping of SOX17 mutation carriers will be required to determine whether the presence of ASDs and other congenital heart abnormalities are more common in carriers of these mutations. differentiation35. Moreover, conditional deletion of SOX17 in mesenchymal progenitors leads to impaired formation of lung microvessels36. The demonstration of familial segregation of the SOX17 p.Y137* PTV with early onset PAH provides additional evidence for a causal role for these variants in PAH. The co- existence of a patent ductus arteriosus in the index case and an atrial septal defect (ASD) in one of the affected offspring is of interest and suggests an association with congenital heart disease. Small ASDs are not uncommon in idiopathic PAH, and a more detailed clinical phenotyping of SOX17 mutation carriers will be required to determine whether the presence of ASDs and other congenital heart abnormalities are more common in carriers of these mutations. regulatory regions to PAH aetiology remains to be determined. This will require functional annotation of regulatory and other non-coding regions speciﬁc for relevant cell types, further case- control analyses of these regions and ultimately functional studies of gene regulation to assess the pathogenicity of non-coding variants. Our ﬁndings to date provide support for a central role of the pulmonary vascular endothelium in disease pathogenesis, and suggest new mechanisms that could be exploited therapeutically in this life-limiting disease. Methods Ethi d Following read alignment to the reference genome (GRCh37), variant calling and annotation of whole-exome data for individual II:1 were per- formed using GATK UniﬁedGenotyper40 and ANNOVAR41, respectively. Anno- tations included minor allele frequencies from other control data sets (i.e. ExAC42, 1000 Genomes Project43 and UK10K44) as well as deleteriousness and conservation scores (i.e., CADD45, SIFT46, PolyPhen-247 and Gerp48) enabling further ﬁltering and assessment of the likely pathogenicity of variants. To take forward only high quality calls, the pass frequency (proportion of samples containing alternate alleles that passed the original variant ﬁltering) and call rate (proportion of samples with reference or alternate genotypes) were combined into the overall pass rate (OPR: pass frequency × call rate) and variants with an OPR of 80% or higher were retained. Taken together, this study identiﬁes rare sequence variation in new genes underlying heritable forms of PAH, and provides a unique resource for future large-scale discovery efforts in this disorder. Mutations in previously established genes accounted for 19.9% of PAH cases. Including new genes identiﬁed in this study (GDF2, ATP13A3, AQP1, and SOX17), the total proportion of cases explained by mutations increased to 23.5%. It is likely that independent conﬁrmation of the expanded list of putative genes identiﬁed in this study will increase further the proportion of cases explained by mutations, but this will require larger inter- national collaborations. The results suggest that the genetic architecture of PAH, beyond mutations in BMPR2, is char- acterised by substantial genetic heterogeneity and consists of rare heterozygous coding region mutations shared by small numbers of cases. The contribution of rare variation within non-coding Estimation of ethnicity and relatedness. We estimated the population structure and relatedness based on a representative set of SNPs using the R package GEN- ESIS to perform PC Air49 and PC Relate50, respectively. The selected 35,114 autosomal SNPs were present on Illumina genotyping arrays (HumanCoreExome- 12v1.1, HumanCoreExome-24v1.0, HumanOmni2.5–8v1.1), do not overlap quality control excluded regions or multiallelic sites in the 1000 Genomes (1000 G) Phase 3 data set43, do not have any missing genotypes in NIHR BR-RD, had a MAF of 0.3 or above and LD pruning was performed using PLINK51 with a window size of 50 bp, window shift of 5 bp and a variance inﬂation factor threshold of 2. \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications ARTICLE Of the 9110 NIHR BR-RD samples, we assigned 80.2% to Non-Finish European (n = 7307), 7.2% to South Asian (n = 649), 2.3% to African (n = 213), 0.08% to East Asian (n = 78), 0.02% to Finnish-European (n = 19) and 9.2% to Other (n = 844) and retrieved a maximum set of 7,493 unrelated individuals (UWGS10K), representing 82.2% of the entire NIHR BR-RD cohort. Production of pGDF2 wild type and variant proteins. The cloning of human wild-type pro-GDF2 (pGDF2) in pCEP4 has been described previously59. Site- directed mutagenesis was performed according to the manufacturer’s instructions (QuickChange Site-Directed Mutagenesis Kit, Agilent Technologies). Mutations were conﬁrmed by Sanger sequencing. HEK-EBNA cells were transfected with plasmids containing either wild-type or mutant pGDF2 for 14 h. The transfecting supernatant was removed and replaced with CDCHO media (Invitrogen) for 5 days to express the proteins. The conditioned media containing GDF2 and the variants were collected and snap-frozen on dry-ice before being stored at −80 °C. For each variant, conditioned media from three independent transfections were collected for further characterisation. Cohort deﬁnition and allele frequency calculation. Based on the relatedness analysis, we deﬁned the following sample subsets: (a) the maximum number of unrelated non-PAH controls (UPAHC, n = 6385), (b) all affected PAH cases (PAHAFF, n = 1048), and (c) all unrelated PAH index cases (PAHIDX, n = 1038). These subsets were used to annotate the variants in the multi-sample VCF ﬁle with calculated minor allele frequencies using the ﬁll-tags extension of BCFtools52. GDF2 ELISA. High binding 96-well ELISA plates (Greiner, South Lanarkshire, UK) were coated with 0.2 µg/well of mouse monoclonal anti-human GDF2 antibody (R&D Systems, Oxfordshire, UK) in PBS (0.1 M phosphate pH7.4, 0.137 M NaCl, 2.7 mM KCl, Sigma) overnight at 4 °C in a humidiﬁed chamber. Plates were washed with PBS containing 0.05% (v/v) Tween-20 (PBS-T), followed by blocking with 1% bovine serum albumin in PBS-T (1% BSA/PBS-T) for 90 min at room temperature. Recombinant human GDF2 standards (1–3000 pg/ml) or conditioned media samples (100 µl/well of 1:30, 1:100, 1:300, 1:1000, 1:3000 and 1:10,000 dilutions) were then added and incubated for 2 h at room temperature. After washing, plates were then incubated with 0.04 µg/well biotinylated goat anti-human GDF2 (R&D Systems) in 1% BSA/PBS-T for 2 h. Plates were washed, then incubated with ExtrAvidin(r)-Alkaline phosphatase (Sigma) diluted 1:400 in 1% BSA/PBS-T for 90 min. ARTICLE The derivation of human tissues and cells was approved by Papworth Hospital ethical review com- mittee (Ref 08/H0304/56 + 5) and all subjects provided informed and written consent. Burden analysis of protein-truncating and missense variants. Filtered variants were grouped per gene and consequence type (predicted PTV/missense) and subjects with at least one variant were counted (no double counting) per group and tested for association with disease. We applied a one-tailed Fisher’s exact test with post hoc Bonferroni correction to calculate the P value for genome-wide signiﬁcance. 37 °C in 95% air/5% CO2 until PASMCs had formed conﬂuent monolayers. Cells were then trypsinized, and for subsequent passages cells were maintained in DMEM supplemented with 10% FBS. The cellular phenotype of PASMCs was conﬁrmed by positive immunoﬂuorescence staining with anti-smooth muscle speciﬁc alpha-actin (Clone IA4 Sigma-Aldrich; 1:100 dilution). The derivation of human tissues and cells was approved by Papworth Hospital ethical review com- mittee (Ref 08/H0304/56 + 5) and all subjects provided informed and written consent. Rare variant analysis using SKAT-O. To further investigate the aggregated effect that rare variants contribute to PAH aetiology, we applied a Sequence Kernel Association test (SKAT-O). SKAT-O increases the power of discovery under dif- ferent inheritance models by combining variance-component and burden tests. Variants were ﬁltered based on MAF as speciﬁed above, and only PTV and mis- sense variants were included. For the analysis we implemented SKAT-O in RvTests v1.9.953 with default parameters and weights being Beta(1,25), and applying a correction for read length, gender and the ﬁrst ﬁve principal components of the ethnicity PCA. Variants were collapsed considering only the protein-coding region in the canonical transcript of the protein-coding genes in the genome assembly GRCh37. Human blood outgrowth endothelial cells (BOECs) were derived from 40–80 ml of peripheral venous blood isolated from healthy subjects. The study was approved by the Cambridgeshire 3 Research Ethics Committee (Ref 11/EE/0297), and all subjects provided informed and written consent. BOECs were cultured in 10% FBS supplemented with EGM-2MV (Life Technologies, Carlsbad, CA). Cells were used between passages 4 and 860. The endothelial phenotype of BOECs was determined by ﬂow cytometry for expression of endothelial surface markers, as described previously25. Cells were routinely tested to exclude mycoplasma infection. Analysis of large deletions. Copy number variation was identiﬁed using Canvas54 and Manta55. Deletions called by both Manta and Canvas with a reciprocal overlap of ≥20% were retained. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 data were used to perform a principal component analysis (PCA) using PC Air. We modelled the scores of the leading ﬁve principal components as data generated by a population speciﬁc multivariate Gaussian distribution and estimated the corre- sponding mean and covariance parameters. Genotypes from the NIHR BR-RD samples were projected onto the loadings for the leading ﬁve principal components from the 1000 G PCA and we computed the likelihood that each sample belonged to each subpopulation under a mixture of multivariate Gaussians models. Each sample was allocated to the population with the highest likelihood, unless the highest likelihood was similar to likelihood values for other populations, as might be expected for example under admixed ancestry or if the sample came from a population not included in 1000 G. Such ambiguous samples were labelled as “other”. PC Relate was used to to identify related individuals in NIHR BR-RD. We used the ﬁrst 20 PCs from PC Air to adjust for relatedness and extracted the pairwise Identity-By-State distances and kinship values. The pairwise information was used by Primus to infer family networks and calculate the maximum set of unrelated samples. Conﬁrmation of variants. Variant sequencing reads for SNVs, indels and deletions were visualised for validation on Integrative Genomes Viewer (IGV)57, and were conﬁrmed by diagnostic capture-based high-throughput sequencing, if the IGV inspection was not satisfactory. For the familial segregation analysis, linkage to the BMPR2 locus was ﬁrst examined by microsatellite genotyping analysis. Mutation screening of the BMPR2, ACVRL1, ENG, AQP1 and SOX17 genes was conducted by capillary sequencing using BigDye Terminator v3.1 chemistry. All DNA frag- ments were resolved on an ABI Fragment Analyzer (Applied Biosystems). All primer sequences are listed in Supplementary Table 9. The family trees were drawn using the R package FamAgg58. Structural analysis of novel variants. The domain structures and the functional groups of the novel PAH genes were plotted according to the entry in UniProtKB. Clustal Omega was used for sequence alignment. Structural data were obtained from RCSB Protein Data Bank and analysed according to published reports. Fig- ures were generated using PyMOL Molecular Graphics System. ARTICLE Plates were washed with PBS-T followed by water. The ELISA was developed with a colorimetric substrate comprising 1 mg/ml 4-Nitrophenyl phos- phate disodium salt hexahydrate (Sigma) in 1 M Diethanolamine, pH 9.8 con- taining 0.5 mM MgCl2. The assay was developed in the dark at room temperature and the absorbance measured at 405 nm. Rare variant ﬁltering. Filtering of rare variants was performed as follows: (1) variants with a MAF less than 1 in 10,000 in UPAHC subjects, UK10K and ExAC were retained (adjusted for X chromosome variants to 1 in 8000); (2) variants with a combined annotation dependent depletion deleteriousness (CADD) score of less than 15 were excluded. CADD scores were calculated using the CADD web service (http://cadd.gs.washington.edu) for variants lacking a score; (3) premature trun- cating variants (PTVs) or missense variants of the canonical transcript were retained; 4) missense variants predicted to be both tolerated and benign by SIFT and PolyPhen-2, respectively, were removed. y y To identify likely causative mutations (as reported in Supplementary Table 3), variants in previously reported and putative genes, identiﬁed in this study, were examined in more detail to exclude variants that did not segregate in families (where data available). Furthermore, variants shared between cases and non-PAH controls, as well as variants of uncertain signiﬁcance that co-occurred with previously reported causative mutations or high impact PTVs were also excluded. Cell culture and treatments. Distal human pulmonary artery smooth muscle cells (PASMCs) were cultured from explants dissected from lung resection specimens. Small pulmonary arterioles (0.5 to 2 mm diameter) were dissected and divided into small pieces before plating in T25 ﬂasks. Explants were left to adhere for 2 h and then incubated in DMEM/20% FBS plus amino acids at Cell culture and treatments. Distal human pulmonary artery smooth muscle cells (PASMCs) were cultured from explants dissected from lung resection specimens. Small pulmonary arterioles (0.5 to 2 mm diameter) were dissected and divided into small pieces before plating in T25 ﬂasks. Explants were left to adhere for 2 h and then incubated in DMEM/20% FBS plus amino acids at 37 °C in 95% air/5% CO2 until PASMCs had formed conﬂuent monolayers. Cells were then trypsinized, and for subsequent passages cells were maintained in DMEM supplemented with 10% FBS. The cellular phenotype of PASMCs was conﬁrmed by positive immunoﬂuorescence staining with anti-smooth muscle speciﬁc alpha-actin (Clone IA4 Sigma-Aldrich; 1:100 dilution). Methods Ethi d The 2,110 samples from the 1000 G Project including the European (EUR), African (AFR), South Asian (SAS) and East Asian (EAS) populations (excluding the admixed American population) were ﬁltered for the selected SNPs and the ﬁltered Estimation of ethnicity and relatedness. We estimated the population structure and relatedness based on a representative set of SNPs using the R package GEN- ESIS to perform PC Air49 and PC Relate50, respectively. The selected 35,114 autosomal SNPs were present on Illumina genotyping arrays (HumanCoreExome- 12v1.1, HumanCoreExome-24v1.0, HumanOmni2.5–8v1.1), do not overlap quality control excluded regions or multiallelic sites in the 1000 Genomes (1000 G) Phase 3 data set43, do not have any missing genotypes in NIHR BR-RD, had a MAF of 0.3 or above and LD pruning was performed using PLINK51 with a window size of 50 bp, window shift of 5 bp and a variance inﬂation factor threshold of 2. The 2,110 samples from the 1000 G Project including the European (EUR), African (AFR), South Asian (SAS) and East Asian (EAS) populations (excluding the admixed American population) were ﬁltered for the selected SNPs and the ﬁltered NATURE COMMUNICATIONS\| (2018) 9:1416 12 \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 RNA using High Capacity Reverse Transcriptase kit (Applied Biosystems, Foster City, CA). Quantitative PCR reactions employed MicroAmp optical 96-well reac- tion plates (Applied Biosystems). 50 ng µl−1 cDNA was used with SYBR Green Jumpstart Taq Readymix (Sigma-Aldrich), ROX reference dye (Invitrogen) using custom made sense and anti-sense primers (all 200 nmol l−1). Primers for human ACTB (encoding β-actin), AQP1, ATP13A3, B2M, HPRT and SOX17 were designed using PrimerBLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/; Supplementary Table 9). Reactions were ampliﬁed on a Quantstudio 6 Real-Time PCR system (Applied Biosystems). The relative abundance of each target gene in different cell lines was compared using the equation 2−(CtGOI-Ct3HK), where Ct3HK 5. Deng, Z. et al. Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene. Am. J. Hum. Genet. 67, 737–744 (2000). 6. Evans, J. D. et al. BMPR2 mutations and survival in pulmonary arterial hypertension: an individual participant data meta-analysis. Lancet Respir. Med. 4, 129–137 (2016). 7. Trembath, R. C. et al. Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia. N. Engl. J. Med. 345, 325–334 (2001). 8. Harrison, R. E. et al. Transforming growth factor-beta receptor mutations and pulmonary arterial hypertension in childhood. Circulation 111, 435–441 (2005). corresponded to the arithmetic mean of the Cts for ACTB, B2M and HPRT for each sample. For expression analysis of siRNA knockdown, the 2−(ΔΔCt) method was used and fold expression determined relative to the DH1 control. 9. Nasim, M. T. et al. Molecular genetic characterization of SMAD signaling molecules in pulmonary arterial hypertension. Hum. Mutat. 32, 1385–1389 (2011). siRNA transfection. Prior to transfection, cells were preincubated in Opti-MEM-I reduced serum media (Invitrogen) for 2 h before transfection with 10 nM siRNA that had been lipoplexed for 20 min at RT with DharmaFECT1 (GE Dharmacon, Lafayette, CO). Cells were then incubated with the siRNA/DharmaFECT1 com- plexes for 4 h at 37 °C before replaced by full growth media. Cells were kept in growth media for 24 h before further treatment. Knockdown efﬁciency was con- ﬁrmed by mRNA expression or immunoblotting. For proliferation assays, parallel RNA samples were collected both on day0 and day6, conﬁrming that ATP13A3 expression was reduced by >90% on Day 0 and still reduced by >70% at Day 6. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 For all other assays, parallel RNA samples were collected on the day of the experiment to conﬁrm knockdown, which was >90%. The siRNAs used were oligos targeting ATP13A3 (SASI_Hs02_00356805) from Sigma-Aldrich and ON-TARGET plus non-targeting Pool (siCP; GE Dharmacon). 10. Shintani, M., Yagi, H., Nakayama, T., Saji, T. & Matsuoka, R. A new nonsense mutation of SMAD8 associated with pulmonary arterial hypertension. J. Med. Genet. 46, 331–337 (2009). 11. Austin, E. D. et al. Whole exome sequencing to identify a novel gene (caveolin-1) associated with human pulmonary arterial hypertension. Circ. Cardiovasc. Genet. 5, 336–343 (2012). 12. Ma, L. et al. A novel channelopathy in pulmonary arterial hypertension. N. Engl. J. Med. 369, 351–361 (2013). 13. Kerstjens-Frederikse, W. S. et al. TBX4 mutations (small patella syndrome) are associated with childhood-onset pulmonary arterial hypertension. J. Med. Genet. 50, 500–506 (2013). 14. Eyries, M. et al. EIF2AK4 mutations cause pulmonary veno-occlusive disease, a recessive form of pulmonary hypertension. Nat. Genet. 46, 65–69 (2014). 15. Best, D. H. et al. EIF2AK4 mutations in pulmonary capillary hemangiomatosis. Chest 145, 231–236 (2014). Flow cytometric apoptosis assay. BOECs were plated 150,000/well into 6-well plates and transfected with siATP13A3 or siCP lipoplexed with DharmaFECT1. Cells were then serum-starved in EBM-2 (Lonza) containing 0.1% FBS and A/A for 8 h before treating with EBM-2 and A/A containing either 0.1%FBS or 5% FBS for another 24 h. Cells were then trypsinized and after washing with PBS, stained using the FITC Annexin V Apoptosis Dectection Kit I (BD Biosciences). For each condition, dual-staining of 5 µl FITC conjugated Annexin V and 5 µl propidium iodide (PI) were added and incubated at room temperature for 15 min. For the single staining controls for compensation, either 5 µl FITC Annexin V or 5 µl PI was added into non-transfected cells. All samples were analysed on BD Accuri™C6 Plus platform (BD Biosciences). Data were collected and analysed using FlowJo software, with AnnexinV+/PI−cells deﬁned as early apoptotic (Treestar). 15. Best, D. H. et al. EIF2AK4 mutations in pulmo hemangiomatosis. Chest 145, 231–236 (2014). 16. Abenhaim, L. et al. Appetite-suppressant drugs and the risk of primary pulmonary hypertension. International Primary Pulmonary Hypertension Study Group. N. Engl. J. Med. 335, 609–616 (1996). y p g 17. Machado, R. D. et al. Pulmonary arterial hypertension: a current perspective on established and emerging molecular genetic defects. Hum. Mutat. 36, 1113–1127 (2015). 18. Hadinnapola, C. et al. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 Phenotypic characterization of EIF2AK4 mutation carriers in a large cohort of patients diagnosed clinically with pulmonary arterial hypertension. Circulation 136, 2022–2033 (2017). yp 19. Hamid, R. et al. Penetrance of pulmonary arterial hypertension is modulated by the expression of normal BMPR2 allele. Hum. Mutat. 30, 649–654 (2009). 20. David, L., Mallet, C., Mazerbourg, S., Feige, J. J. & Bailly, S. Identiﬁcation of BMP9 and BMP10 as functional activators of the orphan activin receptor-like kinase 1 (ALK1) in endothelial cells. Blood 109, 1953–1961 (2007). Caspase-Glo 3/7 assay. BOECs were seeded at a density of 150,000/well into 6- well plates and transfected with siATP13A3 or siCP lipoplexed with Dharma- FECT1. For each condition, cells were trypsinized from 6-well plates and reseeded in triplicates into a 96-well plate at a density of 15,000–20,000/well and left to adhere overnight. Cells were quiesced in EBM-2 containing 0.1% FBS for 24 h before treating with or without EBM-2 and A/A containing either 0.1% FBS or 5% FBS for 16 h. For measuring caspase activities, 100ul Caspase-Glo® 3/7 Reagent (G8091 Promega) was added into each well, incubated and mixed on a plate shaker in the dark for 30 min at room temperature. The lysates were transferred to a white-walled 96-well plate and luminescence was read in a GloMax® luminometer (Promega). 21. Mi, L. Z. et al. Structure of bone morphogenetic protein 9 procomplex. Proc. Natl Acad. Sci. USA 112, 3710–3715 (2015). 22. Sui, H., Han, B. G., Lee, J. K., Walian, P. & Jap, B. K. Structural basis of water- speciﬁc transport through the AQP1 water channel. Nature 414, 872–878 (2001). 23. Sinner, D., Rankin, S., Lee, M. & Zorn, A. M. Sox17 and beta-catenin cooperate to regulate the transcription of endodermal genes. Development 131, 3069–3080 (2004). 24. Remenyi, A. et al. Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers. Genes Dev. 17, 2048–2059 (2003). Data availability. WGS data of PAH cases included in this manuscript and eligible for public release according to the UK Research Ethics rules have been deposited in the European Genome-phenome Archive (EGA) at the EMBL—European Bioin- formatics Institute under accession number EGAD00001003423. 25. Toshner, M. et al. Transcript analysis reveals a speciﬁc HOX signature associated with positional identity of human endothelial cells. PLoS ONE 9, e91334 (2014). 26. Schultheis, P. J. et al. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 Characterization of the P5 subfamily of P-type transport ATPases in mice. Biochem. Biophys. Res. Commun. 323, 731–738 (2004). Received: 5 September 2017 Accepted: 2 March 2018 Received: 5 September 2017 Accepted: 2 March 2018 27. Madan, M. et al. ATP13A3 and caveolin-1 as potential biomarkers for diﬂuoromethylornithine-based therapies in pancreatic cancers. Am. J. Cancer Res. 6, 1231–1252 (2016). 28. Taraseviciene-Stewart, L. et al. Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension. FASEB J. 15, 427–438 (2001). ARTICLE Of these, deletions were excluded if both failed standard Illumina quality metrics or overlapped with known benign deletions in healthy cohorts56. Deletions with a reciprocal overlap of ≥50% between samples were merged and ﬁltered for a frequency of less than 1 in 1000 in WGS10K and overlapping exonic regions of protein-coding genes (GRCh37 genome assembly). The number of subjects with deletions were added up by gene (no double counting of subjects) and tested for association with the disease. We applied a one-tailed (greater) Fisher’s exact test with Bonferroni post hoc correction for multiple testing to determine the P values for genome-wide signiﬁcance. y p Human pulmonary artery endothelial cells (PAECs) were purchased from Lonza (Cat. No. CC-2530; Basel, Switzerland). Cells were maintained in EGM-2 with 2% FBS (Lonza). PAECs were used for experiments between passages 4 and 8. For experiments cells were cultured in the presence of EBM-2 containing Antibiotic-Antimycotic (Invitrogen, Renfrewshire, UK). Cells were routinely tested to exclude mycoplasma contamination. RNA preparation and quantitative reverse transcription-PCR. Total RNA was extracted using RNeasy Mini Kit with DNAse digestion (Qiagen, West Sussex, UK), according to the manufacturer’s instructions. cDNA was prepared from 1 µg of 13 \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications NATURE COMMUNICATIONS\| (2018) 9:1416 \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications Acknowledgements 35. Corada, M. et al. Sox17 is indispensable for acquisition and maintenance of arterial identity. Nat. Commun. 4, 2609 (2013). g The UK National Institute for Health Research BioResource—Rare Diseases (NIHR BR- RD) and the BHF/MRC UK National Cohort of Idiopathic and Heritable PAH made this study possible. We gratefully acknowledge the participation of patients recruited to the NIHR BR-RD. We thank the NIHR BR-RD staff and co-ordination teams at the Uni- versity of Cambridge, and the research nurses and coordinators at the specialist pul- monary hypertension centres involved in this study. The UK National Cohort of Idiopathic and Heritable PAH is supported by the NIHR, the British Heart Foundation (BHF) (SP/12/12/29836), the BHF Cambridge Centre of Cardiovascular Research Excellence, the UK Medical Research Council (MR/K020919/1), the Dinosaur Trust, BHF Programme grants to R.C.T. (RG/08/006/25302) and N.W.M. (RG/13/4/30107), and the UK NIHR Cambridge Biomedical Research Centre. Funding for whole-exome sequencing was provided through a Bart’s Charity award (MGU0205) to R.C.T. and D.v. The UK National Institute for Health Research BioResource—Rare Diseases (NIHR BR- RD) and the BHF/MRC UK National Cohort of Idiopathic and Heritable PAH made this study possible. We gratefully acknowledge the participation of patients recruited to the NIHR BR-RD. We thank the NIHR BR-RD staff and co-ordination teams at the Uni- versity of Cambridge, and the research nurses and coordinators at the specialist pul- monary hypertension centres involved in this study. The UK National Cohort of Idiopathic and Heritable PAH is supported by the NIHR, the British Heart Foundation (BHF) (SP/12/12/29836), the BHF Cambridge Centre of Cardiovascular Research Excellence, the UK Medical Research Council (MR/K020919/1), the Dinosaur Trust, BHF Programme grants to R.C.T. (RG/08/006/25302) and N.W.M. (RG/13/4/30107), and the UK NIHR Cambridge Biomedical Research Centre. Funding for whole-exome sequencing was provided through a Bart’s Charity award (MGU0205) to R.C.T. and D.v. H. N.W.M. is a BHF Professor and NIHR Senior Investigator. CH is a NIHR Rare Disease Translational Research Collaboration Clinical PhD Fellow. L.S. is supported by the Wellcome Trust Institutional Strategic Support Fund (204809/Z/16/Z) awarded to St. George’s, University of London. C.J.R. is supported by a BHF Intermediate Basic Science Research Fellowship (FS/15/59/31839). A.L. is supported by a BHF Senior Basic Science Research Fellowship (FS/13/48/30453). 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If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/ licenses/by/4.0/. 60. Ormiston, M. L. et al. Generation and culture of blood outgrowth endothelial cells from human peripheral blood. J. Vis. Exp. 106, e53384 (2015). g cells from human peripheral blood. J. Vis. Exp. 106, e53384 (2015). 61. David, L. et al. Bone morphogenetic protein-9 is a circulating vascular quiescence factor. Circ. Res. 102, 914–922 (2008). p p p 61. David, L. et al. Bone morphogenetic protein-9 is a circulating vascular quiescence factor. Circ. Res. 102, 914–922 (2008). quiescence factor. Circ. Res. 102, 914–922 (2008). 62. Thever, M. D. & Saier, M. H. 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Additional information Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-03672-4. 1Department of Medicine, University of Cambridge, Cambridge CB2 0QQ, United Kingdom. 2Department of Haematology, University of Cambridge, Cambridge CB2 0PT, United Kingdom. 3NIHR BioResource—Rare Diseases, Cambridge CB2 0PT, United Kingdom. 4Molecular and Clinical Sciences Research Institute, St George’s, University of London, London SW17 0RE, United Kingdom. 5Division of Genetics & Molecular Medicine, King’s College London, London WC2R 2LS, United Kingdom. 6Royal Papworth Hospital, Papworth Everard, Cambridge CB23 3RE, United Kingdom. 7Institute of Medical and Biomedical Education, St George’s University of London, London SW17 0RE, United Kingdom. 8Addenbrooke’s Hospital, Cambridge CB2 0QQ, United Kingdom. 9Cleveland Clinic, Cleveland, Ohio 44195, United States. 10VU University Medical Center, Amsterdam 1007 MB, The Netherlands. 11Golden Jubilee National Hospital, Glasgow G81 4DY, United Kingdom. 12Royal Free Hospital, London NW3 2QG, United Kingdom. 13Shefﬁeld Pulmonary Vascular Disease Unit, Royal Hallamshire Hospital, Shefﬁeld S10 2JF, United Kingdom. 14University of Newcastle, Newcastle NE1 7RU, United Kingdom. 15Department of Molecular Medicine, University of Pavia, Pavia 27100, Italy. 16Fondazione IRCCS Policlinico San Matteo, Pavia 27100, Italy. 17Département de génétique, hôpital Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, and UMR_S 1166- ICAN, INSERM, UPMC Sorbonne Universités, Paris 75252, France. 18University of Giessen and Marburg Lung Center (UGMLC), member of the German Center for Lung Research (DZL) and of the Excellence Cluster Cardio-Pulmonary System (ECCCPS), Giessen 35392, Germany. 19Imperial College London, London SW7 2AZ, United Kingdom. 20National Heart & Lung Institute, Imperial College London, London SW3 6LY, United Kingdom. 21Université Paris-Sud, Faculté de Médecine, Université Paris-Saclay; AP-HP, Service de Pneumologie, Centre de référence de l’hypertension pulmonaire; INSERM UMR_S 999, Hôpital Bicêtre, Le Kremlin-Bicêtre, Paris 94270, France. 22Ludwig Boltzmann Institute for Lung Vascular Research, Graz 8010, Austria. 23Medical University of Graz, Graz 8036, Austria. 24Royal United Hospitals Bath NHS Foundation Trust, Bath BA1 3NG, United Kingdom. 25Great Ormond Street Hospital, London WC1N 3JH, United Kingdom. 26Blizard Institute, Queen Mary University of London, London E1 2AT, United Kingdom. 27Royal Brompton Hospital, London SW3 6NP, United Kingdom. 28Wellcome Trust Sanger Institute, Hinxton CB10 1SA, United Kingdom. 29Department of Infection, Immunity & Cardiovascular Disease, University of Shefﬁeld, Shefﬁeld S10 2RX, United Kingdom. These authors jointly supervised this work: Stefan Gräf, Nicholas W. Morrell. These authors contributed equally: Stefan Gräf, Matthias Haimel, Marta Bleda, Charaka Hadinnapola, Laura Southgate. Competing interests: The authors declare no competing interests. Bioinformatic characterization of p-type ATPases encoded within the fully sequenced genomes of 26 eukaryotes. J. Membr. Biol. 229, 115–130 (2009). 63. Kanai, R., Ogawa, H., Vilsen, B., Cornelius, F. & Toyoshima, C. Crystal structure of a Na+−bound Na+, K+−ATPase preceding the E1P state. Nature 502, 201–206 (2013). 64. Palasingam, P., Jauch, R., Ng, C. K. & Kolatkar, P. R. The structure of Sox17 bound to DNA reveals a conserved bending topology but selective protein interaction platforms. J. Mol. Biol. 388, 619–630 (2009). © The Author(s) 2018 NATURE COMMUNICATIONS\| (2018) 9:1416 15 \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications ARTICLE \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-03672-4 Stefan Gräf 1,2,3, Matthias Haimel 1,2,3, Marta Bleda 1, Charaka Hadinnapola 1, Laura Southgate 4,5, Wei Li1, Joshua Hodgson1, Bin Liu1, Richard M. Salmon1, Mark Southwood6, Rajiv D. Machado7, Jennifer M. Martin 1,2,3, Carmen M. Treacy1,6, Katherine Yates1,2,3, Louise C. Daugherty2,3, Olga Shamardina 2,3, Deborah Whitehorn2,3, Simon Holden8, Micheala Aldred 9, Harm J. Bogaard10, Colin Church11, Gerry Coghlan12, Robin Condliffe13, Paul A. Corris14, Cesare Danesino15,16, Mélanie Eyries17, Henning Gall18, Stefano Ghio16, Hossein-Ardeschir Ghofrani18,19, J. Simon R. Gibbs20, Barbara Girerd21, Arjan C. Houweling10, Luke Howard19, Marc Humbert21, David G. Kiely13, Gabor Kovacs22,23, Robert V. MacKenzie Ross24, Shahin Moledina25, David Montani 21, Michael Newnham1, Andrea Olschewski22 Horst Olschewski22,23, Andrew J. Peacock11, Joanna Pepke-Zaba6, Inga Prokopenko 19, Christopher J. Rhodes 19, Laura Scelsi16, Werner Seeger18, Florent Soubrier17, Dan F. Stein 1, Jay Suntharalingam24, Emilia M. Swietlik1, Mark R. Toshner 1, David A. van Heel 26, Anton Vonk Noordegraaf10, Quinten Waisﬁsz10, John Wharton 19, Stephen J. Wort27,19, Willem H. Ouwehand2,3, Nicole Soranzo 2,28, Allan Lawrie 29, Paul D. Upton 1, Martin R. Wilkins19, Richard C. Trembath 5 & Nicholas W. Morrell 1,3 NATURE COMMUNICATIONS\| (2018) 9:1416 \| DOI: 10.1038/s41467-018-03672-4\| www.nature.com/naturecommunications 16
https://openalex.org/W4392187117	https://ejournal.poltekkesjakarta1.ac.id/index.php/gemakes/article/download/1527/458	Indonesian	null	PENYULUHAN KESEHATAN GIGI TENTANG CARA MENYIKAT GIGI PADA ANAK STUNTING	Gemakes	2,024	cc-by-sa	2,953	eISSN : 2798-4826 DOI: 10.36082/gemakes.v4i1.1527 Open Access: https://ejournal.poltekkesjakarta1.ac.id/index.php/gemakes GEMAKES: Jurnal Pengabdian Kepada Masyarakat Volume 4, Nomor 1 Tahun 2024, pp. 98-105 eISSN : 2798-4826 DOI: 10.36082/gemakes.v4i1.1527 GEMAKES: Jurnal Pengabdian Kepada Masyarakat Volume 4, Nomor 1 Tahun 2024, pp. 98-105 Open Access: https://ejournal.poltekkesjakarta1.ac.id/index.php/gemakes Corresponding author: applonia1968@gmail.com 1 Jurusan Kesehatan Gigi Poltekkes Kemenkes Kupang, Indonesia 2 Jurusan Kesehatan Gigi Poltekkes Kemenkes Kupang, Indonesia 3 Jurusan Kesehatan Gigi Poltekkes Kemenkes Kupang, Indonesia 4 Jurusan Kesehatan Gigi Poltekkes Kemenkes Kupang, Indonesia enesis Naskah: 31-01-2024, Revised: 06-02-2024, Accepted: 19-02-2024, Available Online: 27-02-2024 DENTAL HEALTH COUNSELING ON HOW TO BRUSH TEETH IN STUNTING CHILDREN DENTAL HEALTH COUNSELING ON HOW TO BRUSH TEETH IN STUNTING CHILDR Abstrak Anak stunting adalah anak yang terhambat pertumbuhan fisiknya karena masalah gizi dan pengasuhan yang kurang. Stunting pada balita akan berdampak buruk jika tidak ditanggulangi segera. Asupan gizi yang diterima tubuh anak sangat penting diperhatikan dan peran orang tua asuh dapat membimbing dan mengawasi anaknya agar kesehatan gigi dapat terjaga. Sehingga mencegah risiko kerusakan gigi dengan rajin menyikat gigi. Melalui pemberian edukasi mengenai perawatan gigi yang benar kepada orang tua asuh anak stunting sangatlah tepat. Pengabdian kepada masyarakat bertujuan untuk meningkatkan pemahaman orang tua asuh tentang cara menyikat gigi pada anak stunting di Kelurahan Liliba Kota Kupang. Metode pelaksanaan pengabdian dengan teknik penyuluhan langsung pada ibu beranak stunting dan pemberian makanan tambahan selama 90 hari. Berdasarkan hasil penyuluhan yang diberikan saat pengabdian, ibu secara umum memahami tentang cara menyikat gigi pada anak stunting dan berkomitmen lebih baik lagi dalam mendampingi anak ketika menyikat gigi. Serta pendampingan yang diberikan selama 4 kali, pertama sebelum diintervensi, kedua pemberian makanan tambahan selama 30 hari, dilanjutkan ketiga 60 hari dan keempat 90 hari.Tim memantau dan mengevaluasi perkembangan kedua anak asuhnya terdapat peningkatan berat badan, tinggi badan serta lingkar lengan atas. Kesimpulan : terjadi perubahan berat badan anak serta ibu memahami cara menjaga kesehatan gigi anaknya. Kata Kunci : Penyuluhan, Makanan Tambahan, Stunting PENYULUHAN KESEHATAN GIGI TENTANG CARA MENYIKAT GIGI PADA ANAK STUNTING Melkisedek O. Nubatonis1, Applonia Leu Obi2, Christina Ngadilah3, Agustinus Wal Melkisedek O. Nubatonis1, Applonia Leu Obi2, Christina Ngadilah3, Agustinus Wali4 Corresponding author: applonia1968@gmail.com Abstract Stunted children are children whose physical growth is hampered due to nutritional problems and inadequate care. Stunting in toddlers will have a bad impact if it is not addressed immediately. It is very important to pay attention to the nutritional intake that a child's body receives and the role of foster parents can be to guide and supervise their children so that dental health can be maintained. So prevent the risk of tooth decay by brushing your teeth diligently. Providing education regarding proper dental care to foster parents of stunted children is very appropriate. Community service aims to increase foster parents' understanding of how to brush the teeth of stunted children in Liliba Village, Kupang City. The method of implementing the service is using direct counseling techniques for mothers with stunted children and providing additional food for 90 days. Based on the results of the counseling given during the service, mothers generally understand how to brush their teeth in stunted children and are committed to being better at accompanying children when brushing their teeth. As well as assistance was provided 4 times, the first before the intervention, the second by providing additional food for 30 days, followed by the third for 60 days, and the fourth for 90 days. The team monitored and evaluated the development of the two foster children, there was an increase in weight, height, and upper arm circumference. Conclusion: there is a change in the child's weight and the mother understands how to maintain the health of her child's teeth. Keywords: Extension, Supplementary Food, Stunting \| 98 Pendahuluan seimbang serta menyikat gigi 2x sehari dengan menggunakan pasta gigi yang ber fluoride juga kontrol kesehatan gigi secara berkala (Clarke, 2017) Masalah gizi di Kota Kupang maupun juga pada umumnya masih mengalami kekurangan gizi maupun kondisi gizi lebih. Data stunting di Indonesia terdapat 37% anak - anak dan di Propinsi Nusa Tenggara Timur terdapat 319.00 anak mengalami stunting (Kemenkes 2013 cit (Paun et al., 2021), tahun 2018 terjadi penurunan stunting sebesar 30,8%. Angka stunting tahun 2019 turun 27,7% dan tahun 2021 turun menjadi 24,4 jika dibandingkan dengan tahun 2022 menurun secara nasional menjadi 21,6%.(Kemenkes, 2022) Stunting adalah kondisi di mana pertumbuhan fisik dan perkembangan anak terhambat akibat kekurangan gizi dan perawatan yang tidak memadai. (Gladys Apriluana, 2017)Kondisi ini dapat berdampak negatif pada kesehatan gigi dan mulut anak-anak. Anak balita stunting cenderung akan sulit dan serius sangat memengaruhi pertumbuhan dan perkembangan fisik dan mental secara optimal. (Nursyamsiyah, Sobrie Y, 2021)Nutrisi sangat berpengaruh terhadap pertumbuhan gigi anak baik dalam bentuk, ukuran gigi ada kaitanya dengan kekurangan kalsium dan fosfor terhadap perkembangan gigi. (Diéguez-Pérez et al., 2022)Untuk mengatasi masalah ini, maka, sebagai kelompok masyarakat peduli kesehatan anak, dengan melaksanakan kegiatan pengabdian masyarakat di Kelurahan Liliba pada bulan April 2023. Kegiatan ini bertujuan untuk memberikan pemahaman kepada masyarakat tentang pentingnya pemberian makanan yang seimbang dan bergizi pada anak stunting dan edukasi ibu anak asuh tentang cara menyikat gigi. Di Nusa Tenggara Timur data hasil pemantauan status gizi (PSG) tahun 2022 berdasarkan tinggi badan menurut umur menunjukkan sebanyak 35,3 % dan menduduki peringkat pertama dari 34 Propinsi yang ada di Indonesia.(Kemenkes, 2022)Faktor penyebab stunting pada anak yang rentan sebaiknya menjadi perhatian penting untuk mendapatkan intervesi yang lebih efektif.(Suratri et al., 2023) Pemerintah daerah tetap menempatkan penanganan stunting sebagai prioritas dan setiap wilayah berupaya mengimplementasikan kebijakan untuk bersama mencapai target penurunan yang signifikan di tahun 2024. Salah satu daerah yang aktif menerapkan strategi penanganan stunting adalah Nusa Tenggara Timur terkhususnya diwilayah Kelurahan Liliba Kota Kupang. Pemerintah Kota Kupang dalam upaya menekan angka prevalensi Stunting dibawah 10% di tahun 2024 melakukan kerja konvergensi atau kerjasama lintas sektoral dalam lingkup Pentahelix (Pemerintah, PT, Swasta, Masyarakat, dan Media Masa). Program orang tua asuh merupakan salah satu solusi kepedulian dan upaya yang dilakukan dengan melibatkan semua sektor Pencegahan penyakit gigi dilakukan sejak dini dengan melalui upaya promotif dan preventif (Ira Liasari et al.,2021). Pendahuluan Promotif kesehatan gigi dan mulut dengan membatasi makanan dan minuman manis dan menjaga pola makan yang \| 99 dalam percepatan pencegahan dan penanganan balita stunting. Kelurahan Liliba Kota Kupang yang dijadikan tempat pelaksanaan kegiatan pengabdian masyarakat. Poltekkes Kemenkes Kupang sebagai salah satu implementasi transformasi kesehatan mendukung program pemerintah dalam memperkuat layanan kesehatan primer khususnya stunting diharapkan dapat terlibat dan menjadi tim dalam percepatan pencegahan dan penanganan stunting di Kota Kupang. Kota Kupang. Koordinasi dilakukan bersama intansi Dinas Kesehatan bersama Poltekkes Kemenkes Kupang. Langkah-langkah yang dilakukan sebagai berikut: 1. Tahap Pra Pelaksanaan a. Survey : pada tahap ini tim pengabdian kepada masyarakat melakukan survei untuk melihat kondisi mitra dan wawancara dengan mitra tentang apa yang sedang dihadapi oleh mitra. b. Koordinasi dan Administrasi : pada tahap ini pihak Poltekkes Kemenkes Kupang melakukan koordnasi dengan : Mitra pengabmas adalah anak stunting di wilayah Kelurahan Liliba Kota Kupang di posyandu Melati sesuai data dari tenaga kesehatan Puskesmas Oepoi, melalui kerjasama Dinas Kesehatan dengan Poltekkes Kemenkes Kupang. Pelaksanaan melalui pembagian anak asuh pada tim pengabdi dalam hal ini adalah dosen untuk pemberian makanan tambahan pada anak stunting. Kegiatan didahului dengan survey dan wawancara langsung pada ibu anak asuh dan kegiatan penyuluhan pada ibu. untuk meningkatkan pengetahuan dalam menjaga kesehatan gigi. Tujuan kegiatan pengabdian kepada masyarakat adalah melakukan penyuluhan kesehatan gigi tentang cara menyikat gigi yang baik dan benar dan pemberian makanan tambahan pada anak stunting. ) Dinas Kesehatan Kota Kupang dalam hal ini diwakili oleh Puskesmas Oepoi untuk mendapatkan data anak stunting serta analisis kebutuhan. ) Kelurahan Liliba dan posyandu Melati mengenai rencana kegiatan pengabdian kepada masyarakat yang akan dilakukan. Kemudian membuat surat pernyataan kesediaan pihak desa dan kader posyandu untuk mendukung kegiatan yang akan dilakukan. ) Kelurahan Liliba dan posyandu Melati mengenai rencana kegiatan pengabdian kepada masyarakat yang akan dilakukan. Kemudian membuat surat pernyataan kesediaan pihak desa dan kader posyandu untuk mendukung kegiatan yang akan dilakukan. c. Persiapan materi yang digunakan dalam kegiatan ini adalah materi tentang cara menyikat gigi yang baik dan benar, penggunaan media penyuluhan berupa panthom dan sikat gigi. Metode Pelaksanaan Pelaksanaan dilakukan diwilayah kerja Puskesmas Oepoi khususnya anak stunting. Kegiatan dilaksanakan selama 90 hari. Sebelum pelaksanaan, tempat kegiatan lokasi kegiatan di Posyandu Melati Kelurahan Liliba d. Tim pengusul melibatkan 3 mahasiswa Prodi Kesehatan Gigi. Poltekkes Kemenkes Kupang dalam pelaksanaan pengabdian masyarakat agar dapat membantu memperlancar pelaksanaan \| 100 kegiatan dan sebagai wadah pembelajaran dalam mengaplikasikan ilmu yang telah di peroleh dibangku kuliah. kegiatan dan sebagai wadah pembelajaran dalam mengaplikasikan ilmu yang telah di peroleh dibangku kuliah. 6) Memberi laporan secara tertulis terkait perkembangan pengasuhan di akhir kegiatan. b. Evaluasi antara lain : b. Evaluasi antara lain : b. Evaluasi antara lain : 2. Tahap Pelaksanaan 2. Tahap Pelaksanaan 1) Mengevaluasi kondisi anak (mengukur BB, TB, dan LILA) setiap bulan. a. Pemberian makanan tambahan dengan melibatkan kader dan pkk kelurahan Liliba untuk posyandu Melati. 2) Penilaian untuk mengetahui peningkatan pengetahuan dan status status gizi balita stunting dan kesehatan gigi. b. Penyuluhan kesehatan gigi dan mulut tentang cara menyikat gigi yang baik dan benar. c) Penyusunan laporan yang telah dilaksanakan setelah pelaksanaan hingga selesai. 3. Tahap Pasca Pelaksanaan : a. Monitoring antara lain : 1) Mengontrol pemberian makanan tambahan untuk anak asuh stunting. 2) Mengidentifikasi kondisi antropometri terkini sebelum dan setelah pendampingan serta pemantauan mingguan anak asuh stunting. 1. Advokasi Pelaksanaan kegiatan pengabdian kepada masyarakat dengan mendapatkan ijin dari kelurahan Liliba Kabupaten Kupang serta kegiatan dilaksanakan pada bulan April tahun 2023. Tim pengabdian masyarakat mendatangi keluarga yang beranak stunting untuk pertemuan dengan lurah di Kelurahan Liliba Kota Kupang pada tanggal 25 April 2023, dengan menyampaikan beberapa kegiatan yang akan dilaksanakan diantaranya dengan memberikan informasi mengenai pentingnya kesehatan gigi pada anak stunting. Kegiatan pemberian makan pada anak stunting ini bekerja sama dengan dinas kesehatan Kota Kupang dengan kelurahan liliba dalam rangka pencegahan stunting. 3) Memberikan edukasi tentang makanan bergizi seimbang bagi anak stunting kepada ibu yang beranak stunting. 4) Mengevaluasi kondisi anak (mengukur BB, TB, dan LILA) setiap bulan yang akan dilakukan bersama oleh Kader Posyandu dan ibu asuh termasuk tim kader dan tim orang tua asuh dalam hal ini dosen dari Poltekkes Kemenkes Kupang. 5) Mendokumentasikan kegiatan pengasuhan dalam logbook di setiap kegiatan. \| 101 Gambar 1. Kunjungan Ke Posyandu Melati pemberian makanan tambahan. Pelaksanaan kegiatan penimbangan berat badan, pengukuran tinggi badan dan lingkar lengan atas (LiLA) dan penyuluhan kesehatan gigi. Kegiatan ini dilakukan atas kerjasama antara tim pelaksana dengan tenaga kesehatan dari Puskesmas Oepoi dan kader dari Posyandu Melati di Kelurahan Liliba Kota Kupang. Kegiatan dilaksanakan selama 4 kali, dimana tahap pertama dilakukan sebelum intervensi dengan mendapatkan data anak stunting dari Puskesmas Oepoi, lalu tahap kedua diberikan makanan tambahan selama 30 hari, tahap ketiga 60 hari dan tahap keempat dilaksanakan selama 90 hari. Selama 3 bulan terus ada monitoring dan evaluasi untuk melihat perkembangan dari anak asuh tersebut. Data sebelum intervensi anak asuh stunting dapat dilihat pada tabel 1 berikut ini : makanan Gambar 1. Kunjungan Ke Posyandu Melati Gambar 1. Kunjungan Ke Posyandu Melati 2. Kegiatan Pengabdian Kepada Masyarakat Dilaksanakan Kepada Anak Stunting Di Kelurahan Liliba Kota Kupang. a. Sebelum Intervensi Pada Anak Asuh Stunting Kegiatan ini, tim mendapatkan anak asuh sebanyak 2 anak yang berlokasi di Kelurahan Liliba sebanyak 2 anak (Inisial B. M dan R.B). Anak asuh yang direkomendasikan oleh Puskesmas berdasarkan data hasil kegiatan timbang bulan Februari 2023 memiliki resiko stunting dan gizi kurang. Oleh karena itu tim pelaksana bekerjasama dengan Dinas Kesehatan dalam hal ini diwakili oleh tenaga kesehatan dari Puskesmas Oepoi untuk bersama sama melakukan koordinasi dan monitoring serta evaluasi terkait perkembangan anak asuh. Tabel 1. Data Sebelum Intervensi Anak Asuh Stunting Anak Asuh Bulan Mei (Sebelum Intervensi) BB (Kg) TB/PB( cm) LiLA (cm) B M 6.6 68.4 12.4 R B 7.4 67.9 13.4 Tabel 1. Data Sebelum Intervensi Anak Asuh Stunting Dalam upaya mengurangi resiko stunting dan gizi buruk bagi anak asuh, tim melakukan kegiatan diantaranya yaitu \| 102 Gambar 2. Penimbangan Berat Badan, Tinggi Badan dan Pengukuran Lila asuh tersebut. Namun untuk anak asuh RB sebelum diintervensi didapat berat badannya 7.4 kg dengan tinggi badan hanya 69.4 cm dengan pengukran lila 13,4 cm. Setelah 30 hari dengan pemberian makanan tambahan ada penambahan berat badan 7.9 kg tinggi badan berubah 72,6 cm dan lingkar lengan 13,5 cm seperti terlihat pada tabel 2 dibawah ini. b. Pemberian Makanan Tambahan Untuk Pada kegiatan ini, tim pengabdian kepada masyarakat melakukan pemberian makanan tambahan (PMT) selama 3 bulan (90 hari) dan tim melakukan monitoring dan evaluasi untuk melihat perkembangan dari 2 (Dua) anak asuh. Anak asuh yang berisial B.M, sebelum diintervensi memiliki berat badan 6.6 Kg, tinggi badan 68,4 cm dan lila 12,4 cm dan anak asuh R.S.B sebelum diberikan intervensi 7,4 kg dengan tinggi badan 67,9 serta pengukuran lila 13,4 cm dan di monitoring dan evaluasi pada 30 hari pertama terjadi kenaikan berat badan dan tinggi badan pada kedua anak Tabel 2. Data Intervensi I Selama 30 Hari S Anak Asuh Monev I ( Intervensi 30 hr) BB (Kg) TB/PB (cm) LILA (cm) B M 6.9 72.6 12.4 R B 7.9 72.6 13.5 Gambar 2. Penimbangan Berat Badan, Tinggi Badan dan Pengukuran Lila Tabel 2 dilakukan interfensi di bulan ke 2 (60 hari) pada anak B.M terlihat berat badanya sedikit berkurang hanya terjadi perubahan pada lingkar lengan sedangkan untuk anak asuh R.B terus bertambah berat badan, tinggi badan dan lingkar lengan atas. c. Penyuluhan Kesehatan Gigi Penyuluhan dilaksanakan pada tanggal 25 Juli 2023 di posyandu Melati Liliba Kota Kupang. Tema : cara menyikat gigi pada anak stunting. Proses penyuluhan direspon baik dan ada juga beberapa pertanyaan yang diajukan. Edukasi dilakukan untuk meningkatkan pengetahuan ibu. Setelah itu dilanjutkan demonstrasi cara menyikat gigi dan mulut yang baik dan benar dengan menggunakan phantom dan sikat gigi. Diharapkan ibu dapat membimbing anaknya di rumah untuk menyikat gigi secara baik dan benar sehingga dapat meningkatkan keinginan anak dalam menyikat gigi agar bebas dari karies gigi. Pendidikan kesehatan gigi dengan mengutamakan teknik menyikat gigi serta penggunaan pasta gigi yang berfloride, perilaku sehat dan menghindari kebiasaan makan yang tidak sehat dan Gambar 3. Penyuluhan Kesehatan Gigi Tabel 3. Data Intervensi II Selama 60 Hari Anak Asuh Monev II (Intervensi 60 hr) BB (Kg) TB/PB (cm) LILA (cm) B M 6.7 72.6 13 R B 8.5 74 14.2 Tabel 3 menunjukkan pemberian makanan tambahan setelah 2 bulan (60 hari) \| 103 ada peningkatan berat badan,tinggi badan dan lila anak. pemeriksaan gigi secara berkala (Fraihat et al., 2019) pemeriksaan gigi secara berkala (Fraihat et al., 2019) Tabel 4. Data Intervensi II Selama 90 Hari Gambar 3. Penyuluhan Kesehatan Gigi Tabel 4. Data Intervensi II Selama 90 Hari Anak Asuh Monev III (Intervensi 90 hr) BB (Kg) TB/PB (cm) LILA (cm) B M 7.1 75.5 13.8 R B 9 79.1 14.9 Gambar 3. Penyuluhan Kesehatan Gigi Tabel 4 terlihat kenaikan berat badan, tinggi badan dan lila setelah 3 bulan pada kedua anak asuh tersebut. Kesimpulan dan Saran Berdasarkan kegiatan pengabdian masyarakat program kemitraan masyarakat yang telah dilaksanakan kepada kedua anak asuh stunting maka dapat disimpulkan sebagai berikut : 1. Kegiatan ini dapat terlaksana dengan baik sesuai dengan metode pelaksanaan yang dirancang. Kegiatan ini berlangsung selama 90 hari dengan pemberian makanan tambahan (PMT) dan edukasi terkait pencegahan stunting melalui penyuluhan kesehatan gigi dan mulut kepada orang tua dari anak asuh stunting. 1. Kegiatan ini dapat terlaksana dengan baik sesuai dengan metode pelaksanaan yang dirancang. Kegiatan ini berlangsung selama 90 hari dengan pemberian makanan tambahan (PMT) dan edukasi terkait pencegahan stunting melalui penyuluhan kesehatan gigi dan mulut kepada orang tua dari anak asuh stunting. \| 104 Sekolah Dasar Makassar. Darmabakti Cendekia:, 3, 45–48. https://doi.org/10.20473/dc.V3.I2.2021.45-48 2. Adanya peningkatan berat badan, tinggi badan dan lingkar lengan dari kedua anak asuh stunting. Kemenkes. (2022). Hasil Survei Status Gizi Indonesia (SSGI) 2022. Kemenkes, 1–7. 3. Adanya perubahan pengetahuan pada ibu dari anak asuh stunting tentang cara menyikat gigi sebagai upaya pencegahan stunting. Nursyamsiyah, Sobrie Y, B. S. (2021). Faktor-Faktor yang Berhubungan dengan Kejadian Stunting pada Anak Usia 1 – 24 Bulan. Syntax Literate ; Jurnal Ilmiah Indonesia, 6(10), 5061. https://doi.org/10.36418/syntax- literate.v6i10.4363 Kegiatan pengabdian masyarakat program kemitraan masyarakat yang telah dilaksanakan kepada anak asuh stunting perlu dilaksanakan secara berkelanjutan dengan berbagai inovasi sehingga dapat meningkatkan status gizi anak dan perilaku ibu dalam pemeliharaan kesehatan gigi dan mulut anak stunting sebagai upaya pencegahan stunting. Paun, R., Bia, M. B., Shagti, I., Gunawan, Y. E. S., Krisyudhanti, E., Dafroyati, Y., & Mau, F. (2021). the Relationship Between Intestinal Worm Infection and Stunting in Elementary School Children in South Central Timor Regency, East Nusa Tenggara. ICPH E- Proceeding, 328. https://doi.org/10.26911/ICPHepidemiology.FP. 08.2021.11 Suratri, M. A. L., Putro, G., Rachmat, B., Nurhayati, Ristrini, Pracoyo, N. E., Yulianto, A., Suryatma, A., Samsudin, M., & Raharni. (2023). Risk Factors for Stunting among Children under Five Years in the Province of East Nusa Tenggara (NTT), Indonesia. International Journal of Environmental Research and Public Health, 20(2). https://doi.org/10.3390/ijerph20021640 Daftar Pustaka Clarke, R. D. (2017). Miami , Florida Parental Attitudes , Beliefs And Behaviors About Caries Prevention Among Their Black Preschool Children A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor Of Philosophy in Public Health by. Diéguez-Pérez, M., Paz-Cortés, M. M., & Muñoz- Cano, L. (2022). Evaluation of the Relationship between the Weight and Height Percentiles and the Sequence and Chronology of Eruption in Permanent Dentition. Healthcare (Switzerland), 10(8). https://doi.org/10.3390/healthcare10081363 Fraihat, N., Madae’En, S., Bencze, Z., Herczeg, A., & Varga, O. (2019). Clinical effectiveness and cost- effectiveness of oral-health promotion in dental caries prevention among children: Systematic review and meta-analysis. International Journal of Environmental Research and Public Health, 16(15). https://doi.org/10.3390/ijerph16152668 Gladys Apriluana, S. F. (2017). Analisis Faktor-Faktor Risiko terhadap Kejadian Stunting pada Balita. Jurnal Departemen Gizi Fakultas Kesehatan Masarakat, Vol. 28 No, 247–256. Ira Liasari, Ardian Priyambodo, Munadirah, Jumriani, Nurhaeni, A. (2021). Pencegahan Karies Melalui Aplikasi Pit Dan Fissure Sealant Pada Murid \| 105
https://openalex.org/W4309458592	https://sosains.greenvest.co.id/index.php/sosains/article/download/518/1077	English	null	The Association Of Adolescent Pregnancy With Stunting Incidence In Child Under Five Years Old; Literatur Review	Jurnal sosial dan sains/Jurnal Sosial dan Sains	2,022	cc-by-sa	5,048	JURNAL SOSAINS JURNAL SOSIAL DAN SAINS VOLUME 2 NOMOR 11 2022 P-ISSN 2774-7018, E-ISSN 2774-700X THE ASSOCIATION OF ADOLESCENT PREGNANCY WITH STUNTING INCIDENCE IN CHILD UNDER FIVE YEARS OLD JURNAL Adelbertha A. B. Namita Candra Devi., Rita Oktavia Harahap, Astika Gita Ningrum Faculty of Medicine, Universitas Airlangga, Indonesia Email : adelbertha.apriana.berkanis-2021@fk.unair.ac.id, namita.can.devi- 2021@fk.unair.ac.id, rita.oktavia.harahap-2021@fk.unair.ac.id, astika.gita.n@fk.unair.ac.id ABSTRACT ABSTRACT Background: Nowadays early marriage is a worldwide issue and influences maternal outcome and increases the incidence of stunting. Stunting is known as one of the most significant barriers to human development and globally affects around 162 million children under the age of 5 years. The Global stunting incidence in children under 5 years is 149.2 million or about 22.0% of all children under five. The prevalence of stunting in children aged less than 5 years old in Africa is around 31.7%, Southeast Asia is 30.1%, the Eastern Mediterranean Region is 26.2% and in Indonesia it is 24.4%. It is known that children born of women who are less than 20 years have a 1.3 times risk of experiencing stunting and the prevalence of stunting in adolescent pregnant women is around 44.4% compared to mothers who are old enough, namely 35.6%. Keywords: adolescent pregnancy, adolescent mother, teen pregnancy, stunting, Indonesia Purpose: This study aims to determine the relationship between teenage pregnancy and the incidence of stunting INTRODUCTION Stunting is one of the most significant barriers to human development and globally affects about 162 million children under the age of 5 years (WHO, 2014), and is a growth and development disorder in children due to malnutrition, recurrent infections and inadequate psychosocial stimulation (WHO, 2014). And have negative impacts both in the short and long term (Yadika, Berawi, & Nasution, 2019). Stunting is caused by malnutrition since the baby is in the womb and in the early days after the baby is born, but the stunting condition only appears after the child is 2 years old (Wemakor, Garti, Azongo, Garti, & Atosona, 2018). Stunting is one of the nutritional problems that are prone to occur in toddlers due to chronic low food intake (Pertiwi, Lestari, & Ulfiana, 2019). In 2020 the global incidence of stunting in children under 5 years of age is 149.2 million or around 22.0% of all toddlers who are estimated to be too short for their age. and the prevalence of stunting in children aged less than 5 years in 2020 is highest in Africa, which is around 31.7%, Southeast Asia at 30.1% and the Eastern Mediterranean Region at 26.2% (WHO-COVID, 2022). In Indonesia, the prevalence of stunting in children under 5 years in 2020 is 24.4%. Stunting is caused by several factors. One of the factors that cause stunting is teenage pregnancy. It is known that teenage pregnancies have an 8 times greater risk than adult women. Stunting can cause children to get sick easily, can cause economic losses to both the family and the country, the form of body posture cannot be maximized as an adult, body functions become unbalanced, and the worst impact that is very worrying is the cognitive ability of children is reduced (Eka, Krisnana, & Husada, 2020). Adolescent pregnancy is a pregnancy in women aged 15-19 years (WHO-COVID, 2022) with an incidence of around 12 million pregnant adolescents who give birth every year or about 777,000 girls under 15 years old give birth every year in developing countries. y g y g y y p g Adolescent pregnancy is associated with stunting and can increase the prevalence of stunting. The prevalence of stunting is higher in under-fives from teenage pregnant women, namely 44.4% compared to mothers who are old enough, namely 35.6%. The Association Of Adolescent Pregnancy With Stunting Incidence In Child Under Five Years Old The Association Of Adolescent Pregnancy With Stunting Incidence In Child Under Five Years Old INTRODUCTION Married adolescent women have a 1.2 times risk, and women who are less than 20 years pregnant have a 1.3 times risk of experiencing stunting under five . Children of adolescent mothers are 8 times more likely to be stunted compared to adult mothers because they are 13 times more likely to be Stunting (Wemakor et al., 2018). In Indonesia Teenage pregnancy is known as one of the causes of stunting. According to Cindy Ega Fiolentina and Rini Ernawati (2021) teenage pregnancy has an impact on stunting, which is 40%. According to (Irwansyah, Ismail, & Hakimi, 2016) teenage pregnancy is associated with the incidence of stunting in children aged 6-23 months by controlling the variables of maternal education, birth weight, and maternal stature. Teenage pregnancy is known to have a negative impact on pregnancy outcomes and infant/toddler health. Therefore, this study aims to explore some of the literature on the relationship between teenage pregnancy and the incidence of stunting in children aged 0- 59 months. This study is expected to provide useful information about the impact of teenage pregnancy, stunting and minimize the incidence of stunting caused teenage pregnancy. Purpose: This study aims to determine the relationship between teenage pregnancy and the incidence of stunting Method: This was a literature review collected from four e-databases search was conducted by PubMed (Medline), Web of science, Scopus and ScienceDirect for articles published between 2019 and 2021 that examine teenage pregnancy and stunting. Existing articles will be filtered and eliminated according to the inclusion criteria and analyzed to find the conclusions from the entire study. Results:. The search led to primary research publications including qualitative and quantitative research. The articles were published between 2019 and 2021. Most of the articles showed that young maternal age influences maternal outcome and we need to pay attention to stunting incidence. Even though there are a lot of factors that play a role in stunting such as nutrition, disease, parent height, etc. but five articles that have been filtered agree that adolescent pregnancy is the main factor that plays a role in stunting.. Conclusion : Our review found the closely related factors that related with stunting are early marriage, clean-healthy behaviors and mental emo-disorder of adolescent mothers. However, there are other factors causing stunting namely direct and indirect factors. Robust programs to support pregnant women and monitor children’s heights from birth will help prevent intergenerational stunting. Nevertheless, it is also necessary to review the policy on how stunting criteria are set in Indonesia based on socio- demographic conditions. 1241 http://sosains.greenvest.co.id 2022 Adelbertha A. B. Namita Candra Devi., Rita Oktavia Harahap, Astika Gita Ningrum Volume 2, Nomor 11, Oktober 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Inclusion and exclusion criteria Inclusion criteria: Reputable journal (ScienceDirect, Pubmed, Web of Science, Scopus) with quantitative and qualitative research conducted in Indonesia and open access. The exclusion criteria were Thesis, Thesis, Final Project, Health profile, booklet. RESEARCH METHODS The research was carried out by identifying the Medical Subject Headings (MeSH) specific to this topic, in the thesaurus dictionary controlled by the National Library of Medicine (NLM), and non-MeSH words, using them to search for the work—individually or in association with each other—through the use of the boolean operators “AND”, “OR”: Adolescent Pregnancy; Teenage Pregnancy; Adolescent Mother; Stunting; Failure to thrive; Stunted growth; Growth Disorder. 1242 Adelbertha A. B. Namita Candra Devi., Rita Oktavia Harahap, Astika Gita Ningrum 12 Volume 2, Nomor 11, Oktober 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X The articles were selected based on the following criteria: Publication dates: from 1 January 2019 to 2021; Languages: English, Indonesian; Article types: qualitative and quantitative articles with open access option. The articles must give information about the relationship between Adolescent Pregnancy and stunting. The search results were screened by reading the titles and abstracts. All articles that were not related to the scope of this review were excluded. Studies not including human subjects were excluded. g j A literature search was conducted using electronic databases namely, PubMed (Medline), Web of science, Scopus and ScienceDirect. Key words entered into the search in various combinations included “adolescent pregnancy or teenage pregnancy or adolescent mother and stunting.” Limitations placed on each search include the year of publication between 2019 and 2021 in both Indonesian and English language. The results of the articles search will be described more clearly on the flow diagram of the database search process. a. Study Design Selection The journal search strategy was carried out by entering all keywords in each of the intended journals to find the appropriate data and getting the number 2650 journals. After going through the screening of titles and abstracts, only 104 articles were appropriate and 656 articles were declared not in accordance with the research objectives. The selection process was continued by reading the entire article by paying attention to the inclusion and exclusion criteria and then getting 15 suitable articles. However, after we equated it with the research objectives, there were only 5 articles that could be followed up. a. Study Design Selection The journal search strategy was carried out by entering all keywords in each of the intended journals to find the appropriate data and getting the number 2650 journals. After going through the screening of titles and abstracts, only 104 articles were appropriate and 656 articles were declared not in accordance with the research objectives. The selection process was continued by reading the entire article by paying attention to the inclusion and exclusion criteria and then getting 15 suitable articles. However, after we equated it with the research objectives, there were only 5 articles that could be followed up. p b. Inclusion and exclusion criteria Inclusion criteria: Reputable journal (ScienceDirect, Pubmed, Web of Science, Scopus) with quantitative and qualitative research conducted in Indonesia and open access. The exclusion criteria were Thesis, Thesis, Final Project, Health profile, booklet. b. 1. Outline of the results of the journals obtained Based on 5 journals that discuss teenage pregnancy with stunting in children under five years of age, it is concluded that there is a relationship between teenage pregnancy and stunting. In 2016, globally there were 154.8 million children under the age of 5 years suffering from child stunting, which is around 22.9%. The highest prevalence of stunting in the Southeast Asian region is Indonesia. The average prevalence of stunting under five in Indonesia is 36.4% (Kemenkes, 2021). Child RESULTS AND DISCUSSION In this article, we attach a prism flow chart that is carried out as a process of screening articles that will be used in this literature review 1 J Flynn, FF Alkaff, WP Sukmajaya, S Salamah, Tahun 2020 Comparison of WHO growth standard and national Indonesian growth reference in determining prevalence and determinant s of stunting and underweight in children 218 Cross sectional Multivariate analysis indicated that Children with maternal age under 20 years old during pregnancy are more likely to be underweight 1243 http://sosains.greenvest.co.id http://sosains.greenvest.co.id The Association Of Adolescent Pregnancy With Stunting Incidence In Child Under Five Years Old 2022 under five: a cross- sectional study from Musi sub- district 2 Noviati Fuada, Leni Latiifah, Diyah Yunitawat, Hadi Ashar Assessment of Nutritional status of children under-five in families of Adolescent Mothers in Indonesia 2012 978 Families of adolescent mothers of children under-Five cross- sectional study Design This study showed mental emotional disorder of adolescent mother, related with underweight and stunting.Another factor, which is related with nutritional status is clean and healthy behaviors of the 2 Noviati Fuada, Leni Latiifah, Diyah Yunitawat, Hadi Ashar Assessment of Nutritional status of children under-five in families of Adolescent Mothers in Indonesia 2012 978 Families of adolescent mothers of children under-Five cross- sectional study Design This study showed mental emotional disorder of adolescent mother, related with underweight and stunting.Another factor, which is related with nutritional status is clean and healthy behaviors of the mother . Adelbertha A. B. Namita Candra Devi., Rita Oktavia Harahap, Astika Gita Ningrum 1244 3 Ingka K. Pangaribuana, Isyos Sari, Marlina Simbolona, Basaria Manurung, Kosheila Ramuni Relationshi p between early marriage and teenager pregnancy to stunting in toddler at Bangun Rejo Village, Tanjung Morawa District, Tanjung Morawa, Deli Serdang 645 toddler aged between 0 and 59 months cross- sectional study Design The result showed that there was correlation between early marriage and teenage pregnancy to stunting in a toddler. 4 Heru Subaris Kasjono, Agus Wijanarko, Rizki Amelia, Dina Impact of Early Marriage on Childhood 310 children aged 0-59 cross- sectional study Teenage pregnant women who had stunting children Adelbertha A. B. 5 Kencana Sari, Ratu Ayu Dewi Sartika RESULTS AND DISCUSSION Prevention of pregnancy complications is actually possible through early detection carried out with regular and quality inspections (Sulis, Wahyuni, & Prasetyo, 2020) In a study conducted by Jeannie Flynn and friends in 2020, it was found that children born to mothers with maternal age under 20 years during pregnancy had a greater likelihood of being underweight (Flynn, Alkaff, Sukmajaya, & Salamah, 2020).The prevalence of stunting and underweight is significantly lower when measured using Indonesian national benchmarks compared to using WHO standards. In the study, it was stated that the national growth reference of each country is more suitable to reflect the condition of its own population (Mo-Suwan & Choprapawon, 2016). Currently, early marriage has become a global problem, as well as the nutritional status of children under five. At the age of teenagers, they are not ready to get married, both in terms of reproductive health and mental health. Early marriage can contribute to a cycle of poverty and powerlessness in women. A study conducted by Noviati in 2013 discussed factors related to the nutritional status of children under five. The research design used was cross sectional with logistic analysis (chi-square test) on 978 samples. The results of the study found that a significant factor related to malnutrition was the mother's emotional mental disorder. Underweight, stunting, wasting, are closely related to clean and healthy living behavior and emotional mental disorders in adolescent mothers (Fuada, Latifah, Yunitawat, & Ashar, 2020). In another study conducted in Bangun Rejo Village, Tanjung Morawa District, Deli Serdang in 2019 showed that there was a relationship between early marriage and teenage pregnancy with the incidence of stunting in toddlers. Teenage pregnancies have a greater chance of giving birth to premature babies or babies with low birth weight. Teenage pregnancies are usually unplanned and occur more frequently in economically disadvantaged populations. Lack of education in adolescent mothers causes poor parenting in children (Pangaribuan, Sari, Simbolon, Manurung, & Ramuni, 2020). This study revealed that the majority of stunting occurred in the first 2 years of life. The early period of child malnutrition starts from the period of fetal development in malnourished mothers. Maternal nutrition during pregnancy plays an important role in the growth and survival of the child. Mothers who experience pregnancy at a young age are not fully developed biologically. This results in less nutrient flow to the fetus during pregnancy. RESULTS AND DISCUSSION Namita Candra Devi., Rita Oktavia Harahap, Astika Gita Ningrum 1244 Volume 2, Nomor 11, Oktober 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Fadillah, Wahyu Wijanarko, Sutaryono Stunting months design 5 Kencana Sari, Ratu Ayu Dewi Sartika The Effect of the Physical Factors of Parents and Children on Stunting at Birth Among Newborns in Indonesia 756 newborn cross sectional In terms of age, children whose parents were both younger than 20 or older than 35 had the highest proportion of stunted newborns (12.8%), of which the proportion of males (13.7%) was higher than females (11.4%) 5 Kencana Sari, Ratu Ayu Dewi Sartika The Effect of the Physical Factors of Parents and Children on Stunting at Birth Among Newborns in Indonesia 756 newborn cross sectional In terms of age, children whose parents were both younger than 20 or older than 35 had the highest proportion of stunted newborns (12 8%) of 1. Outline of the results of the journals obtained Based on 5 journals that discuss teenage pregnancy with stunting in children under five years of age, it is concluded that there is a relationship between teenage pregnancy and stunting. In 2016, globally there were 154.8 million children under the age of 5 years suffering from child stunting, which is around 22.9%. The highest prevalence of stunting in the Southeast Asian region is Indonesia. The average prevalence of stunting under five in Indonesia is 36.4% (Kemenkes, 2021). Child 1245 http://sosains.greenvest.co.id The Association Of Adolescent Pregnancy With Stunting Incidence In Child Under Five Years Old The Association Of Adolescent Pregnancy With Stunting Incidence In Child Under Five Years Old 2022 2022 stunting can occur in the first 1000 days after conception which is influenced by many factors, namely socio-economic status, food intake, infection, maternal nutritional status, infectious diseases, micronutrient deficiencies and the environment. From WHO data, it was found that women aged 15-19 years who had given birth at least once, can be at risk of having babies with low birth weight and children can be stunted. This happens because the mother's own growth has not been completed at this age (Marques, Loureiro, Avelar-Rosa, Naia, & Matos, 2020). Teenage pregnancy is a high risk pregnancy. Adelbertha A. B. Namita Candra Devi., Rita Oktavia Harahap, Astika Gita Ningrum Volume 2, Nomor 11, Oktober 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Volume 2, Nomor 11, Oktober 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X with low birth weight or premature birth. Both of these risk factors are responsible for the occurrence of stunting in infants. From the journal entitled Impact of Early Marriage on Childhood Stunting, Heri and colleagues conducted a study using the Cross Sectional method. This research was conducted at 8 Public Health Center in West Bangka Belitung Islands, Indonesia in October-November 2018. The research sample consisted of 310 children aged 0-59 months. Based on variables that have risk factors for stunting in West Bangka Regency, the age of early marriage (under 20 years) is still high. Early marriage among adolescents is generally associated with early pregnancy, and pregnancy increases the need for iron and affects iron deficiency and iron deficiency anemia experienced by adolescent girls. Approximately 1,000 mg of iron is needed to support changes associated with pregnancy, such as increased blood volume, ideal fetal growth and development. Most pregnant women, both in developing and developed countries, have low iron stores in early pregnancy. Teenage pregnancy becomes more risky because of the increased need for iron during pregnancy coupled with the need for iron during the mother's own growth spurt. Malnutrition with anemia and underweight can cause low birth weight babies when compared to women of childbearing age who are safe for pregnancy (Kasjono, Wijanarko, Amelia, Fadillah, & Wijanarko, 2020). Kencana Sari and Ratu Ayu Dewi in 2021 conducted a study by analyzing secondary data from the 2018 Indonesian Basic Health Survey conducted in 34 provinces and 512 regencies/cities. The results showed that the incidence of stunting could be associated with maternal age at the time of first pregnancy, parity, parental height, parental age, and gestational age. Children of mothers who gave birth first at the age of 25 years or older were less likely to be stunted at birth compared to the age group of mothers under 25 years. Children whose parents are both < 20 years old are twice as likely to be stunted at birth. RESULTS AND DISCUSSION Physically, teenage pregnant women are also still not fully developed, so there can be a struggle for nutrients between the fetus and the mother's own metabolism. This situation will lead to insufficient intake of maternal nutrients so that the fetus will experience growth retardation. This stunted growth can increase the risk of the fetus being born Adelbertha A. B. Namita Candra Devi., Rita Oktavia Harahap, Astika Gita Ningrum 1 1246 Volume 2, Nomor 11, Oktober 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X a. Direct and indirect factors of stunting g Stunting is a chronic nutritional problem caused by multifactorial and is intergenerational. There are two factors causing stunting, namely direct and indirect factors. Direct factors were determined by the intake of food, birth weight, and disease, while the indirect factors were such as economic, cultural, education and employment, health-care facilities. Education is one of the indirect factors that can also affect the occurrence of stunting because the mother's low knowledge in providing nutrition to her child is also not optimal. The age of parents who are too young, which is less than 20 years old, has twice the risk of stunting compared to pregnancy at the ideal age, in boys the incidence is higher than in girls. Children whose parents are below average height are six times more likely to experience stunting. b. Parenting Stunting is also caused by several factors, one of which is parenting. Parents in providing parenting to children play an important role in shaping children's eating behavior and eating patterns (Govender, Taylor, & Naidoo, 2020). Parenting patterns are defined as the behavior of parents in caring for toddlers. Negative parenting by parents, especially mothers, increases the risk of stunting in children (Pertiwi, Lestari and Ulfiana, 2019). Negative parenting patterns are often associated with parental age, parental education and knowledge in providing care to children. Parenting patterns are one of the main factors that have a close relationship with the incidence of stunting in the community (Yenita, Thamrin, Amin, & Agrina, 2021). According to (Govender et al., 2020) Maternal parenting is one of the factors causing the high prevalence of stunting in toddlers aged 12-36 months. Mothers with poor parenting have a 1.47 times higher prevalence of stunting under five than mothers with good parenting. In addition, pregnant adolescents have problems in parenting behavior, difficulties in raising children, and physical and mental health problems. A teenage mother still has to be guided because she is not ready to become a mother and a comprehensive education program is needed for teenage mothers in childcare readiness CONCLUSION Adolescent pregnancies that occur in Indonesia in most of the articles are the cause of stunting, but there are also some articles which announce that there is no relationship between teenage pregnancy and the incidence of stunting in children under 5 years of age. However, we need to compare the incidence rates in Indonesia with other developing countries so that we can conclude the main problem causing stunting in developing countries. Volume 2, Nomor 11, Oktober 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Women who gave birth for the first time at the age of less than 27 years had lower levels of maturity (Finlay, Özaltin, & Canning, 2011) Mothers whose first pregnancy was in the 27-29 year age range were more likely to live in better sanitary conditions, have higher levels of education, have higher socioeconomic status, have a partner, and live in urban areas (Sari & Sartika, 2021). At the age of 20 and under, a woman's uterus and pelvis are not fully developed and have a greater chance of developing severe preeclampsia, eclampsia, postpartum hemorrhage, poor fetal growth, and fetal distress. Pregnant women aged 35 years have a greater chance of preterm delivery, hypertension, superimposed preeclampsia, severe preeclampsia, and a reduced risk of chorioamnionitis. Older women (≥40 years) have an increased risk of mild preeclampsia, fetal distress, and poor fetal growth (Kyozuka et al., 2021). Preeclampsia still becomes an important topic related to cardiovascular system health in more than 300 million women worldwide and has both short-term and long-term morbidity (Akbar, Kinanti, Ernawati, & Lestari, 2021). This suggests that maternal age during pregnancy can result in poor birth outcomes that hinder the growth potential of the child. Young mothers are advised to delay pregnancy. Because pregnancy in adolescent mothers poses an adverse risk to the health of the child, one of which is stunting. Midwives play an important role in providing education to young mothers to increase awareness of their own health. 1247 http://sosains.greenvest.co.id The Association Of Adolescent Pregnancy With Stunting Incidence In Child Under Five Years Old 2022 The Association Of Adolescent Pregnancy With Stunting Incidence In Child Under Five Years Old Volume 2, Nomor 11, Oktober 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X births: evidence from 55 low-and middle-income countries. BMJ Open, 1(2), e000226. Flynn, Jeannie, Alkaff, Firas Farisi, Sukmajaya, William Putera, & Salamah, Sovia. (2020). Comparison of WHO growth standard and national Indonesian growth reference in determining prevalence and determinants of stunting and underweight in children under five: a cross-sectional study from Musi sub-district. F1000Research, 9. Fuada, Noviati, Latifah, Leni, Yunitawat, Dyah, & Ashar, Hadi. (2020). Assessment of Nutritional Status of Children Under-Five in Families of Adolescent Mothers in Indonesia 2013. Journal of Nutritional Science and Vitaminology, 66(Supplement), S425–S431. Govender, Desiree, Taylor, Myra, & Naidoo, Saloshni. (2020). Adolescent pregnancy and parenting: Perceptions of healthcare providers. Journal of Multidisciplinary Healthcare, 13, 1607. Irwansyah, Irwansyah, Ismail, Djauhar, & Hakimi, Mohammad. (2016). Kehamilan Remaja dan Kejadian Stunting Anak Usia 6-23 Bulan di Lombok Barat. Berita Kedokteran Masyarakat, 32(6), 209–216. j , , j , g , , , , , j , Wahyu. (2020). Impact of Early Marriage on Childhood Stunting. 1st International Conference on Science, Health, Economics, Education and Technology (ICoSHEET 2019), 172–174. Atlantis Press. Kemenkes, R. I. (2021). Profil Kesehatan Indonesia 2020. Kementrian Kesehatan Republik Indonesia. Https://Pusdatin. Kemkes. Go. Id/Resources/Download/Pusdatin/Profil- Kesehatan-Indonesia/Profil-Kesehatan-Indonesia-Tahun-2020. Pdf. Kyozuka, Hyo, Murata, Tsuyoshi, Fukusda, Toma, Yamaguchi, Akiko, Kanno, Aya, Yasuda, Shun, Sato, Akiko, Ogata, Yuka, Endo, Yuta, & Hosoya, Mitsuaki. (2021). Teenage pregnancy as a risk factor for placental abruption: Findings from the prospective Japan environment and children’s study. Plos One, 16(5), e0251428. Yasuda, Shun, Sato, Akiko, Ogata, Yuka, Endo, Yuta, & Hosoya, Mitsuaki. (2021). Teenage pregnancy as a risk factor for placental abruption: Findings from the prospective Japan environment and children’s study. Plos One, 16(5), e0251428. Marques, Adilson, Loureiro, Nuno, Avelar-Rosa, Bruno, Naia, Ana, & Matos, Margarida Gaspar de. (2020). Adolescents’ healthy lifestyle. Jornal de Pediatria, 96(2), 217– 224. https://doi.org/10.1016/j.jped.2018.09.002 Mo-Suwan, Ladda, & Choprapawon, Chanpen. (2016). Comparison of prevalence of nutritional status of thai children in the first 2 years of life using national and international growth charts. J Med Assoc Thai, 99(1), 58–64. Pangaribuan, Ingka K., Sari, Isyos, Simbolon, Marlina, Manurung, Basaria, & Ramuni, Kosheila. (2020). Relationship between early marriage and teenager pregnancy to stunting in toddler at Bangun Rejo Village, Tanjung Morawa District, Tanjung Morawa, Deli Serdang 2019. Enfermeria Clinica, 30, 88–91. Pertiwi, Melinda Restu, Lestari, Pudji, & Ulfiana, Elida. (2019). Relationship between parenting style and perceived information sources with stunting among children. International Journal of Nursing and Health Services (IJNHS), 2(4), 273–279. BIBLIOGRAPHY Akbar, Muhammad I. A., Kinanti, Hapsari, Ernawati, Ernawati E., & Lestari, Pudji. (2021). Maternal and Perinatal Outcomes of Early-onset and Late-onset Preeclampsia at a Tertiary Center Hospital. Journal of South Asian Federation of Obstetrics and Gynaecology, 13(5), 339. Akbar, Muhammad I. A., Kinanti, Hapsari, Ernawati, Ernawati E., & Lestari, Pudji. (2021). Maternal and Perinatal Outcomes of Early-onset and Late-onset Preeclampsia at a Tertiary Center Hospital. Journal of South Asian Federation of Obstetrics and Gynaecology, 13(5), 339. Eka, Mufidah Binti, Krisnana, Ilya, & Husada, Dominicus. (2020). Risk Factors Of Stunting Events In Toddlers Aged 24-59 Months. Indonesian Midwifery and Health Sciences Journal, 4(4), 374–385. Eka, Mufidah Binti, Krisnana, Ilya, & Husada, Dominicus. (2020). Risk Factors Of Stunting Events In Toddlers Aged 24-59 Months. Indonesian Midwifery and Health Sciences Journal, 4(4), 374–385. Eka, Mufidah Binti, Krisnana, Ilya, & Husada, Dominicus. (2020). Risk Factors Of Stunting Events In Toddlers Aged 24-59 Months. Indonesian Midwifery and Health Sciences Journal, 4(4), 374–385. ( ) Finlay, Jocelyn E., Özaltin, Emre, & Canning, David. (2011). The association of maternal i h i f li hild h i f il di h d i f fi ( ) Finlay, Jocelyn E., Özaltin, Emre, & Canning, David. (2011). The association of maternal age with infant mortality, child anthropometric failure, diarrhoea and anaemia for first Finlay, Jocelyn E., Özaltin, Emre, & Canning, David. (2011). The association of maternal age with infant mortality, child anthropometric failure, diarrhoea and anaemia for first inlay, Jocelyn E., Özaltin, Emre, & Canning, David. (2011). The association of maternal age with infant mortality, child anthropometric failure, diarrhoea and anaemia for first y y g age with infant mortality, child anthropometric failure, diarrhoea and anaemia for first 1248 Adelbertha A. B. Namita Candra Devi., Rita Oktavia Harahap, Astika Gita Ningrum 124 Volume 2, Nomor 11, Oktober 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X The Association Of Adolescent Pregnancy With Stunting Incidence In Child Under Five Years Old The Association Of Adolescent Pregnancy With Stunting Incidence In Child Under Five Years Old 2022 Stunting Incidence In Child Under Five Years Old (WHO/NMH/NHD14. 7). Geneva: World Health Organization. Yadika, Adilla Dwi Nur, Berawi, Khairun Nisa, & Nasution, Syahrul Hamidi. (2019). Pengaruh stunting terhadap perkembangan kognitif dan prestasi belajar. Jurnal Majority, 8(2), 273–282. Yenita, Riski Novera, Thamrin, Thamrin, Amin, Bintal, & Agrina, Agrina. (2021). Identification and Analysis of Stunting Risk Factors in Children under Three Years of Age in the Area of Kampar Watershed. Open Access Macedonian Journal of Medical Sciences, 9(G), 149–157. o This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License. Volume 2, Nomor 11, Oktober 2022 p-ISSN 2774-7018 ; e-ISSN 2774-700X Sari, Kencana, & Sartika, Ratu Ayu Dewi. (2021). The effect of the physical factors of parents and children on stunting at birth among newborns in Indonesia. Journal of Preventive Medicine and Public Health, 54(5), 309. Sulis, Diana, Wahyuni, Chatarina Umbul, & Prasetyo, Budi. (2020). Maternal complications and risk factors for mortality. Journal of Public Health Research, 9(2). Wemakor, Anthony, Garti, Humphrey, Azongo, Thomas, Garti, Helene, & Atosona, Ambrose. (2018). Young maternal age is a risk factor for child undernutrition in Tamale Metropolis, Ghana. BMC Research Notes, 11(1), 1–5. p ( ) WHO-COVID, W. H. O. (2022). 19-statistics. World Health Organization. WHO-COVID, W. H. O. (2022). 19-statistics. World Health Organization. WHO, UNICEF. (2014). Global nutrition targets 2025: breastfeeding policy brief WHO COVID, W. H. O. (2022). 19 statistics. World Health Organization. WHO, UNICEF. (2014). Global nutrition targets 2025: breastfeeding policy brief 1249 http://sosains.greenvest.co.id (WHO/NMH/NHD14. 7). Geneva: World Health Organization. Yadika, Adilla Dwi Nur, Berawi, Khairun Nisa, & Nasution, Syahrul Hamidi. (2019). Pengaruh stunting terhadap perkembangan kognitif dan prestasi belajar. Jurnal Majority, 8(2), 273–282. j y Yenita, Riski Novera, Thamrin, Thamrin, Amin, Bintal, & Agrina, Agrina. (2021). Identification and Analysis of Stunting Risk Factors in Children under Three Years of Age in the Area of Kampar Watershed. Open Access Macedonian Journal of Medical Sciences, 9(G), 149–157. o This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License. 1250 Adelbertha A. B. Namita Candra Devi., Rita Oktavia Harahap, Astika Gita Ningrum 1250
https://openalex.org/W2952084164	http://library.oapen.org/bitstream/20.500.12657/31301/1/631951.pdf	English	null	Publishing Addiction Science: A Guide for the Perplexed	Ubiquity Press eBooks	2,017	cc-by	164,046	3RD EDITION 3RD EDITION EDITED BY Thomas F. Babor, Kerstin Stenius, Richard Pates, Michal Miovský, Jean O’Reilly, and Paul Candon Publishing Addiction Science: A Guide for the Perplexed Third Edition First published 2017 Cover design by Amber MacKay, developed from 2nd Edition cover by Matthew West of Vasco Graphics. Images used in the cover design were sourced from Pixabay and are licensed under CC0 Public Domain. Reproductions of journal covers and logos used with permission. Printed in the UK by Lightning Source Ltd. Print and digital versions typeset by Siliconchips Services Ltd. Printed in the UK by Lightning Source Ltd. Print and digital versions typeset by Siliconchips Services Ltd. ISBN (Paperback): 978-1-911529-08-8 ISBN (PDF): 978-1-911529-09-5 ISBN (EPUB): 978-1-911529-10-1 ISBN (Mobi): 978-1-911529-11-8 DOI: https://doi org/10 5334/bbd ISBN (Paperback): 978-1-911529-08-8 ISBN (PDF): 978-1-911529-09-5 ISBN (EPUB): 978-1-911529-10-1 ISBN (Mobi): 978-1-911529-11-8 DOI: https://doi.org/10.5334/bbd ISBN (EPUB): 978-1-911529-10-1 ISBN (Mobi): 978-1-911529-11-8 Edited by Published by Ubiquity Press Ltd. 6 Windmill Street London W1T 2JB www.ubiquitypress.com Text © The Authors 2017 DOI: https://doi.org/10.5334/bbd This work is licensed under the Creative Commons Attribution 4.0 Interna tional License (unless stated otherwise within the content of the work). To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ or send a letter to Creative Commons, 444 Castro Street, Suite 900, Mountain View, California, 94041, USA. This license allows for copying any part of the work for personal and commercial use, providing author attribution is clearly stated. The full text of this book has been peer-reviewed to ensure high academic d d F f ll i li i h // bi i / The full text of this book has been peer-reviewed to ensure high academic standards. For full review policies, see http://www.ubiquitypress.com/ h p g standards. For full review policies, see http://www.ubiquitypress.com/ Suggested citation: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P (eds.) 2017 Publishing Addiction Science: A Guide for the Perplexed. London: Ubiquity Press. DOI: https://doi.org/10.5334/bbd. License: CC-BY 4.0 Suggested citation: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P (eds.) 2017 Publishing Addiction Science: A Guide for the Perplexed. London: Ubiquity Press. DOI: https://doi.org/10.5334/bbd. License: CC-BY 4.0 To read the free, open access version of this book online, visit https://doi.org/10.5334/bbd or scan this QR code with your mobile device: We dedicate this book to two people who changed addiction science for the better: Lenka Čablová (1986–2016) and Griffith Edwards (1928–2012) Contents Contents Foreword ix Preface xi About the Authors xvii Supporting Institutions xxi Section 1: Introduction 1 Chapter 1: A Guide for the Perplexed (Thomas F. Babor, Kerstin Stenius and Jean O’Reilly) 3 Chapter 2: Infrastructure and Career Opportunities in Addiction Science: The Emergence of an Interdisciplinary Field (Thomas F. Babor, Dominique Morisano, Jonathan Noel, Katherine Robaina, Judit H. Ward and Andrea L. Mitchell) 9 Section 2: How and Where to Publish 35 Chapter 3: How to Choose a Journal: Scientific and Practical Considerations (Thomas F. Babor, Dominique Morisano, Kerstin Stenius and Judit H. Ward) 37 Chapter 4: Beyond the Anglo-American World: Advice for Researchers from Developing and Non–English-Speaking Countries (Kerstin Stenius, Florence Kerr-Corrêa, Isidore Obot, Erikson F. Furtado, Maria Cristina Pereira Lima and Thomas F. Babor) 71 Chapter 5: Getting Started: Publication Issues for Graduate Students, Postdoctoral Fellows, and other Aspiring Addiction Scientists (Dominique Morisano, Erin L. Winstanley, Neo Morojele and Thomas F. Babor) 89 Foreword Preface About the Authors Supporting Institutions Section 1: Introduction 89 vi Contents Chapter 6: Addiction Science for Professionals Working in Clinical Settings (Richard Pates and Roman Gabrhelík) 119 Section 3: The Practical Side of Addiction Publishing 133 Chapter 7: How to Write a Scientific Article for a Peer-Reviewed Journal (Phil Lange, Richard Pates, Jean O’Reilly and Judit H. Ward) 135 Chapter 8: How to Write Publishable Qualitative Research (Kerstin Stenius, Klaus Mäkelä, Michal Miovský and Roman Gabrhelík) 155 Chapter 9: How to Write a Systematic Review Article and Meta-Analysis (Lenka Čablová, Richard Pates, Michal Miovský and Jonathan Noel) 173 Chapter 10: Use and Abuse of Citations (Robert West, Kerstin Stenius and Tom Kettunen) 191 Chapter 11: Coin of the Realm: Practical Procedures for Determining Authorship (Thomas F. Babor, Dominique Morisano and Jonathan Noel) 207 Chapter 12: Preparing Manuscripts and Responding to Reviewers’ Reports: Inside the Editorial Black Box (Ian Stolerman and Richard Pates) 229 Chapter 13: Reviewing Manuscripts for Scientific Journals (Robert L. Balster) 245 Section 4: Ethics Matter 265 Chapter 14: Dante’s Inferno: Seven Deadly Sins in Scientific Publishing and How to Avoid Them (Thomas F. Babor, Thomas McGovern and Katherine Robaina) 267 Chapter 15: The Road to Paradise: Moral Reasoning in Addiction Publishing (Thomas McGovern, Thomas F. Babor and Kerstin Stenius) 299 Section 4: Ethics Matter Contents vii Chapter 16: Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies, and Other Funding Agencies: Holy Grail or Poisoned Chalice? (Peter Miller, Thomas F. Babor, Thomas McGovern, Isidore Obot and Gerhard Bühringer) 323 Section 5: Conclusion 353 Chapter 17: Addiction Publishing and the Meaning of [Scientific] Life (Thomas F. Babor, Kerstin Stenius and Jean O’Reilly) 355 Index 365 Section 5: Conclusion Index Foreword to the Third Edition The health and social burden attributable to psychoactive substance use is enormous. Alcohol, tobacco and illicit drug use taken together are by far the most important preventable risk factors to a population’s health. Accord ing to the latest WHO estimates, the harmful use of alcohol alone results in around 3.3 million deaths every year. With rapid social and cultural changes taking place in many countries, alcohol and drug use are becoming increas ingly embedded in social matrices, often with strong commercial forces playing a role in promoting the use of legal intoxicating and dependence-producing substances. A number of jurisdictions have undertaken major changes in the regulation of psychoactive substances controlled under international drug trea ties. New Psychoactive Substances (NPS), with their health effects and distri bution channels, present new challenges for public health authorities. Debates around alcohol and drugs are at the forefront of social policy processes in many countries, with significant variations in societal responses. Unfortunately, these debates are often not based on solid data or research evidence, and in many cases the relevant data simply does not exist. Significant caveats exist in the evaluation of existing policy responses and policy changes made in different jurisdictions. There is an urgent need to strengthen the evidence base for the development of adequate program and policy responses to substance use and substance use disorders at different levels.fii f It is difficult to overestimate the role of research and scientific data in shap ing policy and program responses at all scales, from local communities to the x Foreword to the Third Edition international level. A consistent and common issue is the lack of sufficient resources for research on substance use and substance use disorders, and very often even those resources available are not utilized to their maximum poten tial. One of the biggest problems is when investment in research does not result in the publication and dissemination of results, preferably in peer-reviewed journals. This is a particularly prevalent issue in less-resourced countries where opportunities for publishing results of research on substance use and substance use disorders are limited, and where no specialized journals on addiction exist.h p j The third edition of Publishing Addiction Science: A Guide for the Perplexed is an important resource for researchers around the world, especially for those who work in low and middle-income countries. Foreword to the Third Edition It is hoped that this resource will facilitate the dissemination of new data and knowledge in this area, given that research remains very much skewed towards a limited number of high- income countries with well-developed research and publishing infrastructures. The International Society of Addiction Journal Editors (ISAJE) continues to work towards increasing the publishing competence of researchers from all over the world, with this work often being implemented in consultation with our program in the World Health Organization. Such efforts make a significant and much needed contribution to capacity building in research on substance use and substance use disorders, particularly in less-resourced countries, and the WHO Department of Mental Health and Substance Abuse welcomes the third edition. We look forward to continued collaboration with ISAJE in this area. Dr Shekhar Saxena Director Department of Mental Health and Substance Abuse World Health Organization An Idea Whose Time Has Come The development of this book had many complex motives but a single pur pose. The motives include improving scientific integrity in the field of addic tion studies, sharing information with junior investigators, and strengthening addiction specialty journals. The single purpose of this volume, however, is to provide a practical guide to scientific publishing in the addiction field that is used often enough to affect personal decisions, individual careers, institutional policies, and the progress of science. The time is ripe for such an ambitious undertaking: The field of addiction research has grown tremendously in recent years and has spread to new parts of the world. With that growth has come a concomitant increase in competition among researchers, new bureaucratic regulations, and a growing interest in addiction research by health agencies, policy-makers, treatment and prevention specialists, and the alcohol industry. New professional societies, research centers, and university programs have taken root, and regulatory responsibilities such as conflict of interest declara tions, human and animal subjects assurances, and the monitoring of scientific misconduct are now common.hi The journal-publishing enterprise, the main organ of scientific communica tion in the field, has an important role to play in all of these developments, and xii Preface the third edition of Publishing Addiction Science is designed to meet this need. The inspiration for the first edition of this volume came from the International Society of Addiction Journal Editors (ISAJE), which is not only the first society for addiction journal editors, it is also the first international organization spe cifically devoted to the improvement of scientific publishing in the addiction field. i From its inception, ISAJE has recognized a need for ethical guidelines for member journals. There are several reasons why ethical issues are particularly important in the addiction science field. Strong industries, such as pharmaceu tical manufacturers, tobacco companies, and alcohol producers, have impor tant financial interests to protect, and they pay special attention to the work of addiction scientists. Further, many addiction-related issues are politically loaded, a situation that could affect the objectivity of researchers. Many of the individuals who are the object of addiction research are vulnerable and in need of special protections. Finally, the field of science has become much more ethi cally challenging because of its growing importance and complexity. An Idea Whose Time Has Come Although ISAJE offers a set of ethical guidelines, abstract policy statements and moral pronouncements are rarely read carefully or applied to the day-to-day business of conducting research and communicating ideas to the scientific community. This book aims to improve transparency in addiction publishing and, in the process, show how young investigators can negotiate the complex and some times bewildering ethical challenges faced on the path to a successful career in the field. E-Attachments e-Attachments are additional supplementary materials that can be used to deepen your understanding of the concepts in Publishing Addiction Science. e-Attachments comprise additional information sources, readings, examples and exercises that can improve your skills and help you practice your first steps in the publishing world. You can find 6 different kinds of e-Attachments on our websites: readings, exercises, examples of good practice, simple Power Point presentations, videos and full e-learning lessons. Some items are used for more than one chapter while others are quite specific to their chapters. For your effective use of the e-Attachments and the book, please follow the instructions on our website. e-Attachments are additional supplementary materials that can be used to deepen your understanding of the concepts in Publishing Addiction Science. e-Attachments comprise additional information sources, readings, examples and exercises that can improve your skills and help you practice your first steps in the publishing world. You can find 6 different kinds of e-Attachments on our websites: readings, exercises, examples of good practice, simple Power Point presentations, videos and full e-learning lessons. Some items are used for more than one chapter while others are quite specific to their chapters. For your effective use of the e-Attachments and the book, please follow the instructions on our website. All e-Attachments are free to download from the website of the International Society of Addiction Journal Editors (ISAJE) on www.isaje.net. e-Attachments will be updated continually.hf There are six kinds of e-Attachments, each with a different purpose: • Readings provide additional information about a chapter or issues discussed in more than one chapter. Some of these documents provide more contex tual information or are original documents to which the chapter refers.h • Exercises are materials for practicing and training. They are appropriate for individual or group application. • Examples of good practice provide a better understanding of topics or themes discussed in the chapters. • Simple PowerPoint presentations are mainly designed for use by teachers and lecturers but students and readers may find them useful as simple e-learning documents that provide well-structured information complementary to the full chapter text. • Videos, like the PowerPoint presentations, provide actual presentations or workshop/training lectures given by the chapter author(s) or one of more of their colleagues from ISAJE.h • Full e-learning lessons provide more sophisticated e-leaning support. Rationale for the Third Edition There are several reasons why a third edition of Publishing Addiction Science is necessary. First, rapid developments in the field of addiction publishing neces sitate revisions of parts of this book, particularly the move to online and open- access publication options, the launching of many new addiction specialty journals, and the new ethical and technological challenges facing addiction publishing. For example, more than 30 new journals have been identified since the second edition of the book was published in 2008, many of them launched by for-profit enterprises with little appreciation for scientific quality or peer review. Another reason for the third edition is related to experience from our Pub lishing Addiction Science workshops, which have been conducted during the past few years in many parts of the world, including Denmark, Finland, Greece, Jordan, Nigeria, South Korea, Uganda, the United Kingdom, and the United States. The workshops identified new areas of interest that needed attention. To make Publishing Addiction Science even more relevant to its target market of advanced students and young professionals, the third edition has accordingly Preface xiii Preface xiii added new material on publication issues faced by postdoctoral researchers, the ethical challenges of research funding, how to write a research paper, and procedures for peer-reviewing manuscripts,. The development of new online training material will enable the book to continue to be used as a textbook for research ethics in colleges and universities and in training workshops at scien tific meetings. Sponsorship/Acknowledgements The publication costs for revising and reprinting this book were covered by the book’s primary sponsors: the international journal Addiction, the (U.K.) Society for the Study of Addiction, and the International Society of Addiction Journal Editors (ISAJE). We are also grateful to the academic institutions that enabled the authors and editors to work on this collaborative effort, includ ing The University of Connecticut Alcohol Research Center (Farmington, Connecticut, USA, NIAAA Grant # 5P60AA003510-39), the Nordic Welfare Centre (Helsinki, Finland), the Rutgers University Center of Alcohol Studies (Piscataway, New Jersey, USA), and the Department of Addictology, Charles University in Prague (Czech Republic; Grant No. PRVOUK-P03/LF1/9). A number of individuals provided key contributions to the third edition; in par ticular, we thank Deborah Talamini and Melissa Feulner.i We also thank sponsors who have provided financial support for develop ing online supplementary materials, training materials, workshops and trans lations related to Publishing Addiction Science. These include the American Academy of Addiction Psychiatry (AAAP), Charles University in Prague’s Department of Addictology, the International Order of Good Templars (IOGT International), the U.S. National Institute on Drug Abuse (NIDA), the Research Society on Alcoholism (RSA), Substance Abuse Librarians & Information Spe cialists (SALIS), and Wiley. y Finally, we thank an even larger number of organizations that have helped us to disseminate the book’s contents and its online materials. These include all organizations mentioned in the two paragraphs above as well as the American Society of Addiction Medicine (ASAM), the College on Problems of Drug Dependence (CPDD), the Nigerian Centre for Research and Infor mation on Substance Abuse (CRISA), the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), the International Confederation of Addiction Research Associations (ICARA), the International Society for the Study of Drug Policy (ISSDP), and the Kettil Bruun Society (KBS). E-Attachments They combine PowerPoint slides with the full text of a presentation and fin ish with a knowledge test that lets you check your understanding of the lesson. xiv Preface xiv Preface xiv Preface Closing We hope that the third edition of this book will aid the training of young research ers and the continuing education of seasoned addiction scientists around the world. Given the book’s continued focus on supporting young scientists who are entering the field and its goal of improving the integrity and ethicality of addic tion science, we dedicate this edition of the book to Lenka Čablová (1986–2016) and Griffith Edwards (1928–2012). Lenka was the lead author of Chapter 9. She was a promising young scientist whose short professional life was nevertheless filled with creative work on the interconnections among substance use, ADHD and nutrition, and an overarching concern with addiction and risk to families. Preface xv Preface xv Griffith’s career as an addiction scientist, master clinician, research center direc tor, and policy analyst served not only as an inspiration for this third edition of Publishing Addiction Science, but also as a model for the kind of addiction scientist the book’s content would like to inspire. The Editors About the Authors Thomas F. Babor is Professor and Chair, Department of Community Medicine and Health Care, University of Connecticut School of Medicine, USA. He is Editor-in-Chief of the Journal of Studies on Alcohol and Drugs. Robert L. Balster is the Luther A. Butler Professor of Pharmacology and Toxicology and Research Professor of Psychology and Psychiatry at Virginia Commonwealth University in Richmond, Virginia, USA. He is former Editor- in-Chief of the journal Drug and Alcohol Dependence. Gerhard Bühringer is Professor for Addiction Research in the Department of Clinical Psychology and Psychotherapy at the Technische Universität in Dresden, Germany. Lenka Čablová, now deceased, was a Postdoctoral Research Fellow at the Department of Addictology, First Faculty of Medicine, Charles University in Prague, and the General University Hospital in Prague, Czech Republic. Paul Candon is Managing Editor of the Journal of Studies on Alcohol and Drugs, based at the Center of Alcohol Studies at Rutgers, The State University of New Jersey, USA. xviii About the Authors Erikson F. Furtado is a full-time tenured Assistant Professor of Child and Ado lescent Psychiatry in the Department of Neurosciences and Behavior in the Faculty of Medicine at Ribeirão Preto, University of São Paulo, Brazil. Florence Kerr-Corrêa is Professor of Psychiatry, Department of Neurology, Psychology and Psychiatry, Botucatu Medical School, São Paulo State Univer sity (UNESP), Brazil. Roman Gabrhelík is Assistant Professor, Department of Addictology, First Medical Faculty, Charles University in Prague, Czech Republic. He is Executive Editor of the Czech journal Adiktologie (Addictology). Tom Kettunen is Editor of the journal Nordic Studies on Alcohol and Drugs (Nordisk alkohol- & narkotikatidskrift). Phil Lange has retired. He was Editor of the Journal of Gambling Issues. Klaus Mäkelä, now deceased, was Research Director of the Finnish Foundation for Alcohol Studies. Thomas McGovern is Professor Emeritus of Psychiatry and Founder Emeritus of the Center for Ethics/ Humanities/ Spirituality at the School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas, USA. He is Editor-in-Chief of Alcoholism Treatment Quarterly. Peter Miller is Professor of Violence Prevention and Addiction Studies at the School of Psychology, Deakin University, Australia. He was also the Commis sioning Editor of the journal Addiction from 2006–2016. Peter Miller is Professor of Violence Prevention and Addiction Studies at the School of Psychology, Deakin University, Australia. He was also the Commis sioning Editor of the journal Addiction from 2006–2016. About the Authors Michal Miovský is Head of the Department of Addictology, 1st Faculty of Med icine at Charles University in Prague, Czech Republic. He is also a clinical psy chologist, psychotherapist and supervisor. He is Deputy Editor-in-Chief of the Czech Journal Adiktologie (Addictology) and leads a creative team to establish academic study programs in addictions in Prague. Andrea L. Mitchell is the Executive Director of Substance Abuse Librarians & Information Specialists (SALIS). Andrea L. Mitchell is the Executive Director of Substance Abuse Librarians & Information Specialists (SALIS). Dominique Morisano is a clinical psychologist and research/evaluation consultant and appointed as Assistant Professor (Status-only), Dalla Lana School of Public Health, University of Toronto; Collaborator Scientist, Institute for Mental Health Policy Research, Centre for Addiction and Mental Health; About the Authors xix About the Authors xix Visiting Scholar, Rotterdam School of Management, Erasmus University (Netherlands); and Faculty, Centre for Mindfulness Studies. Neo Morojele is Deputy Director of the Alcohol, Tobacco & Other Drug Research Unit of the South African Medical Research Council. She is an Asso ciate Editor for the African Journal of Drug and Alcohol Studies and the Interna tional Journal of Alcohol and Drug Research and Associate Editor for Africa of the Journal of Substance Use. Jonathan Noel is a doctoral candidate in the Graduate Program in Public Health, Department of Community Medicine and Health Care, University of Connecticut School of Medicine, USA. Jean O’Reilly is Editorial Manager for the journal Addiction and a consulting book editor. Isidore Obot is Professor, Department of Psychology, University of Uyo, and Director, Centre for Research and Information on Substance Abuse (CRISA), Uyo, Nigeria. He has been Editor-in-Chief of the African Journal of Drug and Alcohol Studies since 2000. Richard Pates is a consultant clinical psychologist who worked in treatment of addiction problems in the NHS in the UK for 30 years. He now works at a secure children’s home. He has been Editor of The Journal of Substance Use for the past 16 years. He holds an honorary post at the University of Worcester. Maria Cristina Pereira Lima is an Associate Professor of Psychiatry, Depart ment of Neurology, Psychology and Psychiatry, Botucatu Medical School, São Paulo State University (UNESP), Brazil. About the Authors Katherine Robaina is a researcher at the Department of Community Medicine and Health Care, University of Connecticut School of Medicine, USA and is a member of Delta Omega, the Honorary Society in Public Health (Beta Rho chapter). Kerstin Stenius is guest professor at the Centre for Social Research on Alco hol and Drugs (SoRAD) at Stockholm University, Sweden. Until 2017 she was Editor-in-Chief of the journal Nordisk alkohol- & narkotikatidskrift (Nordic Studies on Alcohol and Drugs) at The Nordic Welfare Centre, Helsinki, Finland. Ian Stolerman is Emeritus Professor of Behavioural Pharmacology at the Insti tute of Psychiatry, Psychology and Neuroscience, King’s College London, UK. xx About the Authors He served as President of ISAJE and as co-editor of the journal Drug and Alcohol Dependence. Judit H. Ward is Science Reference/Instruction Librarian at Rutgers, The State University of New Jersey. She is Field Editor of the Journal of Studies on Alcohol and Drugs. Robert West is Professor of Health Psychology and Director of Tobacco Stud ies, Cancer Research UK Health Behaviour Research Centre, Department of Epidemiology and Public Health, University College London, UK. He is Edi tor-in-Chief of the journal Addiction. Erin L. Winstanley is an Assistant Professor of Health Outcomes at the James L. Winkle College of Pharmacy at the University of Cincinnati and the Director of Health Services Research, Mercy Health, USA. Supporting Institutions saje www.isaje.net INTERNATIONAL SOCIETY OF ADDICTION JOURNAL EDITORS 4/a Publication was supported from institutional programme of Charles University No. PRVOUK-P03/LF1/9 ıcara • International Confederation of ATOD Research Associations I O GT I N T E R N A T I O N A L SECTION 1 SECTION 1 How to cite this book chapter: Babor, T F, Stenius, K and O’Reilly, J. 2017. A Guide for the Perplexed. In: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P. (eds.) Publishing Addiction Science: A Guide for the Perplexed, Pp. 3–8. London: Ubiquity Press. DOI: https://doi.org/10.5334/bbd.a. License: CC-BY 4.0. Introduction CHAPTER 1 What is a Journal? According to Lafollette (1992, p. 69), “a journal is a periodical that an identifi able intellectual community regards as a primary channel for communication of knowledge in its field and as one of the arbitrators of the authenticity or legitimacy of that knowledge.” Journals establish intellectual standards, pro vide a forum of communication among scientists, bring valuable information to the public, set the agenda for a field of study, provide an historical record of a particular area of knowledge, and confer implicit certification on authors for the authenticity and originality of their work (Lafollette, 1992). In addition, journals have the potential to serve the interests of career advancement and personal reward for scholarly achievement. Journals are joint enterprises typically managed through a division of labor among owners, publishers, editors, reviewers, and authors. How this cast of characters is organized into an integrated set of players varies from one jour nal to another. The owners of a journal can be nonprofit organizations (such as learned societies, universities, or professional organizations), government agencies, or private publishers. The publishers of a journal range from small printers to large-scale, multi-national organizations that distribute often hun dreds of journals. Journal editors tend to be appointed by the owners, society officers, or publishers. Editors of some of the larger scientific and medical jour nals are paid for their services and have full-time staff at their disposal. Editors of smaller journals are generally unpaid and have a small editorial staff with some volunteer assistant editors. Reviewers are usually established investigators who have specialized knowledge of the subject matter. Without remuneration and as a service to the field, reviewers provide critical and often anonymous evaluations of manuscripts written by their peers. Without journals, addiction science—or any science—would have a limited audience and a short half-life. Therefore, scientists who wish to search for truth and to help humankind must understand the inner workings and current com plexities of the journal publication process. Thomas F. Babor, Kerstin Stenius and Jean O’Reilly Thomas F. Babor, Kerstin Stenius and Jean O’Reilly “I do not presume to think that this treatise settles every doubt in the minds of those who understand it, but I maintain that it settles the greater part of their difficulties.” fi Maimonides, Guide for the Perplexed (ca. 1190) To be perplexed is to be puzzled or even confused by the intricacy of a situation. One way to deal with perplexing situations is to find a guide who can provide advice, information, and direction. Many such guides have risen to the occasion throughout the ages, providing useful knowledge for the perplexed students of literature, religion, philosophy, and science. One of the most influential philo sophical treatises, for example, was Maimonides’ Guide for the Perplexed. In a time of religious, moral, and political change, Maimonides (1135–1204) sought to harmonize Greco-Roman, Christian, Jewish, and Arabic thought into a phil osophical guide for those seeking meaning in life. In a sense, Publishing Addic tion Science is intended to be a similar (albeit less ambitious!) guide for those of us who from time to time are perplexed about how to find our way through the complex world of addiction science. The chapters in this book constitute a virtual guide through the practical, scientific, moral, and even philosophical issues with which we must become acquainted if we are to succeed, either as temporary visitors to the field or as career scientists dedicating our lives to the study of addiction. It is our contention—and a guiding theme of the book—that the key to suc cessful publishing in addiction science is to understand not only how to write a scientific article and where to publish it but also how to do these things hon estly and ethically. Therefore, in addition to the practical business of publishing 4 Publishing Addiction Science scientific articles in both multi-disciplinary and addiction specialty journals, the ultimate goal of this book is to enhance scientific integrity in the publica tion process, giving special consideration to the main organ of scientific com munication, the scholarly journal. Purpose of the Guide The addiction field has grown tremendously in the past 35 years, and addic tion publishing has been no exception. Currently there are more than 120 journals devoted primarily to the dissemination of scholarly information about A Guide for the Perplexed 5 A Guide for the Perplexed 5 addiction and related health problems, and many more journals publish addic tion science as part of their broader mission. Despite the growing amount of published material in addiction science and the increasing opportunities for publication, there exists no other guide designed to inform prospective authors about the opportunities, requirements, and challenges of publishing addiction science. Moreover, the addiction field has become perhaps one of the first areas of science in which interdisciplinary collaboration between biomedical and psychosocial researchers is essential to progress (see Edwards, 2002). At the same time, however, as Matilda Hellman (2015) argues, we appear to be mov ing into an age of academic compartmentalization, with increasingly narrow fields of study in which researchers are encouraged to specialize. It is therefore important that addiction science, a field that is perhaps unfashionably collabo rative, has a publishing guide that looks at the field as an inter-related whole rather than as a collection of separate disciplines. Within this context, the primary purpose of Publishing Addiction Science is to advise potential authors of articles in the addiction field of the opportunities for publishing their work in scholarly journals, with an emphasis on addiction spe cialty journals. Although all prospective authors will find such a guide useful, it should be particularly helpful to students, younger investigators, clinicians, and professional researchers.hi The book’s broader purpose is to improve the quality of scientific publishing in the addiction field by educating authors about the kinds of ethical and profes sional issues with which the International Society of Addiction Journal Editors (ISAJE) has long been concerned: scientific misconduct, ethical decision mak ing, the publication process, and the difficulties experienced by authors whose first language is not English. Guide to the Guide Publishing Addiction Science is organized into five sections. The first section provides an overview of this book and a chapter (“Infrastructure and Career Opportunities in Addiction Science”) describing the development and under lying structure of the field of addiction science.hh i The second section covers general issues of how and where to publish. The initial overview chapter (Chapter 3, “How to Choose a Journal: Scientific and Practical Considerations”) deals with choosing where to submit your article, a very important decision in the publication process. The chapter describes the range of journals that publish articles related to addiction and psychoactive substances; summarizes the growth in addiction journals, including the move into open-access journals; and explains 10 steps to choosing a journal. It also provides two tables containing practical information about 45 addiction spe cialty journals (e.g., areas of interest, acceptance rates, author fees) to assist authors with the selection of an appropriate journal. The next chapter in this 6 Publishing Addiction Science section (“Beyond the Anglo-American World: Advice for Researchers from Developing and Non-English-speaking Countries”) describes the practical and professional issues addiction scientists face in countries that are less resourced or in which English is not the main language, how authors who come from these countries can improve their chances of publishing in English-language journals, the possibilities for authors to publish in both English and an addi tional language so they can communicate with different audiences, and how to decide whether an article may better serve the public by being published in the author’s mother tongue. Chapter 5 (“Getting Started: Publication Issues for Graduate Students, Postdoctoral Fellows, and other Aspiring Addiction Scien tists”) describes the challenges and rewards of publishing early in one’s profes sional career, including authorship issues, timetables, ethical dilemmas, and the pressure to publish. Lastly, Chapter 6 (“Addiction Science for Professionals Working in Clinical Settings”) looks at research and publication issues specific to clinicians who work in the field of addiction. It offers advice for identifying types of clinical research that lend themselves to research articles, planning and funding such research, and avoiding common pitfalls in the journey to publication.h The third section provides a detailed guide to the practical side of addic tion publishing. Guide to the Guide Chapter 7 (“How to Write a Scientific Research Article for a Peer-reviewed Journal”) describes the development of a typical data-based research article from the planning stage to the completion of the final draft, emphasizing scientific writing techniques, the structure of a scientific article, common reporting guidelines for specific types of articles, effective methods of scientific communication, and resources for improving one’s writing. The fol lowing chapter (“How to Write Publishable Qualitative Research”) explores the differences and commonalities between qualitative and quantitative research, identifies the hallmarks of exemplary qualitative research, and offers practical advice not only for writing a qualitative article but also for getting it published. Chapter 9 (“How to Write a Systematic Review Article and Meta-analysis”) provides a step-by-step process for designing, researching, and writing a com prehensive synthesis of existing research—typically a much larger undertak ing than a single research article—and describes some of the best databases and guidelines available to authors. Chapter 10 (“Use and Abuse of Citations”) describes appropriate and less-appropriate citation practices with recommen dations for good behavior and gives a critical appraisal of citation metrics, particularly the journal impact factor, which is used to evaluate the impor tance attributed to different journals. Chapter 11 (“Coin of the Realm: Practical Procedures for Determining Authorship”) deals with the often vexing question of how to assign authorship credits in multi-authored articles. We offer prac tical recommendations to provide collaborating authors with a process that is open, fair, and ethical. Chapter 12 (“Preparing Manuscripts and Respond ing to Reviewers’ Reports: Inside the Editorial Black Box”) focuses on how to negotiate the peer-review process. It describes how the process works and how A Guide for the Perplexed 7 journal editors make decisions about publishing an article. It also considers editors’ criteria for selecting articles and explains how to revise an article when an editor asks for a response to the reviewers’ comments. The final chapter in this section (“Reviewing Manuscripts for Scientific Journals”) covers the peer- review process, what journal editors expect from reviewers, and how to prepare a constructive critical review.h The fourth section of Publishing Addiction Science is devoted to ethical issues. The first article in this section (Chapter 14, “Dante’s Inferno: Seven Deadly Sins in Scientific Publishing and How to Avoid Them”) reviews seven types of scien tific misconduct in the context of a broader definition of scientific integrity. Guide to the Guide The seven “sins” are carelessness in citing and reviewing the literature, redundant publication, unfair authorship, failure to declare a conflict of interest, failure to conform to minimal standards of protection for animal or human subjects, plagiarism, and scientific fraud. We discuss these ethical improprieties in terms of their relative importance and possible consequences and suggest procedures for avoiding them. Chapter 15 (“The Road to Paradise: Moral Reasoning in Addiction Publishing”) discusses the same issues in the context of a framework for making ethical decisions. We use case studies to illustrate the seven ethical topics, with a commentary on each case that demonstrates a practical approach to making sound decisions. Chapter 16 (“Relationships with the Alcoholic Bev erage Industry, Pharmaceutical Companies, and Other Funding Agencies: Holy Grail or Poisoned Chalice?”) reviews recent trends in the funding of addiction research and the ethical risks involved in accepting funding from industry as well as nonindustry sources. The fifth and final section contains the book’s concluding chapter (Chapter 17: “Addiction Publishing and the Meaning of [Scientific] Life”), in which the editors describe the pursuit of scientific integrity as a journey worth taking, as much for the joy of honest discovery as for the achievement of fame and fortune. How to Use This Guide Effectively The authors have collectively striven to present practical advice as well as “best practices.” In most cases, such as in resolving authorship disputes or ethical problems, the solutions are not always simple or obvious but rather depend on the situation and on an open dialogue among colleagues. For these cases, we offer advice on how to use effective problem-solving techniques that will allow the reader to develop skills that can be applied to a variety of situations. The authors emphasize that no researcher, no matter how experienced in the game of science, can argue that she or he has all the right answers. This book is best seen as providing a basis for discussions about concrete problems in various research environments. Although the book’s chapters can be read in sequence, each chapter also functions as a self-contained unit and can be downloaded and read separately. 8 Publishing Addiction Science As a result, there is some repetition among chapters, more so that would occur in a book designed to be read from cover to cover, as more than one chapter may discuss similar issues in slightly different ways.h f The chapters are also meant for use as background readings for lectures, workshops, and practical exercises that accompany many of the chapters. The ISAJE website (www.isaje.net) contains supplementary readings, exercises, slides, and other materials for each chapter, all free to download. Recognizing that there are important institutional responsibilities in the ethical conduct of addiction research, we hope that this book will also inspire research institutions to develop guidelines and policies that support the ethical practices considered in these chapters. Although we have subtitled the book as A Guide for the Perplexed, we point out that its chapters will be helpful as well to those who believe they have all the answers, including established investigators at professional organizations and scientific institutions. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. How to cite this book chapter: Babor, T F, Morisano, D, Noel, J, Robaina, K, Ward, J H and Mitchell, A L. 2017. Infrastructure and Career Opportunities in Addiction Science: The Emergence of an Interdisciplinary Field. In: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P. (eds.) Publishing Addiction Science: A Guide for the Perplexed, Pp. 9–34. London: Ubiquity Press. DOI: https://doi.org/10.5334/bbd.b. License: CC-BY 4.0. References Edwards, G. (Ed.). (2002). Addiction: Evolution of a specialist field. Oxford, UK: Blackwell Publishing.h Hellman, M. (2015). The compartmentalisation of social science: What are the implications? Nordic Studies on Alcohol and Drugs, 32, 343–346. Lafollette, M. C. (1992). Stealing into print: Fraud, plagiarism and misconduct in scientific publishing. Berkeley CA: University of California Press.h i Maimonides, M. (2004). The Guide for the Perplexed. Translated by M. Friedländer [1903] (Barnes & Noble Library of Essential Reading) Paperback. CHAPTER 2 Introduction During the latter part of the 20th century, there was rapid growth in the number of people employed in the societal management of social and medical problems associated with the use of alcohol, tobacco, and illicit drugs (Edwards & Babor, 2012). At the same time, similar growth occurred in the number of institutions and individuals engaged in addiction science. The current worldwide infra structure of addiction science includes numerous research funding sources, more than 90 specialized scholarly journals, scores of professional societies, over 200 research centers, more than 80 specialty training programs, and thou sands of scientists. The purpose of this chapter is to describe the global infrastructure support ing addiction science and the career opportunities available to addiction sci entists. The current global infrastructure is evaluated from two perspectives: (a) its ability to produce basic knowledge about the causes of addiction and the mechanisms by which psychoactive substances affect health and well-being and (b) its ability to address substance-related problems throughout the world at both the individual and the population levels. The first perspective speaks 0 Publishing Addiction Science 10 to the mission of science to produce fundamental knowledge. The second is a public health mission that is often used to justify societal investments in clinical and translational research.h This chapter begins with a discussion of the meaning of addiction science as an interdisciplinary field of study. We then consider six areas of infrastruc ture development: (a) specialty journals; (b) research centers; (c) professional societies; (d) specialized libraries and documentation centers; (e) training and education programs; and (f) funding agencies. We close with a discussion of the career opportunities and future directions of addiction science. What is Addiction Science? The multidisciplinary area of “addiction studies” (variously called addictology, narcology, alcohology) is generally devoted to the understanding, manage ment, and prevention of health and social problems connected with the use of psychoactive substances. Within this area of addiction studies, addiction sci ence represents a more specialized subarea of research activity applying the scientific method to the study of addiction. Over the past 150 years, addic tion science has developed its own terminology, concepts, theories, methods, workforce, and infrastructure. Addiction science merges biomedical, psycho logical, and social perspectives within a transdisciplinary, issue-driven research framework. The goal is sometimes stated as an attempt to advance physical, mental, and population health by contributing to prevention, treatment, and harm reduction.hi The field of addiction science, like other interdisciplinary areas of research, often requires expertise and collaborations across traditional disciplinary boundaries as well as transdisciplinary research efforts (Choi & Pak, 2006) that involve scientists trained in the basic sciences, medicine, and public health, as well as the social, biological, and behavioral sciences. It also encourages integration of nonacademic participants, such as policymakers, service providers, public interest groups, and persons in recovery from substance use disorders. The basic underlying framework, or infrastructure, of current addiction science consists of research centers, scholarly journals, professional societies, education programs, specialized services, specialized libraries, fund ing agencies, and the people to populate these institutions and services. Box 2.1 provides an abbreviated chronology of major events in the development of addiction science in North America, Europe, and other parts of the world.hi The first wave of activity consisted of establishing organizational and commu nication structures such as the American Association for the Study and Cure of Inebriety in 1870, and its British counterpart, the Society for the Study and Cure of Inebriety in 1884. The emergence of addiction science was driven primarily by societal concerns about the problems of alcohol and, later, about cocaine and opiates. What is Addiction Science? Addiction science initially flowered and then nearly expired in concert Infrastructure and Career Opportunities in Addiction Science 11 • First Wave: Organizational and Communication Structures ––1870 – American Association for the Study and Cure of Inebriety ––1884 –Society for the Study and Cure of Inebriety (United Kingdom) ––1907 – International Bureau Against Alcoholism • Second Wave: Institutional Support for Research ––Early 1940s – Yale Center of Alcohol Studies, New Haven, Connecticut, United States ––1949 – Addiction Research Foundation, Toronto, Canada ––1950 – Finnish Foundation for Alcohol Studies, Helsinki, Finland ––1960 – National Institute for Alcohol Research, Oslo, Norway ––1967 – Addiction Research Unit, London, United Kingdom ––1971 – U.S. National Institute on Alcohol Abuse and Alcoholism ––1973 – U.S. National Institute on Drug Abuse • Third Wave: The Modern Era ––Addiction research centers ––Addiction specialty journals ––Addiction-focused professional societies ––Addiction-focused education and training programs ––Addiction-focused libraries Box 2.1: Major milestones in the history of addiction science. • First Wave: Organizational and Communication Structures • Third Wave: The Modern Era ––Addiction research centers ––Addiction specialty journals ––Addiction-focused professional societies ––Addiction-focused education and training programs ––Addiction-focused libraries Box 2.1: Major milestones in the history of addiction science. with the rise and fall of the temperance movement in America and Europe. Dur ing a 40-year period (1875–1915), an international cadre of addiction special ists emerged from various areas of medicine and science to advance knowledge about addiction problems. This was done by means of professional societies, international meetings, scientific journals, scholarly books, and expert com mittee reports (Babor, 1993a,b; 2000; Billings et al., 1905; Bühringer & Watzl, 2003; Sournia, 1996). Although the research produced by these organizations was unsophisticated by current standards, there were some notable advances in toxicology, clinical diagnosis, epidemiology, and policy research during this time (Babor, 1993a, 2000; Billings et al., 1905; Sournia, 1996), especially in the United Kingdom, the United States, France, Germany, and Sweden. The demise of addiction studies followed the imposition of prohibition legislation in the United States, Scandinavia, the United Kingdom, and many other countries in the aftermath of the First World War. It was not until the 1940s that addiction research regained a sense of identity and purpose and not until the 1970s when it gained enough scientific respectability to be considered a legitimate part of society’s public health response to alcohol and other drug problems. with the rise and fall of the temperance movement in America and Europe. Dur ing a 40-year period (1875–1915), an international cadre of addiction special ists emerged from various areas of medicine and science to advance knowledge about addiction problems. This was done by means of professional societies, international meetings, scientific journals, scholarly books, and expert com mittee reports (Babor, 1993a,b; 2000; Billings et al., 1905; Bühringer & Watzl, 2003; Sournia, 1996). Although the research produced by these organizations was unsophisticated by current standards, there were some notable advances in toxicology, clinical diagnosis, epidemiology, and policy research during this time (Babor, 1993a, 2000; Billings et al., 1905; Sournia, 1996), especially in the United Kingdom, the United States, France, Germany, and Sweden. The demise of addiction studies followed the imposition of prohibition legislation in the United States, Scandinavia, the United Kingdom, and many other countries in the aftermath of the First World War. It was not until the 1940s that addiction research regained a sense of identity and purpose and not until the 1970s when it gained enough scientific respectability to be considered a legitimate part of society’s public health response to alcohol and other drug problems. Box 2.1: Major milestones in the history of addiction science. 12 Publishing Addiction Science The second wave of addiction science is characterized by the growth of insti tutional support for research, beginning with the establishment of the Yale Center of Alcohol Studies in New Haven, Connecticut, in the United States in the early 1940s; the Addiction Research Foundation, Toronto, Canada, in 1949; and similar organizations in Finland, Germany, Norway, and other countries. With the creation of government funding agencies at the federal level in the United States in the early 1970s, the stage was set for the modern era. As part of the developing biomedical establishment in the United States, addiction science experienced phenomenal growth, which was paralleled by similar developments in Europe. That growth—the third wave—can be char acterized by at least four megatrends (Babor, 1993b): (a) the rapid expansion of scientific publishing of addiction research, (b) the development of addiction research centers and related organizational structures, (c) international col laboration in research, and (d) the development of significant scientific break throughs in addiction science and medicine. We now consider these trends in the context of the seven types of infrastructure that have emerged in the modern era described above. Addiction Specialty Journals One indication that addiction science has emerged as a separate discipline is the appearance of specialty academic journals that serve as a medium of communication among clinicians and scientists. The first journals specifically publishing addiction science were the (quarterly) Journal of Inebriety (1876–1914), the British Journal of Inebriety (1884–present; now Addiction) and the International Monthly Journal for the Fight against Drinking Practices (1890-present with two World War interuptions; now SUCHT). After a relative lapse of interest in addiction science, the Quarterly Journal of Studies on Alcohol (now the Journal of Studies on Alcohol and Drugs) was established in 1940 and revived scientific interest in alcoholism, a development that began the modern era of addiction research. Figure 2.1 traces the cumulative growth of addiction specialty journals since 1884. The journals are characterized in terms of their language of publication (English and non-English), but there are other important distinctions that are discussed in more detail in Chapter 3. The dominance of English as the inter national language of science has facilitated communication far beyond national boundaries. With the development of online publishing and the “open access” trend to make scientific research freely available to the scientific community and the general public, there has been a proliferation of online open-access English-language journals that have transformed the way that scientific infor mation is published and distributed. However, as discussed in Chapter 3, many of the new online open access journals that have been established in the last decade are produced by “predatory publishers,” organizations that engage in Infrastructure and Career Opportunities in Addiction Science 13 Fig. 2.1: Growth of addiction specialty journals. 0 10 20 30 40 50 60 70 80 90 100 1884 1890 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010 Total English Non-English 2015 Number of journals Fig. 2.1: Growth of addiction specialty journals. questionable practices with regard to journal management, marketing activi ties, peer review, and page fees (Beall, 2012). Addiction specialty journals provide a communication forum for scientists and clinicians. They deliver valuable information to practitioners, scientists, and the general public. They set the agenda for a field of study and maintain ethical and quality standards. Another function is to archive the historical record for an area, allowing permanent access to articles for future use by scien tists, clinicians, administrators, policymakers, and historians. Addiction Specialty Journals Finally, by means of the peer-review process, journals certify the authenticity and originality of an author’s work (LaFollette, 1992). For these reasons, scientific journals are the institutional memory of a field. i In addition to the growth in specialty journals, addiction science is also pub lished by discipline-oriented journals dealing with medicine, pharmacology, biochemistry, neurobiology, psychology, sociology, and epidemiology. When the addiction articles of these journals are combined with the publications in addiction specialty journals, it becomes possible to estimate trends in the vol ume of research in addiction science by means of historical records and bib liometric analyses. Between 1900 and 1950, for example, approximately 500 scientific articles were published per year on alcohol (Keller, 1966). Between 1950 and 1970, the number of publications doubled each decade. By the late 1980s, more than 3,000 scholarly publications on alcohol were appearing per year, and the trend has continued unabated until the present.i To estimate the current output of scientific publications, we used bibliometric procedures to extract journal publications in SCOPUS from 2000 through 2014 that dealt with addiction research (e.g., “alcohol use disorder” and “tobacco use disorder”). We then categorized the publications by area of focus across four areas of research: alcohol, tobacco, other drugs, and gambling. The SCOPUS 14 Publishing Addiction Science database was selected for its inclusion of all MEDLINE journals. It should be noted that there is no single database that covers the entire output of schol arly publications in addiction science, after the major databases that previously collected, indexed, and abstracted addiction literature ceased operations over the past 15 years (ETOH in 2003, Rutgers Alcohol Studies Database in 2007, CORK in 2015). In the absence of a comprehensive database, it is difficult to estimate the number of articles published in the field, and it is not possible to give an accurate account of other addiction-related publications (e.g., books, reports). The estimates provided in this chapter should therefore be considered conservative and better suited to the identification of relative growth trends than to the estimation of the absolute number of publications.h The four searches yielded 233,970 results published since the year 2000. We identified 212,891 unduplicated journal publications for all four areas of research, of which 79,585 were published between 2010 and 2014. Figures 2.2 and 2.3 show the trends in document production. y y g y Rates based on unduplicated totals from total population estimates from 2013; Table 2.1: Publications by country and research category. p p p Source: World Bank (2013). Addiction Specialty Journals The trend is generally posi tive for all areas until 2009 when a decline begins for tobacco and nicotine research, followed by lesser declines in 2013 for alcohol and other drugs. The decline in publications may be attributed to reductions in public research fund ing in the major research-producing countries as well as the global economic recession that began in 2008. This interpretation is supported by the absence of a decline in gambling research, which is mainly supported by the gambling industry or by tax revenues from state lotteries.h The geographical dispersion of the research publications was also examined. The country of origin of each article was determined from the address of the first or corresponding author. Publication contributions between 2010 and 2014 from the most research-prolific countries are shown in Table 2.1. Fig. 2.2: Total number of addiction articles per year (2000–2014). 9655 10172 11275 12832 13577 14382 15030 14872 15771 15740 15759 16368 16530 15921 15007 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 Number of articles Fig. 2.2: Total number of addiction articles per year (2000–2014). Infrastructure and Career Opportunities in Addiction Science 15 15 Fig. 2.3: Total number of addiction articles, by year and category (2000–2014). 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 Alcohol Tobacco Other Drugs Gambling Year Fig. 2.3: Total number of addiction articles, by year and category (2000–2014). 0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 Alcohol Tobacco Other Drugs Gambling Year Fig. 2.3: Total number of addiction articles, by year and category (2000–2014). Addiction Specialty Journals Alcohol Tobacco Other drugs Gambling Population- adjusted publication rate United States 12,479 9,115 14,201 1,067 10.45 United Kingdom 2,421 2,236 2,601 382 10.99 Australia 1,674 1,027 1,723 345 18.71 Germany 1,430 879 1,280 206 4.35 Canada 1,297 1,252 1,738 399 12.04 Italy 996 780 1,233 159 4.90 France 995 686 1,137 134 4.03 Spain 978 661 1,322 108 5.99 The Netherlands 902 707 817 105 13.83 Brazil 838 303 786 64 0.90 China 791 649 1,010 148 0.18 India 755 614 553 18 0.14 Switzerland 568 367 693 59 19.06 16 Publishing Addiction Science When looking at the number of publications across all four categories com bined (totals not shown), the top five producing countries are the United States, United Kingdom, Australia, Canada, and Germany. The United States accounts for approximately 42% of the total production, but on a population-adjusted basis several other countries (Australia, the United Kingdom, Canada, the Netherlands, and Switzerland) make even greater contributions. In the emerg ing economies of the world, China, India, and Brazil are beginning to produce significant amounts of the research published in the English-language literature as well. An important consideration regarding the geographic concentration of research in the United States and Europe is that the findings may not general ize to other parts of the world—especially nations in Africa, Asia, and Latin America—facing epidemics of alcohol abuse, nicotine dependence, other drug dependence, or pathological gambling. In general, these analyses indicate that the steady growth of addiction science during the latter part of the 20th century has continued unabated into the first part of the 21st century. Addiction Research Centers Although addiction research in many countries is conducted by independent scientists whose primary affiliation is to an academic department in a univer sity or by clinicians who work in treatment facilities, in recent years there has been an expansion of specialized centers whose primary purpose is to support alcohol, tobacco, and other drug research. As such, they provide a good indica tor of growth trends in research infrastructure. Centers provide dedicated facilities to groups of scientists and supporting staff so that long-term programmatic research can be carried out. Centers constitute an optimal environment for researchers, one that is relatively free of administrative, clinical, and teaching responsibilities. Not only are the posi tions dedicated exclusively to research, but the centers also provide the prospect of long-term support and career advancement. Training of junior investigators is another important function of research centers. Building on earlier estimates of the annual growth in research centers (Babor, 1993b), we conducted an Internet search to identify the location and other characteristics of addiction research centers, including the dates they were established. We estimate that the number of research centers devoted to addiction research now number approximately 275 worldwide. The largest number of centers is located in the United States, the Nordic countries, the United Kingdom, Russia, Brazil, Canada, and Japan.h The growth of research centers is indicative of a more general trend in addic tion science and clinical services. Over the last 45 years, the number of research centers has increased exponentially, from fewer than 20 before 1970 to more than 150 at the end of the century. By the year 2000, the multi-disciplinary research center had become the dominant setting for basic, clinical, and Infrastructure and Career Opportunities in Addiction Science 17 Fig. 2.4: Cumulative growth of addiction research centers (1940–2015). 0 50 100 150 200 250 300 1940 1942 1944 1946 1948 1950 1952 1954 1956 1958 1960 1962 1964 1966 1968 1970 1972 1974 1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014 US Interna9onal Total Number of research centers Fig. 2.4: Cumulative growth of addiction research centers (1940–2015). psychosocial research on addictive substances. Figure 2.4 shows the exponen tial growth in addiction research centers in both the United States and globally over a 75-year period. Addiction Research Centers The scope of these centers varies, with 70.5% focusing on drugs and alcohol, 57.4% on alcohol alone, 36.0% on tobacco, and 2.9% on other addictions (e.g., problem gambling).h g g g The type of addiction research varies across centers, with 55.6% conducting studies on addiction treatment, 54.2% on the psychosocial factors involved in addiction, 51.3% on policy or prevention programs, and 33.1% on the biological underpinnings of addiction. Approximately 8% of research cent ers are known to have more than 50 affiliated research scientists; 50% house fewer than 25 investigators; and 21% have fewer than 10. As the number of centers has grown, collaborative networks have been formed to better leverage existing resources, conduct cross-national projects, train doctoral and postdoctoral candidates, write scientific publications, pro vide policy consultations, and increase the media coverage of addiction science. In Germany, the Federal Ministry of Education and Research implemented a long-term research funding program (1994–2008 with nearly 35 million euros) to enhance drug research and collaborations, disseminate findings, improve addiction-science information exchange across professionals, and advise the public and policymakers on addiction-related topics. The program supported 18 single projects and, from 2001 onwards, four consortia among 12 research centers (composed of MDs and psychologists) engaged in behavioral, clini cal, neurobiological and genetic research (Mann, 2010). In that context, the first chair in addiction research was created in 1999 at the Central Institute of 8 Publishing Addiction Science 18 Mental Health Mannheim (University of Heidelberg) and the second in 2005 at the University of Dresden. In part because of the success of these networks, Germany is now investing substantially more in addiction research. y g y In the United States, the National Institute on Alcohol Abuse and Alcoholism (NIAAA) and the National Institute on Drug Abuse (NIDA) support research centers and research networks through several funding mechanisms. NIAAA supports 20 research centers through its National Alcohol Research Centers Program and also funds large-scale cooperative agreements among research ers collaborating on high-priority projects such as Project MATCH (Matching Alcoholism Treatments to Client Heterogeneity; Babor & DelBoca, 2003), the multisite trial of Combined Pharmacotherapies and Behavioral Interventions for Alcohol Dependence (COMBINE; Anton et al., 2006), and the Collaborative Study on Genetics of Alcoholism (COGA) project (Agrawal & Bierut, 2012). NIDA also supports a Clinical Trials Network (Wells et al., 2010) devoted to treatment research. Addiction Research Centers These kinds of large-scale, cross-site collaborations facili tate rapid, standardized data-collection projects that would not be possible at a single small site, and they permit more generalizable conclusions and data applications. pp Addiction research centers provide core facilities and laboratories, training opportunities for new scientists, and resources to sustain career investigators. In addition, research centers facilitate links between scientists, policymakers, and the general public. During the 75-year period depicted in Figure 2.4, there was parallel growth in governmental institutes and private funding agencies devoted to the sponsorship of addiction research. The combination of cat egorical support for addiction research and academic freedom to engage in addiction science as a career contributed substantially to the information and productivity explosion in the addiction field discussed in subsequent sections of this chapter (Babor, 1993a,b; Babor et al., 2008). Professional Societies In the addiction field, professional societies have been operating for almost 150 years, with the oldest continuing society being the Society for the Study of Addiction, established in 1884 in the United Kingdom. These societies include national and international organizations and sections of larger organizations that are devoted to addiction treatment, prevention, policy, and research. Mem bership comprises clinical, prevention, and research professionals, including psychologists, physicians, psychiatrists, social workers, addiction counselors, and other professional groups. Figure 2.5 documents the growth of profes sional societies, based on an earlier compilation of alcohol-related associations (NIAAA, 1985) and a review of Internet sources. The number of professional societies grew dramatically between 1970 and 2005, particularly in the United States. A more recent trend has been the growth of international organizations and confederations of societies. Infrastructure and Career Opportunities in Addiction Science 19 Fig. 2.5: Cumulative growth of professional societies. 0 20 40 60 80 100 120 140 160 180 200 Year Established USA World International Total Number of societies Year Established Fig. 2.5: Cumulative growth of professional societies. A minority of these societies, perhaps no more than 40 in number, can be classified as addiction research organizations because their mission statements suggest primary involvement in issues related to research on alcohol, tobacco, other drugs, and behavioral addictions. Twelve countries have national-level research societies, and there are 14 international organizations. Only a few societies are located in developing countries. These organizations can be classi fied into three broad categories: multi-disciplinary, professional specialty, and research societies. Multi-disciplinary societies are open to professionals of all disciplines who work in the addiction area, including treatment, prevention, research, policy, and education. The Brazilian Association for the Study of Alcohol and Other Drugs (ABEAD) is a good example of a multi-disciplinary national society, as is the British Society for the Study of Addiction. Professional specialty societies are typically special-interest groups organized within larger disciplinary societies, such as the Alcohol, Tobacco and Other Drugs section of the American Public Health Association. Several of these specialty societies are international in scope, such as the International Society of Addiction Journal Editors. Research societies provide a forum for new scientific developments and networking for potential investigative collaborations, usually within the context of an annual meeting. membership, and journal sponsorship. Professional Societies The Research Society on Alcoholism, College on Problems of Drug Dependence, and International Society for Biomedical Research on Alcoholism are examples of this type of organization.i Table 2.2 shows professional societies that sponsor scientific journals in terms of their year of foundation, membership numbers, and journal (adapted from Edwards & Babor, 2008). These are among the largest societies devoted to research, representing more than 7,000 members, even taking into account multiple memberships by the same individuals across societies. 20 Publishing Addiction Science 20 Name of organization Year established Number of members Society journal(s) Society for the Study of Addiction (United Kingdom) 1884 478 Addiction, Addiction Biology SOCIDROGALCOHOL: Spanish Scientific Society for the Study of Alcohol, Alcoholism and other Drug Dependencies 1969 816 Adicciones Association for Medical Education and Research in Substance Abuse (United States) 1976 300 Substance Abuse Research Society on Alcoholism (United States) 1977 1,500 Alcoholism: Clinical and Experimental Research ABEAD, Brazilian Association for the Study of Alcohol and Other Drugs 1978 840 Society Bulletin and the Brazilian Journal on Chemical Dependence (Jornal Brasileiro de Dependências Químicas) German Society for Addiction Research and Addiction Treatment 1978 400 SUCHT Société Française d’Alcoologie et Addictologie(French Society of Alcoholism and Addiction) 1978 807 Alcoologie et Addictologie Japanese Society of Alcohol- Related Problems 1979 543 Journal of the Japanese Society of Alcohol-Related Problems Australasian Professional Society on Alcohol & Other Drugs 1981 382 Drug and Alcohol Review Kettil Bruun Society for Social and Epidemiological Research on Alcohol 1987 197 International Journal of Alcohol and Drug Research Society for Research on Nicotine and Tobacco 1994 1,000 Nicotine & Tobacco Research Table 2.2: Selected addiction societies according to year of foundation, membership, and journal sponsorship. Infrastructure and Career Opportunities in Addiction Science 21 Infrastructure and Career Opportunities in Addiction Science 21 Infrastructure and Career Opportunities in Addiction Science 21 Although the activities of professional societies are diverse, first and fore most they run meetings, ranging from large annual events to small topic-based workshops and thematic conferences. Networking—encouraging professionals to communicate and work with each other—is a major function, if not primary purpose, of these organizations. As noted in Table 2.2, many sponsor scientific journals. Some organizations influence national policy. ABEAD (Dias da Silva et al., 2002), for example, is close to the Brazilian government. Professional Societies Others stay clear of political involvement and focus on “science as science”; the German Soci ety for Addiction Research and Addiction Treatment (Mann & Batra, 2008) has supported the renaissance of the national addiction science base. Publica tions are another significant product of many societies, highlighting relevant research and achievements in the form of journals, yearbooks, bulletins, guide lines, and educational materials. Some societies provide continuing education to interested parties, with several offering professional certifications in addic tion medicine or other relevant topics. Most societies share a common concern for enhancing the addiction field’s status as an important area of research and clinical practice, with the aim of overcoming patient stigma and government neglect. g Some countries have just one major body dealing with alcohol and other drugs, whereas others have a plethora. Japan, for instance, has the Japanese Society of Alcohol-Related Problems, the Japanese Medical Society on Alcohol and Drug Studies, the National Society of Biomedical Research on Alcohol, the Society of Psychiatric Research on Alcohol, and a society focused on addiction behavior (Maruyama & Higuchi, 2004). Rather than being the products of government intention, many addiction societies were formed spontaneously by small groups of professionals who identified an emerging need and resolved to work together to address it. The British Society for the Study of Addiction, for example, was formed by an alliance of physicians in 1884 (Tober, 2004) to mobilize parliamentary sup port for the compulsory treatment of “inebriates.” The impetus to the foun dation in 1977 of the Research Society on Alcoholism was the expansion in research funding following the initiation of NIAAA (Israel & Lieber, 2002). The Italian Association on Addiction Psychiatry (SIPDip) (Nizzoli & Foschini, 2002) was established in 1989 to create a role for psychiatry in the face of political chaos and the neglect of addiction-related problems. Each of these societies was shaped by national trends in substance use, assumptions about the proper role of voluntary action, and the role of professional disciplines in the national response to addiction problems. In the late 19th and early 20th centuries, when the world temperance move ment and specialized asylums for addiction treatment had reached a high level of maturity, large umbrella organizations or confederations were formed to facilitate communication among diverse addiction-related entities around the world. Professional Societies The first example of such a coalition of individuals and organizations was the The International Bureau Against Alcoholism, founded in 1907, which Publishing Addiction Science 22 became, in 1964, the International Council on Alcohol and Alcoholism. More recently, confederations of research organizations have again begun to take shape in the addiction field with the creation of the European Federation of Addiction Societies (EUFAS) and the International Confederation of Addic tion Research Associations (ICARA) (Stenius, 2012). The aim of ICARA is to provide a forum for the discussion of issues such as governance, organizational management, relationships with governments, advocacy for addiction science, and the promotion of treatment services. Another sign of the consolidation of infrastructure is the formation in 2001 of the International Society of Addic tion Journal Editors (Edwards & Babor, 2001). According to Krimsky (2003), professional societies, along with a network of academic journals, define “acceptable scholarship and certifiable knowledge” (p. 107). Professional organizations, especially research societies, are a major resource for scientists working in biomedical and psychosocial research. They distribute news and scientific information to their members, publish journals and newsletters, engage in advocacy for research, coordinate scientific meet ings, and at times facilitate collaborative research. These organizations, in turn, provide a means of networking and communication for their members. They confer prestige and often serve as advocates for professional issues such as research funding, the training of scientists, and evidence-based policy. Specialized Libraries and Databases Information services—including libraries, resource centers, and clearing houses—are an integral part of any research program. A specialized library in the addiction field provides information resources, such as books and journals on addiction, as well as reports, pamphlets, and historical documents. Addic tion libraries are usually managed by universities, government agencies, and nongovernmental organizations. With the growth of digital databases, addic tion libraries have provided easy access to the international addiction literature. y Substance Abuse Librarians & Information Specialists (SALIS) is a profes sional organization established in 1978 with assistance from NIDA and NIAAA. As an international association of individuals and organizations interested in the exchange of information on alcohol, tobacco, and other drugs (ATOD), SALIS provides a good example of the growth of specialization in addiction science. A major aim of SALIS is to promote the dissemination of accurate knowledge about the use and consequences of ATOD. Figure 2.6 shows the cumulative growth and decline of specialized addic tion libraries over the past 85 years in the United States and other parts of the world. The figure is based in part on an inventory compiled by SALIS (Mitchell, 1991) to document ATOD libraries, clearinghouses, and resource centers. From it, specialized libraries and collections that primarily serve an academic or research purpose were identified, although some documentation Infrastructure and Career Opportunities in Addiction Science 23 centers were also included. Libraries and other collections reporting fewer than 500 books were not included, nor were mental health libraries, those with no identifiable start date, resource centers, clearinghouses, or trade/ industry libraries, unless they served an academic purpose. The figure plots the cumulative number of functioning libraries by year established, subtract ing any documented closures, based on a 2015 review that identified closures over the past 25 years.hi y The first specialized libraries were established in Europe (1907) and the United States (1940) during the early part of the 20th century. Starting in the 1940s, more ATOD libraries were added, a trend that accelerated in the 1960s. The global network of specialized libraries that SALIS now represents has fol lowed a growth curve similar to other parts of the addiction science infrastruc ture, but there have also been signs of decline. Specialized Libraries and Databases The decline in the number of libraries after 1995 could be because of budget cuts that have affected libraries and databases in both North America and Europe, resulting in downsizing, service reduction, and closures. Another explanation is a change in informa tion-seeking habits, with more professionals using the Internet to access infor mation through their computers and smartphones (McTernan, 2016). Regardless of the reason, specialized addiction libraries are declining in num ber, as are the number of specialized librarians. For example, in 2006, NIDA closed its library—which contained a collection dating from 1935. The U.S. Substance Abuse and Mental Health Services Administration (SAMHSA) also closed its prevention library and cut support to Regional Alcohol and Drug Awareness Resource (RADAR) centers, which were created to disseminate Fig. 2.6: Cumulative growth of specialized addiction libraries. Year Number of Libraries Number of Libraries Year Fig. 2.6: Cumulative growth of specialized addiction libraries. 24 Publishing Addiction Science 24 government agency publications related to alcohol and other drugs. Europe joined the culling effort with library closures or downsizing at the Trimbos Institute (the Netherlands); Alcohol Concern, Drug Scope, and the Temper ance Alliance (United Kingdom); Toxibase (France); and Gruppo Abele (Italy). Some of these organizations maintain online information portals, but collec tions have been packed up, databases and catalogues terminated, and staff positions eliminated. More than 25 libraries or databases have closed in the past decade (Mitchell et al., 2012). Not only have these closures resulted in a reduction in the ATOD information base, but they also have reduced the pool of librarians who have expertise and knowledge of valuable historical material. Print collections have been de-funded and neglected without ensuring archival preservation (Mitchell et al., 2012).i Budget reductions have been justified by the assumption that online access is “free,” but the majority of scholarly literature cannot be accessed readily through search engines or websites because of copyright and the proprietary nature of information. Excluding PubMed, most research databases are avail able only through paid subscription. Furthermore, most do not provide full- text articles without a fee. In addition to specialized libraries, more than 100 companies and institu tions currently offer abstracting and indexing services that provide digital access to abstracts and titles pertaining to the world literature on alcohol, other drugs, tobacco, and the behavioral addictions (e.g., problem gam bling). Specialized Libraries and Databases There are approximately 20 main electronic databases that index the published literature by author, topic, and bibliographic reference and pro vide abstracts of articles for potential readers in search of particular types of information (see Chapter 3). Abstracting and indexing services provide detailed information about the content of scientific journal articles, includ ing abstracts, which are invaluable for those without immediate access to the full text of the article. Some of the more specialized databases were estab lished before the digital revolution in the 1990s, and, as their functions have been taken over by more generic databases, they have fallen into decline and neglect. For example, the Alcohol and Alcohol Problems Science Database, informally known as ETOH, was a comprehensive online resource cover ing all aspects of alcohol abuse and alcoholism, including journal articles, books, conference papers and proceedings, reports and studies, dissertation abstracts, and chapters in edited works. Unfortunately, it ceased operations in 2003. Two other specialized databases, Project CORK and DrugScope, were closed in 2015, leaving the addiction field without a comprehensive digital repository of the world’s addiction literature. To the extent that library closures and downsizing of other information sources could be a bellwether of the future of addiction science, they are per haps an indication that the exponential growth of the field has begun to slow or even decline. Infrastructure and Career Opportunities in Addiction Science 25 Education and Training Programs in Addiction Studies Without career professionals to populate its infrastructure and develop its products, the addiction field would not exist. To fill the need for a growing professional workforce in treatment, prevention, and research, specialized edu cation and training programs have been created throughout the world. Most of them focus on the training of clinicians, but several are devoted to addiction science. In general, the concept of addiction studies can be used as a framework to describe the emerging education programs that focus on the interactions between science, clinical practice, and social policy and across a range of addiction topics (e.g., opiate addiction, nicotine dependence, gambling behav ior, alcoholism). Figure 2.7 shows the cumulative growth in university-based degree programs in addiction studies. Some of these programs offer under graduate- or graduate-level degrees, and they are often interdisciplinary, involving training in genetics, neuroscience, psychology, epidemiology, and public health. Other programs, not included in the figure, offer postbaccalaureate, postdoc toral, or even single-workshop–based training options geared toward a variety of individuals interested in improving their clinical skills, research methods, and professional qualifications for positions in research, clinical services, pre vention, and policy. The aim of addiction studies programs is not to replace other professions but to work with them to promote the integration of research findings, prevention activities, and clinical approaches. Table 2.3 describes some of the training programs in addiction studies. Fig. 2.7: Cumulative growth in degree programs in addiction studies. 0 5 10 15 20 25 Number of programs Fig. 2.7: Cumulative growth in degree programs in addiction studies. Publishing Addiction Science 26 Table 2.3: Examples of specialized addiction-studies programs. Infrastructure and Career Opportunities in Addiction Science 27 Infrastructure and Career Opportunities in Addiction Science 27 The ultimate goal of this new academic area is to advance research-based knowledge, practice, and policies to further improve prevention and treatment of disorders and problems related to substance use. Despite the growth of pro grams for the training of addiction psychiatrists, narcologists, psychologists, social workers, psychiatric nurses, and addiction counselors, there has been little attention to the development of specialized training programs for addic tion scientists. The value of having specially trained addiction scientists is to maintain, if not expand the global infrastructure for social, behavioral, biologi cal, epidemiological and health services research.h The size of the addiction science workforce needed in a country will depend on the extent of addiction-related problems, the delegation of professional responsibilities, and the funding provided by governments to manage the problems of addiction. Globally, there is now a network of perhaps 10,000 peo ple worldwide who identify addiction science as part of their career identity (Babor, 2012). Membership in the 10 professional societies listed in Table 2.2, which includes both basic and clinical scientists, is comparable to this number. Without more systematic attention to workforce monitoring, it is impossible to say whether the current number of addiction scientists is sufficient to meet the needs and the demands for scientific information about addiction. Education and Training Programs in Addiction Studies University Country Degree Program Middlesex University, Aarhus University and University del Piemonte Orientale “A Avogadro” England, Denmark and Italy Master’s degree European Masters in Drug and Alcohol Studies National Addiction Centre at the Institute of Psychiatry, Maudsley Hospital, King’s College London England Master of science degree Clinical and Public Health Aspects of Addiction King’s College London, Virginia Commonwealth University and University of Adelaide England, United States and Australia Joint master’s- level degree International Programme in Addiction Studies Department of Addictology, First Faculty of Medicine, Charles University Czech Republic Bachelor’s, master’s, and doctoral degrees Academic Study Programs in “Addictology” (Addiction Science University of Auckland, School of Populations Sciences New Zealand Postbaccalaureate certificate, postbaccalaureate diploma, full master’s degree Postbaccalaureate specialization in addiction science: Alcohol and Other Drugs Program Center for Addiction Science Specialties, Sahmyook University South Korea Connective major for bachelor degree in Substance Addiction and Behavioral Addiction Prevention Departments of Nursing, Health Management, Counselling and Physical Therapy University of Dresden (TUD) and Dresden International University (DIU) Germany, open for PhD/MD students from Europe Certificate as basis for the MD/ PhD degree at the home university European Graduate School in Addiction Research (ESADD) An Internet search conducted by Charles University (Pavlovska et al., 2015) identified 79 university study programs at 24 different universities. The pro grams were distributed across all education levels, that is, bachelor’s, master’s, and doctorate, with 35 programs located in Europe, 34 in the United States and Canada, 7 in Australia and New Zealand, and 3 in Asia. Infrastructure and Career Opportunities in Addiction Science 27 Funding Sources and Patronage How society allocates its resources to support the infrastructure of addiction science is not only testimony to its values, but it also is an indication of current priorities in relation to the management of society’s addiction-related prob lems. As in other areas of science, the addiction field relies on patronage. In some cases, the support and sponsorship comes from private sources, such as when a philanthropist creates an endowment for a research center or an aca demic chair. More often, however, the patronage comes from public sources. During the past 50 years, a variety of funding mechanisms across the globe have provided support for addiction research and research infrastructure, which in turn has made possible much of the growth in professional careers (Babor, 2012). National research institutes, for example, have been created in many high- and middle-income countries to plan, support, and conduct sci entific research on addiction (Babor, 1993b). Examples of such organizations include the Norwegian National Institute for Alcohol and Drug Research, the Indian National Drug and Alcohol Institute, the National Institute of Pub lic Policy for Alcohol and Other Drugs (INPAD) in Brazil, and the National Research Centre on Addictions (Russian Federation). Many of these organiza tions have been established to support the development of scientific expertise with a clinical and sometimes a public health orientation, via the direct funding of research scientists, research training, public education, and the coordination of international activities. 8 Publishing Addiction Science 28 Another source of support for addiction research comes from the private sector, especially pharmaceutical companies. There has also been an increase in funding opportunities from the alcohol and gambling industries, both through direct support for research projects and programs and indirect support from organizations funded by these industries. As described in Chapter 16, there are some important ethical considerations involved in the acceptance of industry funding, not the least of which is financial conflict of interest. il Another issue is the role of funding agencies in the determination of the research agenda. Increasingly, the dollars dictate the science. Alcohol industry funding has been questioned because the agenda is often set by commercial objectives rather than by public health priorities. But even in the public sector, governments can shape the research agenda toward topics that may not address the most effective solutions for addiction problems.i f Midanik (2006), for example, identified a bias in U.S. Addiction Science as a Career Option As described in the infrastructure areas reviewed in this chapter, the field is built around institutions that help to define its roles and responsibilities. Professional societies, research centers, national institutes, addiction journals, specialized libraries, and specialized treatment programs constitute the major ingredients of the addiction field’s infrastructure, but, as previously suggested (Edwards & Babor, 2012), addiction careers constitute its building blocks and its human capital.i Today, the field of addiction science is populated by a variety of creative peo ple: basic scientists in pursuit of knowledge for its own sake, clinical investigators searching for new or better treatments, and applied researchers trying to solve dif ficult social problems (Edwards & Babor, 2012). How do people select a career in an emerging field that for most of its existence had no name or identity? As sug gested by personal accounts derived from a long series of interviews published in the journal Addiction (Edwards & Babor, 2012), the answer is as varied as the field itself. Personal experience with substance misuse, the influence of a mentor, the need to make a living, and the love of science are all mentioned. Some researchers and addiction professionals developed their interest in the field from personal, even tragic, experience. Others describe serendipity or “opportunity knocking.”i With an identity defined by the work of a diverse group of career scientists and the prominence of mentors from a wide variety of disciplines, the career of an addiction scientist is no longer a risk or a mystery. Addiction science as such can now be perceived as an independent, professional career (Babor, 2012; Edwards, 2002). Funding Sources and Patronage research-funding agen cies’ priorities toward biomedical (vs. psychosocial) approaches to alcohol-related problems. This has led to the majority of U.S. publications on drugs and alcohol being devoted to basic science and clinical interventions, which conflicts with the interests of policymakers on research related to supply control and demand reduction. In the European Union as well, there is a relative disconnect between research published on illicit drugs and the priorities advanced by policymak ers who are responsible for funding research and using its results to lessen the 53% 2% 7% 33% 18% 31% 14% 10% 30% 12% 0% 10% 20% 30% 40% 50% 60% Basic Research Epidemiology Demand Reduction Supply Reduction Policy Analysis Publications Priorities 53% 2% 7% 33% 18% 31% 14% 10% 30% 12% 0% 10% 20% 30% 40% 50% 60% Basic Research Epidemiology Demand Reduction Supply Reduction Policy Analysis Publications Priorities Fig. 2.8: Percentage distributions of research publications (N = 3,028) and research priority ratings (N = 57) across five research areas, based on data from European Union Member states (N = 27). (Source: Bühringer et al., 2009). Fig. 2.8: Percentage distributions of research publications (N = 3,028) and research priority ratings (N = 57) across five research areas, based on data from European Union Member states (N = 27). (Source: Bühringer et al., 2009). Infrastructure and Career Opportunities in Addiction Science 29 29 suffering of those who experience addiction-related problems (Bühringer et al., 2009). This disconnect between research and policy is reflected in the data pre sented in Figure 2.8, which contrasts the distribution of research publications in Europe with research priority ratings obtained from 57 policymakers from 27 European Union Members States. The figure shows an inverse relationship between the types of scientific evidence being published and the priorities of policymakers who fund the research behind the publications. Conclusion In the past 50 years, there has been dramatic growth in the demand for and production of addiction science, both globally and in specific countries. Addic tion science has evolved to become part of a specialized academic field, with its own training programs, professional organizations, research centers, funding mechanisms, and communication channels. It is devoted both to the pursuit of basic knowledge about addiction and the application of that knowledge to treatment and prevention activities. 0 Publishing Addiction Science 30 By integrating itself with the postwar biomedical establishment (particu larly psychiatry), the addiction field experienced phenomenal growth. As sug gested by the information presented in this chapter and elsewhere, that growth has been characterized by a number of “megatrends” (Babor, 1993b, 2000), as depicted in Figure 2.9. These trends include the following: (a) the emergence of public and private financing mechanisms to support treatment, prevention, and research programs; (b) development of an institutional base consisting of research centers, specialized clinical facilities, and related organizational struc tures; (c) the growth of professional societies to give the field a sense of identity and purpose; and (d) the rapid expansion of scientific communication outlets and publication opportunities to facilitate information exchange and dissemi nation. The final ingredient of the addiction field depicted in the figure is the result of all this effort—that is, basic and applied knowledge about addiction.f f Although opinions will differ as to what constitutes the collective “products” of professional careers in academia and the health sector, from a societal per spective, the tangible products of the addiction field can be measured in terms of scientific knowledge, evidence-based clinical and prevention services, and policy interventions designed to address the consequences of psychoactive substance use. Ultimately, the cumulative and collective impact of these efforts should be the reduction of substance-related harm, suffering, and mortality.h f The growth of addiction science has fostered increasing communication and collaboration on an international level. Part of this has been the result of the explosion of communications technology and the ease of international travel, Fig. 2.9: “Megatrends” in addiction science. Societies Basic and Fig. 2.9: “Megatrends” in addiction science. Infrastructure and Career Opportunities in Addiction Science 31 but it may also be the result of the globalization of alcohol and other drug dis tribution networks, which are bringing addictive substances to locations and populations that were previously unexposed. Conclusion Examples include the market ing by transnational alcohol producers of new alcohol products to women and young adults and the growth of illicit drug use in the major population areas of Africa, Latin America, and Asia. Perhaps most importantly, what impact does the modern addiction research infrastructure have on the health of the populations it is intended to serve? Countries invest in research on alcohol and other drugs for a reason. Typically, the purpose is to reduce human suffering caused by psychoactive substance use and to prevent further problems. In most low- and middle-income countries, however, in which addiction presents the same harms as in more developed countries, addiction-research infrastructure is weak or absent. That a journal series on addiction societies and addiction research centers (Edwards & Babor, 2008) could locate in the developing world only a few societies, centers, and journals devoted to the addictions suggests the need to support addiction science in less-resourced countries that have substantial addiction problems. Established groups could aid the development of such societies in large parts of the world that do not at present have this kind of resource. Any such initiatives would need to be cul turally sensitive. Even in countries in which resources might not easily allow development of specialist treatment services, specialist research centers, or the publication of national journals, international collaboration combined with voluntary action catalyzed by local associations may constitute entirely feasible kinds of initiatives capable of considerable impact. p p If research were the main vehicle for the development of a cure for addiction- related problems, however, by now there should have been breakthroughs in translating research findings into effective prevention policy. As previously men tioned, there is a gap between the bulk of scientific research currently conducted and the interests of policymakers who set the agenda for prevention and treatment funds. Despite the field’s apparent growth in many areas, the question of whether the modern infrastructure (surveillance, treatment, prevention, research) has a population-level impact remains unanswered. Until policymakers and addiction experts achieve a greater sense of mission and purpose, nation states will continue to struggle with the question of how best to configure a rational response to the problems of substance abuse. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Conclusion Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. 32 Publishing Addiction Science 32 References References Agrawal, A., & Bierut, L. J. (2012). Identifying genetic variation for alcohol dependence. Alcohol Research and Health, 34(3), 274–281. Anton, R. F., O’Malley, S. S., Ciraulo, D. A., Cisler, R. A., Couper, D., Donovan, D. M., . . . , Zweben, A. (2006). Combined pharmacothera pies and behavioral interventions for alcohol dependence: The COM BINE study: A randomized controlled trial. JAMA, 295, 2003–2017. 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Search Terms Used in SCOPUS Search of Addiction Publications (2000–2014) Search Terms # of results 2010–2014 2000–2014 Alcohol (“alcohol drinking” OR “alcohol-related problems” OR “alcohol intoxication” OR “alcohol abuse” OR “alcohol- induced disorder$” OR “alcohol use” OR “alcohol-related harm$” OR “alcoholism” OR “alcohol use disorder$”) 28,667 74,921 Tobacco (“tobacco smoking” OR “smoking cessation” OR “cigarette smoking” OR “tobacco use disorder$”) 21,528 64,346 Drugs (“street drug$” OR “illicit drug$” OR “illegal drug$” OR “drug dependence” OR “drug use disorder$” OR “drug abuse” OR “marijuana” OR “heroin” OR “hallucinogens” OR “cocaine” OR “cannabis”) 31,425 86,402 Gambling (“gambling” OR “pathological gambling”) 3,192 6,115 Appendix A. Search Terms Used in SCOPUS Search of Addiction Publications (2000–2014) SECTION 2 How to cite this book chapter: Babor, T F, Morisano, D, Stenius, K and Ward, J H. 2017. How to Choose a Journal: Scientific and Practical Considerations. In: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P. (eds.) Publishing Addiction Science: A Guide for the Perplexed, Pp. 37–70. London: Ubiquity Press. DOI: https://doi. org/10.5334/bbd.c. License: CC-BY 4.0. How and Where to Publish CHAPTER 3 Introduction One of the most important and least understood decisions made in the course of publishing a scientific article is the choice of a journal. The decision influ ences the audience reached, the context in which work is presented, and the time it takes to achieve formal publication. At best, the right choice of a jour nal results in the rapid publication of an article that achieves the exposure it deserves. At worst, the wrong choice results in rejection, delay, and even loss of an author’s motivation to persist in seeking publication for a potentially valu able scientific contribution. And in some cases the choice of a journal operated by a predatory publisher can embarrass an author when it is learned that the journal does not conduct peer review and will publish anything for a fee. Journal choice is little understood even by those who have spent decades in the field of addiction research. One reason for this state of affairs is that the field is rapidly changing, with new publication opportunities and formats constantly being added (e.g., electronic journals; e-pub ahead of print; open access; or interactive, supplemented with media options such as audio and video) and more traditional organs of communication (e.g., print journals) adapting to new technology. Another reason for the difficulty in choosing a publication outlet is that, until recently, there was little communication between journal editors and their potential authors. As indicated in Chapter 12, the process by which a journal decides to accept or reject a given article has been mysterious. Publishing Addiction Science 38 Most journals have carefully preserved the mystery within the “black box” of editorial decision making. With virtually no formal training programs on how to write for and publish in scholarly journals, novices often find that the learn ing process for them has been left to chance and to the luck of finding an expe rienced mentor.h This chapter provides guidance on how to choose a journal for a scholarly publication on the subject of addiction, broadly defined as any topic dealing with psychoactive substances as well as behavioral addictions, such as gam bling. A basic assumption of this chapter is that the primary purpose of pub lishing is to communicate findings and ideas to a broader audience than one’s immediate circle. Introduction Our focus is on scholarly journals, which have become the primary organ (in addition to conference presentations, posters, books, and abstracts) of the scientific communication system that has evolved over the past century. Our main interest is in the addiction specialty journals, which limit their subject matter to research on psychoactive substances and related addic tive behaviors. To the extent that many articles on addiction topics are also published in disciplinary journals devoted to psychology, biology, sociology, medicine, and other relevant professional disciplines, we will also consider how to choose among these journals as well. Growth of Addiction Specialty Journals and Other Publication Sources A scientific journal has multiple roles and functions. Journals provide a forum for scientific communication and should certify the scientific value of an indi vidual author’s work. They provide access to reliable knowledge and, at the same time, confer scholarly prestige and facilitate career advancement (see Lafollette, 1992). The number of journals focusing on addiction-related articles since the late 19th century, when addiction publishing first began, accelerated during the 1970s and 1980s, and has continued to grow dramatically since 2007. By the year 2016, there were more than 120 addiction specialty journals operating throughout the world. A majority of the peer-reviewed addiction journals are published in English, which has emerged as the main language for international scientific communi cation (Babor, 1993). Details about the member journals of the International Society of Addiction Journal Editors (ISAJE) are provided in Tables 3.1 and 3.2. The data in these tables are based on the results of a 2015 survey of ISAJE jour nal editors. The survey results were supplemented by a review of public infor mation sources, such as the journal’s webpage (if available), print copies of the journal, and its instructions to authors. Growth of Addiction Specialty Journals and Other Publication Sources Table 3.1 lists the titles of the English-language journals along with informa tion about the substances or addictive behaviors they are concerned with (e.g., alcohol, tobacco, licit and illicit drugs, pathological gambling, other behavioral Journal Name Substances (1) Areas of Interest (2) Issues per Year Acceptance Rate Impact Factor (3) Print/ Online Fees Open Access (4) Abstracting & Indexing Services (5) Addicta: The Turkish Journal on Addictions A,D,T,O T,P,Po,CE, H,PM,S 2 25% – Both Free Full 2 Addiction A,D,T,O HR,T,P,G,Po, SE,PE,H,S 12 15% 4.97 Both Free Hybrid 1 PsycINFO; Web of Science; Scopus; MEDLINE 76 Addiction Science & Clinical Practice A,D,T HR,T,Po N/A 50% – Online Only Processing fee Full Scopus; MEDLINE 9 Addictive Behaviors A,D,T,O HR,T,P,N,Po, SE,PE,PM,S 12 40% 2.80 Both Free Hybrid 1 PsycINFO; Web of Science; Scopus; MEDLINE 20 Advances in Dual Diagnosis A,D HR,T,P,Po, PE,B,PM,S 4 – – Both Free Hybrid 1 Scopus 5 African Journal of Drug and Alcohol Studies A,D,T HR,T,P,Po,SE, PE,PM,S 2 60% – Online Only Free Full PsycINFO; Scopus 9 Alcoholism Treatment Quarterly A HR,T,P,Po, SE,PM,S 4 80% – Both Free Hybrid 1 PsycINFO; Scopus 11 Alcoholism: Clinical & Experimental Research A,T,O A,HR,T,P,N, G,Po,SE,PE, CE,B,PM,S 12 + 1–2 supplements 51% 2.83 Online Only Page Fee Hybrid 1 PsycINFO; Web of Science; Scopus; MEDLINE 47 American Journal of Drug and Alcohol Abuse A,D A,HR,T,P,N, G,Po,PE,B 6 – 1.83 Both Free Hybrid 1 PsycINFO; Web of Science; Scopus; MEDLINE 21 Journal Name Substances (1) Areas of Interest (2) Issues per Year Acceptance Rate Impact Factor (3) Print/ Online Fees Open Access (4) Abstracting & Indexing Services (5) Canadian Journal of Addiction/Le Journal Canadien d’Addiction A,D,T,O HR,T,P, Po,PM,S 3 80% – Both Free Full Scopus (in process of applying to PsycINFO, MEDLINE) 9 (3 more in process) Drug and Alcohol Dependence A,T,D A,HR,T, P,N,GPo, SE,PE,H, B,PM,S 12 – 3.35 Both Free Hybrid 1 PsycINFO; Web of Science; Scopus; MEDLINE 20 Drug and Alcohol Review A,T,D HR,T,P,Po, SE,PE, H,B, PM,S 6 – 2.41 Both Free Hybrid 1 PsycINFO; Web of Science; Scopus; MEDLINE 34 Drugs: Education, Prevention & Policy A,T,D HR,T,P, Po,H, S 6 – 0.76 Both Free Hybrid 1 PsycINFO; Web of Science; Scopus 25 Experimental and Clinical Psychopharmacology A,D,T,O A,HR,T,P, N,G,SE, PE,CE,B, PM 6 56% 2.14 Both Free Hybrid 1 PsycINFO; Scopus; MEDLINE 15 International Gambling Studies O A,HR,T,P,N, Po,SE,PE,H, B,PM,S 3 49% 1.23 Both Free Hybrid 1 PsycINFO; Web of Science; Scopus 8 International Journal of Alcohol and Drug Research A,D,T,O HR,T,P,Po, SE,PM,S 2–4 89% – Online Only Page Fee Full PsycINFO 6 Journal of Addiction Medicine A,D,T,O HR,T,P, Po, SE,PE,B, PM,S 6 48% 2.07 Both Free Hybrid 1 PsycINFO; Web of Science; Scopus; MEDLINE 39 Journal of Addictions Nursing A,O HR,T,P, Po,PM,S 4 – 0.48 Both Free Hybrid 1 PsycINFO; Web of Science; Scopus; MEDLINE 18 Journal of Addictive Diseases A,D,T,O HR,T,P,N, G,Po,SE, PE,CE,B, PM,S 4 – 1.78 Both Free Hybrid 1 PsycINFO; MEDLINE 16 Journal of Behavioral Addictions O HR,T,P,N, G,SE,PE, B,PM,S 4 – 2.49 Both Free Hybrid 1 PsycINFO; Web of Science; Scopus; MEDLINE 11 Journal of Drug and Alcohol Research A,T,D A,HR,T, N,G,B,PM 1 90% – Online Only Processing Fee Full 4 Journal of Gambling Issues O HR,T,P, Po,SE,PE, H,B,PM,S 2 (+ 1 supplement) 60% – Online Only Free Full PsycINFO; Scopus 10 Journal of Groups in Addiction and Recovery A,T,D,O HR,T, PM,S 4 – – Both Free Hybrid 1 PsycINFO; Scopus 18 Journal Name Substances (1) Areas of Interest (2) Issues per Year Acceptance Rate Impact Factor (3) Print/ Online Fees Open Access (4) Abstracting & Indexing Services (5) Journal of Psychoactive Drugs A,D,T,O HR,T,N, Po,SE,PE, H,B,PM,S 5 – 1.78 Both Free Hybrid 1 PsycINFO; Scopus; MEDLINE 14 Journal of Studies on Alcohol and Drugs A,D,T HR,T,P, N,G,Po, SE,PE, B,PM,S 6 – 2.20 Both Free Hybrid 1 PsycINFO; Web of Science; Scopus; MEDLINE 27 Journal of Substance Abuse Treatment A,D,T,O HR,T,Po, PE,PM,S 10 – 2.47 Both Free Hybrid 1 PsycINFO; Web of Science; Scopus; MEDLINE 14 Journal of Substance Use A,D,T,O HR,T,P, Po,PM 6 – 0.89 Both Free Hybrid 1 PsycINFO; Scopus 11 Nicotine & Tobacco Research T A,HR,T, P,N,G, Po,SE,PE, CE,B,PM,S 6 38% 3.81 Both Free Hybrid 1 PsycINFO; Web of Science; Scopus; MEDLINE 16 Nordic Studies on Alcohol and Drugs (Nordisk Alkohol & Narkotikatidskrift) A,D,T,O T,P,Po,SE, H,PM,S 6 67% 0.77 Both Free Full PsycINFO; Web of Science; Scopus 36 Psychopharmacology A,D,T,O A,HR, T,P,N,G, PE,B 24 – 3.54 Both Free Hybrid 1 PsycINFO; Web of Science; Scopus; MEDLINE 35 Substance Abuse A,D,T A,HR,T, P,Po,SE, PE,CE,H, B,PM,S 4 – 2.58 Both Free Hybrid 1 PsycINFO; Web of Science; Scopus; MEDLINE 13 Table 3.1: ISAJE-member journals published in the English language. Growth of Addiction Specialty Journals and Other Publication Sources (1) Substances: A = alcohol; D = licit and illicit psychoactive drugs other than alcohol; T = tobacco and other nicotine products; O = other substances and addictive behaviors, including gambling and eating disorders. (2) Areas of Interest: A = animal research; HR = human research; T = treatment; P = prevention; N = neuroscience; G = genetics; Po = policy; SE = social epidemiology; PE = psychiatric epidemiology; CE = chronic disease epidemiology; H = history; B= biological mechanisms; PM = psychological mechanisms; S = social factors. (3) Thomson Reuters 2015 Impact Factor. From 2016 Release of Journal Citation Reports. Source: 2015 Web of Science Data. (4) This column indicates the journal’s open access policy. Full = all articles of the journal are made available to the reader for free online; Hybrid 1 = open access only for those individual articles for which the authors or the author’s institution or funder pay an open access publishing fee; Hybrid 2 = all articles are open access after an embargo period, usually one year. (5) This column lists up to four of the largest abstracting and indexing services, if the journal is included in them (PsycINFO; Web of Science; Scopus; MEDLINE), plus the total number of abstracting and indexing services that are claimed by the journal, includ- ing those listed. Growth of Addiction Specialty Journals and Other Publication Sources Journal Name Language Substances (1) Areas of Interest (2) Issues per year Acceptance Rate Impact Factor (3) Print/ Online Fees Open Access Abstracting/ Indexing Services (4) Addicta: The Turkish Journal on Addictions Turkish & English A,D,T,O T,P,Po,CE, H,PM,S 2 25% Both Free Full 2 Adiktologie Articles submis sion in Czech, Slovak or English A,D,T,O A,HR,T,P,N, Po,SE,PE,CE, H,PM,S 4 + 1–2 supplements 85% Both Free Hybrid 2 Scopus 4 Alkoholizm i Narkomania (Alcoholism and Drug Addiction) Polish & English A,D,T A,HR,T,P, G,Po,SE,PE, H,B,PM,S 4 85% Both Free Full 3 Drogues, santé et société French (English Abstracts) A,D,T H,T,P,N,Po, SE,PE,H, PM,S 2 68% Online Only Free Full PsycINFO 5 Exartisis Greek (English Abstracts) A,D,T,O T,P,PM,S 2 85% Print Only Free No N/A Narcologia Russian A,D,T,O HR,T,P,Po, SE,PE,H,B, PM,S 12 Print Only N/A N/A N/A Nordisk Alkohol & Narkotikatidskrift (Nordic Studies on Alcohol and Drugs) English, Danish, Norwegian, & Swedish A,D,T,O T,P,Pol,SE, H,PM,S 6 67% 0.77 Both Free Full PsycINFO; Web of Science; Scopus 36 SUCHT German (with English abstracts and titles), but some articles in English A,D,T,O HR,T,P,Po, SE,PE,H, PM,S 12 80% Both Free Hybrid 1 PsycINFO; Scopus 10 Suchtmedizin (Addiction Medicine) German (English Summaries) A,D,T,O T,P,Po,SE, PE,B,S 6 – Both Free Full Scopus 2 Table 3.2: ISAJE-member journals published in languages other than English. (1) Substances: A = alcohol; D = licit and illicit psychoactive drugs other than alcohol; T = tobacco and other nicotine products; O = other substances and addictive behaviors, including gambling and eating disorders. (2) Areas of Interest: A = animal research; HR = human research; T = treatment; P = prevention; N = neuroscience; G = genetics; Po = policy; SE = social epidemiology; PE = psychiatric epidemiology; CE = chronic disease epidemiology; H = history; B= biological mechanisms; PM = psychological mechanisms; S = social factors. (3) Thomson Reuters 2015 Impact Factor. From 2016 Release of Journal Citation Reports. Source: 2015 Web of Science Data. (4) This column lists up to four of the largest abstracting and indexing services, if the journal is included in them (PsycINFO; Web of Science; Scopus; MEDLINE), plus the total number of abstracting and indexing services that are claimed by the journal, includ- ing those listed. Table 3.3: Journals publishing the highest annual numbers of articles on alco hol and drug research. Growth of Addiction Specialty Journals and Other Publication Sources Publishing Addiction Science 46 addictions); general topical areas covered (e.g., treatment, prevention, epi demiology, biological mechanisms, history); and details about the journals’ frequency of publication, acceptance rate, impact factor, and dissemination channels (i.e., abstracting or indexing services). Table 3.2 provides similar information for journals published in languages other than English. These tables were last updated in January 2017; the most current list of ISAJE member journals is available on www.isaje.net.h j j The number of specialized addiction journals is only part of the story of how the addiction field has grown in size and complexity. A significant por tion of the addiction literature is also published in scholarly journals that have a more general orientation toward disciplines such as medicine, psychology, biochemistry, sociology, economics, and public health. In an earlier version of this chapter published in the first edition of Publishing Addiction Science, we reported that 58% of the alcohol-related articles prior to 2003 were published in general or disciplinary journals and that 42% were published in addiction specialty journals. When the articles were subclassified as either “biomedical” (i.e., dealing with biological or medical topics) or “psychosocial” (i.e., dealing with topics such as treatment, prevention, epidemiology, psychology, or social policy), the addiction specialty journals published a higher percentage of arti cles on psychosocial topics, whereas disciplinary journals published a greater share of the biomedical articles. Alcoholism: Clinical and Experimental Research Drug and Alcohol Dependence Addictive Behaviors PLOS One Neuropsychopharmacology Addiction Journal of Studies on Alcohol and Drugs Substance use and misuse Psychopharmacology Journal of Substance Abuse Treatment Psychology of Addictive Behaviors Biological Psychiatry Neuropharmacology BMC Public Health Drug and Alcohol Review Table 3.3: Journals publishing the highest annual numbers of articles on alco hol and drug research. How to Choose a Journal 47 Table 3.3 provides a list of the top 15 of journals publishing articles on alcohol and drug research as identified through a search in Web of Science for articles published in 2014, indexed with any of the following terms: alcohol, alcoholism, addiction, drug abuse, drug addiction, substance use, or substance abuse. The table suggests that many disciplinary journals (e. g., Biological Psychiatry) also publish significant amounts of addiction research. Growth of Addiction Specialty Journals and Other Publication Sources i In addition to the expanding array of journals that addiction authors have to choose from, many publishers have increased the standard number of issues released per year, added supplements or special issues, and created new elec tronic formats for submitting articles. With the increased number and breadth of scholarly journals covering addiction-related research, there has probably never been a greater opportunity to publish on the subject. Nevertheless, the plethora of journals has created new challenges for prospective authors, not the least of which is the proliferation of online, open-access journals, some of which have questionable publishing credentials. Other questions that arise in the rapidly changing publishing environment are the following: What are the relative merits of publishing in disciplinary versus addiction specialty journals? How does an author find the most appropriate journal for a particular article? What are the chances that an article will be accepted by a given journal? Which journals have the greatest impact on the field? How does an author know whether a journal will reach the intended audience for a specific article? What are the costs of publishing in pay-per-page journals?i To assist prospective authors in finding answers to these questions, Box 3.1 describes the kinds of decisions that must be made during the search for an 1. Decide first whether the article is primarily of interest to a national or an international audience. 2. Consider the language of publication. 3. Consider whether to publish in a generic, disciplinary, or addic tion specialty journal. 4. Review the journal’s content range (type of drug, clinical/basic science, etc.) and general culture. 5–6. Evaluate the journal’s quality and integrity. 7. Gauge your article’s potential exposure by reviewing the jour nal’s indexing and abstracting services, as well as its open-access policy. 8. Evaluate your chances of acceptance. 9. Take into account time to publication and other practical matters. 10. Consider, but don’t be fooled by, impact factors. 1. Decide first whether the article is primarily of interest to a national or an international audience. 2. Consider the language of publication. 3. Consider whether to publish in a generic, disciplinary, or addic tion specialty journal. 4. Review the journal’s content range (type of drug, clinical/basic science, etc.) and general culture. 5–6. Evaluate the journal’s quality and integrity. 7. Gauge your article’s potential exposure by reviewing the jour nal’s indexing and abstracting services, as well as its open-access policy. 8. 1. Decide First Whether the Article is Primarily of Interest to a National or an International Audience This is partly a matter of the article’s information content and partly a matter of presentation or appeal. If the topic is primarily of local or national interest (e.g., prevalence of substance abuse among Brazilian secondary-school students or an evaluation of a local treatment program) and the presentation is oriented toward professionals in a particular country, then the article should be submit ted to a journal capable of reaching that audience, such as one sponsored by a national professional society. If the topic is likely to appeal to scientists or professionals in many countries and the presentation speaks to this broader audience, then an international journal should be considered. Country- or region-specific case studies of international significance and new advance ments or findings with potential international follow-up or applications would also suggest the choice of an international journal. In general, the best way to determine the scope and audience of a journal is to visit the journal’s website and review its mission statement. Growth of Addiction Specialty Journals and Other Publication Sources Evaluate your chances of acceptance. 9. Take into account time to publication and other practical matters. 10. Consider, but don’t be fooled by, impact factors. Box 3.1: Ten steps in choosing a journal. Publishing Addiction Science 48 appropriate journal. The following sections expand on this outline, discussing each step in the process. It should be noted that although our review focuses primarily on how to publish a standard article based on original research, the publication of other types of articles (e.g., review articles, theoretical articles, case reports) can also be informed by following these steps. 2. Consider the Language of Publication English has become the main language of scientific communication through out the world. Nevertheless, significant numbers of scientific articles are pub lished in German, Russian, Japanese, French, Spanish, Italian, Chinese, and the Scandinavian languages, as indicated by the journals listed in Table 3.2. For most researchers, choosing what language to publish in depends largely on the author’s native tongue, the country in which the study was conducted, and the potential audience. Another limiting factor is the availability of an addic tion journal that publishes in that language and accepts articles on the author’s topic. If one is writing for an international audience, it is wise to choose an English-language journal that can be read by scholars in most countries. Under many circumstances, an article in English will have greater exposure, especially when the journal’s articles are included in major abstracting and indexing ser vices (e.g., MEDLINE, Web of Science), most of which operate in the English How to Choose a Journal 49 49 language. Some journals demonstrate an intentional internationalism that is expressed in a readiness to publish articles and review books submitted from many different countries. f English-language authors can choose between national, more specialized journals or the bigger international journals, depending on the quality of the article, the importance of the findings, and the audience one wishes to reach (see Step 1). If the article is likely to be of interest to an international audience but is not written in English, the author can consider publishing it in English in addition to his or her native language. Multiple publications in different lan guages, however, require permission from both of the journal editors involved. In some cases, reporting research findings in more than one language will result in very different publications, because the target audience will require different perspectives and background information. The rules of academic integrity and plagiarism still apply, as described in Chapter 14. Alternatively, researchers writing in languages other than English should consider publishing in journals that provide English-language abstracts (see Table 3.2), thereby gaining entré into some of the world’s major abstracting services (see Appendix A to this chapter). In general, journals published in languages other than English provide a valuable service to national and regional audiences that have a special interest in addiction studies. 2. Consider the Language of Publication For example, if an article has special relevance to French- speaking populations, the journal Alcoologie et Addictologie (Alcohol and Addiction Studies) provides immediate access to that audience not only because of the language it is written in but also because of the network through which the journal is distributed (i.e., the Société Française d’Alcoologie et Addictol ogie [French Society of Alcohol and Addiction Studies]). Articles written in languages other than English also fulfill an important function by maintaining language use and terminology current and relevant to the addiction field in all of these languages. With the fast-paced changes in addiction science, shifts in use of language and terminology inadvertently mirror the trends in research, society, and scholarly communication. Authors and editors play a significant role in shaping the language of addiction science and promoting use of pre ferred terms such as nonstigmatizing words and phrases in every language. Overall, non–English-language journals serve as a necessary medium for communication among clinicians, scientists, and policymakers within major linguistic areas of the world. They increase the range of cultural and scientific diversity in the addiction field and, in this way, provide new opportunities for authors and readers. In some countries, for instance Finland and Norway, jour nals in national languages have been upgraded as publication channels, even if they do not have an impact factor. Authors whose first language is English should not ignore the advantages of publishing in these journals, which often have a higher acceptance rate and, in some cases, are open to submissions writ ten in English. Depending on the topic and scope of the article, some journals Publishing Addiction Science 50 are willing to either translate into the language of publication or publish the article directly in English. 4. Review the Journal’s Content Range and General Culture 4. Review the Journal’s Content Range and General Culture Every journal has a culture of its own, sometimes developed over many years of serving a particular professional society or through the influence of editors who sometimes place their own particular imprint on the journal. The best way to understand that culture is to review several issues of the journal in their entirety, including editorials, letters to the editor, and scientific articles. A visit to the journal’s homepage will accomplish the same purpose. Prospec tive contributors should also read the journal’s mission statement, which often describes the focus of the journal, its goals, its preferences, and its audience. Although these statements are sometimes dated and written in general terms, they often provide a broad outline of the journal’s traditions, image, priorities, and aspirations. p In Tables 3.1 and 3.2, the first column describes the major substances (and addictive behaviors) that each journal considers part of its purview. Some jour nals (e.g., Nicotine and Tobacco Research) are interested in one particular sub stance, whereas others are quite generic (e.g., Drug and Alcohol Dependence). The topical areas covered by a journal are also an important consideration. Some specialize in treatment research, others in biological effects or mecha nisms, and still others in prevention or policy. The less a particular article meets a journal’s content areas, the more likely it is to be rejected. Even when an arti cle is considered to be scientifically sound and relevant to the addiction field, it may be dismissed by a journal editor because it does not meet with the journal’s current priorities and stated mission. It is therefore important for authors to narrow their choice of journals to those whose history and current contents have demonstrated an interest in (or at least an openness to) the topic, sub stance, and scope of the article being submitted. When in doubt, it is always advisable for authors to talk with colleagues and communicate with journal editors. By asking someone with experience in publishing for advice, younger or less experienced authors can obtain firsthand information about the priori ties and preferences of particular journal editors. 3. Consider Whether to Publish in a Generic, Disciplinary, or Addiction Specialty Journal 3. Consider Whether to Publish in a Generic, Disciplinary, or Addiction Specialty Journal The third step involves examining whether the results of a study are mainly of interest to other addiction researchers or to a more general readership. It is probably easier to get an addiction article accepted in an addiction specialty journal. Publishing in a non–addiction journal may require authors to write the article in a way that is understandable to those who do not speak the “addic tion dialect.” Some journals, such as Nature and Science, are multidisciplinary and are oriented toward the general scientific community. Other journals, such as The Lancet and the Bulletin of the World Health Organization, publish articles deal ing with a specific discipline, such as medicine or public health, respectively. In countries without addiction specialty journals, a journal in psychiatry can, for instance, be an important channel for addiction research.h There are several reasons for considering more broadly oriented generic and disciplinary journals. As noted above, disciplinary journals publish a consid erable amount of the scientific literature on substance-related research. These journals are generally published by and oriented toward professional groups associated with the major disciplines contributing to addiction studies (i.e., biology, neuroscience, genetics, psychology, medicine, psychiatry, public health, sociology, and anthropology). Disciplinary journals are sometimes favored by addiction researchers because they are thought to have greater prestige value within a given disci pline than addiction specialty journals. Professional advancement for academic researchers is often based on such subtle considerations. Moreover, some of the most popular disciplinary journals (e.g., The Lancet, The New England Journal of Medicine) have higher impact factors (discussed below) than addiction spe cialty journals, which adds to their prestige value. Nevertheless, the chances of publishing an article on an addiction-related subject are sometimes reduced if a journal does not have reviewers or editors familiar with the topic. If a particular disciplinary journal rarely publishes articles on addiction, it is advisable to contact the editor before submitting an article. In addition, if a disciplinary journal has a large circulation and a high impact factor, authors should make sure that the article is likely to be seen as important before submitting it for review. In the remainder of this chapter, we discuss the merits of publishing in addiction specialty journals, which offer a range of opportunities to prospective authors that are comparable to those available in the disciplinary journals. 3. Consider Whether to Publish in a Generic, Disciplinary, or Addiction Specialty Journal How to Choose a Journal 51 5–6. Evaluate the Journal’s Quality and Integrity Until recently, scientific journals were usually managed by publishing compa nies and professional societies, which vouched for the quality and integrity of the journal. The most important criterion for quality and integrity is the peer- review process, as overseen by a qualified journal editor and the journal’s edito rial board. A troubling development in scientific publishing is the proliferation of publishing companies that operate online journals of questionable quality and integrity. Twenty-nine journals in the addiction field operate in open- access formats. One third of them are members of ISAJE, which evaluates their Publishing Addiction Science 52 quality and integrity as a condition of membership. Of the remaining jour nals, several have been evaluated by Thomson Reuters and are listed in the Web of Science. Others are listed in Scopus, PsycINFO, and MEDLINE, which are indexing and abstracting services that have standards that must be met before a journal’s articles are listed. And then there are a few online open-access jour nals that fail to fulfill the minimal criteria for a responsible scientific journal. ii Conventional non–open-access journals cover publishing costs through sub scriptions and single-article purchases. Some non–open-access journals pro vide open access after an embargo period of 6–12 months or longer. Some allow authors to post their manuscripts, before final copyediting, on their own or their institution’s website. Some allow no open access (see Tables 3.1 and 3.2 for information about the journals that are members of ISAJE). Open-access journals use a funding model that does not charge readers or their institutions for access. They allow users to read, download, copy, distrib ute, print, search, or link to the full texts of their articles at no cost to the user. Open-access thus provides unrestricted online access to peer-reviewed schol arly research without the need for a journal subscription or use of a univer sity library. A few open-access journals have financial resources, for instance state support, that make it possible to provide open access without any costs for the author (platinum open access). Most open-access journals operate using a business model in which they charge authors fees to publish their articles (gold open access), but some journals waive these charges. Some unscrupulous entre preneurs have discovered that this financial base offers an opportunity to make money by providing a publication channel without any quality control. 5–6. Evaluate the Journal’s Quality and Integrity It seems that the majority of new open-access journals levy page charges or process ing fees as part of a business model in which a publishing company manages scores, sometimes hundreds, of online journals.hf The term predatory publisher was coined by Jeffrey Beall, a University of Colorado librarian (Beall, 2012). The term refers to some of the open-access publishing companies that engage in questionable practices with regard to journal management, marketing activities, peer review, and page fees. Efforts to test the quality of the review process conducted by these journals have not been encouraging. One researcher (Davis, 2009) submitted an article with nonsense text and fictitious authors, who were listed as being affiliated with the nonexist ent “Center for Research in Applied Phrenology.” The author received a letter from the editor of The Open Information Science Journal stating that the article had been “accepted for publication after peer-reviewing process” (quoted in Davis, 2009). A publication fee of $800 was requested, to be sent to an address in the United Arab Emirates. In another case (Bohannon, 2013), a science writer submitted a faked and fab ricated cancer article to 305 online journals. The fictitious authors of the article received 157 acceptance letters and 98 rejections. Would the results have been the same had these fake articles been submitted to traditional, subscription-based journals? One would hope that fraud and mediocrity would not be rewarded as How to Choose a Journal 53 easily, but there is some evidence to suggest that the scientific enterprise is not being protected by the traditional academic publishers either. Until it stopped operating in 2016, Beall’s list of predatory publishers was the main resource for authors to verify the quality of publishers and individual journals. It has also started an entire movement of “journal watching,” a grass roots movement to maintain the integrity of scholarly communication. Infor mation on new and potentially questionable journals is voluntarily submitted as “hat tips” to the website by scholars, authors, and librarians, who report inci dents of inappropriate or unethical practices. Box 3.2 summarizes the characteristics of journals associated with predatory publishers. As indicated in the box, several tactics are used by these journals to take advantage of the situation in which publication in peer-reviewed journals is considered one of the highest distinctions for peer recognition, academic advancement, and personal accomplishment. Box 3.2: Characteristics of journals associated with predatory publishers (adapted from Beall, 2012, 2013). 5–6. Evaluate the Journal’s Quality and Integrity These include flattering authors with invitations to contribute articles to be included in special issues and the promise of rapid publication in a peer-reviewed journal. Many scientists have received email invitations to serve on editorial boards by these publishers. 1. Rapid acceptance of articles with little or no peer review or qual ity control 2. Journal names or website styles that resemble those of more established journals 3. Use of poor English grammar and syntax in the journal’s website and email communications 4. No issue or only single issue has been published before 5. Aggressive email marketing that urges academics to submit arti cles or serve on editorial board, sent to you “because of your eminence in the field” 6. Journal editors who have no academic standing or minimal sci entific credentials in the topical area of the journal, or the editor cannot be identified at all 7. Article fees not apparent at the time of submission 8. Listing academics as members of editorial boards without their permission 9. No ethical guidelines, or guidelines that apply to the entire range of journals the publisher operates 10. Misleading information about the location of the publishing operation 1. Rapid acceptance of articles with little or no peer review or qual ity control 2. Journal names or website styles that resemble those of more established journals 3. Use of poor English grammar and syntax in the journal’s website and email communications 4. No issue or only single issue has been published before 5. Aggressive email marketing that urges academics to submit arti cles or serve on editorial board, sent to you “because of your eminence in the field” 6. Journal editors who have no academic standing or minimal sci entific credentials in the topical area of the journal, or the editor cannot be identified at all 7. Article fees not apparent at the time of submission 8. Listing academics as members of editorial boards without their permission 9. No ethical guidelines, or guidelines that apply to the entire range of journals the publisher operates 10. Misleading information about the location of the publishing operation 1. Rapid acceptance of articles with little or no peer review or qual ity control y 2. Journal names or website styles that resemble those of more established journals 3. Use of poor English grammar and syntax in the journal’s website and email communications 4. 5–6. Evaluate the Journal’s Quality and Integrity No issue or only single issue has been published before 5. Aggressive email marketing that urges academics to submit arti cles or serve on editorial board, sent to you “because of your eminence in the field” i 6. Journal editors who have no academic standing or minimal sci entific credentials in the topical area of the journal, or the editor cannot be identified at all i 7. Article fees not apparent at the time of submission 8. Listing academics as members of editorial boards without their permission 9. No ethical guidelines, or guidelines that apply to the entire range of journals the publisher operates 10. Misleading information about the location of the publishing operation 54 Publishing Addiction Science 54 Prospective board members, regardless of their experience or qualifications, are told that if they decide to publish in the journal, they will receive a discounted fee based on the manuscripts they secure from other authors for the journal. In addition to publishing journals, some predatory publishers host conferences, including the publication of the proceedings, for a fee. The latest development is the appearance of the predatory impact factor, an arbitrary number com puted by for-profit publishers for a fee (see later section on impact factor).i i In the preparation of this chapter, the authors identified several problems with the approximately 20 journals having addiction-related names that are affiliated with predatory publishers and other non-ISAJE, open-access, online, for-profit publishers. Most did not respond to an editors’ survey we conducted. Almost half (n = 9) had no identifiable editor. Some were found to falsely list indexing/abstracting services. Many listed Google as one of their indexing/ abstracting services.hi g What are the risks of publishing in these journals? The first risk is that your article may not reach its intended audience because these journals are poorly indexed and may not be permanently stored or archived. Many of them simply cease to exist after a few issues. A second risk is that your contribution to the publisher’s profit margin may help to perpetuate journals that engage in ques tionable publishing practices, including the publication of fabricated articles accepted with minimal or nonexistent peer review. A third risk is that when an article published in a questionable journal is listed in a person’s curriculum vitae, it may ultimately cause embarrassment to that individual, or there could be worse consequences. 5–6. Evaluate the Journal’s Quality and Integrity As these problems become more apparent in the future, the quality of journals will be evaluated more rigorously by those charged with protecting scientific integrity, as well as university committees charged with hiring, appointments, promotions, and tenure decisions. Publishing in or being listed on the editorial boards of low-quality or unverifiable journals may be a disadvantage in that these publications could count against hiring, promotion, or tenure because they represent such poor scientific quality. i What can be done to protect authors from being exploited and embarrassed by publishing in a journal that does not operate competently, ethically, and scientifically? In the addiction field, quality control is provided by ISAJE, an organization that insists that its 33 member journals subscribe to a set of core principles covering appropriate peer review, conflict of interest policies, edito rial management, and transparency (Farmington Consensus, 1997). As shown in Tables 3.1 and 3.2, ISAJE has several online open access journals that are fully compliant with the Farmington Consensus. Another precaution is to find out whether the journal receives any significant citations by checking Web of Science or the Journal Citation Reports before submitting to an open-access journal. Ulrich’s Periodicals Directory, now available online in most academic libraries (on a subscription basis), has been a trusted resource to find informa tion about scholarly journals, magazines, and newsletters since 1932. The most How to Choose a Journal 55 55 effective precaution is not to submit an article to an open-access journal pub lished by an organization that meets the criteria listed in Box 3.2, sponsorship by a learned society, email or telephone access to an editor who is qualified to manage manuscripts, evidence that there is a rigorous peer-review process, and the existence of a verifiable Thomson Reuters impact factor are other ways to determine whether a journal is reputable.i Finally, the reputation and scientific standing of a journal can be checked by verifying that the journal is indexed in one or more of the key indexing and abstracting services that disseminate information only about journals that meet minimal criteria for quality and integrity (e.g., MEDLINE, PsycINFO, Scopus, Web of Science). This is discussed in the following section. 7. 5–6. Evaluate the Journal’s Quality and Integrity Gauge Your Article’s Potential Exposure by Reviewing the Journal’s Indexing and Abstracting Services, as Well as its Open-Access Policy One of the most important goals of scientific publication is to reach one or more specific audiences, such as the scientific community, clinical practition ers, or policymakers. A journal’s ability to provide exposure to these audiences is determined by its circulation (print and electronic) and its dissemination capabilities, determined by access to abstracting and indexing services. Print circulation refers to the number of copies printed for the journal’s sub scribers as well as those who receive free copies. Scholarly journals have two major types of subscribers: members of professional organizations and aca demic libraries. In addition, there are smaller numbers of personal and nonaca demic institutional subscribers. Before the advent of the Internet, the number of journal copies in circulation was a good indicator of a journal’s exposure. Today, figures describing the number of visits to homepages or the number of downloads may be better measures of how extensively and frequently a journal is read. In addition to traditional circulation data, article-level alternative metrics beyond page visits and download counts provide evidence of the immediate impact of the article (i.e., readership, as reflected in scholarly social media; Weller, 2015). A new field of measuring scholarly performance, called altmet rics, compiles data on the publication’s appearance in the various social media outlets, such as shares or mentions on Facebook and Twitter, or in blogs, as well as in mainstream media (Piwowar, 2013; Priem et al., 2012). As evidence of immediate exposure, the citation analysis computed by altmetrics is said to be a good indicator of an article’s success when compared with traditional bibliometrics, such as citation counts (Ortega, 2015). A few journals, such as Addiction, already indicate these metrics on their sites at the article level, often symbolized with the so-called altmetric donut, with each color representing a different type of alternative metric. 56 Publishing Addiction Science 56 If an article is relevant to the members of a particular learned society (e.g., the British Society for the Study of Addiction to Alcohol or Other Drugs), pro fessional group (e.g., the Canadian Medical Association), or scientific organi zation (e.g., the Research Society on Alcoholism), then it may make sense to submit the manuscript to a journal that is sponsored by that organization. 5–6. Evaluate the Journal’s Quality and Integrity Many of the journals listed in Tables 3.1 and 3.2 are sponsored by professional organi zations or learned societies that provide free subscriptions or reduced rates to their members. For example, Alcoologie et Addictologie (Alcohol and Addiction Studies) is sponsored by the Société Française d’Alcoologie [French Society of Alcohol Studies], which distributes free copies of the journal to its 1,400 mem bers. Psychology of Addictive Behaviors is published by the American Psychologi cal Association, which makes the journal available to members of the Society of Addiction Psychology (American Psychological Association, Division 50) at a reduced subscription rate. See Chapter 2, Table 2.2 for a complete list.f p p p In addition to targeting organizational subscribers, exposure is also affected by the number of library subscriptions. Libraries, especially university libraries, guarantee exposure to students and scholars, thereby providing direct access to perhaps the most important audience for any scientific communication. Cur rently, a single subscription from a large university library might mean exposure to as many as several thousand potential readers, because journal subscriptions are based on full-time equivalents (a calculation of faculty, staff, and students). The world of library and information science has changed rapidly in the past decade, with electronic subscriptions replacing or supplementing print cop ies available on the library shelf. Library subscriptions remain an important conduit for a publication to reach a broader audience than the members of a particular learned society, but now subscriptions are mainly electronic and dis coverability and access have become key components of exposure. University libraries and other large information sources have begun to pool resources to increase electronic availability of full-text journals. This also means that the same journal can be available from various content providers on various plat forms in various subscription packages. Tools provided by modern technology to describe, organize, and access information include versions of the library catalog, the database of the content provider, and the full text of the article from the journal optimized for mobile devices. The result is an increased dis coverability, more exposure, and potentially larger impact. Access to individual articles is no longer limited to content subscribed to by the library. As a result of consortia and interlibrary-loan agreements, those affiliated with an institu tion of higher education can have full-text articles delivered on their desktop, free of charge, as fast as the next day after placing a request. 5–6. Evaluate the Journal’s Quality and Integrity Unaffiliated readers can also benefit from the better discoverability provided by proprietary and subscription databases, as well as from the new access options for a fee, such as previewing or renting an article instead of purchasing it. Beyond the journal’s print circulation and subscriber base, an article’s expo sure is now determined primarily by the electronic databases that index the How to Choose a Journal 57 published literature by author, topic, and bibliographic reference and pro vide abstracts of articles for potential readers in search of particular types of information. Abstracting and indexing services provide detailed information about the content of scientific journal articles and eBooks by adding metadata and abstracts, which are invaluable for those without immediate access to the full text of the article. Proprietary databases use a controlled vocabulary by establishing preferred terms for each word or concept, such as Medical Subject Headings (MeSH) in MEDLINE. Added to the individual articles by a trained indexer as a subject heading or descriptor, these keywords ensure that the main ideas of the articles will become transparent and are appropriately conveyed. As a result, the article will be discoverable and retrievable via a search conducted in the database. Users can locate relevant articles, chapters, or books in the databases enhanced with abstracting and indexing services at a higher rate of precision by searching the metadata, keywords, and the abstract than they can by using a free search engine, such as Google Scholar, which searches the full text of articles, resulting in higher recall and lower precision. Those affiliated with an institution that has access to the service as well as a subscription to the particular journal can download the full text with the help of an article-linker application. If it is an open-access publication, they can immediately use the full text. Otherwise, they are usually shown information from the publisher on how to access the text. More than 100 companies and institutions currently offer abstracting and indexing services, but many may not cover subject areas related to addiction. At present, no single service or database is available to cover the entire addiction literature, as is the case, for example, for the psychol ogy literature (PsycINFO). 5–6. Evaluate the Journal’s Quality and Integrity To the extent that most of the information summa rized in this paragraph applies to the English-language literature, the reader is referred to Chapter 4 (“Beyond the Anglo-American World”) for information and advice related to publishing in other languages. Appendix A lists some of the main abstracting and indexing services used by the addiction specialty journals listed in Tables 3.1 and 3.2. These organizations provide a variety of important services that dramatically increase the poten tial exposure of a scholarly communication. Although some of these databases used to be available in both print and electronic versions, electronic databases have now become the information source of choice for those who are search ing for topical information via the Internet. They are comprehensive and rapid, and at least some of the information is often inexpensive or free. These services differ widely in their subject matter, coverage of the literature, document types included, service features, and content provider. The major databases (e.g., MEDLINE, Scopus, Web of Science, PsycINFO) are available through librar ies that pay a subscription fee and are highly selective in choosing the journals that they list in their index. They permit searches of the current and past lit erature according to author, title, and keywords, often providing the author’s abstract for review. Other abstracting and indexing services (e.g., Sociological Abstracts) are selective and scholarly but tend to reach a smaller distribution Publishing Addiction Science 58 network. Still other services (e.g., Google Scholar) are more general in nature and may not provide the best access to the audience an author is trying to reach. Many of the journals operated by predatory publishers are indexed or listed only in services databases such as the Directory of Open Access Journals or are only crawled by Google and Google Scholar. DOAJ is a reference tool, based on the fact that a journal is available free on the web. Google and Google Scholar are search engines that aggregate information from the internet and are not abstracting and indexing services. From the author’s perspective, a journal’s ability to provide a listing of its journal articles and abstracts to these secondary information sources greatly increases an article’s exposure to scholars and students throughout the world. 5–6. Evaluate the Journal’s Quality and Integrity The greater the number of quality indexing and abstracting services a journal belongs to (as indicated in Tables 3.1 and 3.2), the more likely it is that an article will reach its intended audience. Although many of the non–English-language journals indicate minimal coverage in abstracting and indexing databases, this situation is changing rapidly, and most of these journals now provide English abstracts and keywords, an important first step in reaching an international audience. 8. Evaluate Your Chances of Acceptance A major consideration in the choice of a journal is the likelihood of accept ance. Journals vary tremendously in the criteria they use to select articles for publication and in the competition a given article will encounter in relation to other authors seeking to claim the same journal space. Some journals have high acceptance rates and are often looking for articles to publish. Other journals have a surfeit of submissions, making it necessary for editors to reject articles that would nevertheless be worthy of publication in less competitive journals. A journal’s acceptance rate provides a rough estimate of an author’s chances of eventual acceptance, but the rates listed in Tables 3.1 and 3.2 are subject to a number of limitations. First, some journals do not know or choose not to reveal their acceptance rate. In the ISAJE member journal survey conducted for the preparation of this chapter, we asked journal editors to tell us the proportion of articles accepted that were eligible for peer review (regardless of whether the articles were sent out for review or were returned un-reviewed). It should be noted that several journals (e.g., Alcohol Research: Current Reviews, Addiction Science & Clinical Practice) operate primarily by commis sioning authors to write articles on a topic or theme, which accounts for their high acceptance rates. Beyond a journal’s acceptance rate, an author’s chances of acceptance depend on many other considerations, some of them scientific, some stylistic, others administrative. Stylistic factors include the quality of the writing and the way in which the data are presented. If the article is poorly written or not well organized, 59 How to Choose a Journal 59 reviewers may see this as a limitation, and editors may be reluctant to take the time to work with authors to bring the article up to the journal’s standards. Administrative factors include the length of the article, the amount of revision required, and the appropriateness of the topic to the journal’s mission. If an article is too long, it reduces the amount of space available for equally worthy articles that are written more concisely by competing authors. If the article is not appropriate to the journal’s current priorities or mission statement, it might be rejected even before it is sent out for peer review. Finally, the number of articles published by a journal could affect chances of acceptance. 9. Take into Account Time to Publication and Other Practical Matters There are several other factors that should be taken into account in selecting a journal. One is the lag time to publication. Some journals take longer than others to process their manuscripts. However, most journals do not reveal how long it takes to arrive at a decision; and even when this information is avail able, it should be noted that the average time is affected by the number of manuscripts that are rejected before being sent out for peer review. Another factor is the time between the acceptance of a revised manuscript and its final publication. This will depend in part on the number of issues published by the journal per year, the number of accepted manuscripts, and the efficiency of the publisher. In general, journals that publish more frequently are likely to have a shorter lag time to publication. The best way to obtain information about the review process is to consult the journal’s instructions to authors or the journal’s website. It is best not to rely on hearsay, anecdote, or the journal’s reputation. 8. Evaluate Your Chances of Acceptance Journals that are published monthly or weekly need to accept more articles than journals that publish less frequently. But journals that publish more frequently also tend to be more competitive. See Chapter 12 for further discussion of factors influenc ing the acceptance or rejection of manuscripts. 10. Consider, but Don’t Be Fooled by, Impact Factors The Journal Impact Factor is an attempt to provide an objective measure of how often a scientific journal’s published work is cited. Such a measure has also been used to judge the quality of an author’s work, to the extent that publishing in a high-impact journal may reflect the quality of a particular article. The impact of a journal on a field of study is thus based on the assumption that the more a journal’s articles are cited, the more influence it has on the field. In 1964, the Institute of Scientific Information began publishing the Science Citation Index. By the early 1990s, 3,200 journals belonged to the core or citation journals of Science Citation Index (Seglen, 1998). 0 Publishing Addiction Science 60 Impact factor was originally developed to objectively compare the quality of journals listed in a particular database (i.e., Journal Citation Reports [now of Thomson Reuters], which provides tools for ranking, evaluating, categoriz ing, and comparing journals; Garfield, 1994). Devised by Eugene Garfield, the founder of the Institute for Scientific Information, impact factors are calculated annually based on the data of the previous years. The impact factor of a journal is the average number of citations received per article published in that journal during the two preceding years. Impact factor is widely used to compare jour nals in a particular field, and, as such, its use has generated a lot of debate con cerning its validity as a measure of a journal’s importance as well as a reflection of the quality of an author’s work (for more on the history and use of impact factor, see Garfield, 1994). i Increasingly, the data used to calculate impact factors have been used as a shortcut to compare and rank individual articles, researchers, and research groups. Impact factor has been criticized almost from its inception (Seglen, 1998; Stenius, 2003), partly because its databank covers only a small share of the world’s scientific journals. Different research fields have different coverage in the database. The database has a clear preference for English-language jour nals (particularly those based in the United States). National or regional jour nals in other languages are not well represented (Seglen, 1998), as indicated by only one of the journals listed in Table 3.2 having an impact factor. All jour nals from a field that is underrepresented will receive lower impact factors. 10. Consider, but Don’t Be Fooled by, Impact Factors In addition, citation frequencies and patterns vary among different research fields. Thus, it is not acceptable to compare impact factors for journals from differ ent fields. A journal representing a field that typically favors large numbers of references will automatically get a higher impact factor, especially if the field is quickly developing. Research fields that get references from related disciplines get higher impact factors. This explains why journals focusing on basic science have higher values. The humanities are in a particularly unfavorable position. Disciplines in which national or regional research, or publications in local lan guages, are important also tend to get low impact factors (Rousseau, 2002). As a measure of impact, with its two-year time frame, impact factor is more appropriate for quickly developing research fields, such as molecular medi cine. Applied, clinical, or social sciences do not fare as well with the two-year window (Andersen, 1998; Luukkonen, 1994). Non–English-language journals or bilingual journals (for instance Japanese–English), even if included in Sci ence Citation Index, will on average receive a lower impact factor. The recently introduced five-year impact factor is supposed to provide a more balanced pic ture of the performance of journals. Finally, it is important to note that a citation is not necessarily an indication of research quality. Every researcher knows that there are numerous reasons (apart from its quality) for citing a scientific publication. Authors may cite or quote for polemical reasons, to flatter their readers, or to promote their own research (or that of their friends, colleagues, or patrons). West and McIlwaine How to Choose a Journal 61 (2002) studied 79 articles published in Addiction between 1995 and 1998 and found no correlation between citation frequency (up to the year 2000) and an independent quality rating. Interestingly, West and McIlwaine also found that articles from the developing world received fewer citations than the quality ranking would have led them to expect. (See Chapter 10 for further discussion of citation procedures).il As a response to the narrowly defined yet widely influential impact factor, a new metric called Eigenfactor was created in 2007 to rank journals in a more comprehensive way. Eigenfactor (eigenfactor.org), also available from Thom son Reuters along with impact factor, expands the timeframe to five years and takes into account the influence level of the citing journals in its algorithm. Conclusion Journals differ in the quality of articles they publish, the exposure they provide to an author’s work, and their subject matter. Once an author or a group of authors has a clear idea of the results of a particular study or project, it is often valuable to conduct a preliminary review of the journals most likely to publish an article on that subject. As indicated in Tables 3.1 and 3.2, there are many peer-reviewed addiction specialty journals to choose from, as well as hundreds of disciplinary and multidisciplinary journals. The careful selection of a journal, when one takes into account both scientific and practical considerations, is clearly worth the effort. Not only is the process likely to save valuable time for authors, peer reviewers, and journal editors, but it also will increase the likelihood that an arti cle will contribute as much to science as it does to the author’s curriculum vitae. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. 10. Consider, but Don’t Be Fooled by, Impact Factors 62 Publishing Addiction Science 10. Consider, but Don’t Be Fooled by, Impact Factors l Similarly, a nonproprietary application, SCImago Journal Rank (SJR; scima gojr.com) attempts to rank journals with its SJR indicator based on the popu lar Google PageRank algorithm, which also takes into account the quality of journal citations in addition to quantity (Moed, 2006). Another metric, Source Normalized Impact per Paper (SNIP), factors the amount of potential citing sources based on the size of the field in order to normalize the numbers for direct comparison (Moed, 2010). These recent statistics indicate that no single statistic can definitively rank journals in a comprehensive way. i y j p y Although altmetrics and scholarly social media are promising alternatives to measure scientific impact both at the level of an article or the author (Ward et al., 2015), they are not treated as equivalents in most fields of science, in which impact factor predominates. However, there is a widely accepted indicator of an individual’s scholarly performance, the h-index, which is also based on cita tions. Introduced by Hirsch (2005), this performance indicator computes a scholar’s top-cited articles rather than considering the total citation count. The main problem with this metric is, as with impact factor, the number is com puted within a particular database only and, as such, will be only as accurate as the data input. As an example, an author with 250 total publications will be underrepresented in Scopus if only 75 of these articles are listed under the author’s name due to the coverage of the database. On the other hand, with its duplicates and erroneous author attributions, Google Scholar Citations can dis play an inflated number closer to 400. The discrepancies will lead to an embar rassing h-index in the first case and a falsely high one in the second, with a difference of as many as 20 points. Either way, it is beyond the author’s reach to correct them. In conclusion, impact factors should be treated with caution. Until the deficiencies in the system have been corrected and its limitations are better understood, however, impact factor remains a relatively crude index of the value of a particular journal. According to Jones (1999), authors should not be preoccupied with the impact factor of a journal. Rather, they should give more consideration to the speed and efficiency of the editorial handling of their manuscripts, the selectiveness of its abstracting and indexing services, and the quality and timeliness of the peer review. References Andersen, H. (1998). Acta Sociologica på den internationale arena: Hvad kan SSCI fortelle? [Acta Sociologica on the international arena: What can SSCI tell us?]. Dansk Sociologi, VII, 72–78. Babor, T. F. (1993). Beyond the invisible college: A science policy analysis of alcohol and drug research. In G. Edwards, J. Strang, & J. H. Jaffe (Eds.), Drugs, alcohol and tobacco: Making the science and policy connections (pp. 48–69). Oxford, England: Oxford University Press Babor, T. F. (1993) Megatrends and dead ends: Alcohol research in global per spective. Alcohol Health and Research World, 17, 177–186. Beall, J. (2012). Predatory publishers are corrupting open access. Nature, 489, 179. DOI: https://doi.org/10.1038/489179a Beall, J. (2013). Predatory publishing is just one of the consequences of gold open access. Learned Publishing, 26, 79–84. DOI: https://doi. org/10.1087/20130203 Bohannon, J. (2014). Who’s Afraid of Peer Review? Science, 342, 60–65. DOI: https://doi.org/10.1126/science.342.6154.60 Cohen, J. F., Korevaar, D. A., Wang, J., Spijker, R., & Bossuyt, P. M. (2015). Should we search Chinese biomedical databases when performing system atic reviews? Systematic Reviews, 4, 23. DOI: http://doi.org/10.1186/s13643- 015-0017-3; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374381/ How to Choose a Journal 63 How to Choose a Journal 63 Davis, P. (2009, June 10). Open access publisher accepts nonsense manuscript for dollars. The Scholarly Kitchen. Retrieved from http://scholarlykitchen. sspnet.org/2009/06/10/nonsense-for-dollars/ Farmington Consensus. (1997). Addiction, 92, 1617–1618.ihh Garfield, E. (1994, June 20). The Thomson Reuters Impact Factor. Thomson Reuters. Retrieved from http://wokinfo.com/essays/impact-factor/.i Hirsch, J. E. (2005). An index to quantify an individual’s scientific research output. Proceedings of the National Academy of Sciences of the United States of America, 102, 16569–16572. DOI: https://doi.org/10.1073/pnas. 0507655102 Jones, A. W. (1999). The impact of Alcohol and Alcoholism among substance abuse journals. Alcohol and Alcoholism, 34, 25–34. Lafollette, M. C. (1992). Stealing into print. Fraud, plagiarism, and misconduct in scientific publishing. Berkeley, CA: University of California Press. i Luukkonen, T. (1994). Viiteanalyysi ja tutkimuksen arviointi [Reference analy sis and evaluation of research]. Signum, 27, 130–132.h Moed, H. F. (2006). Citation analysis in research evaluation. Dordrecht, The Netherlands: Springer.h Moed, H. F. (2010). The Source-Normalized Impact per Paper (SNIP) is a valid and sophisticated indicator of journal citation impact. Retrieved from http:// arxiv.org/ftp/arxiv/papers/1005/1005.4906.pdf. t Ortega, J. L. (2015). Relationship between altmetric and bibliometric indicators across academic social sites: The case of CSIC’s members. Journal of Infor metrics, 9, 39–49. DOI: https://doi.org/10.1016/j.joi.2014.11.004 Piwowar, H. (2013). Altmetrics: Value all research products. Nature, 493, 159. DOI: https://doi.org/10.1038/493159a Priem, J., Piwowar, H. References A., & Hemminger, B. M. (2012). Altmetrics in the wild: Using social media to explore scholarly impact. Retrieved from http://arxiv. org/html/1203.4745. g Rousseau, R. (2002). Journal evaluation: Technical and practical issues. Library Trends, 50, 418–439. Seglen, P. (1998). Citation rates and journal impact factors are not suitable for evaluation of research. Acta Orthopaedica Scandinavica, 69, 224–229. Stenius, K. (2003). Journal impact factor - mittari joka vahvistaa tutkimusmaail man hierarkiaa [The journal impact factor—an indicator that strengthens the hierarchy of the research world]. Tieteessä Tapahtuu, 7, 35–39. Retrieved from http://www.tieteessatapahtuu.fi/037/stenius.pdf.i i Ward, J., Bejarano, W., & Dudás, A. (2015). Scholarly social media profiles and libraries: A review. Liber Quarterly, 24, 174–204. DOI: https://doi. org/10.18352/lq.9958/ Weller, K. (2015). Social media and altmetrics: An overview of current alternative approaches to measuring scholarly impact. In I. M. Welpe, J. Wollersheim, S. Ringelhan, & M. Osterloh (Eds.), Incentives and performance (pp. 261–276). Switzerland: Springer International Publishing. 64 Publishing Addiction Science 64 West, R., & McIlwaine, A. (2002). What do citation counts count for in the field of addiction? An empirical evaluation of citation counts and their link with peer ratings of quality. Addiction, 97(5), 501–504. g y Xia, J., Wright, J., & Adams, C. E. (2008). Five large Chinese biomedical bib liographic databases: accessibility and coverage. Health Info Libr J., 25, 55–61. DOI: https://doi.org/10.1111/j.1471-1842.2007.00734.x [PubMed] [Cross Ref] Appendix A: An Inventory of Abstracting and Indexing Services and Databases Relevant to the Scientific Literature on Addiction Subscription databases are available through the author’s institutional sub scription. Please contact your local library for information on how to access them. Chinese Databases According to the China National Knowledge Infrastructure, large numbers of publications may be missed when not searching Chinese databases. These data bases index 2,500 journals largely not familiar to MEDLINE users. Free access, search features, record selection, ease of downloading, and cost of subscription varies considerably between databases. At a minimum, Chinese biomedical databases should be searched when performing systematic reviews. (See Xia et al. (2008); Cohen et al. (2015)) Current Contents (subscription) Current Contents, a current awareness database developed at the Institute for Scientific Information, now part of Thomson Reuters, provides access to bib liographic research information from articles, editorials, meeting abstracts, and other sources from more than 8,000 scholarly journals, with separate edi tions for clinical medicine, life sciences, and social and behavioral sciences. Internet access is provided through Current Contents Connect. Updated daily, it provides access to complete tables of contents, abstracts, and bibliographic information from the most recently published journals and books. CC Connect offers cover-to-cover indexing that provides access to all the valuable informa tion available in journals — not just articles. Directory of Open Access Journals (DOAJ) (Open Access): https://doaj.org/ DOAJ is an online directory that indexes and provides access to high quality, open-access, peer-reviewed journals. Launched at Lund University, Sweden, in 2003, DOAJ is a membership organization, with membership intended to prove a commitment to quality, peer-reviewed open access. The aim of the DOAJ is to promote increased usage and impact of open-access scientific and scholarly journals by increasing their visibility and ease of use. Includ ing more than 10,000 journals from 134 countries, it covers over 2 million articles, of which more than 6,000 are searchable at the article level. Subjects listed include broad areas such as medicine, health sciences, psychiatry, pub lic health, and social sciences, indexing them with top-level Library of Con gress Subject categories only. Keyword search is available for the full text of the article, with high recall and low precision. The directory claims to be com prehensive and cover all open-access academic journals that use an appropri ate quality-control system. DOAJ is independent and is not connected to, or owned by, any other organization or business. To be included, a journal must exercise peer review with an editor and an editorial board or editorial review carried out by at least two reviewers. The DOAJ Seal of Approval for Open Access Journals is a mark of certification awarded by DOAJ to journals that achieve a high level of openness, adhere to best practices, and have high pub lishing standards. CSA Sociological Abstracts (Subscription) CSA Sociological Abstracts provides an index and abstracts of journal articles from the international literature in sociology and related disciplines in the social and behavioral sciences. Major subject areas include evaluation research, family and social welfare, health law, substance abuse, and addiction. Its data base is drawn from more than 2,000 serials publications, including a variety of sources such as journal articles, conference papers, books, dissertations, and conference papers, plus citations to important book reviews related to the social sciences. A backfile that begins in 1952 adds to the coverage with records published by the then print version of Sociological Abstracts. Because 40% of the provided content is published outside of North America, the database also provides a global perspective. The database is updated monthly with approxi mately 30,000 records added per year. How to Choose a Journal 65 EMBASE (Elsevier) (Subscription) Embase is a comprehensive index of the world’s literature on human medi cine and related disciplines. Each record is classified and indexed using terms and synonyms that assist the process of searching for specific subjects. Subject coverage includes AIDS, drug dependence, psychiatry, and public health. EMBASE provides access to articles from more than 2,900 journals from 110 countries. Fetal Alcohol Spectrum Disorders (FASD) Database: http://fasdcenter. samhsa.gov/search/basic/index.aspx Fetal Alcohol Spectrum Disorders (FASD) Database: http://fasdcenter. samhsa.gov/search/basic/index.aspx The FASD Database collects information on thousands of FASD-related resources, including audiotapes, books, CD-ROMs, newsletter, magazine, newspaper, and journal articles, pamphlets and booklets, posters, videos, slide shows, and Web-based materials. It includes a quick-search function activated by typing in a keyword and selecting a media type and an advanced search option for more specific searches. DrugWise: http://www.drugwise.org.uk/ Launched in 2016 as a continuation of DrugScope, DrugWise is a new drug information service located in the United Kingdom. The full range Publishing Addiction Science 66 of DrugScope archival materials is complemented with updates and new reports on drugs, alcohol, and tobacco (including e-cigarettes). In addi tion to drug information, such as the DrugSearch Encyclopedia and Drug Wise reports, a new function, called I-Know, serves as an international knowledge hub. I-Know brings together international and internationally- relevant national reports and reviews covering the range of substances. The plan is to build up a library of information, policy and practice material over time. International Alcohol Information Database (IAID): www.icap.org Launched in 2014, the International Alcohol Information Database is a publicly accessible bibliographic resource created to provide an easily searchable data base of published research on alcohol. It covers multiple disciplines, includ ing biomedical, sociobehavioral, prevention, treatment, policy, and regulatory research fields. Citations are compiled from more than 3,550 peer-reviewed journals from around the world, and the included research is available in 30 languages and from more than 150 countries. The continually updated database has approximately 50,000 citations from peer-reviewed research jour nals dating back to 2003, accessible through simple or advanced search options. The advanced search allows users to refine their results through title, author, journal, or publication date, as well as through an extensive list of keywords, the countries covered in the research, or the original publication language. There is no cost to search, register, or access the database’s content. The database is sup ported by funding from the International Alliance for Responsible Drinking, a consortium of beer, wine, and spirits producers. Google Scholar (Open Access): scholar.google.com Google Scholar provides access to the scholarly literature across many disci plines and sources, including articles, theses, books, abstracts, and court opin ions. Crawling millions of pages of the public and invisible web, it indexes full text of the scholarly literature, gathering information from academic publish ers, professional societies, online repositories, universities, and other websites. Individual authors can be listed in Google Scholar by simply uploading their articles to a website. The main advantage of Google Scholar is the convenience of searching all scholarly publications on one platform. It allows the user to find related articles. It is currently the fastest way to locate a known item and to retrieve the full text(for either open-access items or titles that one’s library subscribes to). Authors can create a publicly accessible author profile in Google Scholar Citation to showcase their work and track citations to their publica tions (scholar.google.com/citations). The main disadvantage of this service derives from the lack of a controlled vocabulary (i.e., instead of index terms describing the articles, as it is customary in the proprietary databases such as MEDLINE or PsycINFO, the search is performed in the full text of the publica tion, resulting in many irrelevant hits.) Google Scholar provides access to the scholarly literature across many disci plines and sources, including articles, theses, books, abstracts, and court opin ions. Crawling millions of pages of the public and invisible web, it indexes full text of the scholarly literature, gathering information from academic publish ers, professional societies, online repositories, universities, and other websites. International Alcohol Information Database (IAID): www.icap.org CORK Database (Open Access): www.projectcork.org Project Cork was founded at Dartmouth Medical School in 1977 through a grant from Operation Cork. The project also resulted in CORK, a searchable bibliographic database of the substance abuse literature and the emerging area of behavioral addictions. Its goal is to provide immediate access to authori tative information and materials on substance abuse and to assist health and human service professionals, educators and their students as well as those in public policy. The CORK database contains 120,500 items including journal articles, books, book chapters, conference proceedings, and special reports on substance abuse, indexed by more than 400 terms. The database was updated quarterly until 2015. How to Choose a Journal 67 Google Scholar (Open Access): scholar.google.com MEDLINE (Subscription), PubMed, PubMed Central MEDLINE (Medical Literature, Analysis, and Retrieval System Online) is the U.S. National Library of Medicine’s journal citation database. MEDLINE Publishing Addiction Science 68 is widely known as the major source for bibliographic and abstract coverage of biomedical literature, covering the topics of medicine, nursing, dentistry, as well as other areas, such as allied health, biological and physical sciences, humanities, and information science as they relate to medicine and health care, communication disorders, population biology, and reproductive biology. Started in the 1960s, MEDLINE now provides more than 22 million references and includes citations from more than 5,600 scholarly journals published in the United States and other countries. The Literature Selection Technical Review Committee reviews and recommends journals for MEDLINE considering the quality of the scientific content, including originality and the importance of the content for the MEDLINE global audience, using the guidelines found on the National Library of Medicine Fact Sheet MEDLINE Journal Selection (http:// www.nlm.nih.gov/pubs/factsheets/jsel.html). Although MEDLINE is restricted to institutional subscribers, such as libraries, the content of the database can be searched free of charge via PubMed (http://www.ncbi.nlm.nih.gov/pubmed) and PubMed Central (http://www.ncbi.nlm.nih.gov/pmc). MEDLINE is the largest subset of PubMed, with the added value of using the National Library of Medicine controlled vocabulary—Medical Subject Headings (MeSH)—to index the citations in MEDLINE. MEDLINE is updated daily. p y PubMed (http://www.ncbi.nlm.nih.gov/pubmed) has been available since 1996. It offers more than 25 million references, including the MEDLINE data base and additional types of records, such as in-process citations, citations to articles that are out of scope, epub ahead-of-print citations, citations to author manuscripts of articles published by National Institutes of Health–funded researchers, and citations for the majority of books available on the National Center for Biotechnology Information Bookshelf. Both MEDLINE and other PubMed records may include links to full-text articles, depending on open- access and subscription-based availability. PubMed Central (http://www.ncbi.nlm.nih.gov/pmc) was launched in 2000 as a free repository of full-text biomedical and life-sciences journal articles. It serves as a collection for scholarly literature deposited by either participating publish ers or authors who submitted their manuscripts in compliance with the National Institutes of Health Public Access Policy and similar policies. Some PubMed Cen tral journals are also indexed in MEDLINE. There are reciprocal links between the full text in PubMed Central and corresponding citations in PubMed. PsycINFO (Subscription) PsycINFO is the electronic version of Psychological Abstracts, which was pub lished by the American Psychological Association monthly for 80 years and ceased in 2006. With nearly 4 million bibliographic records focusing on the scholarly literature in the behavioral sciences and mental health, the PsycINFO database provides a unique resource for locating scholarly literature for addiction How to Choose a Journal 69 69 researchers. It contains bibliographic records and abstracts of English-language articles from journals originating in more than 50 countries, all professionally indexed by American Psychological Association experts. PsycINFO is avail able through library subscriptions and to individual members of the American Psychological Association. Nearly 2,500 journal titles (99% of which are peer reviewed) are covered in the database. Articles are selected based on their rel evance in psychology and related fields, such as psychiatry, management, busi ness, education, social science, neuroscience, law, medicine, and social work. The database also covers books and book chapters, 3% and 8% of PsycINFO records, respectively. Its global perspective is proven by indexing publications from more than 50 countries, journals from 29 languages, and non–English-language titles in Roman alphabets since 1978. Easy discoverability and high precision during literature searches are ensured by 22 major categories and 135 subcategories in the classification system and by the controlled vocabulary describing the articles, with more than 8,400 terms and cross-references. Online access to a Thesaurus of Psychological Index Terms is included. PsycINFO is updated weekly. Scopus (Elsevier) (Subscription) Scopus is the largest abstract and citation database of peer-reviewed literature, covering scientific journals, books, and conference proceedings. It provides a comprehensive overview of the world’s research output in the fields of science, technology, medicine, social sciences, arts, and the humanities. Scopus features tools to track, analyze, and visualize research. It is oriented toward researchers, teachers, and students. Scopus claims that it has twice as many titles and more than 50% more publishers listed than any other abstracting and indexing data base. It contains more than 50 million records with coverage strongest in the physical sciences (7,200+ titles) and health sciences (6,800+ titles), followed by the life sciences (4,300+ titles), and finally the social sciences and humanities (5,300+ titles). More than 25,000 titles (including open-access journals) from around the world are covered in Scopus. Quick searches by document, author, or affiliation are available, but there is also an advanced search option. Scopus offers several methods of analysis, such as the Journal Analyzer, which com pares the citation metrics of different journals using SCimago Journal Rank and other metrics. Authors can benefit from the citation overview function, which includes the h-index, computed from the author’s publications listed in Scopus. It is updated daily. Web of Science (Thomson Reuters) (Subscription) Thomson Reuters provides paid subscribers with comprehensive coverage of the world’s most important journals. Web of Science covers more than 12,000 0 Publishing Addiction Science 70 international and regional journals in the natural sciences, social sciences, the arts, and humanities. Three citation indexes contain the references cited by the authors of the articles: Arts & Humanities Citation Index (from 1975 to the present), the Science Citation Index Expanded (from 1900 to the present), and the Social Sciences Citation Index (from 1900 to the present). The data base provides bibliographic records, searchable abstracts, and cited references. Many factors are taken into account when evaluating journals for coverage in Web of Science, ranging from the qualitative to the quantitative. The journal’s basic publishing standards, its editorial content, the international diversity of its authorship, and the citation data associated with it are all considered. Thomson Reuters also determines if an electronic journal follows international editorial conventions, which are intended to optimize retrievability of source articles. These conventions include informative journal titles, fully descriptive article titles, and author abstracts, complete bibliographic information for all cited references, and full address information for every author. Thomson Reu ters editors look for international diversity among the journal’s contributing authors, editors, and editorial advisory board members. For more information, see: http://wokinfo.com/essays/journal-selection-process/. Coverage is strong est in the sciences (8,000+ journals), followed by social sciences (almost 3,000 journals), and arts and humanities (approximately 1,600 journals). For impact factor information about specific journals, users are directed to the index Jour nal Citation Reports. How to cite this book chapter: Stenius, K, Kerr-Corrêa, F, Obot, I, Furtado, E F, Lima, M C P and Babor, T F. 2017. Beyond the Anglo-American World: Advice for Researchers from Developing and Non–English-Speaking Countries. In: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P. (eds.) Publishing Addiction Science: A Guide for the Perplexed, Pp. 71–88. London: Ubiquity Press. DOI: https://doi.org/10.5334/bbd.d. License: CC-BY 4.0. Kerstin Stenius, Florence Kerr-Corrêa, Isidore Obot, Erikson F. Furtado, Maria Cristina Pereira Lima and Thomas F. Babor Kerstin Stenius, Florence Kerr-Corrêa, Isidore Obot, Erikson F. Furtado, Maria Cristina Pereira Lima and Thomas F. Babor Kerstin Stenius, Florence Kerr-Corrêa, Isidore Obot, Erikson F. Furtado, Maria Cristina Pereira Lima and Thomas F. Babor For more Information The Substance Abuse Librarians and Information Specialists website offers a more comprehensive collection of abstracting and indexing services and other related databases (salis.org/resources). Maintained by Barbara Weiner of Hazelden–Betty Ford, the lists are monitored by the group and updated fre quently both for U.S. and international services. United States: http://www.hazelden.org/web/public/usdatabaselibrary.page International: http://www.hazelden.org/web/public/lib_cdandaddictions. page United States: http://www.hazelden.org/web/public/usdatabaselibrary.page International: http://www.hazelden.org/web/public/lib_cdandaddictions. United States: http://www.hazelden.org/web/public/usdatabaselibrary.page I t ti l htt // h ld / b/ bli /lib d d ddi ti p g p y p g International: http://www.hazelden.org/web/public/lib_cdandaddictions. page CHAPTER 4 Introduction Today, more than 81% of the world’s population lives in nations categorized as low- and middle-income countries (LMICs) (World Bank, 2014). However, there are still few addiction journals published outside Europe, the United States, and Australia (see Table 3.2, Chapter 3), despite the growing need for specialized knowledge in many countries where addiction problems are prevalent. Presently, between 5% and 9% of the world’s population grows up with Eng lish as their first language. The dominance of English within scientific com munication is, however, overwhelming. It is estimated that 80% of the world’s scientific articles are published in English-language journals (Montgomery, 2004; Van Weijen, 2012). The dominance is particularly strong in the physi cal and life sciences, whereas local languages may still have important roles in social sciences, law, and humanities. In the addiction field, we estimate that at least three fourths of the known addiction journals communicate in English.h This chapter deals with the challenges encountered by addiction scientists who work in countries with few resources as well as those whose first language is not English. The aims of the chapter are to discuss (a) the practical and 72 Publishing Addiction Science 72 professional issues that are faced by these scientists, (b) how authors who come from these countries can improve their chances of publishing in English-lan guage journals, (c) the possibilities for authors to publish in both English and an additional language so they can communicate to different audiences, and (d) how to decide whether an article serves the public best by being published in the author’s mother tongue and/or a local or regional journal. The Skewed Distribution of Scholarly Communications In a subsequent WHO mapping of research capacity for mental health in 114 LMICs (WHO, 2007), 66 countries had produced fewer than five articles between 1992 and 2003 that were indexed in MEDLINE or PsycINFO. On the other hand, a number of countries—Argentina, Brazil, China, India, the Repub lic of Korea, and South Africa—at this time all had substantive and increasing scientific production. More than half of the journals that published most of the indexed mental health research articles from LMICs were also edited in these countries. Most of the problems in research production and indexing could be applied to the addiction field. Many countries with few resources are striving to develop scientific research capabilities in general. Efforts to strengthen addic tion research do not always have sufficient political support. Politicians and decision makers in these countries—as in many others—are not necessarily interested in whether certain alcohol or other drug treatment and prevention measures are evidence based or not. Public support may be more important. Also, research results can be difficult to translate into policy. For these reasons, research and scientific publishing on addiction-specific questions may not be high on the list of political priorities. Turci et al. (2010) analyzed for instance the trends of epidemiological production in Brazil from 2001 to 2006. The authors observed that the main themes were public health nutrition, maternal and infant health, and infectious diseases; in short, there was a lack of epide miological research on alcohol in Brazil. Career scientists and professionally trained clinicians are needed, but except in the instance of government-sponsored university programs, there is little support for clinical, epidemiological, and policy research. Few LMIC coun tries have specialist addiction societies in which locally relevant and topical problems can be discussed and solutions developed. Training opportunities are lacking. In some countries, the number of master’s and doctoral students has grown, as have specialization courses at the universities (see Chapter 3). But many addiction professionals entering the work force are clinicians in private practice who may do academic work voluntarily or for a small salary. Under the circumstances, the development of addiction research will be slow.t Further, communication with researchers in other countries is often restricted by lack of resources. Many libraries have run out of journal subscription funds, and addiction journals are seldom a priority. The Skewed Distribution of Scholarly Communications There is a fundamental imbalance between available resources and resource needs in the addiction field. On the one hand, there is as noted above a dispro portionate concentration of addiction science and addiction publishing in the richer and English-speaking areas (North America, Europe, and Australia). On the other hand, the majority of the world’s population and an increasing share of the addiction problems can be found in LMICs and countries where the native language is not English (Room et al., 2002). For example, Russia, Mexico, and many South American countries have high rates of alcohol-related disease and disability (World Health Organization [WHO], 2011), but few addiction journals can be found in these countries. This imbalance between prevalence of problems on the one hand and scientific and publishing possibilities on the other presents a serious challenge to those interested in the most effective and efficient use of resources in the interests of public health on an international level. In November 2003, the WHO arranged a meeting called “Mental Health Research in Developing Countries: Role of Scientific Journals.” The joint state ment by participating journal editors and the WHO (2004) describes the bar riers to scientific publishing experienced by researchers from LMICs in the mental health research field.hi i The document states that the accumulation of scientific knowledge is depend ent on free and accessible communication across the world. The promotion of good research increasingly requires not only the ability to access research from other parts of the world, which in many LMICs still is a problem, but also the opportunity to communicate research results. Researchers from LMICs often have difficulties in publishing their findings in scientific journals. The reasons include limited access to information, lack of advice on research design and statistics, and the difficulty of writing in a foreign language as well as material, financial, policy, and infrastructural constraints. Limited global appreciation of the research needs of LMICs and the comparative anonymity of their research ers may constitute additional barriers. According to the WHO (2004) report, many researchers from LMICs “are daunted by the seemingly insurmountable Beyond the Anglo-American World 73 Beyond the Anglo-American World 73 chasm between their research effort and its publication in international journals” (p. 226). The Skewed Distribution of Scholarly Communications In some countries, influential research-funding agencies are now supporting programs that give most uni versities free access to online periodicals. These programs have improved the availability of international research. For example, the HINARI project was launched in 2002 by the WHO in collaboration with scientific publishers to make health research available in LMICs. Today it covers 13,000 journals and 30,000 e-books in many different languages (see www.who.int/hinari). 74 Publishing Addiction Science The formal communication of locally relevant addiction research is encoun tering other challenges. Local journals are necessary to deal with sociocultural peculiarities and the priorities of different societies. Presently there is a strong movement in several countries to publish good-quality articles, preferably in English. Because competition in the scientific field is intensifying, publication in indexed journals is a priority for researchers who need scientific credit for their work. Alcohol and other drug science is, however, a young and relatively small field. Local and non–English-language addiction journals have difficul ties meeting the criteria for inclusion in U.S. and international indexing sys tems, such as Web of Science and MEDLINE. A sign of how problematic the situation still can be is that no addiction jour nal from the Latin American region has been able to establish itself. As a con sequence, many addiction scientists publish in indexed public health or mental health journals when writing for the local or regional audience in this part of the world. Only a small number of these articles are published in English. Pub lishing in these journals is, of course, in itself not a bad thing. But for the devel opment of the addiction field in a particular country or region, a specialized journal can play an important role. In India, addiction researchers have since 2010 had the possibility to publish addiction research in the Indian Journal of Psychiatry (Murthy et al., 2010), but also the Journal of Mental Health and Human Behavior has articles on addiction. Researchers in African countries have the option of publishing in the African Journal of Drug and Alcohol Stud ies. In relation to the population and problems, the local publishing availability is anyhow extremely restricted. In many other countries the only option if you want to publish in an indexed addiction journal is to seek for one from outside your own country. Marginalisation of LMIC Research in the International Discourse In academia, faculty are often evaluated by the number of their publications and the impact of the journals in which their articles are published. Publishing in high-impact journals has become the principal aim for many because grants, positions, and funding go to scientists, faculty, and departments that succeed in this respect (e.g., see Linardi et al., 1996). When research funds are in short supply, resources are concentrated in the hands of a few investigators, and the dominance of impact factors contributes to this concentration.h Thomson Reuters, which publishes the most commonly used impact factors, does not provide complete coverage of the world’s scientific journals. English- language journals and especially U.S. journals are better represented. This means that, in general, research conducted in LMICs and reported in languages other than English is under-represented. However, the situation is improving in several regions. SciELO is a bibliographic database and electronic library focus ing on the developing world. In 2014, it covered more than 1,000 selected jour nals from South America, Spain, Portugal, the Caribbean, and South Africa. The topics include health sciences and social sciences, and every article can be downloaded free. In 2013 SciELO reached an agreement with Thomson Reuters Web of Knowledge that will increase the visibility of Latin American and Por tuguese language research. This development was possibly facilitated by strong efforts to increase the English language publication of Brazilian research. In Brazil, English language scientific articles now are more common than Portu guese, and there are systematic attempts to improve the quality of the published texts (Science for Brazil, 2013). The African Journals Online (AJOL), a data base with nearly 500 journals, has been launched to promote access to African research. About 160 of the journals are devoted to health fields, but only one addiction journal (see above) is listed among them. The European Reference Index for the Humanities and Social Sciences (ERIH PLUS) (which expanded in 2014 to include both humanities and social sciences) is established with the aim to “enhance global visibility of high quality research in the humanities pub lished in academic journals in various European languages all over Europe” (NSD, 2014). In Iran, several electronic databases for scientific publishing were established in 2004. The Skewed Distribution of Scholarly Communications However important national or local journals are, it sometimes can be hard for a researcher from a country with few resources to rely on them. These jour nals often have limited funds, may be published irregularly, or may have long delays between submission and publication of an article. Not infrequently, these journals will find themselves in a vicious circle: They are not regarded as prestigious enough, which means that they will not get enough good articles, which in turn means that they will not get enough resources and not enough good articles. Even if there are still relatively few addiction specialty journals outside of North America and Europe, and even fewer that are well indexed, there are some signs that the inequality in access to scientific publication, and in journals’ relative status, may be leveling out. For instance, the indexing of non–English-language journals, including addiction journals, with English- language abstracts in Scopus has increased. Open-access developments and the possibilities to have online-only publications have improved the pos sibilities to publish without printing costs and also to add non–English- language versions of English-language articles as online-only supporting material (Meneghini & Packer, 2007). This is not yet an established practice Beyond the Anglo-American World 75 Beyond the Anglo-American World 75 in addiction journals but may be a model for the future. World Psychiatry, the journal of the World Psychiatric Association, is for instance now published not only in English but also in Arabic, Spanish, Chinese, Russian, French, and Turkish, with the aim to improve dissemination of research to clinical psychiatrists in different parts of the world (Maj, 2010). Marginalisation of LMIC Research in the International Discourse Amin-Esmaili and colleagues (2009) showed that the international databases have a low coverage of Iranian addiction research but argue that, by combining the bilingual (Iranian and English) Iranian databases with big international ones such as MEDLINE, PsycINFO, and Embase, it was Publishing Addiction Science 76 possible to cover as much as 80% of the Iranian addiction research publica tions. Similar efforts are seen in Turkey.h f The problems for LMIC researchers who seek to publish internationally may be compounded by structural factors associated with the management of the English-language scientific journals. Around 2000, a survey of the editorial and advisory boards of leading international journals in the field of mental health (e.g., Archives of General Psychiatry, American Journal of Psychiatry, Schizophre nia Bulletin, British Journal of Psychiatry, Adolescent Psychiatry) found only 4 representatives from LMICs among 530 board members (Saxena et al., 2003). The absence of LMIC representation on the editorial boards of the major jour nals may explain why authors from developing countries often feel that their articles do not receive sympathetic treatment. Thus, research from LMICs is likely to be regarded as less relevant in the international discourse. This is sup ported by a study of articles published in Addiction (West & McIlwaine, 2002), which found that articles from LMICs were cited significantly less often than those ranked by independent peer reviewers to be of the same quality as those from the developed world. Other studies have shown that an increase in the number of articles published from LMICs is not paralleled by a similar increase in citation of these articles (Holmgren & Schnitzer, 2004; Volpato & Freitas, 2003). Additional factors that may account for the relatively limited number of publications from these countries include poor research methods, inadequate sample sizes, less-sophisticated statistical analyses, lack of national or regional journals, and limited English-language competence ( see for instance Gosden, 1992) The Language and Culture Trap English is the lingua franca of scientific research today and will be in the fore seeable future. However, as Montgomery (2004) points out, to call it “the uni versal language of science” is ahistorical and possibly inattentive to the complex linguistic developments taking place in the world. In the future, more and more people will be bilingual, and languages other than English will grow in impor tance. For the present, however, the English language has a dominant position in addiction science.hi The scientific world today is dominated by a small group of rich countries. The United States is in the lead, followed by the United Kingdom, Canada, Australia, and the European nations, which are oriented toward a similar sci entific tradition and in which English-language training is well developed. The disproportionate influence of research from these countries extends to basic science, prevention, epidemiology, and treatment research. American research ers tend to cite American researchers (see further discussion in Chapter 7 and in Babor, 1993). The same applies to other countries, but with the dominance Beyond the Anglo-American World 77 Beyond the Anglo-American World 77 of journals from the United States and other English-language countries (and English-oriented countries such as Sweden), there is a citation bias across the research field as a whole. Research that is performed in the United States may represent a priori for many Anglo-American readers and some uncritical read ers as well—that is, such results may appear to represent a more universal truth than results from a study conducted in a country such as India. Researchers in some Western nations (e.g., the Nordic countries) have adapted to the domi nant research paradigms and seem to manage quite well, in terms of citation measurement (Ingwersen, 2002). The under-representation of non–English- speaking nations in indexed journals and in cited research extends to several developed countries, such as Spain, Germany, and France (Maisonneuve et al., 2003), suggesting that general linguistic and cultural influences may be at work. The present dominance of a few countries’ science on an international level may imply a serious bias in the selection of research topics, questions asked, meth ods used, and types of research conducted, and a relative neglect of problems in the developing world. There are other problems inherent in this hierarchy within addiction research. Addiction science has at least two subdivisions— basic and applied research. The Language and Culture Trap The former is more or less universal in its nature, and scientific knowledge from basic research can be applied everywhere in the world. The latter is contextual. Public health research, for instance, belongs to this category. Today, public health research in LMICs suffers from a double dis advantage: (a) the difficulty in getting published and quoted in the influential journals and (b) unfair competition at the national and international level with the much better funded neurobiological research (see Midanik, 2004). In short, this means that the world literature on substance misuse is rarely determined by the research priorities of the developing countries. Commerce plays a role as well and may not favor the public health interests of the poorer parts of the world. Randomized clinical trials of new medicines, with potential markets in richer countries, have a greater probability of being published than brief interventions to treat alcohol and other drug users. Not all policymakers realize that alcohol and tobacco are more important issues than heroin and cocaine in the developing countries (Ezzati et al., 2002). Again, we can see signs of an improvement in the situation. Warner et al. (2014) analyzed published contributions in the international journal Tobacco Control between 1992 and 2011. The proportion of original-article authors from LMICs during 2007–2011 compared with all the earlier years increased from 7.2% to 22.7% and LMIC lead authors increased from 4.0% to 13.7%. There was also a significant increase in articles covering LMIC issues. In another study (Zyoud et al., 2014), a considerable increase of tobacco articles with authors from Middle Eastern Arab countries was reported between 2003 and 2012. For researchers from LMICs, some of the problems in getting published come from not being familiar with the codes of international scientific com munication. In the above-mentioned survey of physics, chemistry, and biology journals (Gosden, 1992), the editors summarized the problems encountered by Publishing Addiction Science 78 researchers who were not native English speakers. The most often mentioned problem was that research results and discussion were not well written: that is, an inability to communicate the importance and relevance of the research. Another important problem was that authors did not know the written and unwritten “rules of the publishing game” (pp. 132–133). For instance, they failed to cite sufficient references to earlier research and were not familiar with the argumentation style or scientific level of the journal (Gosden, 1992). The Language and Culture Trap Writ ing a good scientific article for an international audience demands not only technical skill, such as being able to carefully follow the instructions to authors, but also an acquired competence in social communication. The best way to gain this is by reading some of the journals mentioned in Chapter 3 and get ting feedback on your writing from more experienced researchers. This is not always easy in an LMIC. What Can an Author Do? In this section we turn to some practical suggestions that may help to correct the imbalance, level the playing field, and improve the diversity of addiction science. What Do We Know about Addiction Journals’ Language and Cultural Policies? Unfortunately, we have almost no research to show how addiction journals in general deal with articles from LMICs and only a small, and partly old, amount of information about their language policies. In two surveys con ducted by the International Society of Addiction Journal Editors (ISAJE), Edwards and Savva (2002a, 2002b) mapped the language policies of 14 English- language journals and nine non–English-language journals. Half the editors of the English-language journals who responded had not mastered any lan guage besides English. This is a handicap in a multilingual scientific world. Based on this ISAJE questionnaire, it seems that the English-language addic tion journals outside the United States have greater international represen tation on their editorial boards. The composition of an editorial board can give an indication of the internationalism of a journal. We have no exact knowledge of how the LMICs are represented on the editorial boards, but representation is likely to be low. Among the responding English-language journals in the 2002 survey, the share of research articles from non–English-language countries varied from 0% to 57% at this point of time. In this sample, about one third of the journals had a policy to give special support to authors with mother tongues other than English. Only three of the 14 journals declared that they could not give any language-editing support. Of the non–English-language journals responding to the questionnaire, the majority published only in the language of the country of publication. Several published articles that had already been published in English. Several journals were regional or had international ambitions. All the editors knew English, and several were competent in more than one foreign language. All journals had English summaries. The editorial boards often had representatives from other countries. Beyond the Anglo-American World 79 Beyond the Anglo-American World 79 Beyond the Anglo-American World 79 In general, because ISAJE is an international organization with particular sensitivity to the language issue, it is possible that addiction journal editors are more conscious than editors in general of the importance of supporting research from non–English-language cultures. Crossing the Cultural Border to the English-language Publications As noted above, it may be particularly difficult for authors from LMICs and non–English-speaking countries to get an article accepted in an English- language journal. It is thus especially important for LMIC authors to show that they have mastered the rules of the game: to carefully follow the instructions to authors, checking that the structure, the language, and the presentation of the study and its results are clear and logical and that the references are correct. If the formalities are not followed, even a study containing strong and origi nal findings might immediately be turned down. Cultural bias may put higher demands on research from countries where resources are few. The famous Chilean pharmacologist Jorge Mardones concluded in an interview (Edwards, 1991, p. 392) after a long career: I do not know why there is a generalized attitude of doubt concerning results reported in papers coming from Latin American laboratories. In order to overcome this situation, we need to be extremely certain about the accuracy and high significance of our results, before submitting a paper for publication. I feel that this is an advantage, because the worst thing a scientist can do is to pollute the scientific environment with data of poor value. Before submitting a manuscript, an author would be wise to find a mentor or an experienced investigator who could read through the article and give advice on the presentation of the results. This may however be difficult in many countries where the addiction research milieu is very small. ISAJE is able in some cases to provide support to unexperienced authors through its mentoring program, in which experienced editors and researchers will help authors to produce pub lishable manuscripts (see ISAJE’s website, www.isaje.net). Collaborative studies should be encouraged. A survey of Nigerian articles published in a psychology journal showed that more than 75% of the articles Publishing Addiction Science 80 were published by single authors, a figure that was much higher than that found in American journals at the time (I. Obot, personal communication, 2004). One suggestion is to try to work in a team that includes people with expertise in different areas, such as statistics and social science. This may help to improve the quality of the study and enhance its appeal to a greater number of readers. Crossing the Cultural Border to the English-language Publications Another possibility is to work within a joint project with researchers from non- LMICs or within a large, international network. This is in most cases only pos sible if you have already published in an international English-language journal or work with other researchers who have international contacts and reputa tion. International conferences can provide possibilities for networking, but to attend them you need financial resources. In Brazil, it has been possible to document publishing success with this kind of cooperation and international exchange (Barata, 2010). Technical requirements are relatively easy to identify and follow. A more dif ficult challenge is that conventions about how to write an article differ among countries. Burrough-Boenisch (2013), in a text on editing problems, gives some examples that show how culturally embedded our scientific writing endeavors are. For an Anglo-American, the author states, the German tradition of writ ing may seem both pretentious and less well organized. The traditional writ ing style of some Asian cultures, such as China, Japan, Korea, and Thailand, may give an incoherent impression. Further, when French scientists transfer the French convention of reporting science in the present tense to their English writing, they seem to be stating general truths, rather than describing their own procedures and findings. i In most cases it is not possible for an author to communicate with the readers of a journal if the author cannot talk to them in the “scientific dialect” of that particular publication. (This is of course also true when you choose a publica tion channel within one linguistic area.) This requires that the author is fairly well acquainted with the specific journal and knows what types of articles are published and in what format. Some English-language journals are more sympathetic than others to articles from other countries and cultures. Crossing the Language Border Montgomery (2004) points out that the linguistic future of the world will be one of diversity, bilingualism, or even multilingualism. An important goal in this world will therefore be “to increase tolerance towards variation in scientific English—to avoid the imperial attitude that one standard must be obeyed” (p. 1335). Until this tolerance is developed, however, authors of scientific arti cles have to take the language issue seriously.t As noted above, the way in which authors present their results is often crucial to how the editor and reviewers will view the research report. The importance of good English-language usage cannot be over-emphasized. The presentation of the study and the results is particularly important when the topic or setting may seem new and exotic to the editor and reviewers. It is not just a matter of using the right terminology. Many English-speaking editors and reviewers (similar to many French-, German-, or Swedish-speaking editors) will have a rather strict idea of what constitutes good language. g g g Should one do a professional language check before sending in an article? Although it is expensive and time consuming, the answer is YES. If research ers are certain that they have a good case, a more experienced person has read the article and found it good, and the authors want to publish it in a journal with no resources to help with language editing, it will definitely increase the chances of acceptance. There is also the risk that if the article is considered to be a “borderline case,” it will be rejected if there are language problems. However, in rare cases, if the authors know that the journal and the editor have a policy of accepting articles by non–English-language authors and the journal has the resources to do a language check, it may not be necessary to have perfect Eng lish at the time of the first submission. But this is a case where contacting the editor beforehand is definitely worthwhile. i A few words about editing services: in most countries, there are English language manuscript editing services available for academic research papers written by non-native English speakers. These manuscript editors are gener ally native speakers of English with substantial experience in editing scholarly articles, and many of them are accomplished authors in the field. Crossing the Cultural Border to the English-language Publications This is possible to find out by doing the following: • looking at the journal’s mission statement to see if it has any policy regard ing articles submitted from different countries or cultures; f • checking whether the journal has previously published articles by non– English-language authors; • checking to what extent the editorial board is international, which may imply a greater understanding of cultural diversity and a more multicul tural peer-reviewer pool; andi • contacting the editor to find out if the journal may be interested in your work—pointing out its particular importance and the possible mitigating circumstances of being from an LMIC or non–English-speaking country. Beyond the Anglo-American World 81 Killing Two Birds with One Stone: Dual-Language Publication Where the topic of the article is such that it would be important to publish both at the national level and in an international journal, the author could consider trying to publish the same text in more than one language. In fact, if authors feel that their results should be considered in the development of local policy, publication of the results in an international journal may very well give the findings more prestige among the politicians of their country. Some addiction journals will agree to publish an article that has already been published in another language or to simultaneously publish the article in sev eral languages. These practices do not violate ethical codes regarding duplicate publication (see Chapter 14) as long as the editors agree and the simultaneous publication is mentioned along with the source of the original. If there is an interest in presenting the article to several audiences, the general rule for the author is to find out the policy of the journal(s). If the journal is published with open access or provides the option to publish additional material online only, there is a possibility that the same journal can publish an English–language and another language version of the same article. Check this with the editor. Crossing the Language Border English edit ing services usually assure that the most important points, ideas, and opinions are communicated in the appropriate style of scientific writing and using the appropriate vocabulary for the context. The text is also checked for typographi cal and spelling errors, including punctuation. Services range from a simple language check through to highly detailed copyediting. Additional options may include formatting according to the par ticular journal’s standards, adjusting the word count to meet journal require ments, and writing a cover letter. Many services use an English language expert to complete a substantive edit first, then pass the text on to a profes sional English proofreader who makes sure the text flows well and the mean ing is clear. Of course, all of the options also raise the price of the service, but Publishing Addiction Science 82 even a basic language check can be very useful for teams of non-native Eng lish authors, when it can be difficult to maintain a consistent style throughout a document. Killing Two Birds with One Stone: Dual-Language Publication Importance of National and Local Publications As a researcher, one should not be blinded by the prestige of internationalism but instead try to protect the diversity and applicability of research. The diffu sion of relevant research to a national audience fulfils important democratic, social, and health policy aims. Brazil has been prioritizing this as well, and there is good research available in Portuguese but not in English with relevance to policies. (Bastos & Bertoni, 2014; INPAD, 2012). The development of cultur ally specific research is also important for the global development of addiction research. Nevertheless, some research may lack universal relevance. Research on spe cific treatment systems, on special treatment modalities, or on effects of nation ally implemented policy measures in LMICs may sometimes be irrelevant outside their national or regional audience. In parallel, some of the research published in the big international journals, based on findings in North America or Europe, may not be relevant in other cultural circumstances or in developing countries. Beyond the Anglo-American World 83 As long as most of the important databases and indexing systems favor English-language journals and journals from the affluent countries, jour nals published in LMICs and non-English journals may be regarded as less-prestigious publication channels. However, in some countries, such as Nigeria, there has been a growing acceptance of locally published articles as important parts of a person’s academic curriculum vitae The African Journal of Drug and Alcohol Studies was set up in response to the number of addic tion researchers in Africa having grown and some of the issues of national importance not being of interest to international journals, the only channels for African researchers in earlier times.h The wider acceptance of local publications also recognizes the reality that it is difficult for many researchers to get published in international journals. The number of scientists has increased but not the resources and support—such as libraries and translation services—that are needed to conduct the kind of research and produce the kind of articles that would be interesting for an inter national journal. This does not mean that the research is not valuable. h For researchers from LMICs, pragmatism in the choice of a publication chan nel seems essential. As noted above, it can sometimes be problematic to rely on only national or local journals, especially those with few resources, but the situation may be improving. Conclusions Addiction problems and their solutions have strong local, national, and cul tural characteristics. Addiction research needs to communicate within these milieus. It is important to preserve linguistic and cultural diversity in the communication of scientific findings. Addiction problems are an unfortu nate fact of life in many countries and are growing in Latin America, Africa, and Asia. International communication is clearly necessary for the spread of information and can be personally rewarding, as indicated in Box 4.1. The research communities in LMICs need support and encouragement. In a world of increasing globalization, the English-speaking developed world can easily become isolated, not recognizing that it has much to learn from experience in other parts of the world. In this chapter we have noted some signs that the global balance in science is improving. We know that many international and English-language journals are sympathetic toward publishing research from other countries and linguistic areas (see Edwards & Savva, 2002a, 2002b). The activities within international organizations such as ISAJE will hopefully further increase the awareness of resource, language, and cultural issues among journal editors and the research community in general through fostering networks and striving to change the discriminative practices of the databases and indexing systems. This is the good Publishing Addiction Science 84 The following quotation from an interview with Professor Mustapha Soueif, an Egyptian psychologist, cannabis researcher, and internation ally recognized addiction expert, shows how exciting it can be to con front the challenges of publishing in multiple languages and different cultures (Edwards, 1991): I have to be “bilingual” if I care for international readership and acknowledgement. And bilingualism is not an easy job. You can not reduce it to a pendular movement from Arabic to English and vice versa. Rather, you switch off a whole way of thinking, feeling and mode of expression; and tune yourself to a totally different wave length. At the start of your career you find that this exercise is really tough, and overloaded with frustrating moments. But you accept it the way it is, because you chose to have it this way. Gradually, you attain higher levels of relevant skills; your troubles decrease, yet they never disappear. Box 4.1: Professor Mustapha Soueif on “Bilingualism” in addiction publishing. Conclusions In this chapter we have pictured the unique challenges faced by addiction scientists who work outside the cultural and linguistic mainstream. It will take a great deal of skill, persistence, and courage to get to the top of your field. But the rewards awaiting you at the summit may be that much greater, because you will have acquired the skill to read the map and orient yourself both in your country of origin and in the world that lies beyond. i In this chapter we have pictured the unique challenges faced by addiction scientists who work outside the cultural and linguistic mainstream. It will take a great deal of skill, persistence, and courage to get to the top of your field. But the rewards awaiting you at the summit may be that much greater, because you will have acquired the skill to read the map and orient yourself both in your country of origin and in the world that lies beyond. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. Conclusions Another implication is that you have to accept a double load of responsibilities most of the time; I mean your local duties (the university, the private clinic, sharing in national meetings and writing in periodicals) and international requests (usually meet ings and writings). Sometimes you have to turn down a request from one side or the other. But you have to be very careful if you intend to play the two roles with optimum smoothness. It takes creative effort to find points of convergence between both, and it is, therefore, highly rewarding. A third implication is that gradually your role is redefined for you. You are no more just a local scientist with international reso nance. You are transformed into a culture-transmitter or a bridg ing factor. You are expected to behave as a medium for commu nication between two cultures. Whenever you cross the fence you should do something useful and interesting to the people on the other side. Of course what you carry with you should always be relevant to scientific endeavour. But it is sometimes peripheral. Yet it proves to be quite instrumental in promoting mutual under standing between investigators trying to transcend national and/ or cultural barriers. This is all the more important when it comes to an area like research in drug abuse. (pp. 438–439) Box 4.1: Professor Mustapha Soueif on “Bilingualism” in addiction publishing. Beyond the Anglo-American World 85 Beyond the Anglo-American World 85 news for researchers from less-resourced countries and non–English-language cultures.h The bad news is that the competition within research is hardening, strength ening existing hierarchies in the world of science and putting increasing demands on researchers from LMICs. Researchers from these countries face special challenges. General advice and rules of conduct are of limited value. Hard work and a good dose of pragmatism are needed if you want to commu nicate your research to the appropriate audience and get scientific credit for it. The bad news is that the competition within research is hardening, strength ening existing hierarchies in the world of science and putting increasing demands on researchers from LMICs. Researchers from these countries face special challenges. General advice and rules of conduct are of limited value. Hard work and a good dose of pragmatism are needed if you want to commu nicate your research to the appropriate audience and get scientific credit for it. References Amin-Esmaili, M., Nedjat, S., Motevalian, A., Rahimi-Movaghar, A., & Majdzadeh, R. (2009). Comparison of databases for Iranian articles; Access to evidence on substance abuse and addiction. Archives of Iranian Medi cine, 12, 559–565. Babor, T. F. (1993). Megatrends and dead ends: Alcohol research in global per spective. Alcohol Health and Research World, 17, 177–186. Barata, R. B. (2010). International cooperation: Initiatives in graduate stud ies in public health [Editorial]. Cadernos de Saúde Pública, 26, 2008–2009. DOI: https://doi.org/10.1590/S0102-311X2010001100002 Bastos, F. I., & Bertoni, N. (2014). Pesquisa Nacional sobre o uso de crack: Quem são os usários de crack e/ou similares do Brasil? Quantos são nas capitais bra sileiras? Rio de Janeiro, Brazil: ICICT. Retrieved from http://www.obid.senad. gov.br/portais/OBID/biblioteca/documentos/Publicacoes/329797.pdf. Burrough-Boenisch, J. (2013). Editing texts by non-native speakers of English. In P. Smart, H. Maisonneuve, & A. Polderman (Eds.), Science Editors’ Hand book (2nd ed.). Surrey, England: European Association of Science Editors. Retrieved from http://www.ease.org.uk/sites/default/files/1-2.pdf.li i Edwards, G. (Ed.). (1991). Addictions: Personal influences and scientific move ments. New Brunswick, NJ: Transaction. Publishing Addiction Science 86 Edwards, G., & Savva, S. (2002a). Hur icke-engelskspråkiga tidskrifter på ber oendeområdet arbetar med språkliga frågor: En enkät av ISAJE. [How non–English language journals in the addiction work with language ques tions: An ISAJE questionnaire.]. Nordisk Alkohol- & Narkotikatidskrift, 19, 207–210. Edwards, G., & Savva, S. (2002b). Hur engelskspråkiga tidskrifter på beroende området arbetar med språkliga frågor: En enkät av ISAJE. [How English language journals work with language questions: An ISAJE questionnaire.]. Nordisk Alkohol- & Narkotikatidskrift, 19, 211–215. ft Ezzati, M., Lopez, A. D., Rodgers, A., Vander Horn, S., & Murray, C. J. L. (2002). The comparative risk assessment collaborating group (2002). Selected major risk factors and global and regional burden of disease. The Lancet, 360, 1347–1360. Gosden, H. (1992). Research writing and NNSs: From the editors. Journal of Second Language Writing, 1, 123–139. Holmgren, M., & Schnitzer, S. A. (2004). Science on the rise in developing countries. PLOS Biology, 2(1), e1. DOI: https://doi.org/10.1371/journal. pbio.0020001 Ingwersen, P. (2002). Visibility and impact of research in psychiatry for north European countries in EU, US and world context. Scientometrics, 54, 131–144. INPAD (Instituto Nacional de Ciência e Tecnologia para Politicas Publicas do Álcool e Outras Drogas). (2013). II Lenad: Levantamento nacional de álcool e drogas. Retrieved from http://inpad.org.br/lenad/resultados/alcool/ resultados-preliminares.h Linardi, P. M., Coelho, P. M. Z., & Costa, H. M. A. (1996). References The ‘impact fac tor’ as a criteria for the quality of scientific production is a relative, not absolute, measure. Brazilian Journal of Medical and Biological Research, 29, 555–561. Maj, M. (2010). World Psychiatry and the WPA task force to promote dissemi nation of psychiatric research conducted in low and middle income coun tries. Epidemiologia E Psichiatria Sociale, 19, 204–206. Maisonneuve, H., Berard, A., & Bertrand, D. (2003). International submissions to journals. The Lancet, 361, 1388–1389. jh Meneghini, R., & Packer, A. L. (2007). Is there science beyond English? EMBO Reports, 8, 112–116. Midanik, L. T. (2004). Biomedicalization and alcohol studies: Implications for policy. Journal of Public Health Policy, 25, 211–228.i Monteiro, C. A., & Barata, R. B. (2007). Fórum de Editores Científicos em Saúde Pública [Editorial]. Revista de Saúde Pública, 41, 1–2. DOI: https:// doi.org/10.1590/S0034-89102007000100001i Montgomery, S. (2004). Of towers, walls, and fields: Perspectives on language in science. Science, 303, 1333–1335. Beyond the Anglo-American World 87 Beyond the Anglo-American World 87 Murthy, P., Manjunatha, N., Subodh, B. N., Chand, P. K., & Benegal, V. (2010). Substance use and addiction research in India. Indian Journal of Psychiatry, 52(Supplement S3), 189–199. NSD. (2014). ERIH PLUS. Retrieved from https://dbh.nsd.uib.no/publiserings kanaler/erihplus/about/index (Accessed October 31, 2015). Obot, I. (2014). Personal communication, September 15, 2014 Room, R., Jernigan, D., Carlini-Marlatt, B., Gureje, O., Mäkelä, K., Marshall, M., . . . , Saxena, S. (2002). Alcohol in developing societies: A public health approach. Geneva, Switzerland: World Health Organization. Saxena, S., Levav, I., Maulik, P., & Saraceno, B. (2003). How international are the editorial boards of leading psychiatric journals? [Correspondence]. The Lancet, 361, 6090. DOI: https://doi.org/10.1016/S0140-6736(03)12528-7 Science for Brazil (2014). Web of Knowledge gets Brazilian Link. www. scienceforbrazil.com/web-of-knowledge-gets-brazilian-link/. Accessed October 31, 2015. Targino, M. G., & Garcia, J. C. R. (2000). Ciencia brasileira na base de dados do Institute for Scientific Information (ISI) [Brazilian periodicals in Insti tute for Scientific Information (ISI)]. Ciencia da Informacao Brasilia, 29, 103–117. Retrieved from http://www.scielo.br/pdf/ci/v29n1/v29n1a11. Turci, S. R. B., Guilam, M. C. R., & Câmara, M. C. C. (2010). Epidemiolo gia e Saúde Coletiva: Tendências da produção epidemiológica brasileira quanto ao volume, indexação e áreas de investigação - 2001 a 2006. Ciên cia & Saúde Coletiva, 15, 1967–1976. DOI: https://doi.org/10.1590/S1413- 81232010000400012hi van Weijen, D. (2012). The language of (future) scientific communication. Research Trends, 31. Retrieved from http://www.researchtrends.com/issue- 31-november-2012/the-language-of-future-scientific-communication.i i Volpato, G. L., & Freitas, E. G. (2003). How to cite this book chapter: Morisano, D, Winstanley, E L, Morojele, N, and Babor, T F. 2017. Getting Started: Publication Issues for Graduate Students, Postdoctoral Fellows, and other Aspiring Addiction Scientists. In: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P. (eds.) Publishing Addiction Science: A Guide for the Perplexed, Pp. 89–118. London: Ubiquity Press. DOI: https://doi.org/10.5334/bbd.e. License: CC-BY 4.0. Getting Started: Publication Issues for Graduate Students, Postdoctoral Fellows, and Other Aspiring Addiction Scientists Dominique Morisano, Erin L. Winstanley, Neo Morojele and Thomas F. Babor References Desafios na publicação cientifíca [Challenges in scientific publication]. Pesquisa Odontologica Brasileira, 17(Supplement 1), 49–56. Warner, K. E., Tam, J., & Koltun, S. M. (2014). Growth in Tobacco Control publi cations by author from low- and middle-income countries. Tobacco Control, 23, 231–237. DOI: https://doi.org/10.1136/tobaccocontrol-2012-050762i West, R., & McIlwaine, A. (2002). What do citation counts count for in the field of addiction? An empirical evaluation of citation counts and their link with peer ratings of quality. Addiction, 97, 501–504. World Bank. (2014). World Development Indicators 2014. Washington DC: World Bank Author. World Health Organization. (2004). Galvanizing mental health research in low- and middle-income countries: Role of scientific journals. A joint statement issued by editors of scientific journals publishing mental health research. Department of Mental Health and Substance Abuse, WHO, Geneva, January 2004. Bulletin of the World Health Organization, 82, 226–228. Publishing Addiction Science 88 World Health Organization. (2007). Research capacity for mental health in low- and middle-income countries. Geneva: Global Forum for Health Research and WHO. World Health Organization. (2011). Global status report on alcohol and health. Retrieved from http://www.who.int/substance_abuse/publications/global_ alcohol_report/msbgsruprofiles.pdf?ua=1. i Zyoud, S. H., Al-Jabi, S. W., Sweileh, W. M., & Awang, R. A. (2014). A Scopus- based examination of tobacco use publications in Middle Eastern Arab countries during the period 2003–2012. Harm Reduction Journal, 11, 14. DOI: https://doi.org/10.1186/1477-7517-11-14 General Issues The challenges of publishing early on the academic trajectory include making decisions about authorship and timetables, navigating ethical dilemmas, and balancing publication pressures with training goals. Yet publications can open doors for both career advancement and financial remuneration. Introduction In recent years, there has been increasing pressure on graduate and medical students, postdoctoral fellows, and even research assistants and lab techni cians to write or co-author scientific publications. Some of this pressure has extended to undergraduates (e.g., Trammell, 2014), often before they have had the opportunity to take a statistics course. y The number of publication credits is frequently a key criterion for students’ acceptance into advanced study, postdoctoral opportunities, and internship placements as well as for the receipt of scholarships, fellowships, grants, and employment. For novice academics, publication numbers and authorship order are often at the top of considerations for tenure-track advancement. More competitive universities that value high publication numbers might urge stu dents and junior faculty to compose theoretical papers and review articles or to write reports based on publically sourced unpublished data (e.g., www.apa. org/research/responsible/data-links.aspx) instead of running original studies, which take time and do not always yield publishable results. In some countries, students are advised to publish articles in addition to producing a monograph- style dissertation; in others, they are expected to focus solely on the produc tion of a “compilation thesis” or article-based dissertation that might lead to Publishing Addiction Science 90 multiple publications. Some students must produce dissertations that are based on published articles (possibly with multiple authors). In any case, for post graduate trainees and junior academics, authorship is increasingly at the fore front of issues faced in education and early employment.h This chapter presents issues that are particularly relevant to publishing as a graduate student or postdoctoral fellow, but anyone early in her or his publish ing career might benefit from reading through the topics covered. The chap ter begins with a discussion of general issues related to authorship and then addresses the more specific topic of publishing graduate-level theses. The latter section focuses on the entire process of thesis publication, ranging from issues that might arise before writing one’s thesis all the way to eventual postpublica tion submission to an appropriate journal. Our main sources of information on this topic come from North American and European universities in high- income countries, but the issues and solutions discussed are increasingly rel evant to university students in other regions. Accordingly, special attention is provided to the challenges encountered by students or novice investigators in less resourced countries. Authorship As noted in Chapter 11, authorship of peer-reviewed journal articles is the “coin of the realm” in academic settings, although the ability to write even unpub lished reports is a valuable skill in any work situation. For the great major ity of graduate students and postdoctoral fellows, early-career authorship will come only from collaboration with faculty members,1 senior researchers, and supervisors. As such, both mentors and mentees should consider a number of ethical and practical issues that could arise on joint projects (see Chapters 14 and 15 for a discussion of authorship ethics). At the heart of such trainee– faculty (or even employee–supervisor) collaborations lies an inherent power imbalance (Fine & Kurdek, 1993; Gross et al., 2012). Often, the faculty mem bers with whom students and trainees have the most interactions (and thus the greatest chance to do research) are responsible for providing them with recommendation letters and evaluating their work. These faculty members may even be responsible for trainee salaries, as in the case of graduate assistantships or postdoctoral fellowships. Many students and trainees begin with minimal Getting Started 91 experience and competence in publishing and must rely on faculty support and guidance. Even if students and postdoctoral trainees are consulted during the process of assigning authorship, faculty members generally make the ultimate decisions on where (or whether) students or trainees are placed on the author list. Students who disagree with or misunderstand such decisions might fail to voice their opinions for fear of negatively impacting the ways in which those faculty members will evaluate them.h y The academic level of the collaborating faculty member or supervisor could also influence the authorship decision-making process. Senior faculty with established research grants might be more likely to give students or trainees opportunities for first authorship on co-authored publications. With poten tially bigger labs or projects and greater numbers of volunteers and research assistants, senior faculty might even provide more chances to publish in gen eral, handing over projects, ideas, and datasets to their mentees. In contrast, junior faculty members are frequently under significant pressure to get their own names on publications in order to earn research grants, advance to higher faculty positions, and gain tenure. As a result, they might have more concerns about sustaining and advancing their own careers than about taking time to help their students or trainees to publish. Authorship Figure 5.1 provides a satirical view of authorship situations sometimes encountered by students who work on publications with more experienced or higher ranked investigators. Although the cartoon is a spoof, many academ ics would agree that it is uncomfortably close to the procedures witnessed in some research labs, centers, and departments. The procedures for determining student–faculty co-authorship are likely to vary by discipline, institution, and even culture, but they should ideally reflect a dynamic process that evolves as the authors revise and resubmit their article. Figure 5.1: Authorship credit comic from “Piled Higher and Deeper” by Jorge Cham (www.phdcomics.com, reprinted with permission of author. All rights reserved.). Figure 5.1: Authorship credit comic from “Piled Higher and Deeper” by Jorge Cham (www.phdcomics.com, reprinted with permission of author. All rights reserved.). 2 Publishing Addiction Science 92 Graduate students, postdoctoral fellows, and young professionals working in basic and applied research settings are often uninformed about acceptable procedures for deciding authorship within a given field or discipline. In addi tion, procedures seem to vary so greatly even within departments that it can be difficult to stay abreast of what constitutes acceptable practice. The availability of specific guidelines is indispensable to establish equal opportunities for stu dent authorship and consistent procedures for student–faculty collaborations. As in the case of the more general issue of authorship (discussed above), there are specific guidelines available that can facilitate this process at some institu tions and help prevent problems from arising in the first place. Some examples of these guidelines are discussed below. If they are not readily available at your research center or university, however, it is possible to adopt guidelines from another institution or professional society (see Chapter 11 for an example).i As a rule, graduate students should be the first authors of journal articles based on their thesis or dissertation manuscripts. Many disciplines and insti tutions enforce this broad principle. For example, the American Psycho logical Association’s Ethical Principles of Psychologists and Code of Conduct (American Psychological Association, 2010) explicitly states, “Except under exceptional circumstances, a student is listed as principal author on any multiple-authored article that is substantially based on the student’s doctoral dissertation” (Section 8.12). Further, the American Psychological Association indicates that faculty advisors should discuss publication credit with students as early as feasible and throughout the research and publication process. Authorship However, the “exceptional circumstances” mentioned highlight a universal gray area, and it is often the case that other factors might complicate seem ingly straightforward authorship assignment, for instance when the graduate student’s dissertation is based on part of an advisor’s grant. In line with changing times, several institutions of higher learning have posted general authorship guidelines on their websites. The University of Pennsylvania, for example, has developed a broad policy on fairness regard ing authorship credit for publications co-authored by graduate students and faculty. A university-wide process for determining authorship sets forth simple principles and an appeal process and requires graduate programs to provide more specific guidelines to reflect interdisciplinary and interdepartmental dif ferences in assigning authorship credit (University of Pennsylvania’s Office of the Provost, 2013). Mandating such procedures within each graduate group clarifies expectations about authorship for both students and faculty mem bers. Specific departmental guidelines cover topics such as authorship criteria (specific and general principles regarding the kind of work that warranted a publication credit), whom to consult to resolve disputes, and the issues that faculty should discuss with students when beginning joint projects. Examples of such issues include (a) whether the graduate student will share authorship credit, (b) the expected order of authorship, (c) the division of labor on the project, and (d) when to revisit or review work that is being completed by each Getting Started 93 Getting Started 93 93 collaborating member of the pair or group. The University of Alberta’s website hosts a similar set of guidelines around intellectual property and authorship (University of Alberta, 1996). y In general, with the expansion of the Internet as the primary tool of com munication in most circles of higher education, online policies appear to be an efficient and user-friendly way of spreading authorship and intellectual- property guidelines to junior investigators with adequate access. Harvard Med ical School Office for Research Issues (1999), the University of Toronto (2007), Washington University in St. Louis (2009), and the University of Cambridge (2014), among others, have also provided statements on authorship or intellec tual property for members of their institutions—although some are rather brief in nature, they seem to be evolving. University of Pennsylvania and University of Alberta guidelines provide the best models for the development of similar policies in higher learning institutes across the world. Authorship Such university-wide policies are an excellent way to keep students, postdoctoral fellows, and faculty members informed about the most fair and equitable procedures to follow in joint-authorship situations. j p In what has become a US benchmark article for writings on student–faculty co-authorship, Fine and Kurdek (1993) produced a set of authorship guide lines based on the idea that both faculty and students should meaningfully participate in the authorship decision-making process. Fine and Kurdek rec ommended that, at the very initiation of joint projects, supervisors and faculty collaborators provide new students and postdoctoral fellows with information about how authorship decisions are made. They also put forth a series of specific and potentially controversial recommendations about student authorship, argu ing, for example, that supervisors cannot and should not expect as much from students as from experienced professional colleagues. Instead, the authors sug gested that there should be a different standard for the level of professional con tribution required by students to attain a given level of authorship credit within a student–faculty collaboration. At the same time, however, they maintained that student contributions must be professional in nature: that is, creative, intel lectual, and integral to completion of the paper. Examples of such contributions might include developing the research design, writing sections of the manu script, integrating diverse theoretical perspectives, developing new conceptual models, designing assessments, contributing to data-analysis decisions, and interpreting results. Other tasks—such as entering data, carrying out statistical analyses specified by the supervisor, and typing a manuscript—might warrant a footnoted acknowledgement, but they would not, according to the authors, deserve authorship credit. Fine and Kurdek suggested that supervisors and stu dents decide early in the publication process what combinations of professional activities would merit a given level of authorship credit for both parties. These decisions might now need to be checked against journal or discipline-specific guidelines and standards, many of which have become more detailed over the years in response to authorship confusion and transgressions (see Chapter 11). 94 Publishing Addiction Science 94 Fine and Kurdek (1993) raised a variety of issues and case scenarios sur rounding authorship in student–faculty collaborations that are still relevant more than two decades later. Chapter 11 is a direct response to articles such as this as well as to the diverse but brief and scattered array of individual uni versity guidelines mentioned above. Authorship Students, postdoctoral fellows, and other early investigators in the process of article publication should refer often to the general set of very practical authorship guidelines provided in Chapter 11. These guidelines span the planning, drafting, and finalization stages of author ship. Indeed, the chapter is an ideal source for beginning researchers to con sult as they try to determine where (or if) they should appear within author lists. It touches on potentially controversial issues, such as what constitutes a “substantive” authorship contribution. For example, if a graduate student has developed, coordinated, and carried out a research project for a mentor or supervisor but did not come up with the original idea, analyze or interpret the resulting data, or participate in the writing of the ensuing manuscript, does he or she deserve to be listed as an author on publications arising from the project? According to the recommendations in Chapter 11, the answer is no, because there is no involvement in the writing process (and to be an author, one must write!). However, one might argue that this student should at least be given the option of contributing in a more substantive way to the publication process in order to earn authorship. Students might therefore want to explicitly express their interest in being involved in future publications. In summary, there is a great amount of room for improvement in the realm of early-career publishing. The process has not yet been clearly documented in terms of student and junior investigator rights, responsibilities, and roles. Although progress has been made in clarifying the issues and formalizing some long overdue policies, much remains to be done at both the level of the academic institution and the level of the individual faculty and trainee. Fortunately, there are plenty of opportunities to learn more about this area to improve the pro cess. The mentorship of a seasoned investigator can provide her or his students, postdoctoral fellows, or other trainees with a golden opportunity to ascertain how publication works. At the very least, the sharing of articles such as this chapter might help to raise awareness of the issues and how to deal with them. Publishing One’s Thesis or Dissertation Converting the thesis or dissertation into one or more journal articles is a key publishing opportunity for aspiring researchers. Incentives to early publica tion include building confidence, establishing a pattern of scholarly activity, enhancing student satisfaction, increasing knowledge of the publication pro cess, and advancing or updating the science.2 Sometimes, early publication affords a novice researcher the opportunity to demonstrate the need for a par ticular area of research (Robinson & Dracup, 2008). As noted above, there are Getting Started 95 Getting Started 95 many incentives to begin publishing early or publishing before the research data “shelf life” has expired, particularly for those who are interested in aca demic careers (Resta et al., 2010). Given the amount of work that is invested in the preparation of a thesis or dissertation, this is often the ideal place to begin one’s publication career, and it is important to be strategic about the develop ment of a publication plan. When considering a timeline for publication, there are several questions researchers might ask themselves. For instance, “What is my academic trajec tory?” Or, “How fast is this area of research developing?” “How much informa tion is available in my content area?” “Is the literature up to date or does it need updating?” “What is the potential real-world impact of my research?” “Does current literature support the need for my research, or do I need to build a published case?” “What audience is most interested in my area of research?” Answering these simple questions could help a novice researcher to develop a successful publication plan both during and after thesis or dissertation comple tion. The following section describes additional considerations. Before Writing One’s Dissertation: Format Considerations There are several different doctoral dissertation formats, which vary in accept ability depending on the country and the university in which they are written. Two of the more popular formats are the monograph style (single authored) and the separate manuscript style (multiauthored; Hagen, 2010). Many gradu ate programs increasingly favor dissertations that depart from the traditional monograph style and that instead facilitate the incremental translation of the dissertation into publishable manuscripts.hf The manuscript style of dissertation—although it might have different names—generally requires that chapters be written in article format. For exam ple, at The Johns Hopkins Bloomberg School of Public Health, a student can choose to write a traditional monograph-style (chapter-based) dissertation or a “papers option.” The latter format requires that a minimum of three of the dissertation chapters take the shape of publishable manuscripts, with one chapter usually serving as a critical review of the literature and two chapters comprising empirical analyses. To the extent that the papers are “publishable,” whether they must be submitted or accepted for publication to earn a degree varies across universities. In the Nordic countries (Denmark, Iceland, Finland, Norway, Sweden), most of the dissertation articles must have been published or accepted for publication before the dissertation can be passed. Manuscripts may represent the entire chapter or a portion of a dissertation chapter that is supplemented with a synthesis or independent introduction. An example of the purposive changes that may be made to a manuscript to fulfill the chapter requirements include the addition of regional data and epidemio logical information, the definition of terms for lay readers, a longer and more Publishing Addiction Science 96 in-depth explanation of the phenomenon, the theoretical tenets guiding the proposed study, and a conclusion that illustrates student mastery of the subject.hl The extent to which manuscripts need to be interrelated and reflect a single focus of research, as occurs in a monograph-style dissertation, varies across institutions, departments, and advisors. It is, in part, contingent on the clar ity of the institutional guidelines provided. Anecdotal evidence suggests that the rules are not concrete. Furthermore, if one is writing a literature-review chapter, it is helpful to keep in mind that many addiction journals do not accept unsolicited review articles and that getting this type of manuscript published could be a special challenge. Literature reviews using a systematic or structured approach are more likely to be published. Before Writing One’s Dissertation: Format Considerations If one has the opportunity to choose which dissertation format to take, it is important to consider the benefits and particular challenges of a style that is meant to facilitate the publication process. For example, even if one chooses to write one’s thesis in the manuscript style, resulting chapters might still require significant revision if they need to be shortened and formatted later for a par ticular journal and written with a broader audience in mind than one’s disserta tion committee (Azar, 2006). In the Trenches: Writing One’s Dissertation with Publication in Mind In the Trenches: Writing One’s Dissertation with Publication in Mind While writing the thesis or dissertation, it is helpful to think about whether chapters or sections will eventually be suitable for journal publication. If the answer is yes, then several issues arise that should be addressed sooner rather than later. For instance, if one hopes to publish one’s data in a particular jour nal, it is important to consider the author guidelines during the drafting stage in order to tailor the writing and formatting style of the dissertation toward specific journal requirements. It is also useful to consider the intended audi ence of that journal early on (see Chapter 3 for issues related to choosing a jour nal). Even if a particular target journal has not yet been identified, the chapter can be written with the potential audience in mind (e.g., clinicians or policy makers), in a way that can help refine the scope of the manuscript. It is also important to remember that if one publishes data or other study-related mate rial before submitting the dissertation or thesis, one must consider which parts of the published manuscript(s) are eligible for inclusion in the final disserta tion. Journals and publishers will often grant permission to students to submit published manuscripts as dissertation chapters, but it is wise to request written confirmation. i Furthermore, for many, a considerable amount of time can elapse between creating initial drafts of the thesis or dissertation and preparing to publish the content in a journal. It is therefore important to maintain adequate documenta tion of all analyses and datasets. The lengthy dissertation-writing process plus the journal-submission process could result in a situation in which, months Getting Started 97 or years after data collection, a journal reviewer requests that data analyses be revised or substantially expanded. Although this issue is generally relevant for the authors of any research study, the significant time that it takes to complete the dissertation amplifies the importance of keeping an adequate record of completed work. In sum, the forward-thinking student will strategically balance disserta tion requirements with potential journal submission requirements. This is not always easy. Dissertations typically require a much greater level of detail than most journal manuscripts. This means that significant portions of the disserta tion will need to be cut, edited, and fine-tuned for publication. In the Trenches: Writing One’s Dissertation with Publication in Mind Writing style might also need adjustment, depending on the intended audience (e.g., dis sertation committee vs. journal editors, and reviewers vs. the scientific com munity at large). There are benefits to this conversion exercise, however. The process of transforming dissertations into publishable articles teaches graduate students not only how to summarize research findings in a succinct manner, but also how to communicate to a broader audience than faculty and commit tee members. In the long and sometimes dark days of creating one’s dissertation with publication in mind, it is key to remember that publication presents multiple rewards. In addition to fulfilling degree requirements and contributing to sci entific advancement, all of one’s hard work can be directly applied to making progress on the career front. Publication is, after all, the coin of the realm. Preparing for Publication Once the dissertation has been approved, and the appropriate celebrations have concluded, the time for publication is nigh.3 Frequently, suggestions made dur ing the final dissertation defense will be relevant to the initial stages of prepar ing for publication. During this phase, several issues inevitably will come to the surface. The first is authorship. As previously discussed, the student should be the first author the majority of the time. In the case of multiple authors, institu tional and disciplinary guidelines or even our own recommendations (see Chapter 11) can help to determine authorship order. If committee members are to be invited as potential co-authors, it should be made clear that all authors are required to have made substantial contributions to the journal manuscript itself, as opposed to simply “being a part” of the dissertation-development conversation. Given that many journals now require written statements that specify authorship contributions, this is no longer just a traditional courtesy. Assignment of authorship is a dynamic process that will depend on the amount of time that has lapsed since graduation, the extent of revisions required for publication, and the context in which those revisions are made. For example, revisions are sometimes required at the final stage of the dissertation-approval Publishing Addiction Science 98 process, and it might not be feasible to anticipate the target journal until after graduation. If substantial revisions are requested, the opportunity might arise to seek expertise outside of the dissertation committee. The recruitment of external co-authors can offer several advantages. First, fresh insight might facilitate the process of tailoring a manuscript for a particular target audi ence. Alternatively, external experts might be able to address weaknesses in a manuscript that fall outside the student’s field of knowledge. Sometimes new graduates might recruit the co-authorship assistance of a former labmate or graduate-student peer to make broad cuts in superfluous content that might be difficult for the primary author to do. This offers the added opportunity or benefit of publication experience for a peer. i One should also consider publication of the dissertation itself, with or without an accompanying short-form article. This is a requirement at many European institutions, where dissertations often result in published books. Some graduate programs might provide structured guidance regarding the process of indexing the dissertation, copyrighting dissertation materials, and publishing the disserta tion as a complete document. Some university libraries now do this automatically (e.g., McGill University: www.mcgill.ca/library/find/theses). Preparing for Publication Alternatively, there are an increasing number of low-cost opportunities to publish one’s full work online. A sampling of websites offering this possibility is presented in Box 5.1. For example, Dissertation Abstracts Online indexes dissertation abstracts and disseminates them across a wide range of literature search engines. ProQuest Dissertations and Theses allows graduates the option to purchase a permanent link for dissertation abstracts; this can be useful for citation purposes. Other sites offer interested readers the choice to either download or receive a .pdf or paper copy of a dissertation for a nominal fee. If one is looking to reach the widest audience, writing the dissertation in manuscript style can facilitate the process of achieving one or more first-author publications. Finding the time for even one article can be difficult after gradu ation, when important life changes (e.g., finding or starting a new job, starting a family, catching up on things that might have been on hold during graduate school) are often inevitably competing for one’s time. This is why a postdoctoral position, when available, offers an ideal solution: the very nature of the job often includes the development of publications as a primary goal. Furthermore, depending on the area of research, postdoctoral positions of 1–3 years might not allow sufficient time to be a part of a new project from inception to publica tion. Entering the position with one’s own dissertation provides an immediate publishing goal. Publication Timelines Graduate students sometimes lose interest in publishing project data after their theses have been defended (or even before!), and impor tant or interesting scientific results are often buried under more salient tasks at hand (e.g., seeking full-time employment). Regarding specific policies, this is something that supervisors and dissertation committee members should dis cuss with their students early in the collaboration process. A reasonable solu tion for the various parties in these cases might be to designate a mutually agreeable time period together and then sign a written agreement that would bind them to it. One example of an individual professor’s policy that was put together and published online is that of Professor Karl Wuensch (2008,4 East Carolina University). On his website, Wuensch clearly states his policy regarding timeli ness of publication for student theses. For example, if the thesis is the student’s idea, the student does most of the work (e.g., collects and analyzes the data, writes the manuscript), and the manuscript is prepared within 18 months from the date of the research initiation or one year from the date of the thesis defense, then the student is first author. If warranted by their contributions to the jour nal manuscript, the thesis director and other committee members might also be listed as authors. However, if the student does not complete the research, including defending and depositing the thesis and preparing the manuscript for submission for publication within the time limits mentioned above, then all rights to use that thesis data revert to the thesis-committee director. Wuensch also indicates procedures for other situations that might arise, for example, if the student-submitted manuscript is not accepted upon initial submission to a journal. Guidelines such as these might also be adapted for postdoctoral fel lowship projects. In the discussion of publication timelines, it is important to remember that exceptions (e.g., illness) can always be considered if one fails to publish within the agreed-upon period—one need not despair. As long as steady progress is shown and good communication among co-authors is in place, the pressure that might come from thesis advisor(s), co-authors, and committee members can be reduced. Sometimes the issue lies not with one’s own progress but with getting co-authors to respond in a reasonable amount of time. Publication Timelines Some supervisors and faculty members feel it is important to set formal limits, policies, and procedures regarding the time that students have to publish their Getting Started 99 1. UMI (University Microfilms International) Dissertation Pub lishing: www.proquest.com/products-services/dissertations P Q t Di t ti & Th Gl b l d t b 1. UMI (University Microfilms International) Dissertation Pub lishing: www.proquest.com/products-services/dissertationsh b. American Doctoral Dissertations c. Masters Abstracts Internationalh d. ProQuest Dissertations and Theses—United Kingdom (UK) & Irelandh e. Dissertations & Theses @ e. Dissertations & Theses @ e. Dissertations & Theses @ f. Dissertation Abstracts International/Dissertation Abstracts Online/Comprehensive Dissertation Indexh f. Dissertation Abstracts International/Dissertation Abstracts Online/Comprehensive Dissertation Indexh 2. OCLC WorldCat Dissertations and Theses (includes manuscripts from OCLC member libraries): http://www.oclc.orgh 3. Networked Digital Library of Theses and Dissertations: www. ndltd.org 3. Networked Digital Library of Theses and Dissertations: www. ndltd.org 4. DART-Europe (28 countries): www.dart-europe.eu/basic-search. phph 5. BNF: Thèses et écrits académiques (France): http://signets.bnf. fr/html/categories/c_011theses.htmlh 5. BNF: Thèses et écrits académiques (France): http://signets.bnf. fr/html/categories/c_011theses.htmlh 6. EThOS (UK): http://ethos.bl.uk 6. EThOS (UK): http://ethos.bl.uk 7. theses.fr (France): www.theses.frh 8. Theses Canada Portal (Canada): www.bac-lac.gc.ca/eng/services/ theses/Pages/theses-canada.aspx 8. Theses Canada Portal (Canada): www.bac-lac.gc.ca/eng/services/ theses/Pages/theses-canada.aspx 9. DissOnline (Germany): www.dnb.de/DE/Wir/Kooperation/ dissonline/dissonline_node.html 9. DissOnline (Germany): www.dnb.de/DE/Wir/Kooperation/ dissonline/dissonline_node.html 10. Tesionline (Italy): www.tesionline.com/intl/index.jsp 11. Tesis doctorales: TESEO (Spain): www.educacion.es/teseo 12. dissertations.se (Sweden): www.dissertations.seh 13. Database of African Theses and Dissertations (Africa): www.aau. org/page/database-african-theses-and-dissertations-datad 14. Networked European Deposit Library (France, Norway, Finland, Germany, Portugal, Switzerland, Italy, and the Netherlands): www.ifs.tuwien.ac.at/~aola/publications/thesis-ando/NEDLIB. html 15. Google Scholar: http://scholar.google.com 15. Google Scholar: http://scholar.google.com 16. Amicus (Canada): http://amicus.collectionscanada.ca/aaweb/ aalogine.htm 16. Amicus (Canada): http://amicus.collectionscanada.ca/aaweb/ aalogine.htm Box 5.1: Online dissertation indexing and publishing resources. 0 Publishing Addiction Science 100 thesis or project data in a scholarly journal. When this timeline is expired, there might be a debate over whether the right to publish the data should be forfeited to the supervisor or members of the dissertation committee. It is a common belief that if work is not published in a timely manner, it is unlikely to be pub lished at all (Rudestam & Newton, 1992).i In most cases, students should have the right to publish their results as first author, even with considerable delays. If the timely dissemination of important scientific findings is at the root of such policies, however, then these procedures might be warranted. Publication Timelines Although all authors might struggle with the multiple-author publication process, novice writers in particular must learn to develop effective communication strate gies, ideally from their advisors. It can be useful to set specific time frames for Getting Started 101 101 co-authors with concrete deadlines and frequent email reminders. If response time becomes unreasonable, a direct conversation with these co-authors about their place on the manuscript might become necessary. If motivation or writer’s block is an issue, it might be useful to take advantage of some of the strategies presented in Box 5.2. Publication Contracts and Guidelines Several attempts have been made to develop formal procedures to address the ethical, practical and logistical issues discussed above. Professor Bruce M. Shore, an educational psychologist and professor emeritus at McGill University (Montreal, Canada), developed a formal supervision contract (Shore, 2014) for use with students. This contract covers matters such as authorship order, pub lication credit, and general responsibilities of both the advisor and the student within the supervisee–supervisor relationship. As a supervisor, he required that all of his students read, discuss, and sign the contract before agreeing to work with him, and he often raised the issues involved with authorship before pro jects even germinated. He agreed to share his contract as an example of an advi see–advisor agreement for the purposes of this chapter (see Appendix A). The process recommended in the agreement is refreshing. Regardless of whether a student agrees with the various conditions of the contract, the issues are trans parent and open to discussion at the onset of the mentee–mentor relationship. A similar guideline was developed by graduate students at the University of Connecticut School of Medicine (Cornell et al., 2014; Authorship Rights of Graduate Students, see Appendix B) to protect graduate students working in various areas of health science by clearly defining student–faculty authorship criteria and the ethical responsibilities of each party. The procedures described in the guideline (as well as Professor Shore’s contract) can be adopted by department chairs, center directors, student organizations, and individuals to protect graduate students from negligence or mistreatment related to scientific authorship. Financial Remuneration Conversations about financial remuneration can arise in the creation of a man uscript. Some faculty and supervisors feel that students or other individuals who are paid as research or graduate assistants should not be given author ship because credit for performed work is being given in the form of a salary. These same faculty members might express that publication credit replaces the need for financial remuneration, because the individuals will ultimately benefit from having their names listed on a paper. Fine and Kurdek (1993) are firm in their position that paying a research assistant or graduate student should not 02 Publishing Addiction Science 102 Even if you love writing, sometimes it takes great effort to put a line down on paper. With an infinite array of potential distractions on the Internet (e.g., social media), especially when one must make use of online resources (e.g., Google Scholar, PubMed) to write, writing time can suffer. Add to that the existence of smartphone apps and offline “dis tractions” (e.g., work tasks with deadlines, that new novel you can’t put down, television, family or household obligations, social invitations), and finding time to write can be nearly impossible. Some potential solutions: 1. Make a writing schedule and stick to it. Mark the time in your cal endar, and treat it like you are getting paid by the hour. If an extra incentive is needed, take a cue from behavior-modification experts and give yourself a small reward when you successfully follow through with your writing goals for the day (or even the hour!).i 2. Find a great place to write. Many new scholars find that writing at a local cafe or public library is easier than writing at home. Alter natively, designating an area of your home for “writing” might help to keep you on task. p p y 3. Do something about your smartphone/tablet while you write. Put it on “airplane” mode; take it offline; or, at the very least, turn off notifications. i 4. Take advantage of free, online writing tools and apps. 5. Give yourself a few minutes each day to de-stress. Often, our most creative ideas arise when we pull ourselves out of “go mode” and take a moment to sit and think, relax, take a walk, close our eyes, exercise, or meditate (e.g., with Jon Kabat-Zinn at www.youtube. com/watch?v=iZIjDtHUsR0). Financial Remuneration Do a quick web search for “free writing tools,” and you will encounter a bevy of computer- and smartphone-based applications that will allow you to do such things as (a) keep you offline (e.g., “Freedom” app), (b) block you from specific sites (e.g., “Self-control” app), (c) organize your thoughts (e.g., “Evernote” app), (d) monitor writ ing breaks (e.g., “Time Out” app), or (e) be rewarded or “pun ished” for progress (e.g., “Write or Die” app). The popular website The Huffington Post has even designated an entire section of their site for keeping up to date with the latest writing apps: www.huff ingtonpost.com/news/writing-apps. For those without computer access, setting frequent, proximal, and challenging yet achievable short-term goals has been closely linked to achievement success (see Morisano, 2013). 5. Give yourself a few minutes each day to de-stress. Often, our most creative ideas arise when we pull ourselves out of “go mode” and take a moment to sit and think, relax, take a walk, close our eyes, exercise, or meditate (e.g., with Jon Kabat-Zinn at www.youtube. com/watch?v=iZIjDtHUsR0). Getting Started 1 103 6. Keep up-to-date on the latest research by subscribing to relevant listservs such as the one maintained by the Kettil Bruun Society for Social and Epidemiological Research on Alcohol (instructions at www.kettilbruun.org/Listserve.htm). They are often the source of good ideas and occasionally an inspiration for future articles. 6. Keep up-to-date on the latest research by subscribing to relevant listservs such as the one maintained by the Kettil Bruun Society for Social and Epidemiological Research on Alcohol (instructions at www.kettilbruun.org/Listserve.htm). They are often the source of good ideas and occasionally an inspiration for future articles. Box 5.2: Writing strategies. substitute for authorship credit, when credit for professional and intellectual contributions is due.5 This extends to the hiring of consultants to contribute to the research and writing of an article; payment is not a substitute for author ship. The extent of controversy surrounding financial remuneration indicates that this topic should be covered when creating institutional and departmental guidelines surrounding authorship procedures. In light of the authorship crite ria discussed elsewhere in this chapter and in Chapter 11, it is clear that neither financial reimbursement nor its absence should be considered in the determi nation of authorship credits. The Nitty Gritty: Submitting a Manuscript and Responding to the First Rejection After carefully choosing a target journal (see Chapter 3 for advice), one should normally write a cover letter to the journal’s editor and a brief description of the manuscript. Some journal editors might have sympathy for novice writers when sending written feedback (e.g., by providing more detail), so one’s inex perience could be worth noting here. One should be mindful that some jour nals require specific cover-letter content (e.g., word count, conflict-of-interest statement); therefore, author guidelines must be consulted in advance. These are most often found under Author Guidelines or Instructions for Authors on journal websites, or in the paper copy of the journal itself. Some journal edi tors (particularly of smaller journals) are also open to receiving presubmission emails to gauge interest in potential submissions; this is worth considering. Even for the most fastidious researchers and stellar writers, the day will likely arrive when a rejection letter is received. If the rejected work is based on one’s dissertation data, the decision can be particularly devastating, given the time and energy invested (and other issues previously discussed). It is important to under stand that rejection is simply a part of the writing and publication process—even senior and experienced researchers have manuscripts rejected.6 It is surprisingly easy to forget that if one is reaching for the stars and submitting to a competitive 104 Publishing Addiction Science 104 journal, acceptance rates are low. Even lower ranked journals are increasingly incorporating rigorous standards that might require a decent paper to go through a “revise and resubmit” round or two before acceptance. The most productive step to take post-rejection is to read and incorporate reviewer feedback as much as possible into a new draft, and try, try again (at another journal, unless resubmis sion is specifically invited). Chapter 12 provides guidance on how to respond to editors’ requests for revised manuscripts. Special Issues of Relevance to Students and Junior Investigators from Low- and Middle-income Countries Thus far, this chapter has focused on publication issues that are likely to be most relevant to those from well-resourced countries with an established sci entific community in the addiction field. Students and junior investigators in less resourced countries face a number of different issues related to conducting and disseminating research (see Chapter 4 for a discussion of broader issues related to addiction research in developing countries). The following section addresses some of the special challenges encountered by students and novice addiction researchers from low- and middle-income countries (LMICs) as well as those from LMICs who earn their degrees from universities in developed countries and then return home. There is an imperative at both the national and international levels to publish research on addiction issues that is relevant to populations outside of Europe, Australia, and North America. High-quality dissertation research in general has the potential to significantly impact addic tion science. Further, individuals from LMICs have especially strong obliga tions (and pressures) to conduct research and publish the results. In many LMICs, research is used to shape both the policy agenda and prevention/inter vention programs. But most of the evidence on what policies and interven tions “work” to reduce substance-related harms is based on studies conducted in developed countries. Indeed, the notion that research is limited in LMICs is highlighted by the shocking 10/90 gap statistic, according to which, only 10% of global research spending is directed to health problems that comprise 90% of the world’s disease burden (Global Forum for Health Research, 2004). A Word on Predatory Publishers With the dramatic expansion of open-access and online journals (see Chapter 3), a number of for-profit enterprises have created new “journals” that will publish almost any article submitted for a processing fee ranging from $500 to several thousand dollars (Beall, 2012). The name “predatory publisher” has been applied to this type of business because it involves charging publication fees to authors without providing the editorial and publishing services associated with legiti mate journals. Several new addiction-science journals have been launched by these publishers, raising serious questions about their impact on a field that is already plagued by conflict-of-interest threats from the alcohol, tobacco, gam bling, and pharmaceutical industries (see Chapter 16).h The characteristics of these journals include rapid acceptance of articles with little or no peer review; aggressive marketing, often using poor grammar and syntax; journal editors with no academic standing in the addiction field; misleading or nonexistent publication metrics (e.g., impact factors, indexing services); and publication fees that are not revealed until after the article is accepted. p It is easy to understand both the frustration of a new investigator who might receive multiple rejection letters and the appeal of an online journal that levies page charges after a cursory review. If early-career scientists or trainees choose to publish in such journals, however, the most likely consequence is to appear to peers, grant reviewers, potential employers, and promotion committees to be naive, unethical, or desperate for authorship credits. Many researchers are not familiar with the complicated and often confusing developments in jour nal publishing and may be easily scammed and embarrassed. Fortunately, resources on how to protect the integrity of science and avoid these unscru pulous phantom publishing operations masquerading as addiction journals are available, including Jeffrey Beall’s (2015) list of predatory publishers (see References for a link). Prospective authors should also consult Chapter 3 of this book and the updated website of the International Society of Addiction Journal Editors (ISAJE; www.isaje.net), which provide a list of journals that subscribe to the Farmington Consensus, a code of ethics for journals and journal editors (Farmington Consensus, 1997). Getting Started 105 105 General Capacity Challenges The capacity of individuals to conduct and publish research varies consider ably within and across LMIC academic institutions. In many university envi ronments, salaries may be low, with both high teaching loads and competing demands. Personal financial constraints might compel academics to undertake other activities, such as seeing private patients or conducting various types of consulting work to supplement their incomes. Academics in LMICs often have minimal staff support and must conduct the bulk of their research work unas sisted. In better resourced environments, investigators are more likely to have staff assistance for many of the activities that are required to write and submit papers for publication (e.g., literature reviews, data collection, entry, and data analysis) and for other aspects of research (including grant writing). Publishing in countries with minimal research infrastructure outside of an academic institution is a special challenge, because writing is often lower on the priority list than tasks that are directly related to conducting the research, Publishing Addiction Science 106 running prevention and intervention programs, or moving on to the next pro ject. The final product of research is often a report for a local or national agency rather than a formal journal article. Although reports are an important mecha nism for disseminating research findings, redrafting them into journal articles is necessary for the data to reach a broader scientific audience, to influence work in other LMICs, and to contribute to global knowledge. Publishing in a peer-reviewed outlet might also provide the author(s) with helpful feedback and ways to improve the work and thus the contribution. Converting reports into journal articles under intensive work constraints can be a difficult, albeit surmountable, challenge. ISAJE has developed a writing mentorship program for this purpose (see http://www.parint.org/mentor_1. htm for more information; Miller, 2011). It provides novice researchers with the opportunity to be mentored by senior researchers, which can be useful if the immediate work environment does not provide sufficient opportunities to learn how to write for peer-reviewed academic journals. Some LMIC researchers might sometimes fear that their work does not meet the standards of certain journals. With the development and use of increasingly sophisticated equipment and statistical techniques in high-income countries, the perception might arise that any research that is not state-of-the-art is not publishable. This is absolutely not the case. General Capacity Challenges As suggested in Chapter 4, LMIC research may provide drug and alcohol policymakers with regionally specific data and evidence-based interventions. When implementing new laws, treat ment policies, or programs anywhere, it is imperative that they are culturally appropriate and relevant. Furthermore, it is useful for researchers in Europe, Australia, and North America to have a more global perspective on research and prevention or intervention outcomes when developing their own proto cols and policies. Exposing addiction scientists from non-LMICs to research ers from LMICs might lead to important investigative collaborations and cross-cultural research. Some of the most valuable studies of alcohol and drug screening, brief intervention, treatment, and epidemiology were conducted as cross-national collaborations between researchers from LMICs and high- income countries (Humeniuk et al., 2012; Rehm et al., 2010; Saunders et al., 1993). By regularly reading journal articles, attending conferences, and joining international research societies, LMIC researchers can gain exposure to diverse international research and build the confidence, skills, and connections that could lead to opportunities for international collaborative research. Research Topics Although there is still a significant underrepresentation of LMIC publications in scientific journals, improvements have been observed in recent years (Large et al., 2010; Warner et al., 2014). Large and colleagues demonstrated that the proportion of psychiatric publications from LMICs, as identified via PubMed, Getting Started 107 107 increased from 8.0% in 1998–2002 to 12.5% in 2003–2007. Similarly, Warner and others reported an increase in LMIC research publications in a leading addiction journal (Tobacco Control), from 10.1% in 1992–2006 to 30.9% in 2007–2011.h increased from 8.0% in 1998–2002 to 12.5% in 2003–2007. Similarly, Warner and others reported an increase in LMIC research publications in a leading addiction journal (Tobacco Control), from 10.1% in 1992–2006 to 30.9% in 2007–2011.h increased from 8.0% in 1998–2002 to 12.5% in 2003–2007. Similarly, Warner and others reported an increase in LMIC research publications in a leading addiction journal (Tobacco Control), from 10.1% in 1992–2006 to 30.9% in 2007–2011.h The relative lack of studies emerging from many developing countries in a multitude of research areas, however, provides ample topics for publication. Recent graduates have an easy publication target: their dissertations or theses. Academics will likely conduct new research. Further, people in government agencies, clinical settings, and nongovernmental organizations, who may not have access to original data, might consider alternative publication routes such as narrative or systematic reviews that involve synthesizing the results of mul tiple research studies on a specific subject. The Cochrane Collaboration site (www.cochrane.org) can be consulted for information on potential review top ics and systematic-review writing procedures. Similarly, it is possible to write case studies, letters, or policy and opinion pieces, all of which can stimulate public debate and influence policies. Language Many times a manuscript is rejected by a journal not because of the quality of the research but because of the authors’ failure to express their ideas clearly. For authors whose first language is not English, translating one’s work for English- language journals can be difficult. Writing in English can even be a challenge for individuals who have attended English-language academic institutions and who have written their theses or dissertations in English. Converting the dis sertation or thesis to the shortened format required for most journals can add to the difficulties of working in a second or third language and can lengthen the time to publication. To manage such language constraints, it is advisable to invite a native English speaker to serve as a co-author and help with editing, as long as she or he meets all the key authorship criteria. International conferences and meetings can be a good forum for networking with potential co-authors. Alternatively, authors may consider using English-language editing services, which usually entail a fee (e.g., http://webshop.elsevier.com/languageservices/ languageediting, http://wileyeditingservices.com/en/english-language-editing). Selecting an Appropriate Journal As indicated in previous chapters (e.g., Chapter 3), there are many addiction journals, but the selection of the “right” journal can present special challenges for those from LMICs. A number of competing considerations might influence the choice. As indicated in previous chapters (e.g., Chapter 3), there are many addiction journals, but the selection of the “right” journal can present special challenges for those from LMICs. A number of competing considerations might influence the choice. First, in both developed and developing countries, many academics are under pressure to publish in “high-impact” peer-reviewed journals (see Chapter 3 for a discussion of impact factors). In South Africa, for example, academic insti tutions receive government subsidies based on the number of peer-reviewed publications produced in journals that have been accredited by the Department of Higher Education and Training. Moreover, in many LMIC institutions, the academic evaluation of faculty and their potential for career advancement is dependent on their publication record. Second, authors might be faced with having to choose between publishing in a high-impact international journal that furthers their research careers or pub lishing in a low-impact local journal that reaches the public health audience of interest. In some cases, a middle-ground solution can be reached (e.g., publish ing one’s papers in both types of journals under agreed-upon conditions; see Chapters 3 and 4 for a discussion).hfl Third, the topical foci or missions of different journals must also influence one’s choice. Some journal reviewers and editors might not have an interest in studies of non-American or non-European populations. Advance familiariza tion with the contents of the journal under consideration can help to gauge the likelihood that an LMIC health or addiction issue would engage the journal’s editors and readership. 108 Publishing Addiction Science 108 Finally, one could consider publishing in an open-access journal, which is usually accompanied by payment (although it is important to take heed of the predatory publishers previously mentioned!). Advantages to authors might include increased accessibility and citations, which contribute to researchers’ rankings and assessments. However, submission or publication costs can be high and difficult to justify in the case of limited research funds. Access to Literature and the Internet In numerous academic and other institutions in LMICs, access to journal articles, books, and other relevant literature is a major challenge that hinders research, writing, and publishing. Paper copies of articles and other literature often have to be ordered via slow, costly, and unreliable interlibrary loan sys tems. Furthermore, many academics do not have easy access to online journals because (a) they have unreliable Internet connections, (b) their institutions do not own subscriptions to the required journals, or (c) they might be unaware of free or reduced-cost options for accessing journal articles. In 2002, the World Health Organization and a number of major publishers established the Health Inter-Network Access to Research Initiative (HINARI) to directly address such difficulties. HINARI provides free or reduced-cost online-journal access to health workers and researchers from local, not-for-profit institutions in many LMICs. More information about the initiative, including eligible countries, instructions for access, and related initiatives is available on the HINARI web site (www.who.int/hinari/en). Getting Started 109 Getting Started 109 109 The lack of consistent and reliable Internet access also causes problems at the online article-submission stage for authors from LMICs. This process can be lengthy even for those with good Internet connections. Establishing collabora tions with researchers who have better access to these resources might, in some cases, help to address this challenge. Comment: Be Optimistic Despite the significant challenges for novice scientists from LMICs, there are advantages to the relative lack of existing research for those just setting out on their research and publication careers. One might be able to claim truthfully that the research has never been conducted or replicated outside of the developed world. Furthermore, the presence of numerous academics from LMICs who con tinue to be prolific despite the under-resourced settings in which they work pro vides evidence that many of the aforementioned difficulties are surmountable. Challenges of Rejection As noted above, it is quite common for manuscripts to be rejected for pub lication after initial submission. Papers from non-European and non–North American settings are sometimes rejected because the reviewers or editors are not aware of the significance of the research in its cultural or local context. In such cases, authors may exercise their right to appeal the rejection if they believe it is based on the editors’ or reviewers’ lack of appreciation of the impor tance of the topic. It might also be useful to precede submission with an email to the journal editor about the topic and its importance before sending it in. Conclusions: Take the Long View A career in addiction science is not for everyone, but it can be very reward ing for those who have the motivation and the aptitude (Edwards, 2002). The best way to begin is to attempt publication of one’s thesis or dissertation, work closely with one or more well-published investigators, employ the writing strat egies discussed, and find a place for postdoctoral research or clinical training. The writing process from student to postdoctoral fellow to junior researcher is generally the same, although the level of autonomy increases with each tran sition. Greater autonomy is usually accompanied by more security regarding one’s place in the publication process and an increased ease in negotiating authorship order. Further, full-time research scientists are not the only ones who enjoy the rewards of publishing. Those who work in clinical settings, government agencies, and other organizations often find that while journal 110 Publishing Addiction Science 110 publications are not rewarded by their employers, neither are they likely to be discouraged. The pros of publishing one’s work usually outweigh the cons. h In this chapter, some basic guidelines have been outlined for inexperienced authors. Although there is no magic formula for guaranteed publication, find ing a mentor, learning to persevere in the face of rejection, and never ceasing to believe in addiction science are key elements to the process. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. Notes 1 For the general purposes of streamlining and efficiency, the term faculty member or faculty (as an adjective) is used throughout this chapter to repre sent any kind of higher education advisor, supervisor, teacher, or researcher who might otherwise be called a researcher, a lecturer (junior or senior), a professor, etc. It should be noted that, depending on the country and/or institution, different terminology may be used.i 1 For the general purposes of streamlining and efficiency, the term faculty member or faculty (as an adjective) is used throughout this chapter to repre sent any kind of higher education advisor, supervisor, teacher, or researcher who might otherwise be called a researcher, a lecturer (junior or senior), a professor, etc. It should be noted that, depending on the country and/or institution, different terminology may be used.i f 2 Let us not forget that this is the true purpose of scientific publishing! 3 For students at some institutions, the dissertation articles must be published before the dissertation can be approved, and publication must therefore be prepared at an earlier stage.h 3 For students at some institutions, the dissertation articles must be published before the dissertation can be approved, and publication must therefore be prepared at an earlier stage.h 4 See http://core.ecu.edu/psyc/wuenschk/Help/ThesisDiss/thauth.htm 5 And of course, those faculty members are usually getting paid as well— publication is an expected part of the job. 6 Including all of the authors of this chapter! References American Psychological Association. (2010, June 1). Ethical principles of psy chologists and code of conduct: Including 2010 amendments. Retrieved from http://www.apa.org/ethics/code/index.aspx. American Psychological Association. (2010, June 1). Ethical principles of psy chologists and code of conduct: Including 2010 amendments. Retrieved from http://www.apa.org/ethics/code/index.aspx. Azar, B. (2006, March). Publishing your dissertation. gradPSYCH Magazine. Retrieved from http://www.apa.org/gradpsych/2006/03/dissertation.aspx. Azar, B. (2006, March). Publishing your dissertation. gradPSYCH Magazine. Retrieved from http://www.apa.org/gradpsych/2006/03/dissertation.aspx. Beall, J. (2012). Predatory publishers are corrupting open access. Nature, 489(7415), 179. DOI: https://doi.org/10.1038/489179a Beall, J. (2015, January 2). Beall’s list of predatory publishers 2015. Retrieved from http://scholarlyoa.com/2015/01/02/bealls-list-of-predatory-publishers-2015/. Getting Started 111 Getting Started 111 Getting Started 111 Cham, J. (2005, March 13). The author list: Giving credit where credit is due. Piled Higher & Deeper: A Grad Student Comic Strip. Retrieved from http:// phdcomics.com/comics.php?f=562. Cornell, E., Doshi, R., Noel, J., & Rusch, L. (2014). Authorship rights of graduate students. Unpublished guideline prepared for the Department of Commu nity Medicine and Health Care, University of Connecticut School of Medi cine, Farmington, CT.i Edwards, G. (Ed.). (2002). Addiction: Evolution of a specialist field. Oxford, England: Blackwell Publishing. g g Farmington Consensus. (1997). Addiction, 92, 1617–1618.l Fine, M. A., & Kurdek, L. A. (1993). Reflections on determining authorship credit and authorship order on faculty-student collaborations. American Psychologist, 48, 1141–1147.h Global Forum for Health Research. (2004). The 10/90 report on health research 2003–2004. Geneva, Switzerland: Author. Retrieved from http:// announcementsfiles.cohred.org/gfhr_pub/assoc/s14789e/s14789e.pdf. ih Hagen, N. T. (2010). Deconstructing doctoral dissertations: How many papers does it take to make a PhD? Scientometrics, 85, 567–579. Gross, D., Alhusen, J., & Jennings, B. M. (2012). Authorship ethics with the dissertation manuscript option. Research in Nursing & Health, 35, 431–434.fi Harvard Medical School Office for Research Issues. (1999, December 17). Authorship guidelines. Retrieved from http://hms.harvard.edu/about- hms/integrity-academic-medicine/hms-policy/faculty-policies-integrity- science/authorship-guidelines. g Humeniuk, R., Ali, R., Babor, T., Souza-Formigoni, M. L. O., de Lacerda, R. B., Ling, W, McRee, B., Newcombe, D., Hemraj, P., Poznyak, V., Simon, S., & Vendetti, J. (2012). A randomized controlled trial of a brief intervention for illicit drugs linked to the Alcohol, Smoking and Substance Involvement Screening Test (ASSIST) in clients recruited from primary health-care set tings in four countries. Addiction, 107, 957–966. Large, M., Nielssen, O., Farooq, S., & Glozier, N. (2010). Increasing rates of psy chiatric publication from low- and middle-income countries. International Journal of Social Psychiatry, 56, 497–506. Miller, P. M. (2011). Introducing the ISAJE-PARINT Online Mentoring Scheme. References Journal of Groups in Addiction & Recovery, 6, 272. Morisano, D. (2013). Goal setting in the academic arena. In E. A. Locke & G. Latham (Eds.), New developments in goal setting and task performance (pp. 495–506). New York, NY: Routledge. Rehm, J., Baliunas, D., Borges, G. L. G., Graham, K., Irving, H., Kehoe, T., Parry, C. D., Patra, J., Popova, S., Poznyak, V., Roerecke, M., Room, R., Samokhvalov, A. V., & Taylor, B. (2010). The relation between different dimensions of alcohol consumption and burden of disease—an overview. Addiction, 105, 817–843. 112 Publishing Addiction Science 112 Resta, R. G., Veach, P. M., Charles, S., Vogel, K., Blase, T., & Palmer, C. G. (2010). Publishing a Master’s thesis: A guide for novice authors. Journal of Genetic Counseling, 19, 217–227. g Robinson, S., & Dracup, K. (2008). Innovative options for the doctoral disserta tion in nursing. Nursing Outlook, 56, 174–178. Rudestam, K. E., & Newton, R. R. (1992). Surviving your dissertation: A compre hensive guide to content and process. Newbury Park, CA: Sage. Saunders, J. B., Aasland, O. G., Babor, T. F., de la Fuente, J. R., & Grant, M. (1993). Development of the Alcohol Use Disorders Identification Test (AUDIT): WHO collaborative project on early detection of persons with harmful alcohol consumption--II. Addiction, 88, 791–804.h Shore, B. M. (2014). The graduate advisor handbook: A student-centered approach. Chicago, IL: The University of Chicago Press. DOI: https://doi. org/10.7208/chicago/9780226011783.001.0001hi Trammell, A. (2014, October 14). The benefits of publishing as an undergraduate. Retrieved from https://publish.illinois.edu/ugresearch/2014/10/14/the- benefits-of-publishing-as-an-undergraduate/. i University of Alberta Faculty of Graduate Studies and Research Council. (1996, November 15). Intellectual property policies: Guidelines for authorship. Retrieved from https://uofa.ualberta.ca/graduate-studies/about/graduate- program-manual/section-10-intellectual-property/10-2-guidelines-for- authorship. University of Cambridge. (2014, November). Good research practice guidelines (Section 9): Dissemination and publication of results. Retrieved from http:// www.research-integrity.admin.cam.ac.uk/sites/www.research-integrity. admin.cam.ac.uk/files/good_research_practice_guidelines_11.14.pdf.fi i University of Pennsylvania’s Office of the Provost. (2013, February 15). Fair ness of authorship credit in collaborative faculty-student publications for PhD, AM, and MS students. Retrieved from https://provost.upenn.edu/policies/ pennbook/2013/02/15/fairness-of-authorship-credit-in-collaborative- faculty-student-publications-for-phd-am-and-ms-students. University of Toronto. (2007). Intellectual property guidelines for graduate students & supervisors. Retrieved from http://www.sgs.utoronto.ca/currentstudents/ Pages/Intellectual-Property-Guidelines.aspx. Warner, K. E., Tam, J., & Koltun, S. M. (2014). Growth of Tobacco Control publications by authors from low- and middle-income countries. Tobacco Control, 23, 231–237. Washington University in St. Louis. (2014, November 21). Policy for author ship on scientific and scholarly publications. Retrieved from https://wustl. edu/about/compliance-policies/intellectual-property-research-policies/ scientific-scholarly-authorship/.h i y Wuensch, K. (2008, November 30). Thesis authorship. A. Advisor’s Responsibilities A. Advisor’s Responsibilities 1. Meet regularly with students and be contactable at other times. 2. Arrange substitute advising during extended absences. 3. Advise on course selection. 3. Advise on course selection. 4. Assist in the preparation for comprehensive or oral examinations. 5. Help prepare conference and journal presentations based on work done in the program and assist with applications for support to attend suitable conferences at a reasonable distance and on whose programs students earn a place. p 6. Help apply for funds to cover direct research costs and to provide sti pends to full-time students. 7. Provide feedback within a mutually agreed time-frame on written work submitted for review. Mutual Expectations Regarding Research Advising High Ability and Inquiry Research Group Department of Educational and Counselling Psychology, McGill University These notes are designed as guidelines to facilitate positive and mutually ben eficial student-advisor relationships and to avoid problems on matters such as authorship and credits on publications, the extent of participation in activities other than the Thesis, Research Project, or Special Activity, and future access to data collected in the course of our work together. Some of the activities described below may be conducted in groups. Where these notes hinder rather than help, they should be amended to meet mutually acceptable needs, in gen eral or as occasions arise. References Retrieved from http:// core.ecu.edu/psyc/wuenschk/Help/ThesisDiss/thauth.htm. Getting Started 113 113 Getting Started 1 B. Students’ Responsibilities 1. Regularly pursue work and keep the advisor informed of progress or problems. 2. To a mutually agreed degree that respects other responsibilities and pri orities, contribute to advancing team activities that further the common good of all of us working together—e.g., workshops for teachers, parent contacts, library orders, data bases, maintaining bibliographies and mail ing lists, convening meetings, maintaining computers and supplies. These tasks will be equitably distributed. y 3. Join in the preparation of conference presentations and publications on research and other activities done with faculty members. 114 Publishing Addiction Science 4. With appropriate guidance, prepare a draft version of the thesis or major report, normally within 3 months of its final presentation for master’s degrees, or 6 months for doctoral degrees; after that point the advisor may take over such preparation and the order of authorship may be changed (within CPA, APA and McGill authorship guidelines). 5. Apply for scholarships and bursaries, especially FQRSC, McGill, and SSHRC (where eligible) [this list of funding sources should be amended to match local availability]. 6. Participate to a mutually agreed extent in teaching-related activities such as the TA course. 7. Take a professional role in one’s discipline by undertaking at least one student or regular membership in an appropriate professional or aca demic organization. 8. Keep at McGill a copy of raw data, coding sheets, instruments, and sub ject-identification data. i 9. Upon graduation, leave with the advisor a printed copy of the main research report, and an electronic copy in modifiable form (e.g., not PDF) of any data and the text of the thesis or project.t 10. Use Microsoft Word and APA [or other, as appropriate] style for written submissions. 11. Report annually in writing on progress and contributions (department and university forms). y 12. Regularly attend and participate in research-team meetings. C. Joint Responsibilities 1. Give full credit for the contributions of others and to research funding in all products. 2. Assign authorship according to the latest APA publication guidelines. (For example, if a thesis topic or report is entirely the student’s original contribution, then the advisor’s contribution is due a footnote. Shared scientific responsibility calls for co-authorship, with the student as first author on the main points of the student’s research of those for which the student took primary creative responsibility, and the advisor as first author on any specific subpoints which the advisor contributed or a broader study of which the student is part.) 3. Both have unlimited access to the data collected on or about the topic of a thesis or project during the time worked together, plus any other that may be agreed to, giving due credit to its origin either by footnote or reference to previous publications. D. Degree Covered by this Agreement Check-mark all that apply [and revise this list as needed for your institution]: o PhD Thesis or Dissertation o MA Thesis o MA Research Project D. Degree Covered by this Agreement Check-mark all that apply [and revise this list as needed for your institution]: o PhD Thesis or Dissertation o MA Thesis o MA Research Project Getting Started 115 o MEd “Special Activity” Project o Undergraduate Honors Thesis o Independent Graduate Student Project o Independent Undergraduate Student Project o Other (specify): _______________________________________ o Not for formal credit E. Comments, Additions, or Special Notes [expand this space as required] F. Signatures We agree to work together in an advisory relationship in accord with the above guidelines. _______________________________ _______________________________ Advisor Date Student Date _______________________________ _______________________________ Printed Name Printed Name One copy for each. Note: a This sample contract was also reproduced in: Shore, B. M. (2014). The gradu ate advisor handbook: A student-centered approach. Chicago, IL: The University of Chicago Press (in the series Chicago Guides to Academic Life). DOI: https:// doi.org/10.7208/chicago/9780226011783.001.0001 It is agreed that. . . 1. Graduate students are a vulnerable population with regard to authorship issues in scientific publications because of their junior status in the aca demic hierarchy. 1. Graduate students are a vulnerable population with regard to authorship issues in scientific publications because of their junior status in the aca demic hierarchy. 2. Graduate students rely on principal investigators, faculty members, and other individuals in positions of power for funding and for access to research opportunities and data. 3. Graduate students rely on principal investigators, faculty members, and other individuals in positions of power for successful completion of any graduate program. 4. Graduate students who participate in research studies often fulfill neces sary roles and provide vital support toward the completion of research projects conducted by teams of faculty, students, and staff. 116 Publishing Addiction Science 116 5. Principal investigators, faculty members, and other individuals in posi tions of power can influence, directly or indirectly, positively or nega tively, the credit given for work done by students following the success ful completion of a research study.t 6. Authorship credits are often important for graduate students’ careers. 7. Students may be given inappropriate and unethical authorship credits to enhance the student’s chances of success. Conversely, students may be denied appropriate and ethical authorship credit.h 8. There is little recourse for a graduate student should a principal investi gator, faculty member, or other individual in a position of power nega tively influence deserved authorship credit.i yl 9. A set of rights and guidelines to protect graduate students and to define faculty–student authorship criteria are needed.h 10. The rights and guidelines listed in the sections “General Research Stud ies” and “Dissertation or Thesis Research” listed below shall be adopted to protect graduate students from negligence or mistreatment and to define graduate student authorship. General Research Studies 1. A graduate student who has participated in a research study conducted by a faculty member who is affiliated with graduate student’s program or who supervises the graduate student has the right to be invited to become an author on any report, abstract, journal manuscript, or other document developed based on the results of the study, provided the student has completed sufficient training. fi a. Study participation may include, but is not limited to, the following: recruitment of study subjects, providing an intervention, data col lection, data entry, questionnaire coding, supervision and training of study personnel, writing of the research protocol, or the provision of other technical services.i b. Authorship is defined as providing a major contribution to a report, abstract, journal manuscript, or other document including, but not limited to, the following: writing the final version of the submission, designing the study, interpreting the results, study coordination, sta tistical analysis, laboratory analysis, data management, or providing informative advice on study design and analysis.fi c. Sufficient training may include, but is not limited to, the following: completion of specific coursework, knowledge of the subject matter, or knowledge of the study design. The extent of training is to be agreed upon prior to the student’s involvement in the research study and occurs between the student, the study’s principal investigator, and/or the student’s major advisor. Getting Started 117 2. A graduate student’s role in the drafting of a report, abstract, journal manuscript, or other document, as well as possible authorship position, is to be discussed prior to the first draft of a report, abstract, or journal manuscript. 3. Financial compensation, whether through graduate assistantships or by other means, is not a replacement for authorship credit. 4. Acknowledgement is not a replacement for authorship credit. 5. A graduate student’s role on a report, abstract, journal manuscript, or other document shall not change without notifying the student, allow ing the student to respond to the notification, and agreement of all co-authors. 6. A graduate student has the right to refuse authorship on a report, abstract, journal manuscript, or other document for any reason. 7. If a disagreement over authorship occurs between a graduate student and a principal investigator, the graduate student may appeal to the Director of their graduate program or the Chair of the department with which the principal investigator is affiliated to appoint an unbiased arbitration com mittee to resolve the conflict. General Research Studies This committee will be comprised of three individuals and will consist of at least one student.h 8. The principal investigator or any other faculty member shall not penal ize a graduate student by eliminating future authorship opportunities, removing study responsibilities, assigning an excessive workload, with holding monetary compensation, or imposing any other punishment, directly or indirectly, should the student disagree with the principal investigator over authorship or invoke independent arbitration.ht 9. These guidelines shall apply for an agreed upon amount of time after the student graduates, changes institutions, or otherwise is no longer affiliated with the graduate program. The time limit shall be agreed upon by the stu dent, the study’s principal investigator, and/or the student’s major advisor. 9. These guidelines shall apply for an agreed upon amount of time after the student graduates, changes institutions, or otherwise is no longer affiliated with the graduate program. The time limit shall be agreed upon by the stu dent, the study’s principal investigator, and/or the student’s major advisor. Dissertation or Thesis Research 1. Research and analyses conducted by a graduate student for the purposes of fulfilling doctoral dissertation or master’s-thesis requirements is con sidered the property of the graduate student, regardless of who is listed as principal investigator on funding, regulatory documentation, or other documentation.i 2. A graduate student has the right to first authorship on any report, abstract, journal manuscript, or other document that is created based on the results of dissertation or thesis research conducted by said graduate student. 3. The principal investigator listed on funding, regulatory documentation, or other documentation that supports a graduate student’s dissertation 118 Publishing Addiction Science 118 or thesis research shall in no way impede, and will support, said gradu ate student in creating a report, abstract, journal manuscript, or other document. 4. Data generated from dissertation or thesis research will revert to the prin cipal investigator if, and only if, a graduate student has not produced a first draft of a report, abstract, journal manuscript, or other document within a previously agreed upon time window. a. If no window is agreed upon, then the data generated from disser tation or thesis research shall not revert to the principal investigator under any circumstances.it b. If the first draft of a report, abstract, journal manuscript, or other doc ument is not produced by the student within the previously agreed upon time window, the principal investigator must include the gradu ate student in the drafting of a report, abstract, journal manuscript, or other document using the guidelines specified in the “General Research Studies” section, unless the graduate student agrees to be excluded from the process. 5. A graduate student has a right not to publish, and not to have published, dissertation or thesis research. a. A graduate student may invoke this right at any time prior to, during, or after the previously agreed upon publication window, unless the pre viously agreed upon window has already been exceeded, the graduate student has been included in the authorship process, and the results have already been published in a peer-review journal; or the graduate student has previously agreed to be excluded from the process. 6. How to cite this book chapter: Pates, R and Gabrhelík, R. 2017. Addiction Science for Professionals Working in Clinical Settings. In: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P. (eds.) Publishing Addiction Science: A Guide for the Perplexed, Pp. 119–131. London: Ubiquity Press. DOI: https://doi.org/10.5334/bbd.f. License: CC-BY 4.0. Dissertation or Thesis Research If a disagreement over authorship occurs between a graduate student and a principal investigator, the graduate student may appeal to the Director of their graduate program or the Chair of the department with which the principal investigator is affiliated to appoint an unbiased arbitration com mittee to resolve the conflict. This committee will be comprised of three individuals and will consist of at least one student.h 7. The principal investigator or any other faculty member shall not penal ize a graduate student by eliminating future authorship opportunities, removing study responsibilities, assigning excessive workload, with holding monetary compensation, or by imposing any other punishment, directly or indirectly, should the student disagree with the principal investigator over authorship or invoke independent arbitration. bThis guideline was developed by Erin Cornell, Riddhi Doshi, Jonathan Noel, and Lisa Rusch in April 2014, when they were graduate students at the Univer sity of Connecticut in the Graduate Program in Public Health. CHAPTER 6 Introduction In many professional fields, the number of well- trained staff who never do research or publish anything after they have quali fied is surprising (e.g., Jowett et al., 2000; Salmon et al., 2007), especially given that the work clinicians perform, whether in the statutory or non-statutory sector, is usually based (or should be based) on proven results and methods founded on research-related best practices.h This lack of willingness to undertake research or to publish research results may result from lack of confidence, opportunity, or willing collaborators. But as will be seen in this chapter, there are plenty of opportunities and sub jects appropriate to study systematically in the clinical setting. Although this chapter is not meant to teach research methods, it is aimed at those who have previously had some research training and who have had the opportunity to undertake research projects. It is also aimed at those interested in evaluating their work or investigating some aspect of their work that may be worthy of publication. We cannot take for granted that the majority of professionals have the skills for conducting scientifically sound studies. To conduct a research study using appropriate design, adequate measures, and correct statistics can sometimes be difficult. Further, there are additional problems of trying to publish the findings in peer-reviewed journals. j Early addiction practice was based on a clinical approach (problems were observed, described, and explained), and this slowly started to shift toward empiricism (allowing for testing hypotheses through observation and experi ment). More recently, an evidence-based approach to addiction services has been promoted and widely accepted. Evidence based practice (or applied addiction science) means that the nature and method of addiction services is based on findings from research studies. The level and quality of clinical work is quantified. Quantified results serve as an evidence of effectiveness or inef fectiveness of any interventions provided.1 In practice, this means a range of things, including treatment of addiction problems, prevention of relapse, and provision of aftercare and other post treatment interventions aimed at helping those in recovery get back to a regular lifestyle. Over time, addiction profes sionals began to ask questions about the effectiveness of the methods being used in the treatment of addiction problems, the prevention of relapse, and the provision of aftercare and other post treatment interventions aimed at reintegrating the person into daily life. As a consequence, interest in appropri ate interventions grew. Introduction This chapter is aimed at doctors, psychologists, social workers, therapists, and other staff in the health sector, social care sector, and criminal justice system (e.g., prisons, probation) working in addiction. It is also written for workers in the nongovernmental (non-statutory or “third”) sector with some professional training or expertise. These clinical workers often are the first to identify new trends in substance use, effects, problematic consequences, and problems that may support or hinder rehabilitation. Therefore, clinicians can play an impor tant role in research. In many developing countries or in countries without a history of alcohol and other drug research, clinicians may be the only people who are able to document problems. At the same time, they also have a duty to identify and collect this information and distribute it. This chapter will discuss what sort of research might be suitable for clinicians, how to approach it, where to publish, and pitfalls in addiction research and publishing. The purpose is to encourage professionals who work in the field of addiction, not primarily as researchers, but as clinicians who have conducted work or research projects that could be worthy of publication. This chapter also provides instruction on how clinicians can collaborate with researchers. Historically, clinicians have played an important role in research. It is worth remembering that the early pioneers in alcohol and other drug research were often doctors such as Trotter, Rush, and Huss (in alcohol research) as well as Dole and Nyswander (in research on the use of methadone in the treatment of 20 Publishing Addiction Science 120 heroin addiction). It is also of note that, today, many of the people working at the top of large research institutes and public health bodies such as the World Health Organization have clinical backgrounds in psychology and medicine. Although a research component is included in many (or most) undergradu ate and postgraduate clinical courses, it is sometimes seen as a process that must be passed before qualification rather than as an exciting opportunity to expand a professional role. Why bother Doing Research? For many clinicians, the idea of undertaking research may seem to be yet another demand on their time and not part of their job. But clinicians should always be asking whether what they do is effective and the best practice. As will be discussed later, research can take many forms in terms of evaluating inter ventions, trying to find the cause of a problem, studying individual cases or reviews of a subject area. Many of these areas may be too complex and involved for the professionals in clinical practice to undertake, but there are some types of research that are well within the capability of clinical staff. Examples of this are research into brief interventions with alcohol and tobacco, which has had an impact on clinical work.ih Many benefits can be derived from taking part in research. There is an intrin sic satisfaction in undertaking a good piece of research, especially if it produces results that may affect your work and make it more effective. There is also the respect that you will earn from your colleagues. But most importantly, per forming research can help to further your career. Even if your work has been mainly clinical, having publications on your résumé or curriculum vitae will do no harm and will probably enhance your career. Future employers will respect your endeavors into research. Introduction Professionals from the field started to search for new ways to achieve better results in less time but with a longer duration of action. Addiction Science for Professionals Working in Clinical Settings 121 Studying the effective factors in addiction services and monitoring the benefits of different interventions became the domain of research. What Research is Appropriate for You? Choosing a research project that is suitable is very different if you are working in a clinical field rather than in an academic institution. In a clinical field, you will need to choose a research subject that permits access to participants (if it is a person-based project) and something that is manageable in the context in which you are working. Many clinical services perform regular audits of their work, and these are already simple forms of research. Of course, if this type of research is undertaken, it needs to be more rigorous than a standard audit and should conform to a research protocol. Sometimes research questions may come from your search for a solution to a problem—you find that little work has been published in that subject area. In the 1990s, when the first author (R.P.) wanted to find treatments for compulsive injecting (needle fixation), a literature search revealed just one article published 20 years before that described three cases. This nevertheless led to a number of research collaborations in a clinical setting in which the problem was stud ied and psychological theories and treatment options were developed (Pates & Gray, 2009; Pates et al., 2001). 122 Publishing Addiction Science If the work you do routinely is common practice and already described in the literature, then it is unlikely to be of interest to journals. However, if you are doing something innovative or have noticed unusual results, this may well be worth formalizing and investigating. If you are planning innovative work, this should be investigated carefully following proper designs and ethical considerations.hfi The late Griffith Edwards, a great champion of addiction science and some one who was influential in encouraging junior researchers to publish their work, made an interesting observation that many clinicians will recognize. In a book of his to be published posthumously (Edwards, in preparation), he asked the question of where addiction research ideas came from. He observed that clinical research often comes from something said by a patient but also noted that the clinician “must have ears with which to listen. It is often too easy to ignore what patients may be saying by believing that expertise lies with the expert! What Research is Appropriate for You? 123 Addiction Science for Professionals Working in Clinical Settings Addiction Science for Professionals Working in Clinical Settings This can be exciting work but requires a lot of extra effort to ensure that the interventions are the same in each center and that all the protocols are being followed in the same way. This can be exciting work but requires a lot of extra effort to ensure that the interventions are the same in each center and that all the protocols are being followed in the same way. Another type of investigation clinicians can do is historical research con ducted by extracting data from case notes. For example, the first author of this chapter (R.P.) wanted to examine whether outcomes had changed in the clinic in which he was working from the establishment of the service 20 years pre viously to the present. This was performed by asking a number of questions that he formulated based on case notes and by taking a cohort of the first 200 patients registered with the clinic to establish things such as morbidity, mor tality, recovery, and loss to the service. These data were then compared with data obtained from another cohort some 15 years later. This study evaluated a span of time when changes in practice were occurring in service delivery, and it was important to see if outcomes had improved. The study results actually had important consequences in terms of delivering services and learning lessons from practices that were found to be too rigid. Good quantitative research is worth pursuing if the topic is original and not just repeating previous research. But, of course, many topics that have been researched are the product of an original idea that was investigated and then later research added to the findings and expanded it. In this way, individual studies become a body of research. Sometimes it is worth investigating a previ ously published research topic by adding a new dimension or helping to gener alize a finding through the study of a different group. It must be borne in mind that, if the study is using a control group for comparison, it would be unethical to withhold a recognized treatment from the control group, even in the inter ests of the research.i Qualitative research is becoming more common in the addiction field. What Research is Appropriate for You? He went on to describe a situation in which a patient of his commented that he (Edwards) had previously given the patient very bad advice: Edwards had told the patient that, to become sober, the patient would need a lengthy in-patient stay—the current practice at that time. The patient said he did not need that sort of help, would not accept it, and that it would mean the end of his business if he chose that path. Inspired by this man, Edwards went on to conduct a comparison trial of in-patient versus out-patient treatment and found, at the 12-month follow– up, there was no significant difference between the two groups. This evidence helped to overturn the conventional consensus at the time—that in-patient treatment for a significant drinking problem is essential for recovery. This is a good example not only of the need to listen to patients but also of the need to challenge conventional ideas in places where they may be rigidly held. In additional to quantitative reports, some journals will accept case reports or series of case studies (see also Chapter 8 on qualitative research), in which unusual findings may be reported (e.g., uncommon manifestation of diseases, “off-label” uses of medication, previously unreported effects of medications, unexpected effects of treatment). These studies can be of great interest because you may be the first to report a phenomenon—only make sure you are see ing and understanding cause and effect. These can add to the literature in an incremental but important way. It is often in clinical settings that these unusual practices come to notice, which could be the beginnings of a phenomenon or just unusual outliers in the field. i In addition to working within a centre there is the opportunity to work with other professionals doing similar work. This might entail being part of a multi- centre trial, in which a number of treatment centres work on the same project to increase the numbers of people being treated and provide greater statistical power to the analyses. A multi-centre trial also allows for comparison across sites and thus increases the generalizability of findings. This sort of trial is usu ally expensive because it needs coordination, usually from a research center. What Research is Appropriate for You? Twenty-five years ago, it was difficult to get qualitative research published because it was often not seen as “proper” research. That view has changed, and qualitative research is becoming more common. The advantage of conducting qualitative research is that you can investigate questions more deeply and fol low up information that comes out of the research. It is often undertaken with fewer participants than quantitative research but still requires a rigid method ology and the same safeguards. (See Chapter 8 in this book for a full discussion on carrying out qualitative research.)f One major difference between working in a clinical setting and undertaking academic research is that, often in randomised controlled trials, there is a set of exclusion criteria that is used to remove what may be confounding factors for research. The problem for clinicians is that the people they treat are not subject to exclusion criteria. Storbjörk (2014) has written about this in a large piece of research on alcohol problems with 1,125 participants. She asked the following question: If 10 of the most common exclusion criteria were operationalized and applied to this group, what would be the percentage of real-world prob lem alcohol users excluded from her study and how would this exclusion, bias 124 Publishing Addiction Science treatment outcomes. She found that 96% would have been excluded by at least one exclusion criterion. She found that on average, participants fulfilled 2.56 of the less exclusive criteria (eg unemployed or homeless) and 3.99 of the more exclusive criteria (Currently medicated for psychiatric problems or overdose recently). The percentage of treatment seekers excluded because of not meet ing the less exclusive individual criteria ranged from 5% being excluded for lack of education to 80% excluded for past or current addiction treatment. The importance of these results is that if our clinical work with real-world popula tions is informed by biased results, we will not see the same clear results that are published in some academic journals. p j One example of this is in research undertaken in the United Kingdom on the treatment of amphetamine problems by substitute prescribing. This is now a common practice in the United Kingdom. However, one of the exclusion cri teria has always been the presence of comorbid mental health problems, spe cifically because heavy use of stimulants such as amphetamine can produce paranoia and psychosis. What Research is Appropriate for You? Carnwath and colleagues (2002) challenged this by a piece of retrospective research examining the case notes of eight patients with schizophrenia who had been prescribed dexamphetamine for co-existing amphetamine dependence. The authors commented that the patients with co- existing problems had poorer treatment outcomes, often did not comply with treatment plans, and had frequent periods of hospitalization. However, they found that, in four of the eight cases examined, the prescription of dexamphet amine led to good progress in terms of both substance use and mental health. In two cases, progress was more equivocal although there had been some ben efit, and two cases were deemed to be treatment failures but the condition of the patients was no worse at the end of treatment than at beginning. There was greater adherence to neuroleptic regimes, and none of the patients suffered an exacerbation of their psychotic symptoms as a result of treatment. This is an example of where exclusion criteria for being part of the trial were ignored and good results followed. It is also true that, although randomised controlled trials are seen as the “gold standard” for research, use of a randomized controlled trial sometimes may be unethical if it means depriving one group of potentially advantageous treatment. An example of this can be seen in a research design in which needle exchanges are established in one city and not in another to measure the incidence of new viral infections among injection drug users. This, of course, would be entirely unethical and would have other methodological problems, unless there were only enough resources to establish programs in one of the cities. How to get Started Before starting on a project, you should discuss it with other colleagues to get their approval and cooperation. If this is seen to be feasible, then a thorough Addiction Science for Professionals Working in Clinical Settings 125 research protocol should be written with a description of the scientific need for the study (a literature search and an explanation of your hypothesis), methods of recruiting your sample of participants, methods of measurement, interven tion, and statistical analysis. If you have any doubts or questions, discuss them with colleagues or other people who are active in the field that you wish to conduct research in. More- experienced colleagues are often interested in what you might be doing and will be happy to answer questions and make suggestions about your line of research. Establish a coordinating committee that can provide advice and discuss the project as it progresses. This committee can include members of your depart ment, but it is often useful to have someone from outside to ask the awkward questions and raise points you might not have thought about before. Another option might be to seek collaboration with doctoral students and postdoctoral students. Doctoral students and postdocs may offer their time, knowledge, and skills while supervised by their mentors. Always make sure all the staff involved in the unit are aware of the research, understand the process, and have any queries answered satisfactorily. These may be the people who refer suitable subjects for your research or whose coop eration you may need to get to the project running smoothly. Any research that involves human or animal subjects will require ethical approval. Where to obtain this will vary from country to country, but usually universities or major health centers will have a standing ethics committee. An application to the ethics committee will have to follow its standards and will possibly involve a personal appearance in front of the committee during which you will answer questions, provide assurances, and discuss potential changes to the research protocol. Research usually requires extra funding. Such funds may be obtained as research grants, obtained as small grants from the employing authority, or absorbed in the normal running costs of the unit. Some research may be con ducted in house with no extra costs by putting in place research protocols that allow other staff and colleagues to know what is being done. How to get Started You will still need to be thorough and objective in your research, but it can be undertaken as part of clinical work. Investigators working in academic institutions will routinely be applying for research grants and will know the main funding bodies avail able in their field. These are likely to be less familiar to clinicians, but research funds are available from small charitable bodies as well as national funding bodies (e.g., the U.S. National Institute on Drug Abuse) and major organiza tions (e.g., the Gates foundation) who have a huge commitment to solving major world social and health problems. To be approved, it is important that you are working in an area covered by the funding body’s activities and that, when you complete the application form, you answer all the questions and explain exactly what you are doing and why.i Make sure you have identified someone experienced in statistics who may be able to guide you on statistical techniques. Collaboration with a statistician Publishing Addiction Science 126 from the beginning, when writing the project proposal, is encouraged (e.g., when focusing on patients, power sample analyses should be calculated before conducting research or when choosing appropriate data-collection tools). This is also true for someone experienced with quantitative methods when conduct ing qualitative research As an example, an on-going project in the Czech Republic was conducted in therapeutic communities for users of illicit drugs. Research activities are rela tively infrequent in these facilities because of many contextual reasons (e.g., low capacity of individual facility, low interest by staff, no uniform treatment mod els). Within last few years, a new, interesting research problem has emerged (not only) in the context of therapeutic communities: attention-deficit/hyper activity disorder as a comorbid factor and risk factor for significantly higher treatment drop-out and reduction of treatment effect (Miovský et al., 2014). This interesting and important issue was formulated and clarified through a systematic discussion and series of meetings with staff within a two-year pre paratory phase. The Czech team decided to invite the National Association of NGOs and its working group of therapeutic communities to participate. After a selection procedure, they contracted particular therapeutic communities, trained the staff in data-collection methods, and supervised the data-collection procedure. Particular communities were direct partners of the study and had participated since the beginning. How to get Started To stay within the study budget and make the study manageable, however, the original concept had to remain limited because of potential travel costs and technical complications linked to the difficulty of testing all new clients for attention-deficit/hyperactivity disorder (which is an unpredictable and irregular procedure). Nonetheless, it is also a good example of how to create, through networking, a very attractive opportunity for exten sive and sophisticated clinical research with a large number of clients. Where Should I Publish? Choosing the right journal to which you may submit your finished article should be done with care. Chapter 3 of this book discusses this and should be consulted. There are many journals that focus specifically on addiction, but, in addition to these “addiction specialty journals,” scientists and practitioners who work in the field also have a “mother” profession or discipline in which they have been trained (e.g., medicine, psychology). These disciplines also publish many jour nals in their fields, and these journals may publish articles on addiction. There are also journals published in countries in which information may be more local and more relevant to national or local populations. Therefore, there is a wide variety of potential journals to which you may submit your manuscript. You must consider, therefore, whether the subject is of national or interna tional importance. If the subject is mainly of interest to people in your country, it may be more appropriate to submit to a national journal. International jour nals may judge whether an article is of international interest and may not accept an article that is more local. However, it may be that a subject that appears to be local in scope becomes of interest to experts in many another parts of the world. Addiction is a worldwide problem, and practices spread. One example of this is that the use of water pipes to smoke tobacco is very common in the Middle East. Therefore, this form of substance use may be seen to be local. However, the practice does have great potential health risks, and the effects of the diaspora of refugees from this region to many other parts of the world will also export this practice and the concomitant health risks. Both authors and editors need to bear this sort of situation mind. Before submitting, check the impact factor and acceptance rate of the jour nal. This can be found in Chapter 3, Table 3.1, in this book. Typically, journals with higher impact factors have lower acceptance rates. If you are submitting to a high-impact-factor journal, it will be more difficult for you to get your article accepted unless it is of high originality and good-quality science. Furthermore, check the instructions for authors either in the relevant journal or on the website to ensure that the journal accepts the type of work your article describes. Who should be in My Article-Writing Team? Conducting a good-quality research project requires knowledge, skills, and enthusiasm combined with high levels of persistence. Writing a scientific arti cle is, however, a discipline on its own. Many colleagues who are involved in the data-collection phase of your research will not participate in the actual writing of the article (s) for various reasons (e.g., because of a low interest in writing, lack of confidence or time). It is often the case that data are available but that there are only a limited number of people who are willing to write an article based on it. You may end up writing the actual manuscript on your own. To avoid this situation, you may want to start an early search for collaborators who will help you to write and submit articles to save time. In the previous section, we suggested that a statistician be part of your research project. With the advent of modern statistical packages for your com puter, it is often simple to run the statistics, but frequently people are using Addiction Science for Professionals Working in Clinical Settings 1 127 inappropriate statistics for the problem. It is important to get this right before you start. When your article is reviewed, your statistical techniques will be examined. If you have used the wrong technique, the article will be rejected. This will either mean you have wasted much time and effort or that it will take a lot more time to rework the statistics—which may of course then produce different outcomes. Similarly, preparing a high-quality qualitative article is dif ficult without the appropriate experience of a good qualitative researcher. What are the Pitfalls to Doing Research in this Setting? There are many potential pitfalls in doing research in a clinical setting. Yet, if you are well prepared, you may avoid most of these. As has been mentioned above, you need set a clear research question that you want to answer, plan your research design, plan your methodology, decide on your statistical techniques, and make sure you get ethical approval. One impor tant way to ensure the research will be finished and finished correctly is to involve your colleagues. If you share your work with them, they are more likely to cooperate, identify study participants for you, and highlight prob lems you may not have considered. One exercise that you can do is to write an abstract of the research without the results. In doing this brief exercise, you can set out the methodology, subjects, research question, and statistical techniques.fi One of the difficulties in conducting research in clinical areas occurs when there is an ethical conflict between using your clients for research and whether the research or the clinical needs take priority. One must always place clinical need above research interest. Sometimes it is better to undertake research and clinical work in different locations to keep your clinical interests and scientific interests apartf Choosing the right sampling technique is a crucial step that affects the whole study. If sampling is not done appropriately, the results may be flawed, irre spective of how well the study was conducted overall. When you are ready to start your research and wish to recruit study participants, you may find that there were many people who had the problem you are researching at the time you decided to do the research, but, once you start recruiting, they often seem scarce! This is a phenomenon noted by clinical researchers. Therefore, be pre pared to go wider to recruit your participants by perhaps involving another agency or advising colleagues in similar facilities to yours that you are trying to recruit. If you are running a trial with a control group, make sure that your control group is a genuine control and match the experimental group in every way possible, including matching by demographic features and definitions of the problem being researched. Where Should I Publish? Make sure when you submit your article it conforms to the standards of the journal. Follow the instructions for authors regarding word length, style of referencing, and formatting of tables and figures. 128 Publishing Addiction Science 128 What are the Pitfalls to Doing Research in this Setting? Too often, a reviewer on a journal will see that the control group does not match the experimental group and will reject an article on that basis Another important aspect of doing your own research is the choice of appro priate data-collection tools. It is always better to choose standard, standard ized, and well-recognized scales, questionnaires, and other types of measures as opposed to those developed on one’s own. It is not always easy to get research published. But there are some things you can do to increase your chances of getting an article accepted in a well- respected journal. It is well to note the following points: Addiction Science for Professionals Working in Clinical Settings 129 • Scientific writing skills take a long time to acquire, and, with every article pro duced, these skills improve. Endurance and enthusiasm is the key. Also, col laboration with someone who already possesses these skills is encouraged.iifi • Scientific writing skills take a long time to acquire, and, with every article pro duced, these skills improve. Endurance and enthusiasm is the key. Also, col laboration with someone who already possesses these skills is encouraged.iifi • Scientific writing skills take a long time to acquire, and, with every article pro duced, these skills improve. Endurance and enthusiasm is the key. Also, col laboration with someone who already possesses these skills is encouraged.iifi • Scientific journal language is specific and differs among fields. For the begin ner, it may be difficult and timely to write densely, specifically, and clearly. What may help is to read published articles to become familiar with the lan guage style that is used and to ask someone experienced to “polish” the article.i • Scientific literature availability may be a problem for those working in smaller clinical facilities with smaller budgets. Their libraries simply may not be able to purchase access to journal full texts. You may want to invite for collaboration someone from an academic setting or a research facility with access to journal subscriptions. Also, you may ask the study authors for an author’s copy. For more options how to search for scientific literature, see Chapter 7. • Time between having completed the research and actually having an article accepted for publication may take months. The approximate time for receiv ing feedback from a journal is three months. Always try to plan ahead. What are the Pitfalls to Doing Research in this Setting? You can save time by doing literature searches during the data-collection phase. Try to publish outcomes of the pilot phase of your research. • Rejection of an article is common and every author has an experience of receiving negative feedback from the journal on his or her article. Always remember that most rejected articles may be improved based on the feed back that is usually sent together with the letter from the editorial office. Try to learn from the unsuccessful attempts, and do not allow pride or bit terness overcome you. • Fighting frustration should be one of the skills you develop. Research and scientific publishing are very demanding, but getting your article published is very rewarding. All the pain pays off once you see your name connected with an important contribution to the field. Chapters 7 and 8 in this book will help when you write up your work for pub lication. Read them carefully and follow the advice, because this will increase your chances of publication. Acknowledgements We offer many thanks to Richard Saitz for his helpful suggestions in our writing of this chapter. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. Serving as a Reviewer Once you publish your first article, the chances increase that you will be asked by a journal to review someone else’s manuscript. You may want to accept for the following reasons: • reviewing an article will help you see and understand what the journal expects from authors (based, for example, on the reviewer guidelines and • reviewing an article will help you see and understand what the journal expects from authors (based, for example, on the reviewer guidelines and Publishing Addiction Science 130 other requirements) and what the processes are inside the journal’s “black box”; other requirements) and what the processes are inside the journal’s “black box”; • reading a manuscript from a reviewer’s position may help you adopt criti cal scientific thinking that you may later use when you write your own article; and • reviewers are an “endangered species,” and journal editors need competent, expert volunteers when arranging independent evaluation • reviewers are an “endangered species,” and journal editors need competent, expert volunteers when arranging independent evaluation expert volunteers when arranging independent evaluation Conclusion Undertaking research as a clinician can be rewarding both for its intrinsic value and its ability to provide answers to many of those troubling clinical questions. It is also valuable for career development and can be an enhance ment for a department or unit. It will take more time and effort but adds vari ety to the working week. You need to follow proper protocols, gain ethical approval, and obtain advice from colleagues or, if necessary, a local academic department. Do not try to take short cuts or believe that, because you are working in the reality of the treatment setting, you know better than academ ics. Nothing is worse than spending a lot of time and effort on a project for it then failing because of a lack of thorough preparation. Good luck with your research! Note 1 The widely advertised evidence-based approach is viewed by some as too narrow and formalistic. Hjørland (2011) promotes what is called research- based practice that, besides taking into account quantitative approaches, also considers as legitimate theoretical work and qualitative research. Addiction Science for Professionals Working in Clinical Settings 131 131 SECTION 3 SECTION 3 References Carnwath, T., Garvey, T., & Holland, M. (2002). The prescription of dexam phetamine to patients with schizophrenia and amphetamine depend ence. Journal of Psychopharmacology, 16, 373–377. DOI: https://doi. org/10.1177/026988110201600414 Edwards, G. (in preparation). Seeing Addiction (Book). Edwards, G., & Babor, T. F. (Eds.). (2012). Addiction and the making of profes sional careers. New Brunswick, NJ: Transaction Publishers. Hjørland, B. (2011). Evidence based practice: An analysis based on the philoso phy of science. Journal of the American Society for Information Science and Technology, 62, 1301–1310. DOI: https://doi.org/10.1002/asi.21523 Jowett, S. M., Macleod, J., Wilson, S., & Hobbs, F. D. R. (2000). Research in primary care: Extent of involvement and perceived determinants among practitioners from one English region. British Journal of General Practice, 50, 387–389. Miovský, M., Čablová, L., Kalina, K., & Šťastná, L. (2014). The effects of ADHD on the course and outcome of addiction treatment in clients of therapeutic communities: Research design. Adiktologie, 14, 392–400.h Pates, R. M., & Gray, N. (2009). The development of a psychological theory of needle fixation. Journal of Substance Use, 14, 312–324. DOI: https://doi. org/10.3109/14659890903235876 Pates, R. M., McBride, A. J., Ball, N., & Arnold, K. (2001). Towards an holistic understanding of injecting drug use: An overview of nee dle fixation. Addiction Research & Theory, 9, 3–17. DOI: https://doi. org/10.3109/16066350109141769f Salmon, P., Peters, S., Rogers, A., Gask, L., Clifford, R., Iredale, W., Dowrick C., & Morriss, R. (2007). Peering through the barriers in GPs’ explanations for declining to participate in research: The role of professional autonomy and the economy of time. Family Practice, 24, 269–275. DOI: https://doi.org/10.1093/ fampra/cmm015 Storbjörk, J. (2014). Implications of enrolment eligibility criteria in alcohol treatment outcome research: Generalisability and potential bias in 1- and 6-year outcomes. Drug and Alcohol Review, 33, 604–611. DOI: https://doi. org/10.1111/dar.12211 How to cite this book chapter: Lange, P, Pates, R, O’Reilly, J and Ward, J H. 2017. How to Write a Scientific Article for a Peer-Reviewed Journal. In: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P. (eds.) Publishing Addiction Science: A Guide for the Perplexed, Pp. 135–153. London: Ubiquity Press. DOI: https://doi.org/10.5334/bbd.g. License: CC-BY 4.0. How to Write a Scientific Article for a Peer-Reviewed Journal Phil Lange, Richard Pates, Jean O’Reilly and Judit H. Ward Phil Lange, Richard Pates, Jean O’Reilly and Judit H. Ward All the chapters in this book speak to our aspirations to contribute to addiction science and to have a role in the scientific life of this field. In large part, this role comes through being published in peer-reviewed journals. Susan Savva (personal communication) The Practical Side of Addiction Publishing CHAPTER 7 Introduction A career in addiction science is largely built on reputation and the (perceived) quality of publications that a researcher (or a team of researchers) produces. If these publications are numerous and of high quality, they may lead to research funding and advancement. To gauge the contribution of a researcher to addic tion science, fellow researchers may consciously or unconsciously compute the number of worthwhile publications that a colleague has produced in relation to the number of years he or she has published. The greater speed of release for journal articles when compared with books—typically months versus years— means that those who wish to influence their field of study need to publish in peer-reviewed journals to quickly communicate their research results.hf This chapter offers the novice author a step-by-step guide to prepare an arti cle for publication. Annotated bibliographies and references listed at the end 136 Publishing Addiction Science of this chapter suggest further readings worth consulting about specific prob lems. This chapter begins with the proviso that a good manuscript written by a graduate student or a junior investigator may be highly praised by faculty and colleagues and yet fall short of being publishable. Indeed, editors regularly receive poor manuscripts that are accompanied by a letter from a graduate stu dent saying that his or her professor recommended submission. Yet the praise from a professor or colleagues does not obviate the need for novice authors to scrutinize every aspect of their text to see that it conforms to the demands of a scientific article.f i Here, we offer suggestions on how to use the style guide for the journal of your choice (for which there is additional information in Chapter 3), explain how to use a publication manual, and offer step-by-step guidance on the writ ing process itself. We also offer advice about working with colleagues, writing strategies, and maximizing the worth of your article for your selected journal. Some of the steps mentioned here are described in more detail, and sometimes with a valuable differing viewpoint, in Chapter 12.h This chapter is written for readers who have completed graduate or post graduate education and have completed a research project that they want to publish in a peer-reviewed journal but who are unsure of some of the basic steps in preparing the manuscript for submission. Introduction This chapter is also appropri ate for readers who already are proficient in another field of science but want to add articles in addiction science to their list of publications. For this scien tist, we advise caution: Terms may have different meanings for the layperson than for addiction scientists. For example, the word recovery connotes in the popular press and in everyday life that someone has undergone a course of clinical treatment or perhaps an affiliation with Alcoholics Anonymous. But in addiction science, recovery means achieving precise behavioral goals or a given score on a measure and by a given point in time. There are enough such special concepts built around everyday language that scientists new to the addiction field are advised to gather a group of colleagues to advise their research from the beginning.i We assume here that the reader is already competent in writing a scientific article. This chapter aims to fine tune competence in writing rather than to teach the basics of science writing. At the other end of the continuum, research ers whose articles are already often accepted in the journals of their choice will likely find little of interest here. Authors from developing or non-English speaking countries may wish first to read Chapter 4, which explores some of the special challenges encountered by researchers from developing and/or non- English speaking countries.i A successful publishing career means writing for a scientific audience, and authors may have to submit a number of manuscripts to various journals to discover how to do this in a way that results in a high percentage of accepted articles. An early decision researchers must make is whether to work alone or with colleagues. You can work in isolation from colleagues and hope to learn How to Write a Scientific Article for a Peer-Reviewed Journal 137 from rejection letters and from harsh peer reviews (see Chapter 12). Or, you can build an informal team of fellow scientists who are both critical and sup portive and who will read and comment on your manuscripts. This is often a quicker, more efficient, and more stimulating path. Introduction If you are new to a center or department and you want to sort out quickly who will be supportive of your aims versus who may be less than helpful (e.g., those who have reputations for being always harshly critical or for promising and then failing to read and critique manuscripts), ask people you trust this question: “If you were writing on my topic of __________, whom would you trust to help critique your work in a helpful way?” A novice author can learn much from established authors by passing them drafts for their assessments and their recommendations for getting published. For a younger or inexperienced writer it may be sensible to check on the acceptance rate of the journals (see Chapter 3) and go for one with a higher acceptance rate. In this way the chances of your paper being accepted are greater. Writing a scientific article for a peer-reviewed journal can be a creative and enjoyable act. Some people write beautifully and effortlessly, whereas others feel as though they are sweating out each word. But, over time, authors with both writing styles can make successful contributions to addiction science.h This chapter presents one way to write such an article—it is not the only way, of course, but it does offer the advantage of a clear step-by-step method that helps you to plan ahead. If you follow these steps, you will finish with a manuscript worth submitting to the journal of your choice (providing of course that the original science is sound). At the end of this chapter, we also present an annotated bibliography describing other approaches to preparing scientific manuscripts for peer review. Being methodical, let us start with a checklist. When you have decided on where to submit your paper make sure you read thoroughly the instructions to authors and follow them precisely. Virtually all journals will now only accept submissions electronically. This may be daunting for the first time a paper is submitted but it makes the process much easier for the journal and the author. Check the Style Guide for Your Journal of Choice Each journal has its own specific style configuration, and, to be accepted by a journal, you must write to its requirements, not those of another style format and not to your own personal preferences. To do this, have all information on all of the parameters required for the journal that you have (initially) chosen (see Chapter 3 for more information). Many journals offer a one-page style guide. But even the minimal style guides for undergraduate articles issued by university departments typically run to many pages, so clearly a lot will have 8 Publishing Addiction Science 138 been left out of a journal’s one-page summary. The Publication Manual of the American Psychological Association has 66 pages on style alone (American Psy chological Association, 2010, pp. 21–86). Much can be said for simply sitting down and reading at one go these 66 pages for a quick and complete overview of essential topics that are left out of most brief style guides. Read these Ameri can Psychological Association chapters and you will emerge an enlightened initiate knowing what topics to be sensitive to even if you must use a different style guide than this manual. The journal you are submitting to may have other style parameters that will affect your article, such as the preferred length of the manuscript and its abstract; gender-neutral or other styles of preferred lan guage; the maximum number, length, and style of footnotes or endnotes; and the maximum size of tables. Your journal of choice may require or recommend the use of reporting guide lines, depending on the type of paper you intend to write. Even if a journal does not require the use of reporting guidelines, it is worth following or at least con sulting a systematic guideline to establish a framework for your paper. There are hundreds of such guidelines in existence, helping researchers to produce accu rate, complete, and reliable reports. Table 7.1 outlines some common guidelines. An additional guideline that was developed in 2016 is SAGER (Sex and Gender Equality in Research), a comprehensive procedure for reporting sex and gender information in study design, data analyses, results and interpretation of find ings: http://www.equator-network.org/reporting-guidelines/sager-guidelines/i A brief warning about tables and figures: Journals may not specify the size lim its on tables and figures, yet these parameters have a huge effect on what infor mation you can include in them and how you organize your writing. Check the Style Guide for Your Journal of Choice Beginning researchers have a tendency to send wider, longer, and less-interesting tables than seasoned researchers. To create tables that will fit the page in those cases where the journal gives no guidance, (a) estimate the typeface in the table when compared to the textual typeface in the journal and (b) build model “trial” tables (one row, the number of columns needed, longest possible data lines per table cell) that would fit within a typeset page. Then build your tables. This alone may save you from immediate rejection or the work of rewriting the text and reor ganizing the table. If you have tables that require more than one page, check the journal to see if it publishes tables of that size or check with the editor. Editors have horror stories of good articles that arrive with huge tables that could never fit on a page. (The tricks authors use to create such large tables include using tiny typefaces, margins of less than a centimeter, and rows that run off the edge of the page and the monitor as well as carrying on for several pages with landscape orientation while submitting to a journal that does not accept that format. Do not consider any of these, because you will only infuriate the editor.)hi The other problem is tables and figures that are excessive in number or size. These all take up large amounts of space, and this may be a consideration for acceptance of the article. Include only those tables with direct relevance to the article and those that help the comprehension of the work. How to Write a Scientific Article for a Peer-Reviewed Journal 139 Acronym Full name Application Description Link CARE Case Reports Case reports Provides a flow diagram for systematic collection of data when seeing a patient or doing a chart review. http://www.care- statement.org/ CONSORT Consolidated Standards of Reporting Trials Randomised controlled trials Recommendations for reporting randomized trials. There are several extensions, including abstracts, cluster trials, pragmatic trials, and N-of-1 trials. http://www.consort- statement.org/ PRISMA Preferred Reporting Items for Systematic Reviews and Meta-Analyses Systematic reviews and meta-analyses Minimum criteria for reporting in systematic reviews and meta-analyses. There are exten sions for abstracts, equity, protocols, individ ual patient data, and network meta-analyses. Box 7.1: The importance of journal guidelines. Check the journal’s style guide for requirements governing the presentation of figures and make sure that they fit within the journal’s page parameters and tech nical requirements. There is a danger in looking to old copies of a journal to assess table and figure design. If you cannot get a current copy online or at a university library, write to the editor explaining the situation, and the editor—surely pleased at your concern—will likely send a sample copy. Figures are often easily sized by click-and-drag formatting to fit a given space within the correct margins. Check the Style Guide for Your Journal of Choice http://www.prisma- statement.org/ STARD Standards for Reporting Diagnostic Accuracy Descriptions of studies involving diagnostic accuracy or validity Essential items that should be included in every report of a diagnostic accuracy study. http://www. equator-network.org/ reporting-guidelines/ stard/ STROBE Strengthening the Report ing of Observational studies in Epidemiology guidance Observational studies in epidemiology, including cohort, case-control studies, and cross-sectional studies A checklist for articles reporting observational research. There is a draft STROBE checklist for abstracts. http://www.strobe- statement.org/ TIDieR Template for Interven tion Description and Replication Descriptions or evaluations involving interventions A guide for writers to describe interventions in sufficient detail to allow their replication. http://www.consort- statement.org/ resources/tidier-2 TREND Transparent Reporting of Evaluations with Nonrandomized Designs Surveys and longitudinal studies A checklist to guide standardized reporting of nonrandomized controlled trials. http://www.cdc.gov/ trendstatement/ Table 7.1: Guidelines commonly used in reporting health research.* For information on more than 280 reporting guidelines, visit The Equator Network: http://www.equator-network.org/. 140 Publishing Addiction Science 140 Editors agree that far too many authors ignore the crucial step of read ing and following the journal’s submission guidelines. Ask yourself, “Am I 100% confident that I have followed every one of even the small est details in the journal’s guidelines?” If your silent answer to yourself is, “Hmmm, certainly yes, probably 90% or 95%,” then your next step is to conclude that this is not good enough: Go back and fix those few items so that they are correct. The bottom line: Read and follow the journal’s instructions. Writing Step #1 Contact your chosen journal with a working title and abstract, ask if your arti cle is of interest and relevant to the journal’s mandate, and ask any awkward questions (. . . flexibility on article length? average time for the peer-review process?). Now is the time to learn if your article is acceptable to this journal, not after you have spent days writing an article to a specific format when that journal is unlikely to accept it. If the answer is favorable, you are ready to start writing. If the response is unfavorable, look for another journal. Alternatively, you might consider asking knowledgeable colleagues what journal(s) they feel are the best choice(s) for your article. Do a Thorough Literature Review The literature review is a crucial portion of your article. Many beginning researchers have problems with the scope and structure of the literature review. By studying examples of good literature reviews, you can improve your under standing of current standards. See also Chapter 9 on how to write system atic reviews. Wikipedia offers an introduction to the basic points of literature reviews (http://en.wikipedia.org/wiki/Literature_review). Kathy Teghtsoonian offers a useful didactic example explaining alternatives in a review of the litera ture on smoking (http://web.uvic.ca/spp/documents/litreview.pdf). An exam ple of a thorough literature review article that serves as a model for shorter reviews within an article—with exemplary background, definitions of terms and variables, treatment conditions, and results—is this article on quasi-compulsory drug treatment in Germany by Stevens et al. (2005). (But avoid the one-sentence paragraphs frequent in this otherwise fine review. Most editors and reviewers hate one-sentence paragraphs and complain about even one or two.) Cochrane Group reviews also deserve your attention. Not only may a review from the Cochrane Group spark improvements in your research, but reading a collection of reviews can also help you to develop a model for your work. See http://www. health.qld.gov.au/phs/documents/cphun/32103.pdf. 141 How to Write a Scientific Article for a Peer-Reviewed Journal Reviewers will be much more familiar with the literature than you are, and, therefore, your literature review needs to be informed and critical, not naive and accepting of all that is cited. One way to improve your literature review is with a step-by-step approach. Do a Thorough Literature Review Have these materials handy: • all the relevant literature needed to establish the theory or hypothesis that you will examine (it will help you to outline your article and to see what background or literature reviews you need for each section); • all the relevant literature needed to establish the theory or hypothesis that you will examine (it will help you to outline your article and to see what background or literature reviews you need for each section); • all relevant literature for each of the measures that you have used (the initial article describing each measure and crucial articles describing challenges, alterations, refinements, including statistics on validity, reliability, and all other relevant attributes); and • all the data needed for your methods, procedures, and results sections (a good way to assess if you need more literature for a given section is to ask yourself, “If I were challenged to support why I chose this [measure, method, statistic], what literature supports my choice?).” If you are writing about qualitative research for a journal that publishes little of your specialty, be sure to have the latest work on rigor in qualitative research and link it solidly to your work, because the probability is high for a rough ride from reviewers who know little about qualitative research and who may be more biased than they realize. (“I have seen a few good qualitative papers, but very few,” they tell me.) Also, please read Chapter 8, which explains how to write about qualitative research. Writing Step #2 Now settle down to write for colleagues and your posterity your unique contri bution to addiction science. Here are a few specific guidelines for each section of your article: 142 42 Publishing Addiction Science Title: You should know the overall writing style of your chosen journal well enough to know intuitively what is a suitable title for your article. If in doubt, (a) read the table of contents of several issues to get a feel for their style of titles and (b) make up a couple of possible titles and ask for reactions from colleagues who know this journal well. g y y (a) read the table of contents of several issues to get a feel for their style of titles and (b) make up a couple of possible titles and ask for reactions from colleagues who know this journal well. Mistakes to avoid: Trendy and cute titles soon look trivial and dated. An edi tor may allow such a title (especially if rushed), but years from now it will look embarrassing in your curriculum vitae when reviewers read it to determine if you deserve research funding.h Abstract: The abstract summarizes how you carried out your research and what you learned. It is increasingly common and often requested that you use a structured abstract (objective, methods (or) design, sample, results, and con clusion). For example, BMJ (n.d.) requires structured abstracts within a sound framework: objectives, design, setting, participants, interventions, main out come measures, results, and conclusions.f Mistakes to avoid: Do not go beyond what is established in your article: Offer no nonsignificant results, no speculation. Do not use telegraphic style (e.g., omitting articles and other parts of speech to achieve brevity) unless allowed by the journal. Do not go over the abstract size limit set by the journal. Introduction statement: A good introduction tells the reader why the arti cle is important in terms of the problems to be investigated, the context for the research question, what place this research question has in understanding addictions, and what is original about the endeavor. Mistakes to avoid: Do not simply describe the substance or behavior under study. Authors who see this as sufficient too often feel that the problem sub stance or behavior itself implies what research is needed. This is almost never true. Writing Step #2 At no point should the volume of loosely related information make the reader feel lost and wonder, “Why is all of this information here?” Avoid archaic arguments that have been resolved or that are not pertinent to your A frequent mistake made by beginning researchers is to not make clear to the editor and reader what is the original contribution of an article. It is easy to forget that scientific journals exist only to publish original knowledge. Describe the originality of your research analyses in your initial letter to the editor to see if there is interest in your article so that if the article later appears on the editor’s desk, he or she will remember it for the innovative understanding that it offers. For the reader’s benefit, your original contribution(s) should be clear from the title (if possible), mentioned in the abstract, and described in the introduction and in the discussion (and/or conclusion). Box 7.2: The importance of originality. A frequent mistake made by beginning researchers is to not make clear to the editor and reader what is the original contribution of an article. It is easy to forget that scientific journals exist only to publish original knowledge. Describe the originality of your research analyses in your initial letter to the editor to see if there is interest in your article so that if the article later appears on the editor’s desk, he or she will remember it for the innovative understanding that it offers. For the reader’s benefit, your original contribution(s) should be clear from the title (if possible), mentioned in the abstract, and described in the introduction and in the discussion (and/or conclusion). Box 7.2: The importance of originality. How to Write a Scientific Article for a Peer-Reviewed Journal 143 article, even though you may have spent months researching these and you have a fascinating solution to the debate. Avoid formulaic first lines: A sentence such as “Access to legalized gambling has increased greatly in the last two dec ades” begins at least one third of articles on gambling. An occupational hazard of editing is to receive by the dozen manuscripts with opening lines such as “Alcoholism (or drug dependence or tobacco use) is a significant public health problem.” The editor’s eyes glaze.h h Literature review: The literature section of a dissertation is an entire chapter. Writing Step #2 For an article, it should briefly summarize only the most important references that lead directly to understanding the importance of your article and the methods used. Keep the topics of your literature review grouped so that the flow is logical and the reader does not have to move back and forth. Move from the general subject to the more specific studies relevant to your research question. For detailed guid ance on which articles to cite, refer to Chapter 10 (Use and Abuse of Citations). For detailed information about how to use state-of-the-art search technologies to locate articles relevant to your literature review, see Appendix A. When your draft is completed, compare it with the literature reviews in your journal of choice. Mistakes to avoid: If several authors have been involved in writing the litera ture review, then it is likely to be too long and detailed, because each author tends to add what he or she knows are essential works. Keep the review concise.t Method: After readers have gone through this section, they should know the research methods in such detail that they could replicate the study in full with another sample. One way to check the completeness of this section is to have colleagues read it and ask them to verify if they could carry out this research project wholly from the methods section. If there are previously released arti cles using the same methods—whether your article or those of others, and espe cially if the method is described in more detail elsewhere—then you should cite these. This may allow you to shorten the method section. h y y Mistakes to avoid: If some aspect of your methods is suboptimal, it is better to mention it here with the comment “see the limitations section” and then be straightforward in the limitations section. Do not try to hide or disguise poor methods; reviewers will pounce on them. If your research involves randomized control trials, editors may refer you to the CONSORT Statement promoting high standards and uniform methods: http://www.consort-statement.org. Results: Here you describe the outcome(s) from your research. Double check that each novel finding mentioned in the discussion is reported here.h i Mistakes to avoid: This section especially lends itself either to over-writing (excessive detail beyond what is needed for analysis, excessive weight given to nonsignificant results) or to under-writing (cursory attention to important aspects and variables). Writing Step #2 Avoid reporting results as “approaching significance”; if they are not statistically significant, do not quote them as a near result. A mistake to avoid here is opening the results section with a description of the sample or an analysis that is more relevant to the methods, such as the validity of your measures. Start your results section with the main findings. Beginning 144 Publishing Addiction Science 144 researchers often take up too much of their manuscript with nonsignificant results; be ready to drop a result that colleagues or reviewers suggest is unim portant, even if it seems like a wondrous and magical thing to you.ii Discussion and/or conclusion(s): Describe how your specific results fit into the world of addiction science. You may address issues raised in the literature review, address policy issues, or raise new questions that are either unaddressed or rarely addressed by others. y y Mistakes to avoid: A little speculation is allowed, but limit it and ask your supportive colleagues what they think. Restrict your discussion of your future research plans to a line or two. Some authors like to end with the trite conclu sion “More research is needed.” It always is. If you wish to write in this vein, be as specific and creative as possible in tracing what original work needs to be done and what interesting hypotheses it will test. Limitations: Describe in brief detail the suboptimal aspects of your research. This newish trend has come as a result of demand for more transparency in research publishing. Junior authors are often afraid that being open about the limitations of their research will create prejudice against an article. In fact, the opposite is true. Senior researchers (i.e., editors and reviewers) will see flaws in your work that you will likely not see. Reviewers and the editor ask only that you acknowledge limitations. To do so is not a sign of weakness in you or your approach, but much to the contrary: It shows that you are an author who is on top of what are best practices and that you are a person who sees the need for better methods (as opposed to one who stumbles along pleased with his or her inadequate work). In concise, simple, and unapologetic language, describe the shortcomings that kept your work from being optimal. Writing Step #4 Submit your article to the editor. It might be useful to read Chapter 12 on man uscript preparation at this point. Bon voyage on this first step in becoming a contributor to the world of addiction science. Writing Step #2 Some journals allow an author to note limitations throughout the text (i.e., not as a subheading toward the end of the article). You may wish to check to see if your journal of choice allows or prefers this alternative. Mistakes to avoid: Do not be ingratiating (e.g., do not apologize, promise to avoid these mistakes in the future, or offer excuses), for this creates the impres sion of servility. You are not groveling You are only signaling to your peers that you know what is better practice in research. References: It is easy to forget that the function of references is to allow any reader to retrace the evidence you cite. Electronic sources that become una vailable threaten this openness. You must check that all the references in the text are cited in the reference section and that all the references in the refer ence section are cited in the text. Too often, authors neglect to check this, and these mistakes may be found by reviewers. You should be completely fluent in the minute details of proper reference style for your chosen journal. Too many errors tell the editor that an author has been careless, and this suggests careless ness perhaps elsewhere Mistakes to avoid: Verify if translation of foreign language titles is required. If it is, translate foreign-language titles even in the first version you send to the editor. How to Write a Scientific Article for a Peer-Reviewed Journal 145 145 How to Write a Scientific Article for a Peer-Reviewed Journal Appendices: If your journal of choice seems not to have published appendi ces, then check with the editor to see if they are allowed. Appendices represent an excellent solution to the problem of presenting background information (e.g., legislation, policy statements, questionnaires and measures, speeches, protocols) that is too long for the body of the article. They are also easy for a reader to skip: a blessing. Online, some journals allow for the posting of appen dix materials such as video and sound files, and URL access, as well as more traditional yet space-consuming items that are difficult or impossible to include in print journals. Note: Such data may not have peer-review status if not evalu ated by the reviewers. Mistakes to avoid: Omit appendices that you feel are relevant to the article but that colleagues feel are not pertinent. Writing Step #3 You have written this first version early enough to allow you to circulate it to several colleagues whom you can trust to read it and to offer prompt and fair critiques. Once you have their feedback, consider if their assessments warrant rewriting before submitting it to your chosen journal. Writing Step #5 Your article has been accepted for review (whether minimal or extensive) and has come back with the reviewers’ and the editor’s comments. This would be a good time to consult Chapter 12, which describes referees’ reports and how to respond to them. If you decide the referees’ criticisms are too severe for you to answer, then write the editor to tell him or her so and provide your precise reasons for not revising your article. This accomplishes several good things to your benefit: (a) It labels you as someone who takes editing a journal seriously, who knows his or her goals, and who does not let work slide; (b) it signals to editors how serious the criticisms were and may lead them to discuss options with you; and (c) they will remember you as someone who did not leave them hanging and wondering if that article was ever coming back. If you decide to revise your article, you have several choices. Authors should not see themselves as helpless in the face of reviewers’ comments. To reassure Publishing Addiction Science 146 authors of their rights, we at our journal send the following paste-in text to even experienced researchers. As we tell all authors, a reviewer’s comments are not orders that have to be carried out. To the contrary, for each point that a reviewer has made, an author has these three options: (a) discuss/debate/refute a reviewer’s comment(s), (a) discuss/debate/refute a reviewer’s comment(s), (b) rewrite the text in response to a comment(s), or (c) a combination of these so that an author both discusses/debates/refutes a reviewer’s comment(s) and rewrites to accommodate some comments by a reviewer. In many of the articles that you see in print, there are several points that appear just as authors intended, because they debated and defended their approach as written. As editor, I sometimes very much give the author the benefit of the doubt. The last point in answering the reviewers’ comments is practical but often over looked. Be crystal clear in accounting for how you responded to each point made by each reviewer. It is a good idea to provide in a letter to the editor the responses you have made point by point to the reviewers comments and to use track changes in the text of the article. Writing Step #5 If your article is rejected, then carefully read the critiques and see if you feel that submitting it to another journal seems a wise step. If so, be sure to format it thoroughly to that journal’s style and revise it in response to the reviewers’ criticisms. It is worth remembering that if your article is rejected and you submit it to another journal, it may be sent to a reviewer who has already rejected it. Writing Step #6 Once your article is accepted, you may have little more involvement until the editor or publisher sends you the proofs to check. When the proofs arrive and you see how the nuances of your careful writing style have been altered, it is easy to feel lonely and unappreciated. But please respect that copy editors know well what is more readable and credible to the target audience. If you have a hard time deciding on whether to accept a change or not, a criterion is to ask yourself is, “Has my meaning been respected or has it been changed?” If it has been respected, then let it be as edited and trust the copy editor. If you read your article a year later, you will usually see the wisdom of the copy editor’s changes. How to Write a Scientific Article for a Peer-Reviewed Journal 147 Publishing Dissertations Most postgraduates who have successfully completed a master’s thesis or doc toral dissertation will want to have their findings published. (Chapter 5 treats this topic in more detail.) It is important to remember that these dissertations are usually much longer and more detailed than will be required for publication as an academic article. Think carefully about how many articles your disserta tion can be split into: Often a doctoral dissertation has enough material for three or four articles. Do not replicate exactly the methodology or literature review (this will be seen as self-plagiarism; see Chapter 14), and keep the meth odology as simple as is necessary to explain what you did. Often the meth odology in dissertations is much more comprehensive than is required for an academic article, keep it to what is needed to explain your procedure. Editors will get frustrated when presented with an unedited dissertation and may reject it before sending it for review. When writing up a dissertation for publication it is important to bear in mind who should be included as authors (see Chapter 11 for discussion of how to assign authorship credits) and appropriate acknowledgement of supervision etc. Conclusion When your first addiction article is published, you will have made a contri bution to the addiction sciences and to the public arena where the dialectics between what is, what could be, and what will be are in struggle. A proverb: some Inuit say that a man can be only as good a hunter as his wife’s sewing will let him be. In the addiction sciences, the effectiveness of our research, treat ment methods, policies, and advocacy can be only as good as the literature that we publish. For Further Reading Boxes 7.3 and 7.4 describe resources for improving your scientific writing in general (writing style and motivation issues) and in particular areas, respec tively. If they do not contain a work specific to your needs or the books are unavailable, try searching your local university or professional library using terms such as scientific writing or publication manual in a title or subject search.ii fi g j Yet another technique is to find the library classification codes (call numbers) at your nearest university for books on writing psychology and biomedical science (e.g., in academic libraries using Library of Congress call numbers, they are mostly among the books labeled with H61 (social 8 Publishing Addiction Science 148 Alley, M. (1996). The craft of scientific writing (3rd ed.). New York, NY: Springer. Alley, M. (1996). The craft of scientific writing (3rd ed.). New York, NY: Springer. Alley, M. (1996). The craft of scientific writing (3rd ed.). New York, NY: Springer. p g • Lengthy chapters on building competence and curing shortcomings. Greene, A. E. (2013). Writing science in plain English. Chicago, IL: Uni versity of Chicago Press. • A short, focused guide presenting twelve writing principles based on what readers need in order to understand complex information, including concrete subjects, strong verbs, consistent terms, and organized paragraphs. g p g p Matthews, J. R., & Matthews, R. W. (2014). Successful scientific writ ing: A step-by-step guide for the biological and medical sciences (4th ed.). Cambridge, England: Cambridge University Press. Cambridge, England: Cambridge University Press. • Step-by-step advice helps researchers communicate their work more effectively. The fourth edition has been updated to provide more guid ance on writing and organizing each part of the manuscript’s draft.i g g gt Rogers, S. M. (2014). Mastering scientific and medical writing: A self-help guide (2nd ed.). New York, NY: Springer. • A compact guide with exercises as solved problems; good for over coming specific writing handicaps. It also addresses issues trou blesome to authors of a non-English language origin. This second edition answers questions resulting from new developments in sci entific communication. i Silvia, P. J. (2007). How to write a lot: A practical guide to productive aca demic writing. Box 7.3: Annotated bibliography of scientific writing: basic problems of writing style and motivation. For Further Reading Washington, DC: American Psychological Association.h • This breezy guide is especially good for authors who realize that their writing style needs improvement or who have been told that a component of their article (e.g., abstract, introduction, method, or discussion,) misses the point of what it should communicate. Jour nal articles have 23 pages of coverage in this book. p g g Strunk, W., & White, E. B. (1999). The elements of style (4th ed.). New York, NY: Longman. g • Still one of the best and shortest writing guides, easily read and absorbed. Those learning English find its clarity and brevity helpful. The 1918 edition by Strunk is available for free as an e-book from Project Gutenberg at http://www.gutenberg.org/ebooks/37134. West, R. (2002) A checklist for writing up research reports. Addiction, 95, 1759-61.hi • This is an advanced, comprehensive guide to scientific writing pre pared by the Editor of one of the leading addiction journals. Box 7.3: Annotated bibliography of scientific writing: basic problems of writing style and motivation. How to Write a Scientific Article for a Peer-Reviewed Journal 149 Goldbort, R. (2006). Writing for science. New Haven, CT: Yale Univer sity Press. Goldbort, R. (2006). Writing for science. New Haven, CT: Yale Univer sity Press. Box 7.4: Annotated bibliography of scientific writing: focusing on standards for scientific articles and specific scientific areas. References American Psychological Association. (2010). Publication manual of the American Psychological Association (6th ed.). Washington, DC: Author. American Psychological Association. (2010). Publication manual of the American Psychological Association (6th ed.). Washington, DC: Author. BMJ. (n.d.). Resources for authors: Research. Retrieved from http://resources. bmj.com/bmj/authors/types-of-article/research Savva, S. (2007, July 25). Personal communication. Stevens, A., Berto, D., Heckmann, W., Kerschl, V., Oeuvray, K., van Ooven, M., Steffan, E., & Uchtenhagen, A. (2005) Quasi-Compulsory Treatment of Drug Dependent Offenders: An International Literature Review, Substance Use & Misuse, 40, 269–283. Acknowledgements The authors are grateful for contributions from Susan Savva, Ian Stolerman, Kerstin Stenius, Sheila Lacroix, Thomas Babor, and Gerhard Bühringer. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. Goldbort, R. (2006). Writing for science. New Haven, CT: Yale Univer sity Press. Advanced guides include a collection of links to invaluable print resources in house and Publishing Addiction Science 150 links to authoritative and reputable online options on the Internet. Here are a few examples, all from the USA: • Michigan State University – http://libguides.lib.msu.edu/medwriting • Duke University – http://guides.mclibrary.duke.edu/scientificwriting • Wilkes University – http://wilkes.libguides.com/scientific_writing • University of California San Diego – http://ucsd.libguides.com/psyc • Bowling Green State University – http://libguides.bgsu.edu/techwriting • Michigan State University – http://libguides.lib.msu.edu/medwriting • Duke University – http://guides.mclibrary.duke.edu/scientificwriting • Wilkes University – http://wilkes.libguides.com/scientific_writing • University of California San Diego – http://ucsd.libguides.com/psyc • Bowling Green State University – http://libguides.bgsu.edu/techwriting Goldbort, R. (2006). Writing for science. New Haven, CT: Yale Univer sity Press. y • This book offers detailed chapters cover every type of science writing by using numerous examples. The author discusses how to approach various writing tasks as well as how to deal with the everyday com plexities that may get in the way of ideal practice.i y g y Gustavii, B. (2003). How to write and illustrate a scientific paper. Cam bridge, England: The Cambridge Press.h g gh g • This work is oriented to the biological and medical sciences. It is the clearest and most succinct work that we found among all such works at our local university. A marvel of clarity and utility. It is also full of relevant URLs for up-to-date information. p Huth, E. J. (1990). How to write and publish papers in the medical sci ences (2nd ed.). London, England: Williams and Wilkins.hf • This compact work offers practical advice on how to make decisions about what to write and what to leave out for both novice and expe rienced researchers. A highly readable source.h g y Miller, J. E. (2005). The Chicago guide to writing about multi-variate analysis. Chicago, IL: University of Chicago Press.hii y g y g • This work shows how specific the aids available to scientific authors are. The book is a mini-course in writing about numbers (i.e., sta tistical analysis). Schimel, J. (2012). Writing science: How to write papers that get cited and proposals that get funded. Oxford, England: Oxford University Press.h • This book is built upon the idea that successful science writing tells a story. The author discusses every aspect of successful science writ ing, from the overall structure of a paper or proposal to individual sections, paragraphs, sentences, and words Box 7.4: Annotated bibliography of scientific writing: focusing on standards for scientific articles and specific scientific areas. sciences), Q158 (biomedical sciences), R119 (biomedical sciences, and T11communication)), and then scan the shelves in those sections for books that did not come up in your title or subject search. Some would call this a strategy of desperation, but half of the books in the annotated bibliographies below were found this way.f Finally, most academic libraries offer so called LibGuides, i.e., special research guides on scientific writing that are not just for students. Appendix A. How to Locate Articles Relevant to Your Literature Review No matter how easy it seems to Google your topic, the scholarly article you are writing deserves a more in-depth literature search than Google or even Google Scholar provides. On the other hand, it would be very time consuming to check individual journals for relevant articles, even though in certain cases the majority of the pertinent articles seem to have been published in a handful of journals. The purpose of scientific databases is to aggregate all publications from a variety of How to Write a Scientific Article for a Peer-Reviewed Journal 151 151 journals in a single database on a particular topic, such as PubMed and Medline on biomedical and health science and PsycInfo on psychology. These databases abstract and index every article published in the journals in their coverage, mak ing the scientific content easily discoverable through literature searches. Since currently there is no single and comprehensive database in the field of addic tion science, expect to spend a significant amount of time searching scientific databases with various scopes. Please see the more general discussion of relevant databases and abstracting & indexing services in Chapter 3. A literature search can also serve as a great start to conceptualize the topic of your article, since in order to run your search in a database, you will first have to produce a list of search terms. Searching is a skill that can best be learned with the help of a professional searcher. Before you start your literature search, please consult your librarian on the latest trends and, if possible, schedule a one-on-one session to find out which databases are available at your institution and what search strategies would work the best in those resources. Choose the Right Database The first step of the search process is choosing the appropriate databases. The best way to start is by reading the description of a database to define the type, scope, and coverage of the resource. For example, the Rutgers Alcohol Stud ies database is a collection of bibliographic records for books, book chapters, journal articles, government documents, conference papers, and dissertations. Although you will not have access to the full text of any document, you can use the reference to find it elsewhere. The Rutgers Alcohol Studies database is a very comprehensive database; discontinued in 2007, it can be considered an excellent source of articles written before 2007. Other useful resources include the Alcohol and Alcohol Problems Science Database, or ETOH, discontinued in 2003, and the CORK database, updated until early 2015. Because there are currently no comprehensive databases for the addiction field, resources such as Medline or PsycInfo will usually provide the best results at the beginning of your literature search on any addiction science-related topic. Searching a major database also comes with an additional bonus: if your institution subscribes to the journal and has an article linker software applica tion in place (most academic libraries do), you will have instant access to the full text of those articles. Build Your Search Search interfaces vary depending on the platform your institution provides (e.g. Ovid or EBSCO). Spending 45–60 minutes with your local librarian can save you precious time to locate the most important features of the databases 52 Publishing Addiction Science 152 and can allow you to focus on the search strategies. In a nutshell, it is highly recommended to use the “Advanced” search option, if possible, in any database on a platform where you perform your search in multiple search boxes (e. g. EBSCO platforms, Academic Search Premier). It’s important to be comfortable using the Boolean operators, truncation, and wildcards, and familiar with the concept of controlled vocabulary, mapping, and the thesaurus. Each database defines its own preferred terms; for example, Medline, PubMed, and PubMed Central (reiterations of the same collection in slightly different formats) use Medical Subject Headings called MeSH terms, the controlled vocabulary the US National Library of Medicine uses for indexing articles. Another notable collection of terms is the Library of Congress Subject Headings used by aca demic libraries, book publishers and Academic Search Premier, a software application originally designed to allow similar titles to be placed physically close to each other on the shelves of brick-and-mortar libraries. For example, “marijuana” is the preferred term in PsycInfo, while Medline uses “cannabis” as an index term. A keyword search usually searches the full text, for exam ple, searching for the word “ganja” as keyword anywhere in an article. A useful feature of the Ovid platform is “mapping” your term to these preferred terms to achieve a high precision of search results. The Ovid platform also prompts you to build a search line-by-line (or term-by-term), resulting initially in an alarmingly high number of hits. Then, using the Boolean operators AND and OR, you can modify and combine your search with additional terms in as many ways you want to filter articles. Each database offers a variety of filters, such as date range, populations and document types, which are essential in the search process. This comprehensive search strategy will retrieve the relevant articles that were indexed by a subject heading or a descriptor matching your concept. Other searches may target certain parts of the articles the database defines as searchable, such as the author, title, abstract, and keywords, usually in a single search box. Build Your Search This type of search is perfect to locate known items, i.e., to find an article written by an author knowledgeable about your topic, or to retrieve the full text of an article discovered earlier. It should be noted that sometimes old-fashioned methods, such as “footnote chasing” or finding a good review article on your topic, may result in unexpected breakthroughs in your literature search. Many novice searchers take screenshots of their most successful search strategies for future reference or documentation, since most databases do not allow you to save your search and return to it. How to cite this book chapter: Stenius, K, Mäkelä, K, Miovský, M and Gabrhelík, R. 2017. How to Write Publishable Qualitative Research. In: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P. (eds.) Publishing Addiction Science: A Guide for the Perplexed, Pp. 155–172. London: Ubiquity Press. DOI: https://doi.org/10.5334/bbd.h. License: CC-BY 4.0. Benefit from Citation Management Software Applications It’s a good idea to save the results of each search, i.e., the bibliographic records and/or the full texts of the articles, in a citation management software How to Write a Scientific Article for a Peer-Reviewed Journal 153 application. Many authors rely on these applications, such as the proprietary EndNote and RefWorks, or the open source Zotero.h These applications serve multiple functions in the process of conducting research and sharing results in a publication. They are integrated with most of the platforms content providers use for databases and individual journals so that researchers can immediately download the metadata, including links to the full text, of several articles retrieved during the search. They can then share them with collaborators, and can finalize which ones to cite in the article to be published. The in-text citation function, such as Write-N-Cite in RefWorks, allows the author to insert placeholders in paragraphs that serve as the basis of the reference list at the end of the article in the format required by a particular journal, such as APA first author/year or numerical style. Most major citation styles are built into most citation management software applications as output styles. Authors who create their own lists and folders of articles to be cited will benefit from the convenience of creating a list of references, endnotes, or footnotes with one click of a mouse. Should the article be rejected, there is no need to reformat the in-text citations and the entire bibliography to match the required style of another journal. All that needs to be done is to change the output style in the citation management software. CHAPTER 8 How to Write Publishable Qualitative Research Kerstin Stenius, Klaus Mäkelä, Michal Miovský and Roman Gabrhelík Introduction Conducting and publishing qualitative research requires the same principal skills as quantitative research. In addition, there may be special challenges for qualitative researchers. They may have to overcome prejudice and communica tion barriers within the scientific community. This chapter provides advice to authors who wish to publish their research in a scientific journal. The chap ter starts with some remarks on the special characteristics of the processes of qualitative study that can affect the reporting of the results. It then identifies the common criteria for good qualitative research and presents some evalua tion principles used by editors and referees. Finally, it offers practical advice for writing and publishing a qualitative scientific article. i In quantitative research, the observations typically follow a systematic scheme whereby the classification of the observations is already determined to a large extent when the data collection starts. This makes it possible to gather large data sets for numerical analyses, but the understanding of the findings will be restricted by the concepts on which the collection of data was based. One can argue that in qualitative research, in which the observations (e.g., texts, sounds, behaviour, images) are usually fewer, the researcher’s preconception of a social phenomenon does not determine the research results to the same extent as in quantitative research (Sulkunen, 1987) Qualitative research thus is often used 6 Publishing Addiction Science 156 to study social processes or the reasons behind human behaviour (Sulkunen, 1987), or as Wikipedia puts it: The why and how of social matters more than the what, where, and when that are often central to quantitative research. t Qualitative addiction research focuses on topics that range from historical processes to treatment outcomes. Qualitative research is used increasingly to answer questions about alcohol and other drug policy, including rapid assess ment of policy developments (e.g., see Stimson et al., 2006). It is used to study program implementation and to evaluate various policy measures (e.g., see Miovský, 2007; Miovský & Zábranský, 2003). Furthermore, ethnographers have used qualitative methods to increase the understanding of patterns of substance use in various population groups (e.g., see Lalander, 2003).h There is also an important and growing interest in combining qualitative and quantitative research into so-called mixed-methods research, notably within evaluation and intervention research in the clinical and policy fields (Creswell & Plano Clark, 2007). Source: Creswell and Tashakkori (2007). Box 8.1: Criteria for good mixed-methods articles. Source: Creswell and Tashakkori (2007). Box 8.1: Criteria for good mixed-methods articles. Introduction The combination of qualitative and quantitative methods can deepen the understanding of processes, attitudes, and motives. There is fre quent discussion in theoretical mixed-method studies of the relations between various kinds of knowledge and the actual procedure of combining qualitative and quantitative methods (Creswell & Tashakkori, 2007). Box 8.1 presents cri teria for good mixed methods articles. Despite what we believe is an increasing interest in qualitative research, many journals do not publish qualitative studies. In addition, many editors of addic tion journals have noted that qualitative manuscripts are more likely to present the editors with problems and are more often declined for publication than are quantitative research reports. Some of the problems are related to how the articles are written.i In the addiction field, there is no journal dedicated exclusively to qualitative research, and in many journals articles must follow a strict standard format. Qualitative articles tend to break with that format, putting special demands a) The study has two sizeable data sets (one quantitative, one qualita tive), with rigorous data collection and appropriate analyses, and with inferences made from both parts of the study. b) The article integrates the two parts of the study, in terms of com paring, contrasting, or embedding conclusions from both the qualitative and the quantitative strands. c) The article has mixed-methods components that can enrich the newly emerging literature on mixed methods research. Box 8.1: Criteria for good mixed-methods articles. Source: Creswell and Tashakkori (2007). a) The study has two sizeable data sets (one quantitative, one qualita tive), with rigorous data collection and appropriate analyses, and with inferences made from both parts of the study.h b) The article integrates the two parts of the study, in terms of com paring, contrasting, or embedding conclusions from both the qualitative and the quantitative strands.h c) The article has mixed-methods components that can enrich the newly emerging literature on mixed methods research. How to Write Publishable Qualitative Research 157 on the reader. Another problem for a comparatively small research field such as addiction research is that it is difficult to find referees who are competent to evaluate qualitative methods and analyses. A qualitative article thus runs the risk of being reviewed by someone who not only is unqualified but also may be prejudiced against qualitative research. For all of these reasons, qualitative researchers have to be particularly professional in their writing. The Challenges of Publishing Qualitative Research Qualitative methods can be used for pilot studies, to illustrate the results of statistical analysis, in mixed-methods studies, and in independent qualitative research projects (c.f. Denzin & Lincoln, 1998). This chapter will focus on the last category: original research reports that use qualitative methods. We will emphasise the similarities and considerable overlap in the evaluation, and effective presentation, of both qualitative and quantitative research.hi f The first and foremost aim of all social research, quantitative as well as quali tative, is to present a conceptually adequate description of a historically specific topic, subject, or target. In qualitative research, the determination of the subject is as important as the choice of a population in a statistical study. The descrip tion of the subject is always, in both types of study, a theoretical task because it requires a conceptually well-organised analysis.hi The processes of classification, deduction, and interpretation are in their fundamental aspects similar for both qualitative and quantitative research. Quantitative analyzing operations, however, are more clear-cut than qualitative operations. Furthermore, the various steps of quantitative research can be more clearly distinguished than can those of a qualitative study. The first issue is that, in qualitative work, the collection and processing of data are more closely inter twined than in a quantitative study. Especially when the researchers personally collect the data, they will not be able to avoid problems of interpretation during the collection phase. A specific issue in some qualitative research is that the methods used can change during the study, depending on interim results. It is a challenge to explain in a short article why this has happened, and why one has used a different method in the final phase of the data acquisition than in the previous parts; or why one changed a classification scheme and encoded the data in a different manner. The researchers must also carefully consider their relations with the study objects. Many qualitative reports discuss at length the character and psychology of the process of data collection, but are less careful in describing what happened to the interview tapes afterwards. Were they tran scribed in whole or in part, how was the resulting stack of papers handled and sorted out? In qualitative research, these data processing explications may be necessary to render credibility to the analysis. The Challenges of Publishing Qualitative Research A second issue is that qualitative analysis is not restricted to an unambigu ously demarcated data set in the same way that a quantitative study is. Good 58 Publishing Addiction Science 158 researchers may keep a detailed field diary and make notes of all discussions and thus produce a corpus to which they limit their analysis. Nevertheless, dur ing the analysis phase, they may recall an important detail that they had not recorded in their notes but must take into account in the analysis. The qualita tive researchers have to describe this analytical process in an honest and con vincing way.h There are several basic factors that make the publication of qualitative research harder and different from standard journal article models of quantita tive research (Miovský, 2006): • The research design may be less strictly defined from the beginning of the research project, and it is not unusual to have design changes as new ques tions arise and new findings are considered. Redesigning necessitates an especially thorough and sometimes lengthy methodology section to explain those changes.f • Qualitative research uses many different theoretical frames (phenomenol ogy, constructivistic approaches, hermeneutics, etc.) that affect data selec tion, methodology, and presentation. This variance is also to some extent found within quantitative research. But because analysis and reporting are more closely intertwined in qualitative research, the differences in theoreti cal perspectives become even more important. As an author, you will have to argue even more clearly for the choice and sufficiency of your data and their scientific significance.f i gi • Compared with quantitative research, qualitative research uses different concepts of research validity (e.g., credibility), with different theoretical backgrounds (Whittemore, Chase & Mandle, 2001) and different views on correct sampling methods and the representativeness of data (Patton, 1990). Some sampling strategies combine qualitative and quantitative perspectives (e.g., respondent-driven sampling). Qualitative-oriented research can be performed with a single case study but also with sampling methods such as snowball sampling or respondent-driven sampling, which can combine traditional probability sampling methods with qualitative-oriented meth ods. It can be a challenge to describe these data sets and the data collecting methods, as well as why and how they were used, within the length limits usually applied to research reports. The Challenges of Publishing Qualitative Research ii • Compared with quantitative research, qualitative research uses different concepts of research validity (e.g., credibility), with different theoretical backgrounds (Whittemore, Chase & Mandle, 2001) and different views on correct sampling methods and the representativeness of data (Patton, 1990). Some sampling strategies combine qualitative and quantitative perspectives (e.g., respondent-driven sampling). Qualitative-oriented research can be performed with a single case study but also with sampling methods such as snowball sampling or respondent-driven sampling, which can combine traditional probability sampling methods with qualitative-oriented meth ods. It can be a challenge to describe these data sets and the data collecting methods, as well as why and how they were used, within the length limits usually applied to research reports. All these factors present authors with a set of practical difficulties, not only because of technical page limits but also because there are not many review ers with insight into qualitative methods and analysis. Scientific publish ing has also gradually become more streamlined, with a lot of written and unwritten habits and rules that are usually based on quantitative approaches and methods. A qualitative researcher must be prepared to tackle these obstacles. How to Write Publishable Qualitative Research 159 Evaluation Criteria for Qualitative Analysis There are some differences between the evaluation of qualitative and quantita tive research. The replicability of a qualitative study cannot be formulated as a problem of reliability, and the accuracy of a qualitative interpretation cannot be compared with the explanatory power of a statistical model. In the following paragraphs, we propose three main criteria for evaluating qualitative studies: 1) significance of the data set and its social or cultural place; 2) sufficiency of the data and coverage of the analysis; and 3) transparency and repeatability of the analysis. Since in qualitative research the analyses and reporting are very closely intertwined, these criteria are as relevant to researchers and authors as they are to reviewers and editors. 1. Significance of the Data Set and its Social or Cultural Place 1. Significance of the Data Set and its Social or Cultural Place The researchers should be prepared to argue that their data are worth analyz ing. It is not easy to identify criteria for the significance of data. One precon dition can, however, be presented: the researcher should carefully define the social and cultural place (contextualising) and the production conditions of the material. The production conditions can be discussed at several levels. When the data consist of cultural products, their production and marketing mechanisms should be considered. Texts produced by individuals should be related to their social position. Furthermore, the situational aspect of the data production and the researcher’s potential influence on the data should be evaluated. The rela tionship of cultural products to people’s everyday life depends on the produc tion and distribution network. Weekly magazines and movies represent the ambient culture at a number of levels. When doing comparisons over time, it is important to bear in mind that the social and cultural place of one and the same genre may vary from decade to decade. In international comparisons, it is important to be able to exclude demo graphic variation as a factor causing differences. If we wish to identify the dis tinct characteristics of Finnish A.A. members’ stories, we should make sure that we do not compare Finnish farmers with American college professors. The criterion for selecting the target group is not demographic but cultural representativeness.ff Additionally, people speak of the same things in different ways on different occasions, and it is the task of the researchers to decide which discourse they want to study and argue for their decision in the article. Informal interviews are often advocated instead of questionnaires on the grounds that they will produce more genuine information. But, on the other hand, an in-depth inter view is a more exceptional situation for a present-day person than completing 0 Publishing Addiction Science 160 a questionnaire. Possible effects of the power structures and gender relations present in every social situation should be considered in the discourse analysis, since it could affect the outcomes of the qualitative research. f Study of the variations of discourse, i.e., the incorporation of the produc tion conditions into the study design, can be rather laborious. Members of A.A. 1. Significance of the Data Set and its Social or Cultural Place emphasize various sides of their story according to the composition of the audi ence, and depending on whether they talk at a closed or an open A.A. meeting. Furthermore, the life story will change in relation to how long the speaker has been in A.A. Even when variation cannot be incorporated into the actual study design, it is important to consider and discuss the conditions under which the material was produced and their place in the potential situational variation of the discourse. 3. Transparency and Repeatability of the Analysis Transparency of the analysis means that the readers are able to follow the researcher’s reasoning and that they are given the necessary information for accepting the interpretations—or for challenging them. The repeatability of an analysis means that the rules of classification and interpretation have been presented so clearly that another researcher applying them will reach the same results. We may identify three ways of improving the transparency and repeat ability of qualitative analysis and the report: 1) enumerating the data; 2) divid ing the process of interpretation into steps; and 3) making explicit the rules of decision and interpretation.h The best method to decrease arbitrariness and increase repeatability is to enumerate all units on which the interpretation is based. To do this an analyti cal unit must be specified and it should be as small as possible. In other words, do not choose a movie or a group discussion but rather choose a scene, a state ment, or an adjacent pair. The identification of the unit of analysis is in itself part of the process of interpretation.h The process of interpretation and analysis can never be fully formalized. It is above all a question of working step by step so that the process of interpretation can be made visible to both the researchers themselves and the reader. Qualitative analysis is of necessity more personal and less standardized than statistical analysis. Thus, it is even more vital that the reader is given as exact a picture as possible of both the technical operations and the chain of reasoning that have led to the reported results. The reader must not be left at the mercy of the researcher’s intuition alone. The demand for transparency in qualitative research is of crucial importance. 2. Sufficiency of Data and Coverage of Analysis For statistical studies, we are able to calculate in advance the extent of data needed to estimate the parameters accurately enough for the purpose of the analysis. We have no similar methods for estimating the extent of qualitative data required. We usually speak about data saturation: data collection can be terminated when new cases no longer disclose new features (Strauss & Corbin, 1998). The difficulty here, of course, is that the limit is not always known in advance, and the collection of data is rarely a continuing process that can be terminated or extended at will. Only in very special cases can you base your analyses on just a handful of observations. In most cases, you will need to be certain that you cover the vari ation of the phenomenon you are studying. On the other hand, a loose but useful rule is that one should not collect too much data at a time. It is better to analyze a small data batch carefully first and only then determine what addi tional data will be needed. To divide the analyses into smaller parts also helps to produce manageable results for a publishable report.t It is often advisable to group the collection of data according to factors which may prove important as production conditions. The goal is not to explain the variation but to make sure that the data are sufficiently varied. For example, it would be helpful to stratify the collection of A.A. members’ life stories accord ing to the members’ social position, sex, age, and length of sobriety (Arminen, 1998). The only difficulty is that we will have no advance knowledge of which characteristics will decide the type of life stories; the stories may depend more on drinking experiences than on external circumstances, and within A.A. there may be various narrative traditions which have an influence on the life stories. l Proper coverage of the analysis means that the researchers do not base their interpretations on a few arbitrary cases or instances but on a careful reading of the whole material. Qualitative reports are often loosely impressionistic because the excessive amount of material has made it unfeasible to analyze it carefully enough. How to Write Publishable Qualitative Research 161 161 Box 8.2: Assessment Criteria for Qualitative Studies. Source: Des Jarlais, Lyles & Crepaz (2004). Editors’ and Referees’ Assessment of Qualitative Research Reports A discussion of the evaluation criteria for peer review of qualitative research can start with evaluation principles for quasi-experimental research or natu ral experiments. The American Journal of Public Health published an evalua tion system for these types of study (Des Jarlais, Lyles & Crepaz, 2004) entitled TREND (Transparent Reporting of Evaluations with Nonrandomized Designs). TREND was designed specifically for research results in which the randomisa tion principle was somehow restricted. The criterion of transparency, which is central to this evaluation system, emphasises a detailed description of all steps and procedures, as well as a detailed justification of the choice and manner of application of the individual methods and theoretical background (see also Mayring, 1988, 1990). Mareš (2002) analysed quality criteria for research using pictorial docu ments and summarised the findings with the concepts of completeness (how 2 Publishing Addiction Science 162 well the data capture the phenomenon examined), transparency (the accuracy, clarity, and completeness of the description of the individual phases of the study), reflexivity (the ability of researchers to reflect on their different steps and measures during the study and how the investigators may have influenced the research situation), and adequacy of interpretation and aggregation of con tradictory interpretations (the identification and weighting of alternative inter pretations and other validity-control techniques). Des Jarlais, Lyles & Crepaz (2004, pp. 363–365) have drawn up a 22-item list to serve as a general assessment guide for authors and evaluators. Box 8.2 shows some of their requirements and recommendations. Additional recommendations proposed by Gilpatrick (1999) and Robson (2002) are summarized in Box 8.3. a) An article should be provided with a structured abstract (as a minimum: background, aims, sample, methods, results). b) The sampling should be described and justified, including an explanation of criteria used. c) The theoretical background of the entire study, or individual methods, should be described, to show that the sample and data collection were consistent with the study’s theoretical background. d) The context (setting) in which the study was carried out should be described. The authors must describe the characteristics of the field in which the study was carried out, and what made it differ ent from other settings. e) A detailed description of the research intervention should be included, and of how study participants responded during that intervention. Practical Advice for Writing a Publishable Qualitative Article A good way to start the process of improving both your writing skills and your chances of publication is to become familiar with the common reasons why editors reject qualitative articles (see Box 8.4), and then carefully read some examples of well-written qualitative articles (see Box 8.5). Based on our experience as journal editors, referees, and researchers, we now present nine recommendations for potential authors of qualitative articles. Editors’ and Referees’ Assessment of Qualitative Research Reports d) Control tools (e.g., research logs, control points) should be reported and how ethical problems were handled (e.g., use of informed consents, careful adherence to research protocols, man ner of preparing the research team to manage risky or problem situations). Box 8.3: Evaluation Criteria for Qualitative Studies. Sources: Gilpatrick (1999) and Robson (2002). a) The research issue and the research questions and goals derived from it, should be properly presented.h b) The goals should be contextually embedded and put into a theoretical framework, with an analysis of the present state of knowledge. g c) The author should argue for the importance of their study against this background (e.g., what questions or issues the results should contribute to, how they will move the field forward). yi d) Control tools (e.g., research logs, control points) should be reported and how ethical problems were handled (e.g., use of informed consents, careful adherence to research protocols, man ner of preparing the research team to manage risky or problem situations). Box 8.3: Evaluation Criteria for Qualitative Studies. Sources: Gilpatrick (1999) and Robson (2002). The qualitative paper, both in its entirety and in its constituent parts, will be evaluated by and large according to the same criteria and expectations as those applied to a quantitative report. Box 8.3: Evaluation Criteria for Qualitative Studies. Sources: Gilpatrick (1999) and Robson (2002). Editors’ and Referees’ Assessment of Qualitative Research Reports f) A detailed description of the analytical methods applied, how they were used, including the tools used for minimising bias; and a validation of the results should be presented. g) A description of the manner of data processing (e.g., technical aspects and procedures) is needed. h) Description of outcomes and their interpretation are obviously necessary. This includes a discussion of limitations (contextual validity of results), and an analysis of how the design of the study reflects these limitations. Box 8.2: Assessment Criteria for Qualitative Studies. Source: Des Jarlais, Lyles & Crepaz (2004). a) An article should be provided with a structured abstract (as a minimum: background, aims, sample, methods, results).hi b) The sampling should be described and justified, including an explanation of criteria used.h c) The theoretical background of the entire study, or individual methods, should be described, to show that the sample and data collection were consistent with the study’s theoretical background.h d) The context (setting) in which the study was carried out should be described. The authors must describe the characteristics of the field in which the study was carried out, and what made it differ ent from other settings. e) A detailed description of the research intervention should be included, and of how study participants responded during that intervention. f) A detailed description of the analytical methods applied, how they were used, including the tools used for minimising bias; and a validation of the results should be presented. g) A description of the manner of data processing (e.g., technical aspects and procedures) is needed. h) Description of outcomes and their interpretation are obviously necessary. This includes a discussion of limitations (contextual validity of results), and an analysis of how the design of the study reflects these limitations. How to Write Publishable Qualitative Research 163 a) The research issue and the research questions and goals derived from it, should be properly presented. b) The goals should be contextually embedded and put into a theoretical framework, with an analysis of the present state of knowledge. c) The author should argue for the importance of their study against this background (e.g., what questions or issues the results should contribute to, how they will move the field forward). 1. Consider the Format and Structure of Your Article • The author has not related the study to earlier (international) literature.h h • The research question is not clearly stated.h • The structure of the article is not clear or does not respond to the expected structure of articles in the journal.h • Theories, methods, and data analyses are not consistent.h • The central concepts are not clearly presented or used in a consist ent way.h • The size of the data set is not defended in a convincing way.hfi • The data set is not sufficiently contextualised, or there is a clear selection bias.h • The data collection is poor and lacks validity control.h • The methods and analyses are not explained clearly enough, which may lead the referees and the editor to regard the article as too descriptive and the analyses based too much on intuition.h • The author makes unsound conclusions or unfounded generalisations. • Ethical rules are violated or ethical issues are not mentioned or ade quately discussed.h Box 8.4: Common reasons why editors decline qualitative articles. Source: Drisko (2005). you are not a very experienced researcher, it may be wise to choose a traditional structure for your research report. 2. Begin with the Abstract Most addiction journals require the authors to write very short abstracts, cov ering background, aims, data and methods, results, and discussion. It is a good idea for the author of a qualitative article to write a preliminary abstract at an early stage of the writing process to ensure that the text will be coherent and logical. 1. Consider the Format and Structure of Your Article When you get acquainted with various addiction journals, you will realize that qualitative articles can look very different depending not only on their topic but also on where they are published. You can choose to target a specific journal and try to follow closely the format used in that publication. But if you want a greater choice of potential journals for your manuscript, and in particular if 164 Publishing Addiction Science • The author has not related the study to earlier (international) literature. • The research question is not clearly stated. • The structure of the article is not clear or does not respond to the expected structure of articles in the journal. • Theories, methods, and data analyses are not consistent. • The central concepts are not clearly presented or used in a consist ent way. • The methodology is poor. • The size of the data set is not defended in a convincing way. • The data set is not sufficiently contextualised, or there is a clear selection bias. • The data collection is poor and lacks validity control. • The methods and analyses are not explained clearly enough, which may lead the referees and the editor to regard the article as too descriptive and the analyses based too much on intuition. • The author makes unsound conclusions or unfounded generalisations. • Ethical rules are violated or ethical issues are not mentioned or ade quately discussed. • The text is too long. Box 8.4: Common reasons why editors decline qualitative articles. Source: Drisko (2005). 3. Choose a Title that Corresponds to the Content The title of an article is very important. Drisko (2005) gives the following advice: present the research question reshaped into the manuscript title. How to Write Publishable Qualitative Research 165 Amos, A., Wiltshire, S., Bostock, Y., Haw, S., & McNeill, A. (2004). ‘You can’t go without a fag . . . you need it for your hash’ – a qualita tive exploration of smoking, cannabis and young people. Addiction, 99(1), 77–81. Demant, J., & Järvinen. M. ( 2006): Constructing maturity through alcohol experience – Focus group interviews with teenagers. Addic tion Research and Theory, 14(6), 589–602. Herd, D. (2005). Changes in the prevalence of alcohol use in rap song lyrics, 1979–97. Addiction, 100(9), 1258–1269. Maher, L., & Hudson, S. L. (2007). Women in the drug economy: A metasynthesis of the qualitative literature. Journal of Drug Issues, 37(4), 805–826. Maeyer, J. D., Vanderplasshen, W., Camfield, L., Vanheule, S., Sabbe, B., & Broekaert, E. (2011). A good quality of life under influence of meth adone: A qualitative study among opioid-dependent individuals. International Journal of Nursing Studies, 48, 1244–1257. Miovský, M. (2007). Changing patterns of drug use in the Czech Repub lic during the post-Communist era: A qualitative study. Journal of Drug Issues, 37(1), 73–102. Phillips, D., Thomas, K., Cox, H., Ricciardelli, L. A., Ogle, J., Love, V., & Steele A. (2007). Factors that influence women’s disclosures of sub stance use during pregnancy: A qualitative study of ten midwives and ten pregnant women. Journal of Drug Issues, 37(2), 357–376. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. Box 8.5: Examples of well-written qualitative articles. Amos, A., Wiltshire, S., Bostock, Y., Haw, S., & McNeill, A. (2004). ‘You can’t go without a fag . . . you need it for your hash’ – a qualita tive exploration of smoking, cannabis and young people. Addiction, 99(1), 77–81. Demant, J., & Järvinen. M. ( 2006): Constructing maturity through alcohol experience – Focus group interviews with teenagers. Addic tion Research and Theory, 14(6), 589–602. h y Herd, D. (2005). Changes in the prevalence of alcohol use in rap song lyrics, 1979–97. Addiction, 100(9), 1258–1269. y Maher, L., & Hudson, S. L. (2007). Women in the drug economy: A metasynthesis of the qualitative literature. 3. Choose a Title that Corresponds to the Content Journal of Drug Issues, 37(4), 805–826.* Maeyer, J. D., Vanderplasshen, W., Camfield, L., Vanheule, S., Sabbe, B., & Broekaert, E. (2011). A good quality of life under influence of meth adone: A qualitative study among opioid-dependent individuals. International Journal of Nursing Studies, 48, 1244–1257. Miovský, M. (2007). Changing patterns of drug use in the Czech Repub lic during the post-Communist era: A qualitative study. Journal of Drug Issues, 37(1), 73–102.h g Phillips, D., Thomas, K., Cox, H., Ricciardelli, L. A., Ogle, J., Love, V., & Steele A. (2007). Factors that influence women’s disclosures of sub stance use during pregnancy: A qualitative study of ten midwives and ten pregnant women. Journal of Drug Issues, 37(2), 357–376. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. 4. State the Research Question Early and Clearly 4. State the Research Question Early and Clearly It is a common failure in qualitative reports to embed the research question so deeply in the text that the reader cannot find it. The best way to avoid this is to include, at the beginning of your manuscript, a subtitle called “Research ques tion” or “Aim of the study.” An alternative is to present the question at the end of the background or introduction section. g It is not unusual for the reader of a qualitative article to find several differ ent, sometimes even contradictory, research questions presented throughout the various sections of the article: one question in the introduction, another in the methods and data section, and a third in the discussion (Drisko,2005). Even if the research process in qualitative research is often more unpredict able than in quantitative research and you gain new insights during the research process that will affect your perspective, the aim of a research report is as a rule to report not on this exploratory process but instead on specific findings answering a specific question. The reader does not want to be taken through the whole story of the researcher’s mistakes and new choice of ques tions. Focus on a single clear question that will orient the reader’s interest and prepare him for the text to come. It may be that your research project will in fact be able to answer many questions. Perhaps then you should consider producing several shorter and focused articles, rather than trying to squeeze it all into one text. If possible, phrase the research question in a way that reflects the scientific ambition of the study: Is it an article that explores a topic, aims at discovering a new social phenomenon, presents a new perspective, seeks to raise conscious ness about a problem, evaluates a project, or tests a theory (Drisko, 2005)? Box 8.5: Examples of well-written qualitative articles. A title that indicates what you are interested in will generate more readers who really are interested in your research—and probably more citations of your article (see Chapter 10). Sometimes it is possible to formulate the title so that it also describes what kind of data you have used. A title should not promise too much or be too fancy. If the title of the article is “The commer cial discourse on alcohol,” the reader expects that the theoretical contribution will be substantial. If it is “An analysis of alcohol marketing” and you deal only with beer advertisements in a short period in Greece, the reader may be disappointed. 166 Publishing Addiction Science 5. Conduct a Thorough Review of Earlier Research A good review of earlier research on the topic is essential for your claim that you are contributing new knowledge. It also shows that you want to take your place in the research community and engage in serious dialogue with other researchers. If the referees find that you have overlooked important literature, particularly if it is their own work (and since qualitative addiction research is a small field, you will often have a referee that has contributed to your topic), or that you have misinterpreted earlier studies, they will read your study with skepticism. Do not limit yourself to literature from your own country, but be sure to cover what has been written from your own culture.h y The literature review should not be solely descriptive. Use it to position your self in relation to other researchers and to demonstrate that you are doing some thing new. What conclusions about your questions can already be drawn from earlier research? State why you think earlier studies have missed a particular aspect of the topic or have taken a perspective that can be complemented with How to Write Publishable Qualitative Research 167 a new one. Alternatively, say why and in what way you want to use an approach or develop a line of thought presented by someone else. When you have presented a good review of earlier research, you will also have defended your theoretical and methodological position and your choice of data. Be certain to choose the right body of literature with theoretical relevance for your question. If you are studying gender differences in advertisements for tobacco, be sure to cover the literature on gender and media: do not focus exclusively on what we know about gender differences in smoking patterns.f f A thorough review, in which you position yourself clearly, also offers a practi cal way to avoid unfavorable referees. If you state that you disagree with X who has not taken Y into account, the editor will probably not send your text to X, to avoid a conflict of interest. Since the number of possible referees available to the editor usually is limited, this is an important consideration. 6. Present Enough Information in the Methods and Data Section According to Drisko (2005), inadequate methods are among the most common reason for qualitative articles being declined by editors. It is important to justify the choice of methods. If you want to be really convincing, explain your choice in relation to alternative methodologies. If you use several methods, explain how they complement each other. For instance, it is not enough simply to state that you use focus group interviews and a post-structuralist text analysis: You should describe how and why you use them Remember that many readers of addiction journals will not be familiar with qualitative methods. Therefore, you must describe the content of the method quite explicitly. Show that the research methods are suitable for the purpose of the study. It is important to convince the reader that you have used your method(s) systematically and on the entire data set. This includes the consist ent use of crucial concepts. You must argue that the size of the sample is sufficient for your purpose. As noted above, a small sample is one of the factors that raises skepticism among readers of qualitative research. How extensive is your data set? How many inter views with how many persons? How many meetings or observations? Position the sample clearly but without being too wordy: Try to focus on the essential features that will help an uninitiated reader to understand what you are analyz ing and what the sample represents. It is important to explain why your data set is the most illustrative and useful to answer the question you are posing. Be careful to describe how you picked your sample. What criteria did you use? Can you compare the data set with other alternatives and why did you choose this one? Describe the important variations within the data set (e.g., age and gender distributions) so that the reader gets a good picture of it. If you have used only a part of the data you have collected within a project, describe the rest of the data briefly to illustrate the Publishing Addiction Science 168 context or refer to another, already-published, article in which these data are presented. For the interpretation and transparency of your reasoning, it is crucial to describe how the data were produced and collected and how these conditions may have influenced the data. What special conditions, for example, come into play if you collect data from A.A. 6. Present Enough Information in the Methods and Data Section members, for whom anonymity is important? Do they affect the research participants’ willingness to be interviewed or how they talk during an interview? Tell the reader how (or whether) you presented the study to the participants. If you used focus groups, describe the groups’ dynamics. y Describe carefully each step in the analysis so that the reader can accept your conclusions—or argue against them. A good rule is to present the analysis of one observation/item/response in detail. Describe your interpretations during the analysis in a systematic way and in small identifiable steps. Show the fruit fulness of your concepts. Show how you argued for saturation and how you handled diversity and contradictions in the data. A thorough description of how the data were handled is also important. It should be clearly stated, for instance, how and whether the interviews were transcribed, coded, and grouped. 7. Link the Results to the Research Question The presentation of the results is easiest for the reader to follow if the structure is directly linked to the research question, moves in logical steps according to the theory and method, and consistently uses the concepts presented earlier in the article. Present your data in a systematic way in the body of the text, so that quota tions, field notes, and other documentations are easily identifiable. The reader must be certain, for instance, whether you are using direct citations or analyz ing interpretations of what the observed or interviewed persons said. The cita tions or other illustrations must be clearly contextualised. For observational material, state whether you collected the data yourself or if you used data col lected by someone else. Give enough raw data (e.g., direct citations) but not too much. Avoid very short quotations. If you run out of space, ask the editor if you can use online appendices for additional material. Do not refer in the results section to data that you have not already presented in the data and methods section; if you state that you are going to use interviews, do not refer to observations in the results section. If the results are contradictory, declare that fact openly and explain how this may have occurred and what it may mean. If you use grounded theory, you should be able to present a theory as a result. Descriptive statements are not enough. The theory should be a product of the analyses and not just confirm or illustrate earlier theories (Glaser & Strauss, 1967). 169 How to Write Publishable Qualitative Research 8. In the Discussion Section, Restate Your Main Findings and Relate Them to Earlier Research The structure of the discussion in a qualitative article can follow the same struc ture as in quantitative research reports. After a very short summary of your research question (check that it is the same as in the introduction) and the motivation for your wish to explore it, you can repeat in one sentence the main result of your study.i Following this, discuss how your findings relate to earlier research: Do they fill out the picture of what we already know or possibly challenge or even con tradict earlier findings? In this section, you can also, if possible, refer to ear lier quantitative research. In what way has your study been important for the research community or for a larger audience? Can the results change the pic ture of similar phenomena in other cultures? Discuss the extent to which the findings with this data set are relevant to the understanding of other situations. What are the concepts that can be transferred to other settings? As noted in Chapter 12, a good discussion will also contain a consideration of the limitations of your study. What problems with the sample and data col lection restrict the possibility of getting a full answer to your research question? With what other data could the answer have been more complete? Could you have used an additional or alternative method? Finally, consider giving recommendations for further research that will improve knowledge about the topic you have studied. 9. And Finally, Some General Advice First, it is sensible for qualitative as well as for quantitative researchers to save their good data for scientific articles. Many qualitative researchers publish their results as reports, sometimes in series that will have limited distribution, or as longer articles in monographs. If you want to spread your findings to a larger audience, it is often more efficient to publish one or more articles in a scientific journal. Second, choose the right journal—a crucial success factor if you want to get your article published. The first step is to choose among either an addiction journal; a journal for qualitative research; or a scholarly journal for sociology, anthropology, history, etc. (see Chapter 3).i If you choose an addiction journal or a disciplinary journal, find out if it accepts qualitative reports. Table 8.1 presents a list of English-language addic tion journals that publish qualitative research. Non–English-language journals as a rule accept submissions of qualitative articles. Check if the journal has par ticular demands on article length that will make it difficult for your submission to be accepted. Look at the editorial board anddetermine whether it includes members who are familiar with qualitative methods. Finally, look at the content 170 Publishing Addiction Science 170 Addiction International Journal of Drug Policy Addiction Research and Theory Journal of Addictions Nursing Addictive Behaviors Journal of Alcohol and Drug Education African Journal of Drug and Alcohol Studies Journal of Drug Education Alcohol and Alcoholism Journal of Drug Issues Alcohol Research and Health Journal of Ethnicity in Substance Abuse Alcoholism Treatment Quarterly Journal of Gambling Issues American Journal of Drug and Alcohol Abuse Journal of Smoking Cessation Contemporary Drug Problems Journal of Social Work Practice in the Addictions Drug and Alcohol Dependence Journal of Studies on Alcohol and Drugs Drug and Alcohol Review Journal of Substance Abuse Treatment Drugs: Education, Prevention and Policy Journal of Substance Use Nordic Studies on Alcohol and Drugs European Addiction Research Substance Abuse Treatment, Prevention, and Policy Harm Reduction Journal Substance Use and Misuse International Gambling Studies Tobacco Control Table 8.1: English-language journals that publish qualitative articles. Table 8.1: English-language journals that publish qualitative articles. of the journal: To what extent do they publish qualitative articles? Bear in mind that many addiction journals are open to various research methods, even if those journals have a predominantly quantitative orientation. Finally, consider if it would be good to suggest a suitable referee for your article. 9. And Finally, Some General Advice Some journal editors may find it difficult to identify experienced refer ees for your manuscript. As an author, you can always suggest someone whom you would like to review your text, without, of course, any guarantee that the editor will follow your advice. Acknowledgements The authors thank Tom Babor, Phil Lange, Tom McGovern, Peter Miller, Jean O’Reilly, and Betsy Thom for valuable comments on earlier versions of the text. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. Conclusions In this chapter, we have emphasised that the similarities between conducting and writing up quantitative and qualitative research are greater than the dif ferences. We have presented some quality criteria, particularly for qualitative research, discussed criteria for evaluation of journal articles, and given some practical advice to authors. How to Write Publishable Qualitative Research 171 How to Write Publishable Qualitative Research 171 Publishing qualitative research is as least as challenging as getting quantita tive reports accepted. However, it is apparent that the addiction field as a whole is increasingly coming to realise the value of qualitative studies. We believe that, in the future, there will be an even greater interest in good qualitative research and a growing demand for mixed-methods studies. Those who have dug themselves down into the qualitative or quantitative trenches will emerge and start communicating with each other, for their own and everyone’s mutual benefit. References Arminen, I. (1998). Therapeutic interaction. A study of mutual help in the meet ings of Alcoholics Anonymous, Vol. 45. Helsinki: The Finnish Foundation for Alcohol Studies. Creswell, J. W., & Plano Clark, V. L. (2007). Designing and conducting mixed methods research. Thousand Oaks, CA: Sage. h Creswell, J. W., & Tashakkori, A. (2007). Developing publishable mixed meth ods manuscripts. Journal of Mixed Methods Research, 2(1), 107–111.h Denzin, N. K., & Lincoln, Y. S. (Eds.). (1998). The landscape of qualitative research: Theories and issues. Thousand Oaks, London, New Delhi: SAGE Publication. Des Jarlais, D. C, Lyles, C., & Crepaz, N. (2004). Improving the reporting qual ity of nonrandomized evaluations of behavioral and public health interven tions: The TREND statement. American Journal of Public Health, 3(94), 361–366.h Drisko, J. (2005). Writing up qualitative research. Families in Society: The Jour nal of Contemporary Social Services, 86(4), 589–593. Gilpatrick, E. (1999). Quality improvement projects in health care. London, Thousand Oaks, New Delhi: SAGE Publications. 72 Publishing Addiction Science 172 Glaser, B. G., & Strauss, A. L. (1967). The discovery of grounded theory: Strate gies for qualitative research. New York: Aldine.t Lalander, P. (2003). Hooked on heroin: Drugs and drifters in a globalized world. London/New York: Berg Publisher. Mareš, J. (Ed.). (2002). Sociální opora u dětí a dospívajících II [Social support of children and adolescents]. Hradec Králové: Nukleus. Mayring, P. (1988). Qualitative inhaltsanalyse: Grundlagen and techniken. Weinheim: Deutcher Studien Verlag. g Mayring, P. (1990). Einführung in die qualitative socialforschung. München: Psychologie Verlag Union. Miovský, M. (2006). Kvalitativní přístup a metody v psychologickém výzkumu [Qualitative approach and methods in psychological research]. Praha: Grada Publishing. Miovský, M. (2007). Changing patterns of drug use in the Czech Republic dur ing the post-Communist era: A qualitative study. Journal of Drug Issues, 37(1), 73–102. Miovský, M., & Zábranský, T. (2003). Kvalitativní analýza dopadu nové dro gové legislativy na drogovou scénu z perspektivy pracovníků zdravotnick ých zařízení a významných poskytovatelů služeb uživatelům nelegálních drog [Impact of new drug legislation on drug scene from the perspective of health care professionals working with drug users: Qualitative analysis]. Čs. psychologie, 47(4), 289–300. p y g Patton, M. Q. (1990). Qualitative evaluation and research methods. London, Thousand Oaks, New Delhi: SAGE Publications. h Robson, C. (2002). Real world research. Oxford: Blackwell Publishing. Stimson, G. How to cite this book chapter: Čablová, L, Pates, R, Miovský, M and Noel, J. 2017. How to Write a Systematic Review Article and Meta-Analysis. In: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P. (eds.) Publishing Addiction Science: A Guide for the Per plexed, Pp. 173–189. London: Ubiquity Press. DOI: https://doi.org/10.5334/bbd.i. License: CC-BY 4.0. References V., Fitch, C., Des Jarlais, D., Poznyak, V., Perlis, T., Oppenheimer, E., Rhodes, T., & for The Who Phase II Drug Injection Collaborative Study Group. (2006). Rapid assessment and response studies of injection drug use. Knowledge gain, capacity building, and intervention development in a multisite study. American Journal of Public Health, 96(2), 288–295. Strauss, A., & Corbin, J. (1998). Basics of qualitative research: Techniques and procedures for developing grounded theory, Second Edition. Thousand Oaks, CA: SAGE Publications.h Sulkunen, P. (1987). Sosiologian avaimet [The keys to sociology]. Porvoo, Helsinki, Juva: WSOY. Whittemore, R., Chase, S. K., & Mandle, C. L. (2001). Validity in qualitative research. Qualitative Health Research, 4(11), 522–537. CHAPTER 9 How to cite this book chapter: How to Write a Systematic Review Article and Meta-Analysis Lenka Čablová, Richard Pates, Michal Miovský and Jonathan Noel Introduction In science, a review article refers to work that provides a comprehensive and systematic summary of results available in a given field while making it pos sible to see the topic under consideration from a new perspective. Drawing on recent studies by other researchers, the authors of a review article make a critical analysis and summarize, appraise, and classify available data to offer a synthesis of the latest research in a specific subject area, ultimately arriving at new cumulative conclusions. According to Baumeister and Leary (1997), the goal of such synthesis may include (a) theory development, (b) theory evalu ation, (c) a survey of the state of knowledge on a particular topic, (d) problem identification, and (e) provision of a historical account of the development of theory and research on a particular topic. A review can also be useful in science and practical life for many other reasons, such as in policy making (Bero & Jadad, 1997). Review articles have become necessary to advance addiction sci ence, but providing a systematic summary of existing evidence while coming up with new ideas and pointing out the unique contribution of the work may pose the greatest challenge for inexperienced authors. 174 Publishing Addiction Science What is the Relevance of a Review? General definitions are one thing; the practical benefit of writing reviews is another. Why would a novice author/researcher engage in this activity? Why is it important? What benefits can it bring? First, it provides the authors with a gen eral understanding of the subject matter they study as part of their area of exper tise. Each field of study has its own terminology, and the more specific a topic is, the greater the terminological differences that may be found among authors. It is therefore important to produce a good description and critical appraisal of existing evidence concerning the topic being explored. Another objective is to integrate the findings generated by different studies into a meaningful body of evidence. The process of writing a review article will help the authors obtain a unique perspective on the issue and assist them in processing the results from many investigators into a consistent form. It will then be possible to summarize the results and interpret the existing evidence in a new light. To increase one’s chances of having a review article accepted for publication, it is useful to address topical issues in a given field or areas of research featuring a number of hetero geneous and controversial studies where a consistent approach is needed. What is a Review? It is difficult to provide a single definition of a review. Indeed, each journal uses its own—slightly different—definition of a review study. For example, the journal Adiktologie defines a review article as a “cogent summary of topical issues; the author’s own experience is not the underlying theme of the paper. The maximum extent is 16 pages, with not more than 50 bibliographical cita tions. References to recent literature (not more than five years old) should prevail” (Gabrhelík, 2013). Addiction, meanwhile, simply states that “reviews draw together a body of literature to reach one or more major conclusions” and allows review articles to contain up to 4,000 words with no limit on biblio graphic citations (Society for the Study of Addiction, 2015). It is difficult to provide a single definition of a review. Indeed, each journal uses its own—slightly different—definition of a review study. For example, the journal Adiktologie defines a review article as a “cogent summary of topical issues; the author’s own experience is not the underlying theme of the paper. The maximum extent is 16 pages, with not more than 50 bibliographical cita tions. References to recent literature (not more than five years old) should prevail” (Gabrhelík, 2013). Addiction, meanwhile, simply states that “reviews draw together a body of literature to reach one or more major conclusions” and allows review articles to contain up to 4,000 words with no limit on biblio graphic citations (Society for the Study of Addiction, 2015). Despite these limitations, clear distinctions can be made between the types of reviews that can be drafted. The traditional type of review is a narrative lit erature review, which assesses the quality and results of a selection of literature using implicit criteria (Culyer, 2014). The conclusions of traditional narrative reviews are often based on subjective interpretations of the literature and may be biased in unsystematic ways. Importantly, narrative reviews are essentially nonreplicable. In contrast, scientific journals often require reviews to be systematic in nature. Systematic reviews use explicit literature search strategies, inclusion and exclusion criteria, and criteria for determining the quality and reliability of study findings. Systematic reviews are replicable and the conclusions drawn by authors more easily verified. How to Write a Systematic Review Article and Meta-Analysis 175 How to Write a Systematic Review Article and Meta-Analysis 175 175 A systematic review that does not include an evaluation of study findings (i.e. What is a Review? performs only a systematic search using explicit inclusion and exclusion crite ria) is referred to in this chapter as a hybrid narrative review. Hybrid narrative reviews provide authors greater freedom to interpret and integrate study results and conclusions compared with systematic reviews but still allow the reader to determine the authenticity of the author’s findings. These reviews are particu larly important for theory development and problem identification, especially when the peer-reviewed literature may be incomplete and when important studies may not use rigorous experimental or longitudinal designs. Meta-analyses are a step beyond systematic reviews; they require a quantita tive analysis of previously published findings.h i The following sections discuss the steps involved in creating systematic reviews and meta-analyses. Although not explicitly mentioned, much of the information applies to hybrid narrative reviews as well. Because traditional narrative reviews are no longer viewed favorably, they will not be discussed. It is strongly recommended, however, that before writing any article, authors should first choose a journal to which to submit their research because of the subtle differences in journal manuscript definitions. Authors should study thoroughly the guidelines for authors and keep them on hand to reference while writing the article. This may save a great deal of time spent on final revi sions or even make them unnecessary. Main Steps to Successful Systematic Review It is useful to observe the following procedure when designing and writing a systematic review. If the intention is to arrive at a systematic classification of evidence, a well-considered and highly structured procedure should be used. Structure is a crucial requirement, and some specific tools (e.g., PICOS: par ticipants, interventions, comparators, outcomes, and study design) can make this more manageable (Smith et al., 2011). Below, we describe the specific steps involved in creating a systematic review and meta-analysis, using the develop ment of a previously published review as an example of good practice. The fol lowing recommended strategies are based on the published systematic review (Čablová et al., 2014). Inclusion of Research Questions In a review article, the research question is included and expressed in the text, formulated as the problem: the topic and the focus of the work. It can be thought of as a spiral that provides logical connections among the parts of the article; that is, different parts build on and follow up on each other in a logical pattern. In terms of a systematic review, the research question must correspond with the objectives of the study and be aligned with the methodology, which is particularly relevant for the identification of data sources (the literature search) and the determination of study inclusion and exclusion criteria. It represents an imaginary starting point for the selection of key words and other parameters that are looked for in the relevant studies. As an example, we can use an article investigating the quality and type of emotional bonds in young adults who use cannabis and its (implicit) research question: “Can an insecure emotional bond be associated with a higher rate of cannabis use among young adults?” or: “Is there a relationship and difference between the lifetime prevalence of cannabis use among young adults and the individual types of insecure emotional bond?” In a review article, the research question is included and expressed in the text, formulated as the problem: the topic and the focus of the work. It can be thought of as a spiral that provides logical connections among the parts of the article; that is, different parts build on and follow up on each other in a logical pattern. In terms of a systematic review, the research question must correspond with the objectives of the study and be aligned with the methodology, which is particularly relevant for the identification of data sources (the literature search) and the determination of study inclusion and exclusion criteria. It represents an imaginary starting point for the selection of key words and other parameters that are looked for in the relevant studies. As an example, we can use an article investigating the quality and type of emotional bonds in young adults who use cannabis and its (implicit) research question: “Can an insecure emotional bond be associated with a higher rate of cannabis use among young adults?” or: “Is there a relationship and difference between the lifetime prevalence of cannabis use among young adults and the individual types of insecure emotional bond?” Aim of the Review The aim of a systematic review is set in the same way as in an original research study; the article must contribute something new to the given research field. The specific aim should correspond with the research questions. It may be, for example, “to provide a systematic review of the results of studies published from 2000 to 2012 that investigate the specific relationship between the level 176 Publishing Addiction Science 176 of parental control and alcohol use among children and adolescents.” Alter natively, it may be “to classify parenting strategies in relation to alcohol-using children aged 12–15” or “to make a critical appraisal of recent studies of the emotional bond in young adults who use cannabis.”hh The aims are typically stated in the last paragraph of the introduction. The aims then determine the choice of the specific procedure used to search sources and process and present the results. In the concluding section of the study, it should be stated whether and to what extent the aims have been fulfilled. • Web of Science: http://www.webofknowledge.com • Medline/PubMed: http://www.ncbi.nlm.nih.gov/pubmed • EBSCO: http://search.ebscohost.com Identify Data Sources—Quality Literature Search The primary and most important data sources are electronic databases, typi cally accessed through university libraries. Because access to specific papers may be limited as a result of financial constrictions, the levels of access granted to students and staff will depend on the resources of the university subscribing to the journals. Thus, you may find that although you can get into a number of databases, you may be able to access only a few full texts (as the others require payment) and have mostly abstracts available, which may not be sufficient for systematic reviews. This is dealt with in more detail in the next point.i yh p In the field of addictology, we recommend to use following databases: • Web of Science: http://www.webofknowledge.com • Medline/PubMed: http://www.ncbi.nlm.nih.gov/pubmed • EBSCO: http://search.ebscohost.com • Web of Science: http://www.webofknowledge.com • Medline/PubMed: http://www.ncbi.nlm.nih.gov/pubmed • EBSCO: http://search.ebscohost.com How to Write a Systematic Review Article and Meta-Analysis 177 How to Write a Systematic Review Article and Meta-Analysis 177 • SCOPUS: http://www.scopus.com • ProQuest Central: http://search.proquest.com/index • PsycARTICLES: http://www.apa.org/pubs/databases/psycarticles/index. aspx Nevertheless, databases and full-text studies are not the only data sources. It is also possible to include conference presentations if the conference abstracts have been published. At the same time, some journals could have a problem with these types of publications because they did not undergo a standard peer-review process. Also, a quality literature search should not disregard print sources, such as monographs; articles in peer-reviewed, non-indexed jour nals; handbooks and manuals pertaining to the relevant topic; graduate theses; and dissertations. These could be included into a category “Records identified through other sources” in the PRISMA (Preferred Reporting Items for System atic Reviews and Meta-Analyses) study flow diagram (see below). l We recommend keeping scrupulous notes on the articles read, either using Endnote or a separate database of references. This is relevant to all research but particularly to reviews. Process of “Data Collection” The complete literature search process needs to be recorded and documented. When evaluating systematic reviews, peer reviewers pay special attention to the means used to collect the “data” (i.e., specific publications) for the analysis. There are specific methods that can be applied for this purpose, with the PRISMA study flow diagram being the most frequently used one in contemporary sci ence (Higgins & Green, 2008; Moher et al., 2009). Figure 9.1 shows the PRISMA study flow diagram used in the systematic review (Čablová et al., 2014). Determine Selection Criteria The relevant publications, the results of which are to be processed, are selected according to the classification criteria that follow. • Year of publication—designating the period that is under study—may be used as the first criterion. i • Number of citations of the article—this information can be found in data bases, most often under the heading “Times cited.” Articles with a greater number of citations report on more prestigious research.li g • Key words—they reflect the terminology used in the given field and also help identify the most relevant studies. • Relevance of the article—online databases may turn up a number of arti cles but, unfortunately, because of the potential overlap of key words and other parameters, some works may be totally inconsistent with the focus of the review. It is therefore necessary to look through each publication—in most cases the abstract will be enough—and exclude any irrelevant studies. • Type of publications—although you may typically work with original and review studies only, specific topics may require the use of information from annual reports, research reports, or guidelines. It is therefore important to state these factors in the description of the procedure. • Study design—as far as research studies are concerned, these may be fur ther divided into subcategories: for example, reviews versus original works or, with clinical issues in particular, cross-sectional versus longitudinal. 178 Publishing Addiction Science 178 • Language of the publications—the languages that currently predominate in science are English and Spanish, with Chinese emerging as a significant language of science (in addition to English, Web of Science databases pro vide the option of searching studies in Chinese). • Sociodemographic environment—it is useful to describe the sociodemo graphic environment in which the research was conducted because it is a relevant factor that may influence the review’s results. Thus, the review needs to take this into account when presenting the research results.l • Funding source and conflicts of interest—last but not least, the fund ing source of a study and other conflicts of interest may influence how the results are interpreted. As explained in other chapters, significant biases in study reporting have been uncovered when the funding source or authors have a financial stake in the results of the study. Determine Selection Criteria Entered into a database or observed when working with hard-copy sources, these criteria make it possible to focus the work on the research question and the aim of the study you have laid down. Finally, all these criteria/indicators will be considered and interpreted in the subsequent discussion section. Explanation of the Specific Items in the Prisma Study Flow Diagram The first item, Records identified through database searching, shows the number of publications found in databases on the basis of the selection criteria. The item Additional records identified through other sources refers to the number of pub lications found in information sources other than those available online (these are typically print documents, such as research reports, handbooks, and manu als). Another step involves the elimination of duplicate articles. If you work with multiple databases, it is very likely that the same publication will be selected several times. Such duplicates should therefore be removed. This process is very easy if you use a citation manager. When using EndNote, for example, this can be achieved by simply activating the “Find duplicates” function. How to Write a Systematic Review Article and Meta-Analysis 17 179 Records identified through other sources (n = 21) Records identified through database searching (n = 371) Records excluded (n = 22) Records screened (n = 386) Records after duplicates removed (n = 386) Full-text articles assessed for eligibility (n = 364) Full-text articles excluded, with reasons (n = 348) Ineligible sample and subject of interest = 328 Different sociocultural setting = 16 Others = 4 Studies included in qualitative evaluation (n=16) Studies included in quantitative evaluation (n=16) Figure 9.1: PRISMA study flow diagram. Source: Čablová et al. (2014, p. 4). Records identified through other sources (n = 21) Records identified through database searching (n = 371) Records after duplicates removed (n = 386) Records excluded (n = 22) Records screened (n = 386) Full-text articles excluded, with reasons (n = 348) Ineligible sample and subject of interest = 328 Different sociocultural setting = 16 Others = 4 Full-text articles assessed for eligibility (n = 364) Studies included in qualitative evaluation (n=16) Studies included in quantitative evaluation (n=16) Figure 9.1: PRISMA study flow diagram. Source: Čablová et al. (2014, p. 4). Then you can focus on the articles. The item Records screened indicates the number of publications that remained after the exclusion of duplicates and publications rejected after you have read the abstracts. The number of articles eliminated on the basis of the examination of their abstracts is indicated in the Records excluded box. Explanation of the Specific Items in the Prisma Study Flow Diagram On the other hand, articles for which the full text is available (these should make up as large a proportion of the initial set of records as possible) are assessed in the next step and their final number is given under Full-text articles assessed for eligibility. When reading through the stud ies, you should continue to bear in mind the selection criteria (ideally, with a checklist on your desk) and watch carefully for them being met in the studies under scrutiny. If a more rigorous design is applied, you can also create a table Publishing Addiction Science 180 specifically for the selection and assessment of publications. If you come across articles that do not meet the selection criteria, you should state the reasons for such ineligibility and the respective number of studies; see the item Full-text articles excluded with reasons. The last figure shows the final number of articles included in the study. This example contains two alternatives—Studies included in qualitative evaluation and Studies included in quantitative evaluation—but one item only, for example, Studies included in quantitative evaluation, is also possible. For more information about the PRISMA study flow diagram method, including further illustrations of the procedure or the PRISMA checklist that helps in keeping a record of the process, visit http://www.prisma-statement. org/statement.htm. Interpretation of Results The results of the studies you have obtained will be further summarized in a structured form—ideally a table—according to the classification criteria. It is advisable to compare the qualitative and quantitative perspectives of the stud ies when processing the results. (Although meta-analysis is not always the goal, it is useful to take quantitative as well as qualitative approaches into account.) When using a quantitative point of view, you can follow the number of stud ies that used a longitudinal versus cross-sectional design, how many studies applied a standardized methodology versus a methodology developed specifi cally for the purposes of the study, or how many studies had their samples of participants well balanced in terms of representativeness and how many did not. On the other hand, a qualitative perspective makes it possible to look for broader aspects of the works and fine subtleties in the results that have been ascertained. There are a number of available tools that can serve as a guide when examin ing study methodologies and results. The Consolidated Standards of Report ing Trials (CONSORT) statement provides a standardized way to report and interpret the results of randomized clinical trials (Schulz et al., 2010). The pri mary tool is a 25-item checklist that contains questions on how the trial was designed, the data analyzed, and the results interpreted. The Strengthening the Reporting of Observational studies in Epidemiology (STROBE) and Transpar ent Reporting of Evaluations with Nonrandomized Designs (TREND) state ments are similar checklists for studies using observational study designs (von Elm et al., 2007; des Jarlais et al., 2004). If a more quantitative analysis of study design is desired, the recommendations of the Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) working group may be used (Atkins et al., 2004). These recommendations contain a point system that can be used in combination with the CONSORT, STROBE, or TREND state ments to further differentiate among studies. Although useful, the results of using these tools should not be considered as absolute but as guides toward How to Write a Systematic Review Article and Meta-Analysis 18 181 determining the weight that a study’s conclusions should be given. Interpretation of Results In addi tion, systematic reviews should always be attentive to sex and gender issues, as described in the SAGER Guidelines (Heidari et al., 2016).ii Interpretation should always be based on the results and findings specified in a given study; you must refrain from adding any conclusions of your own, because the principal rule is to preserve and express the original author’s idea as precisely as possible. When formulating the ideas and working with other review studies, you should always look up the primary source and interpret its results. Other review studies may serve as an inspiration in classifying your results rather than being their source, functioning rather as “background material.” Any copyright rules should be observed when making citations. You should strictly avoid using findings presented by the original authors in their research as your interpretations; if at all, you can resort to a secondary citation, which in itself may appear rather awkward. Therefore, you should seek to be as accu rate as possible and restate the author’s original argument, looking up other relevant works on the topic that you will cite in the same way. In addition, it is necessary to be attentive and socially sensitive when interpreting the results of studies from different sociocultural settings; you should be careful not to make unreasonable generalizations and ensure that the results are always interpreted in terms of the given social context. This may involve engaging in some addi tional research but, particularly in the social science field, this extra effort is an element that has a major impact on the final product. In Table 9.1 we present an example that illustrates the processing of the results in a published systematic review (Čablová et al., 2014). The left hand column lists the studies according to authors and year, which corresponds with the standard identification of cita tions in text. The selection criteria applied to the studies under consideration are indicated in the heading line. The reader thus has a chance to see the results of the work in aggregate and in a clearly structured way without having to wade through a lot of text. Discussion and Conclusion—was the Aim Really Achieved? Once the results have been processed and interpreted, what is probably the most challenging part comes next. For one thing, you may be quite tired by now, because the previous systematic procedure was rather demanding in terms of attention and endurance, and now you need to think about the results and compare them with the conclusions drawn by other relevant studies and with each other. In particular, this requires you to bring a new perspective to the subject matter under study, singling out and discussing most salient finding from the results. Importantly, the discussion should compare and evaluate the results against other relevant research projects rather than against the presenta tion of the author’s opinions on the issue. Each idea or result presented in the 182 Publishing Addiction Science 182 Research studies Criteria Country Study design1 Age category2 Number of respondents Parental involvement3 Parenting styles4 Methods5 L C 1 2 3 Y N 1 2 3 4 5 1 2 Bahr & Hoffmann (2010) USA + + + + 4,938 + + + + + + Barnes et al. (2000) USA + + + 506 + + + Burk et al. (2011) USA + + 362 + + + CRPR Choquet et al. (2008) France + + + 16,532 + + + Clausen (1996) Norway + + + 846 + + + + + + Total 8 8 6 12 14 6 10 13 9 9 7 3 12 6 Table 9.1: Example of processing and presenting the results. 1 L = longitudinal study, C = cross-sectional study. 2 1 = younger children (9–12 years); 2 = older children (13–15 years); 3 = adolescence (16–22 years). 3 Y = yes; N = no. 4 1 = authoritative style, 2 = authoritarian style, 3 = permissive style, 4 = neglectful style, 5 = other. 5 1 = questionnaires newly developed for the purposes of the research, 2 = standardised questionnaires. Source: Čablová et al. (2014, p.5). How to Write a Systematic Review Article and Meta-Analysis 183 article needs to be properly cited, too. The conclusion consists of a practical evaluation of the study; it should not contain any new findings or evidence. Its purpose is briefly to summarize the results and the contribution of the study as a whole. Discussion and Conclusion—was the Aim Really Achieved? Although this can pose a formidable task to an inexperienced author, it is important to practice the skill of communicating your own views concisely.ht The conclusion often includes recommendations (resulting from the study) for further research and tips for practice. It is also advisable to highlight the unique contributions of your review. In technical terms, it is recommended to study carefully the instructions for authors provided by the journal in which you want to submit the article for publication. Although some journals require the discussion and conclusion to come in two separate sections, others prefer to have them combined. The latter requires a slightly different structure, and it is helpful to be familiar with the format requirements before writing the article. The Most Frequent Pitfalls When trying to pursue as systematic and transparent a procedure as possible, you can encounter several problems. We have already mentioned the poten tial problem with differences in terminology used by the authors who publish research on a given subject in the field. To prevent confusion, it is recommended that you read a reasonable number of articles pertaining to your topic and look for the terminology they use. Databases may be helpful in this. The Web of Sci ence platform, for example, features a “related records” function, which may be used to search for similar articles on a certain topic. You may be confronted with a range of often competing theoretical approaches or backgrounds used by the authors to explore the subject matter in question. Because the literature search may be a challenging and time-consuming task, you may need to allow some time to study the relevant concepts thoroughly (for which the studies you have identified may not provide all the answers, requiring you to do further reading), as well as to reflect on such differences in your own conclusions and interpretations. Other differences may be found in the methodology applied by the studies under scrutiny. There are authors who work with standardized methods and their results can be subjected to a simple and valid comparison; on the other hand, there are authors who use their own methodology and whose results are thus difficult to measure. Another aspect that will consume time is the elimination of duplicate records, because researchers sometimes publish the results of the same study in several parts, divided into various subtopics to meet the foci of different journals. A mechanical “remove duplicates” function cannot do all the work. It is necessary to be alert and watch out for any relevant correlates. Another problem that may be encountered when comparing results between studies is the difference in the number of study participants. Many studies do not use a representative sample of participants, and great differences in their 84 Publishing Addiction Science 184 sizes may strongly affect study generalizability. You may also face your own limitations, particularly regarding the inclination toward a selective choice of studies, where certain studies may not be included, either deliberately or inad vertently. The Most Frequent Pitfalls Because citation bias may significantly compromise the results, you should try to avoid it at all costs if you want to arrive at a conclusion that is relevant to the field. If you fail to do so, it is most likely that reviewers will dis cover such a bias, as it is their job to examine related studies in the given area of research.h The last aspect to consider during the interpretation process is the statisti cal versus clinical significance of studies. In a large number of cases, you will find results that are not reflected in clinical practice, despite being significant. Therefore, it is important to maintain contact with clinical practitioners (or consult other experts) and be able to compare the results with real life. You can then formulate how these significances correlate in the conclusion. i For addiction science, the critical evaluation of systematic reviews is quite important. It is the key to the correct interpretation of selective data from par ticular studies, it provides background for comparing findings, and it can help to identify potentially disproportionate or inhomogeneous interpretations of findings. It has always been a sensitive issue in the context of publishing addic tion science because of potential conflicts of interest, and the history of the field contains examples of published papers in which researchers intentionally distorted data. The tendency to interpret data in a different way and present specific points of view can be a potential source of bias (Bero & Jadad, 1997). For example, there are many examples of contrasting study findings in the area of tobacco policy depending on whether the study was or was not sponsored by the tobacco industry (Glantz, 2005). Meta-Analysis Meta-analysis is a form of systematic review that combines findings from a number of studies to create aggregate effect sizes. To do this, the size of the effect is calculated and indexed. This can be used for a number of purposes in addiction science, including the effects of an intervention (e.g., the use of nal trexone and acamprosate for treating alcohol use disorders [Maisel et al., 2013] or the impact of smoking bans on restaurants and bars [Cornelson et al., 2014]) and epidemiology (e.g., substance use among street children [Embleton et al., 2013]) or seroconversion of hepatitis C in relation to shared syringes [Pouget et al., 2012]). By aggregating the effects and applying a statistical analysis, a bet ter understanding may be obtained for some of these research questions.h This is a complicated and time consuming process, probably not best undertaken by inexperienced researchers, but it may add greatly to the better understanding of science and aid treatment providers and policy makers. The process is not dissimilar to that described above in terms of selecting articles How to Write a Systematic Review Article and Meta-Analysis 185 for systematic reviews but requires a more complicated analysis. There are also similarities with primary intervention trials, in which one focuses on how well an intervention works. However, in a meta-analysis, the researcher looks across studies to determine the magnitude of effects. It is worth following a system atic guideline such as PRISMA to establish a framework for the review (Moher et al., 2009).hi The first step is to formulate the research question. Decide the keywords you will use to search for articles, the date from which you wish articles to be included, and the inclusion and exclusion criteria. Search the databases you have chosen for articles that meet your subject and eligibility criteria. It is also worth looking at reference lists from the articles you have selected to find other articles not so far identified.i i Once the articles for inclusion have been identified they will need to be coded according to the variables chosen for the meta-analysis. Because these coding decisions are not always clear, two raters are often used to obtain some meas ure of reliability either by percent agreement or by a kappa coefficient. Meta-Analysis Enter the data extracted onto a database with relevant details of each study entered including, for example, type of intervention, follow-up periods, sample size, type of control group, and research design. yp g p g One of the problems in comparing a number of studies is that studies will report diverse outcomes according to the model they used. To determine effect sizes so that the meta-analysis is effective, a “common currency” of effects needs to be established in order for comparisons and aggregation to be made. Finney and Moyer (2010) suggest that the most common effect sizes used are stand ardized mean difference, odds ratio, and correlation coefficient. The standardized mean difference is “the difference between means on a continuous outcome variable for an intervention and a comparison condition, typically divided by the pooled standard deviation of the two groups.” (Finney and Moyer, 2010, pp 321). By using standard deviations, one can measure by how many standard deviations, or what proportion of standard deviations, the intervention is per forming better than the control group.f Another method of measuring effect size is by using the odds ratio. By calcu lating the probability of something changing divided by something not chang ing, a ratio may be obtained. An odds ratio of 1.00 would show that there was no difference between treatment and a control condition in which there were two possible outcomes.hfi The third method is the correlation coefficient, which can be used to express the relationship between a continuous intervention dimension (which is unu sual in addiction studies) and the outcome (Finney & Moyer, 2010).fi We have now established a method of calculating effect sizes, and, to find out whether there is indeed an effect and what that effect is, we must now aggregate them across the studies we have reviewed. This can be done with a fixed-effects or a random-effects approach. These two approaches deal with the study sampling errors, with the former assuming that the error in estimating 6 Publishing Addiction Science 186 the population effect size comes from random factors associated with subject- level sampling, whereas the latter assumes that there are study sampling errors in addition to subject-level sampling errors. A random-effects model is used more frequently because of a greater generalizability, although the fixed-effects model has a greater statistical power. Effects from larger sample sizes have less variance across studies and are therefore more precise. Meta-Analysis To test whether the overall effect size varies from zero, it is best to use specific statistical software designed to conduct meta-analyses (Finney & Moyer, 2010). As with systematic reviews, a table should be presented detailing all the arti cles included in the study and describing all the relevant characteristics, includ ing author, date of data collection, the main outcome findings, and methods of collecting the data. A forest plot that shows the range of findings for each study is also often included, detailing in comparison the range of effects in an intervention. Issues with Meta-Analysis There a number of issues that should be considered when conducting a meta- analysis. One may have to determine whether the effect sizes vary more than could be expected from subject-level sampling fluctuations in a fixed-effect model or, in a random-effect model, whether there are study-level random effects in addition to the subject-level sampling fluctuations. Are there addi tional factors that add variation in effect sizes explained by moderator variables? The moderator variables include different methods and participants across the studies and the interventions themselves. To test this, a homogeneity test can be used that will test for whether excess variation exists (Viechtbauer, 2007). Another problem is publication bias. If the articles are selected carefully from peer-reviewed journals and conform to the criteria for inclusion, there is still the problem in that studies that show no positive or neutral results are often not published, either because the researchers do not submit for publication or because the papers are rejected for publication. Therefore, any articles that refute the research question may not be included in the databases searched and therefore the results may be skewed. Selection of the articles needs to be done with great care. Only quantitative articles may be included—qualitative articles will not contribute a statistical outcome—and if the criteria are too strict, then the number of articles on which to base the analysis may be too small. On the other hand, if you the selection criteria are too wide, you may then include studies of poor quality that will affect the outcome of the meta-analysis. The other problem with selection of articles may be agenda bias, whereby the authors of the meta-analysis want to use the results to support a specific issue and may cherry pick the articles they include. Meta-analysis is complicated, and the analysis of the variance across articles is complex; therefore, it is always beneficial to get good statistical advice and to use an established statistical package for analyzing the data. How to Write a Systematic Review Article and Meta-Analysis 187 187 Conclusion and Final Advice As previously mentioned, a good review article is hardly possible without a good literature search. The literature search has its own rules that generally apply to both original and review studies. A systematic review involves a literature search procedure guided by the principle of keeping an accurate and transparent record of the entire process! It is useful to create a sum mary Excel table where citations of studies will be recorded according to the selection criteria. It may seem like extra work at the beginning, but the author will come to appreciate this facility even before the first round of the peer-review process is over. Indeed, peer reviewers very easily notice any shortcomings we have tried to hide. It is therefore strongly recommended to draw up and enclose with the article a diagram in which you document the procedure for selecting the studies. This will help reviewers understand the approach and the results obtained, and, if any queries should arise, this evidence will make it easy to refute and explain any misgivings about the process or the results. For these purposes, it is also recommended to archive the documents in both printed and computerized versions; a physical file for hard copies and a separate electronic folder for computerized counterparts may be a useful option, with the latter providing the extra convenience of the “find” functionality. i To summarize, the ultimate goal when developing a review article is a sys tematic, straightforward, and transparent procedure. Both the reader and the editor must be clear about what the aims and methodology are, and all the results must be in line with the methods used. Although certain variations on standard procedures are possible, they always need to be explained and justi fied in discussion; otherwise you will most likely deal with them in the first round of the peer-review process. There are some specific approaches and tools for quality assessment of reviews (e.g., AMSTAR [Smith et al., 2011]; MOOSE [Stroup et al., 2000]) that can be relevant and very helpful in determining what is assessed and how to make the manuscript better. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. References Atkins, D., Best, D., Briss, P. A., Eccles, M., Falck-Ytter, Y., Flottorp, S., . . ., Zaza, S., & GRADE Working Group. (2004). Grading quality of evidence and strength of recommendations. BMJ, 328, 1490. Atkins, D., Best, D., Briss, P. A., Eccles, M., Falck-Ytter, Y., Flottorp, S., . . ., Zaza, S., & GRADE Working Group. (2004). Grading quality of evidence and strength of recommendations. BMJ, 328, 1490. 8 Publishing Addiction Science 188 Baumeister, R. F., & Leary, M. R. (1997). Writing narrative literature reviews. Review of General Psychology, 1, 311–320. Bero, L. A., & Jadad, A. (1997). How consumers and policymakers can use systematic reviews for decision making. Annals of Internal Medicine, 127, 37–42. Čablová, L., Pazderková, K., & Miovský, M. (2014). Parenting styles and alcohol use among children and adolescents: A systematic review. Drugs: Educa tion, Prevention, and Policy, 1, 1–13. Cornelson, L., McGowan, Y., Currie-Murphy, L., & Normand, C. (2014). Sys tematic review and meta-analysis of the economic impact of smoking bans in restaurants and bars. Addiction, 109, 720–728.h Culyer, A. J. (2014). The dictionary of health economics (3rd ed.). Cheltenham, England: Edward Elgar Publishing Limited. Des Jarlais, D. C., Lyles, C., Crepaz, N., & TREND Group. (2004). Improving the reporting quality of nonrandomized evaluations of behavioral and pub lic health interventions: The TREND statement. American Journal of Public Health, 94, 361–366. Embleton, L., Mwangi, A., Vreeman, R., Ayuku, D., & Braitstein, P. (2013). The epidemiology of substance use among street children in resource- constrained settings: A systematic review and meta-analysis. Addiction, 108, 1722–1733. Finney, J. W., & Moyer, A. (2010). Meta-analysis: Summarising findings on addiction intervention effects. In P. G. Miller, J. Strang, & P. M. Miller (Eds.), Addiction research methods. Oxford, England: Wiley-Blackwell. Gabrhelík, R. (2013). Guidelines for Authors (updated January 2013). Adiktologie. Retrieved from http://www.adiktologie.cz/en/articles/ detail/459/4021/GUIDELINES-FOR-AUTHORS. Glantz, S. (2005, September). Why you should not publish any tobacco funded research. Presented at the annual ISAJE conference, San Francisco, CA. Heidari S., Babor T. F., De Castro P., Tort S., & Curno M. (2016). Sex and Gender Equity in Research: Rationale for the SAGER guidelines and recommended use. Research Integrity and Peer Review, 1:2. DOI: https://doi.org/10.1186/ s41073-016-0007-6 Higgins, J. P. T., & Green, S. (Eds.). (2008). Cochrane handbook for systematic reviews of interventions (Version 5.0.1 [updated September 2008]). The Cochrane Collaboration. Retrieved from www.cochrane-handbook.org. Maisel, N. C., Blodgett, J. C., Wilbourne, P. How to cite this book chapter: West, R, Stenius, K and Kettunen, T. 2017. Use and Abuse of Citations. In: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P. (eds.) Publishing Addiction Science: A Guide for the Perplexed, Pp. 191–205. London: Ubiquity Press. DOI: https://doi.org/10.5334/bbd.j. License: CC-BY 4.0. References L., Humphreys, K., & Finney, J. W. (2013). Meta-analysis of naltrexone and acamprosate for treating alcohol use disorders: When are these medications most helpful? Addiction, 108, 275–293. Moher, M., Liberati, A., Tetzlaff, J., Altman, D. G., & The PRISMA Group. (2009). Preferred reporting items for systematic reviews and meta-analysis: The PRISMA statement. Journal of Clinical Epidemiology, 62, 1006–1012. How to Write a Systematic Review Article and Meta-Analysis 189 Pouget, E. R., Hagan, H., & Des Jarlais, D. (2012). Meta-analysis of hepatitis C seroconversion in relation to shared syringes and drug preparation equip ment. Addiction, 107, 1057–1065. Schulz, K. F., Altman, D. G., Moher, D., & CONSORT Group. (2010). CON SORT 2010 statement: Updated guidelines for reporting parallel group ran domised trials. PLoS Medicine, 7, e1000251. Smith, V., Devane, D., Begley, C. M., & Clarke, M. (2011). Methodology in conducting a systematic reviews of healthcare interventions. BMC Medical Research Methodology, 11, 15. Society for the Study of Addiction (2015). Instructions for Authors. Addiction. Retrieved from http://www.addictionjournal.org/pages/authors (August 11, 2015 ). Stroup, D. F., Berlin, J. A., Morton, S. C., Olkin, I., Williamson, G. D., Rennie, D., . . ., & Thacker, S. B. (2000). Meta-analysis of observational stud ies in epidemiology: A proposal for reporting. Meta-analysis of Observational Studies in Epidemiology (MOOSE) group. JAMA, 283, 2008–2012. Viechtbauer, W. (2007). Hypothesis testing for population heterogeneity in meta-analysis. British Journal of Mathematical and Statistical Psychology, 60, 64–75. von Elm, E., Altman, D. G., Egger, M., Pocock, S. J., Gøtzsche, P. C., Vandenbroucke, J. P., & STROBE Initiative. (2007). The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: Guidelines for reporting observational studies. PloS Medicine, 4, e296. CHAPTER 10 Introduction Research output in the form of articles, books, and book chapters exists to be used by other researchers to inform subsequent research, influence policy deci sions, and improve clinical practice. Authors need to consider how to make appropriate use of their previous publications and the work of others and how to ensure that their own work will be used appropriately. A research article, book, policy document, or treatment manual should refer to other writing that is relevant to its message. Citation is the formal vehicle for doing this. It involves explicit reference to a piece of research output that, in principle, can be anything from an article in a journal to a website. Conventions applying to citation practice regulate the transmission of information, and cita tion conventions vary from one research field to another. The following text focuses primarily on what might be termed cumulative research in which the goal is to accumulate enduring knowledge and understanding.h g g g g There are two main types of citation (Box 10.1). In this chapter we use the term referential citation to refer to the situation in which a piece of research output (which may be empirical or conceptual) is being used for what it con tributes to the field. The term critical citation is used when the citing piece points to what is considered a flaw in some research output.h l The citation serves one or more essential functions: It enables the reader to examine the cited work to check the veracity of a statement that it is being used to support or the correctness of the use of a concept or interpretation of a process. When citing in support of a statement being made in one’s own article, it also acknowledges the contribution made by the cited work. Both 2 Publishing Addiction Science 192 the verification function and the acknowledgement function are important. One may also use citations to document how a political debate, historical process, or specific concept has developed and has been defined. We can call this the documentation function.1i Regarding the verification function and the documentation function, the scope for intentional and unintentional distortion of research through unfounded assertions or misleading statements is enormous. In principle, every nonobvious factual claim should be supported in some way, either by citing direct evidence or by tracing a link through citations and/or inference to that evidence. Introduction Similarly every hypothesis, conceptual analysis, or state ment of a theoretical position that is not advanced for the first time in a given article should trace a link to its source. Citations offer the readers an opportunity to determine for themselves whether the original source of a claim was justified and whether that claim is being accurately represented in the current piece. Regarding the acknowledgement function, it is right and proper that researchers should receive credit for their work, and citation is the primary Types of citations Referential citation: a work or part of a work is cited for what it contributes to the field Critical citation: a work or part of a work is cited because it is believed to mislead the field Functions of citations Verification function: the reader should be able to check the source for its accuracy and the accuracy with which it is reported Acknowledgement function: the source is given credit for its contribution Documentation function: the source is identified as the object of the research in its own right Box 10.1: Types and functions of citations. Functions of citations Types of citations Verification function: the reader should be able to check the source for its accuracy and the accuracy with which it is reported Critical citation: a work or part of a work is cited because it is believed to mislead the field Critical citation: a work or part of a work is cited because it is believed to mislead the field Acknowledgement function: the source is given credit for its contribution Documentation function: the source is identified as the object of the research in its own right Box 10.1: Types and functions of citations. 193 Use and Abuse of Citations 193 means by which this is achieved. This is not merely a matter of etiquette: Employment and promotion of individual researchers are built on reputation, and citations play a crucial role in this. The institutions that employ research ers achieve kudos and in many cases funding on the basis of the reputations of their employees. Moreover, charities and government bodies that fund research must receive credit for the work they support. Their own income may well depend on it. Deviations from Ideal Citation Practice Citation practice often falls far short of the ideal (for a discussion, see Reyes, 2001). There are a number of sources one may use to find out about good prac tice in the use of citations in systematic reviews (e.g., Bannigan et al., 1997; Chalmers et al., 1993; Cook et al., 1995; Moher et al., 2009; Petticrew et al., 2008; Reeves et al., 2002; Stroup et al., 2000; Sutton et al., 1999; see also Chapter 9). Use of citations in less formal reviews, such as to introduce research reports, is subject to greater variability. The following paragraphs examine common devia tions from ideal practice (see also Table 10.1). Selective Citation through need for Conciseness A legitimate reason to depart from ideal practice arises from the need for con ciseness. Many times in a given field, a large number of studies may be cited in support of a given statement. In the absence of other constraints, the acknowl edgement function might dictate that all relevant studies are cited. However, this would be impracticable. This raises the question of which article or articles to cite. There is a case for citing what we might call the discovery article: the first article to record the finding. However, this may be impossible to determine. Moreover, it may not represent the most robust support for the assertion in question. There is a case for citing a review article (an article that summarizes the research on a specific topic). This has the advantage of pointing the reader, at least indirectly, to a body of work rather than one or two studies that might be unrepresentative. The disadvantages are (a) the increased danger of misrep resentation because of hearsay and (b) failure to acknowledge the contribution of the original source. g A possible rule of thumb in determining policy relating to a specific finding is to aim to cite the discovery piece and no more than five other original sources that testify to the generality of the finding, unless there is an authoritative and noncontentious review that can be cited instead. When referring to a concep tual or theoretical exposition, the first major presentation of the current version should be used. 194 Publishing Addiction Science Selective Citation in Support of a Viewpoint A common bias in reporting the literature is to select only (or primarily) stud ies that support a given hypothesis or idea (viewpoint citation). This is harder to avoid and to detect than one might imagine. If there were a well-defined body of literature that examined a particular hypothesis, and numerous high-quality studies conflicting with the hypothesis were ignored in a review, that would amount in the eyes of some to scientific misconduct. A reader who was not familiar with the area would be misled as much as if the author had fabricated data. Less straightforward is the case where there are doubts about the meth odological adequacy of conflicting studies. For example, studies that fail to detect the effect of an intervention may be small or involve inadequate implementation of the intervention. Unless one is explicitly attempting a comprehensive review in which there is the space to explore these issues, the citing author has to make a judgement about how far to go in ignor ing weak studies. Given the realistic possibility that the citing author is not wholly disinterested in the matter, it is good practice to alert the reader to conflicting findings and make a brief comment about the weight that might be attached to these and why.fi Even less straightforward is the case in which it is extremely difficult to deter mine what the corpus of findings on the topic is. This can happen for findings that typically do not form the main focus of articles. In the smoking literature, for example, it has been noted and is widely believed that depressed smokers are less likely to succeed in attempts to stop than are non-depressed smokers. There are certainly studies showing such an association (Covey, 1999; Glassman et al. 1990). However, often buried in reports of clinical trials and other studies are numerous reports of failures to find such an association, and indeed one meta- analysis has reported no association (Hitsman et al. 2003). There is no doubt that there are even more instances in which the association has been looked for and not found, with no subsequent report being made. At the very least, scientific prudence dictates that findings that are susceptible to this kind of phenomenon be cited with suitable caveats. Selective Citation for Convenience Using citations that are easy to find or that happen to have come to the atten tion of the author is not good practice but is probably very common. There may be many ways in which convenience citation can distort the literature. Insofar as more accessible articles may not represent the literature, use of convenience citations would create a biased impression. Searchable electronic databases, in principle, could mitigate the problem, but they can also lead to their own kind of distortion. It would be expected that they would favor English-language arti cles in journals indexed by the main databases. One would also expect more recent articles to gain preference because of the way that electronic databases sort the results of searches. Convenience citation would also be expected to favor the more popular journals. One might argue that this is no bad thing because it would be the better articles that would in general find their way into these journals. However, this is not necessarily so. Selective Citation to Enhance Reputation Self-citation or the citation of colleagues with a view to enhancing one’s own or the colleague’s reputation (reputation citation) is clearly unacceptable. It dis torts science and the process of science and is personally damaging to individu als in less-powerful positions or to those who do not engage in that practice (see e.g. Fowler et al., 2007). One may debate how widespread this practice is, but there can be little doubt that self-serving bias runs at some level throughout the scientific literature (see e.g. Aksnes, 2003). Self-citation or the citation of colleagues with a view to enhancing one’s own or the colleague’s reputation (reputation citation) is clearly unacceptable. It dis torts science and the process of science and is personally damaging to individu als in less-powerful positions or to those who do not engage in that practice (see e.g. Fowler et al., 2007). One may debate how widespread this practice is, but there can be little doubt that self-serving bias runs at some level throughout the scientific literature (see e.g. Aksnes, 2003). 195 Use and Abuse of Citations 195 Self-citation can also apply to journals (articles in journals tending to cite articles from the same journal). This may arise for reasons other than reputa tion citation, some of which may be legitimate, but it can distort the literature. One study found significant differences in self-citation rates among journals of anesthesiology (Fassoulaki et al., 2000). It may be thought that a bias of this kind would be easily detected and an appropriate correction could be applied. However, this is probably optimistic. It is not unreasonable that one’s own name should feature prominently in a refer ence list given that one’s research is presumably to some degree programmatic. A similar principle would hold true for one’s close colleagues. It can be difficult therefore to tell when this bias is operating. Citing Inaccessible Sources It is quite common for authors to cite conference papers or their abstracts, submitted articles, in-house papers, or unpublished reports (the so-called gray literature). The problem with this kind of citation is that it does not fulfill the verification function of citation. Therefore, it is generally to be discouraged. There may be cases where it is the only option and important in fulfilling the acknowledgement or documentation role, but if this is not obvious, the use should be justified. If that citation is more than a few years old, the use becomes increasingly problematic. It is often reasonable to presume that if it is an article or abstract and the finding was robust, it would have found its way into the peer-reviewed literature. It is becoming common to cite websites. This is reasonable but will pose increasing problems over time as websites move or become inaccessible. In general, for any statement intended to have lasting significance, this practice is best avoided until a system is devised for ensuring the longevity of web-based scientific literature. In policy analyses or descriptions of historical processes, though, references to sources such as websites and government documents may be a key part of the research process. Selective Citation by Country of Origin It goes without saying that a tendency to cite articles simply because they are from one’s own country of origin is not good practice. Many researchers are under the impression that this occurs, however. Naturally, the greatest suspicion falls on the U.S. as the main producer of research output, and many non-U.S. researchers can probably recount cases where a U.S. author has cited predomi nantly or exclusively U.S. references, even when more appropriate ones from other countries exist. In fact, this bias has been found among both U.K. and U.S. researchers publishing in major medical journals (Campbell, 1990; Grange, 1999). Another study found that North American journals cite North American journals to a greater extent than did journals from other regions (Fassoulaki et al., 2000), but the opposite has also been found (Lancho Barrantes et al., 2012; Pasterkamp et al., 2007). 196 Publishing Addiction Science Citing Without Reading There is a great temptation to cite a work or part of a work on the strength of a report of what it says without going to the original source. Thus, if an article or a book chapter that we have access to makes a statement that is relevant to our work and cites another article in support of it, it is tempting to repeat the assertion and the citation without reading the original source material. This is clearly unacceptable because of the risk of misrepresentation. Equally, having identified an abstract of an article using an electronic database, an author may be tempted to cite the article without going to the full text. This is risky practice because one has not taken the opportunity to evaluate the research being cited by reading the methods and analyses used. As a general principle, authors should not make reference to research output without having read and evaluated that output directly. Overuse of Citations Much of the earlier discussion concerned selective use of citations. Quite a common problem is the reverse: providing a long list of citations to support a single statement when fewer would be sufficient. If it is important that the work of the authors of all the various works be acknowledged or if the intention is to provide a comprehensive review, then a long list of citations is appropriate. Otherwise it can make an article unwieldy, and the rule of thumb of selective citation described earlier could be adopted. Citing Unevaluated Sources When a citation is used to support a substantive statement, the implication is that the cited reference reports evidence in support of that statement. Inade quate though it is, peer review is the primary gatekeeper for this kind of report. Convenience citation selects citation material that is easy to find Discovery article the article that first puts forward a new concept Gray literature unpublished matter, such as conference presenta tions, submitted articles, and in-house papers and reports Publication lag the time between an article’s acceptance by a journal and its publication Reputation citation cites a work or part of a work with a view to enhancing one’s own reputation or that of a colleague Review article an article that summarizes the research on a specific topic Viewpoint citation cites a work or part of a work because it supports a given hypothesis or idea Table 10.1: Terminology related to deviations from ideal citation practice. Table 10.1: Terminology related to deviations from ideal citation practice. 197 Use and Abuse of Citations 197 However, it is commonplace for statements of fact to be supported by citations to book chapters, letters, conference presentations, abstracts, opinion pieces, and other material that has not been peer reviewed. Although in principle read ers can track down the source and make their own evaluations, this is often impracticable. The only thing that comes close to a safeguard is that cited work has been through a peer-review process. Within the social sciences, though, even non–peer-reviewed books still remain a main source for new analytical concepts. In some cases, however, the review process for books is as rigorous as the peer-review process for journal articles. Coercive Citation During the peer-review process, editors can be tempted to help increase the standing of their journal by encouraging authors to add more citations to the journal, without specifying relevant articles or indicating where more refer ences are needed. This practice is sometimes referred to as coercive self-citation. 98 Publishing Addiction Science 198 Coercive citation is inappropriate as it undermines the integrity of academic publishing and it should be resisted by both authors and editors. Unfortunately, the practice is widespread and strategic. One study found that around 20% of academics in business disciplines, economics, sociology and psychology have experienced coercive citation practices (Wilhite & Fong, 2012). The study also found that editors soliciting superfluous citations are more likely to target man uscripts written by few authors, preferably by scholars of lower academic rank. Getting Cited All the above should suggest that the process of citation is subject to consider able bias, and, although there is a duty on researchers to minimize this, it is unlikely that bias will ever be eliminated. This being said, if one is writing an article that one believes is important, it would seem reasonable to try to ensure that it is drawn to the attention of its intended audience, and that means being cited. The choice of journal is obviously of relevance (see Chapter 3). And it may not be the most prestigious journal that offers the best chance but, rather, the best-quality specialist journal. The most prestigious journals tend to be gen eralist and, as such, may not be routinely read by many potential users of the research. Whatever outlet one uses for one’s research, it can often be a good idea to take other steps to publicize the findings. Some researchers email or send copies of their articles to colleagues. One might post reference to them on list serves or publicize them on social media. With increasing use of Open Access, full text can often be made available on demand. Conference presentations and websites are also potentially useful sources of publicity. Citation Indexes We mentioned earlier that citations are often used as a marker of quality. There is a presumption that the more often an article is cited, in some sense the better it is. This extends to journals, for which the single most widely used measure of quality is the impact factor. The impact factor for a journal in a given year is calculated as the average number of citations in that year to articles in the pre ceding two years. Thus, if a journal published 50 articles in 2013 and 2014 and there were 100 citations to these articles in 2015, the journal’s impact factor for 2015 would be 2.0. Citations of authors to their own work are included. There fore, clearly the more prolific an author is and the more that authors cite their own work, the more useful those authors are to a journal wanting to maximize its impact factor.t Researchers are often judged by the citation counts of their articles and by the impact factors of the journals in which they publish. Funding decisions in many institutions are based in part on members of those institutions publishing Use and Abuse of Citations 199 Use and Abuse of Citations in “high-impact” journals. Unfortunately there are many problems associated with using citation counts as a marker of quality and even more with using the impact factor (Hecht et al., 1998; Jones, 1999; Opthof, 1997; Seglen, 1997; Semenzato & Agostini, 2000). Some researchers have suggested that it may be possible to use citation counts and impact factor with appropriate caveats and corrections (Braun, 2003; Fassoulaki et al., 2002; Rostami-Hodjegan & Tucker, 2001), whereas others have argued that such use should be abandoned (Bloch & Walter, 2001; Ojasooet al., 2002; Walter et al., 2003). j Regarding citation counts, the various biases in the use of citations discussed earlier should give an indication of the problem with using them as a marker of quality. In addition, it should be recalled that critical citation is quite com monplace. Therefore, an article might be cited precisely because it is weak or misleading. One article examined the association between peer ratings of qual ity and the numbers of citations between 1997 and 2000 to articles appear ing in the journal Addiction in 1997 (West & McIlwaine, 2002). Citation Indexes Although two independent reviewers agreed moderately in their ratings of the articles, the correlation between these ratings and the number of citations was almost zero. One factor that was correlated with citation count was the region of origin of the first author of the article: Articles from English speaking countries received more citations than those from continental Europe, which received more than those from the rest of the world. A larger analysis of citations to articles in emergency medicine revealed that the citation count of articles was predicted to some extent by the impact factor of the journal in which they appeared and to a more limited extent by quality of the articles (Callahamet al., 2002). A fur ther study of citations to articles reporting randomized trials in hepatobiliary disease found a significant association with a positive outcome but no associa tion with adjudged quality (Kjaergard & Gluud, 2002). Apart from the biases already discussed, the fact that only a small propor tion of predominantly U.S. journals are indexed in Web of Science would lead to a bias, particularly against non–English-speaking countries. One study reported that exclusion of core journals in emergency medicine had led cita tion counts in the field to remain low despite considerable expansion of the field (Gallagher & Barnaby, 1998). Another noted that the way to improve the impact factors of journals in dermatology was to increase the number of them indexed by Web of Science (Jemec, 2001). Another bias arises from researchers in some fields, such as biosciences, simply using more citations than research ers in other fields. This will disadvantage authors in low-citing fields, typically the social sciences. Another bias pertains to texts such as editorials, letters and book reviews not being included in the denominator of citable documents. When they are cited, this can distort the impact factors of small-volume jour nals. For example, journals publishing mostly “noncitable” book reviews can have surprisingly high impact factors (Jasco, 2009). There are a range of other factors that make citation counts potentially misleading as a marker of quality (Box 10.2). 200 Publishing Addiction Science • Articles are sometimes cited as criticism.t • Articles are sometimes cited as criticism.t • Articles are sometimes cited as criticism. Citation Indexes • Articles describing important original studies are often neglected in favor of reviews.h • There is a bias toward citing articles from one’s own country or research group or articles that are easily accessible.i • Some fields of study generate more citations than others irrespective of how important the articles are, for example, fields with high levels of activity and mature fields.h yi • The importance and quality of a work or part of a work may relate to its policy or clinical implications rather than its use by other researchers. • Other researchers may fail to grasp the importance of a work or part of a work.h • The citation indexes are biased toward U.S. and other English-lan guage journals. Box 10.2: Why citation counts are often misleading as a marker of quality. Addressing some of these criticisms, the Journal Citation Reports introduced a number of augmentations in 2007, such as the five-year journal impact factor and Eigenfactor. The five-year impact factor score is similar in nature to the traditional two-year impact factor but deals with a five-year citation window, which can be more useful for research areas in which articles are published and cited at a slower pace. Eigenfactor is based on the structure of the scholarly citation network (based on incoming citations, weighting citations from highly ranked journals more heavily) and gives a numerical indicator of the overall contribution of a journal to the literature. Eigenfactor is influenced by the size of the journal (the more articles, the higher the score). Other journal-level met rics include an Article Influence Score and the SCImago Journal Rank.h The San Francisco Declaration on Research Assessment (DORA), published in May 2013, arose from concerns within the scientific community regarding how research output is evaluated, and how scientific literature is cited. It is signed by a broad coalition of researchers, editors, publishers, research socie ties, universities and funding agencies. The declaration includes a set of indi vidual recommendations for parties involved in research assessment, as well as one general recommendation: Do not use journal-based metrics, such as Journal Impact Factors, as a surrogate measure of the quality of individual research articles, to assess an individual scientist’s contributions, or in hiring, promotion, or fund ing decisions. (DORA, 2013) Use and Abuse of Citations 201 DORA recommends that publishers use a variety of journal-based metrics to provide a more nuanced picture of how journals are performing. Another rec ommendation is to encourage a shift toward assessment based on the scien tific content of an article, rather than the publication metrics of the journal (DORA, 2013). One way of promoting this shift is to provide article-level met rics, such as downloads, citation counts, and altmetrics. Altmetrics measure science dissemination more broadly than traditional research impact, looking at how articles are discussed in the news and social media, saved and book marked in reference management tools, and recommended in postpublication peer-review systems (such as F1000 rating) (Cheung, 2013; Leydesdorff, 2009). However, the usefulness of altmetrics is limited from a bibliometric perspec tive because they are difficult to standardize and some of the measures can be gamed (Priem, 2013). Box 10.2: Why citation counts are often misleading as a marker of quality. Because the journal impact factor is badly suited for assessing the individual quality and quantity of scientific output by a researcher, a number of author- based bibliometric indicators have been developed. These include indices such as the h-index, hI-index, hm-index, i10-index, n-index, several m-indices, A-index, R-index, and the g-index. The multitude of indices reflects the dif ficulty in developing quantitative measures for assessing the quality of research (Fersht, 2009; Jasco, 2008; West et al., 2010a, 2010b). Note 1 We are grateful to Klaus Mäkelä for this insight. 5. Resist the propensity to do the following: 5. Resist the propensity to do the following: a. prefer citations from your own country of origin unless the finding in question is country specific; i b. prefer citations from yourself and colleagues; c. limit citations to those that support a contention, when in fact there are others that conflict with it; l d. cite output that is readily retrievable if there are more appropriate ref erences; and e. provide an unnecessarily large number of citations for a single state ment. 6. Avoid citing inaccessible sources wherever possible. 7. When using citations in support of substantive statements, either use references that have been through some kind of peer-review process or provide an appropriate caveat. Citation counts are widely used as an index of quality. Given that few if any researchers are able to follow all the above principles, together with the many other factors that influence the number of times a piece is cited, citation counts are a highly problematic index of quality. Journal impact factors are even more problematic. Authors should be aware of this and not be beguiled by their apparent objectivity. Ultimately, there appears at present to be no substitute for peer evaluation of research output, however flawed and subjec tive this might be. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. Conclusions Citations are the primary formal means by which scientific findings are com municated. In terms of full transmission of information, ideally citation prac tice would involve comprehensive and objective use of the whole corpus of relevant published literature. Clearly this is impracticable. However, it should still be possible to approximate this ideal by adopting a few guidelines. These recognize that citation serves the dual function of enabling verification of state ments and acknowledging contributions.h In the case of formal reviews, the principles are well documented: The sources searched and the search rules should be clearly specified, as should the inclu sion and exclusion criteria for articles. The sources should go beyond Web of Science databases and include searching reference lists of articles in the search domain. Regarding informal reviews, such as are used to introduce research reports, the following principles can be applied: 1. Support all nonobvious, substantive claims by citation or direct evidence. 2. 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Coin of the Realm: Practical Procedures for Determining Authorship. In: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P. (eds.) Publishing Addiction Science: A Guide for the Per plexed, Pp. 207–227. London: Ubiquity Press. DOI: https://doi.org/10.5334/bbd.k. License: CC-BY 4.0. Thomas F. Babor, Dominique Morisano and Jonathan Noel Like a coin, authorship has two sides: credit and responsibility. One receives professional credit from his/her publications and takes responsi bility for their contents. Biagioli et al. (1999, p. 2) References Systematic reviews in the social sciences: A practical guide. John Wiley & Sons. Priem, J. (2013). Beyond the paper. Nature, 495, 437–440. Reeves, S., Koppel, I., Barr, H., Freeth, D., & Hammick, M. (2002). Twelve tips for undertaking a systematic review. Medical Teacher, 24, 358–363.h Reyes, H. (2001). [The references in articles published in biomedical journals] [article in Spanish]. Revista Médica de Chile, 129, 343–345. Rostami-Hodjegan, A., & Tucker, G. T. (2001). Journal impact factors: A ‘bio equivalence’ issue? British Journal of Clinical Pharmacology, 51, 111–117. Seglen, P. O. (1997). Why the impact factor of journals should not be used for evaluating research. BMJ 314, 498–502.h Semenzato, G., & Agostini, C. (2000). The impact factor: Deeds and misdeeds [Editorial]. Sarcoidosis, Vasculitis, and Diffuse Lung Diseases, 17, 22–26. Use and Abuse of Citations 205 Stroup, D. F., Berlin, J. A., Morton, S. C., Olkin, I., Williamson, G. D., Rennie, D., . . . , & Thacker, S. B. (2000). Meta-analysis of observational studies in epidemiology: a proposal for reporting. Jama, 283, 2008–2012. Sutton, A. J., Jones, D. R., Abrams, K. R., Sheldon, T. A., & Song, F. (1999). Systematic reviews and meta-analysis: A structured review of the methodo logical literature. Journal of Health Services Research & Policy, 4, 49–55. Walter, G., Bloch, S., Hunt, G., & Fisher, K. (2003). Counting on citations: A flawed way to measure quality. Medical Journal of Australia, 178, 280–281. l West, J. D., Bergstrom, T. C., & Bergstrom, C. T. (2010a). Big Macs and Eigen factor scores: Don’t let correlation coefficients fool you. Journal of the Ameri can Society for Information Science & Technology, 61, 1800–1807.h West, J. D., Bergstrom, T. C., & Bergstrom, C. T. (2010b). The Eigenfactor met rics: A network approach to assessing scholarly journals. College & Research Libraries, 71, 236–244.i West, R., & McIlwaine, A. (2002). What do citation counts count for in the field of addiction? An empirical evaluation of citation counts and their link with peer ratings of quality. Addiction, 97, 501–504. Wilhite, A. W., & Fong, E. A. (2012). Coercive citation in academic publishing. Science, 335, 542–543. CHAPTER 11 Introduction Authorship credit is conceivably the most important and least understood area of professional life for members of the scientific community. Because pro motion, prestige, and productivity are judged largely by publication activity, authorship credit has become the “coin of the realm” in the scientific market place (Wilcox, 1998). The two sides of this coin are credit and accountability. The assignment of individual credit to a publication implies certain ethical and scientific imperatives that are of tremendous importance to the scientific enter prise (Rennie & Flanagin, 1994). These imperatives include the certification of public responsibility for the truth of a publication and the equitable assignment of credit to those who have contributed in a substantive way to its contents.h The need for clear and consistent procedures for the determination of authorship credits comes from two considerations. First, many journals are now demanding that articles be prepared in a way that is consistent with the Publishing Addiction Science 208 principles of responsible authorship. Second, a clear consensus about the con ditions governing authorship decisions would make the work of individual authors much easier. Numerous professional organizations (e.g., American Psychological Associa tion, 2010), expert panels (International Committee of Medical Journal Edi tors, 1991, 2003, 2013), and individual commentators (Rennie et al., 1997) have developed policies and procedures dealing with individual, group, and corpo rate authorship. In this chapter, we review some of these guidelines from both the practical and ethical perspectives, in an attempt to develop workable proce dures that authors can follow during the course of preparing and publishing a scientific article. In addition, we consider authorship problems that sometimes arise in the course of a publication cycle. y Authorship problems seem to be occurring with increasing frequency (Wil cox, 1998). Of 785 authors abstracted from 121 articles published in The Lancet, 44% did not meet the most lenient guidelines for authorship and 60% of the most common contributor’s activities overlapped with those on acknowledgement lists (Yank & Rennie, 1999). Among Cochrane Reviews, 39% of publications had evi dence of honorary authors, and 9% had evidence of ghost authors (Mowatt et al., 2002). Introduction An analysis of ghost and honorary authorship among articles published within six leading medical journals (e.g., JAMA, The Lancet) in 2008 found that, although there appeared to have been a decrease in ghost authorship, specifi cally over the previous decade, the prevalence of articles with honorary and/or ghost authorship was still 21% (Wislar et al., 2011). Within 10 top peer-reviewed nursing journals, an even greater number (42%) of articles published in a two- year period contained honorary authors, and 27.6% had ghost authors (Kennedy et al., 2014). Undeserved authorships; failure to credit collaborating authors; relaxed policies for students, research assistants, and postdoctoral fellows; and an excessive number of co-authors are all serious problems. Some journals have gone so far as to limit the number of authors who can be listed on a submission (e.g., The American Journal of Public Health lists the cap as six).h h The pervasiveness of ethical issues in authorship is suggested by the extent to which scientific readers can be amused by the satirical humor epitomized in the “Ode to multi-authorship” quoted in Box 11.1. All cases complete, the study was over the data were entered, lost once, and recovered. Results were greeted with considerable glee p value (two-tailed) equalling 0·0493. The severity of illness, oh what a discovery, was inversely proportional to the chance of recovery. When the paper’s first draft had only begun All cases complete, the study was over the data were entered, lost once, and recovered. Results were greeted with considerable glee p value (two-tailed) equalling 0·0493. The severity of illness, oh what a discovery, was inversely proportional to the chance of recovery. When the paper’s first draft had only begun All cases complete, the study was over the data were entered, lost once, and recovered. Results were greeted with considerable glee p value (two-tailed) equalling 0·0493. The severity of illness, oh what a discovery, was inversely proportional to the chance of recovery. When the paper’s first draft had only begun All cases complete, the study was over the data were entered, lost once, and recovered. Results were greeted with considerable glee p value (two-tailed) equalling 0·0493. The severity of illness, oh what a discovery, was inversely proportional to the chance of recovery. When the paper’s first draft had only begun Coin of the Realm 209 the wannabe authors lined up one by one. Introduction To jockey for their eternal positions (for who would be first, second, and third) and whom “et aled” in all further citations. Each centre had seniors, each senior ten bees, the bees had technicians and nurses to please. The list it grew longer and longer each day, as new authors appeared to enter the fray. Each fought with such fury to stake his or her place being just a “participant” would be a disgrace. For the appendix is piled with hundreds of others and seen by no one but spouses and mothers. If to “publish or perish” is how academics are bred then to miss the masthead is near to be dead. As the number of authors continued to grow they outnumbered the patients by two to one or so. While PIs faxed memos to company headquarters the bees and the nurses took care of the orders. They’d signed up the patients, and followed them weekly heard their complaints, and kept casebooks so neatly. There were seniors from centres that enrolled two or three who threatened “foul play” if not on the marquee. But the juniors and helpers who worked into the night were simply “acknowledged” or left off outright. “Calm down” cried the seniors to the quivering drones there’s place for you all on the RPU clones. When the paper was finished and sent for review six authors didn’t know that the study was through. Oh the work was so hard, and the fights oh so bitter for the glory of publishing and grabbing the glitter. Imagine the wars when in six months or better The Editor’s response, “please make it a letter”. the wannabe authors lined up one by one. To jockey for their eternal positions (for who would be first, second, and third) and whom “et aled” in all further citations. Each centre had seniors, each senior ten bees, the bees had technicians and nurses to please. The list it grew longer and longer each day, as new authors appeared to enter the fray. Each fought with such fury to stake his or her place being just a “participant” would be a disgrace. For the appendix is piled with hundreds of others and seen by no one but spouses and mothers. If to “publish or perish” is how academics are bred then to miss the masthead is near to be dead. Conventions in Assigning Order of Authorship One of the difficulties in determining the criteria for authorship comes from the different traditions and practices that have been used to distribute author ship credits. Table 11.1 provides definitions of common authorship terms and ethical issues, some of which are also discussed in Chapters 5 and 14. Authors are sometimes listed in alphabetical order to avoid controversy about the relative contributions of different authors, especially when the contri butions have been fairly equal. A related convention is to list authors in reverse alphabetical order, presumably to avoid the preference given to persons whose surname begins with a letter that appears early in the alphabet. Another con vention is to list the laboratory director, center director, or other prominent person last. As noted in other parts of this chapter, this convention is not ethical unless that individual has made a substantial contribution to the publication and is not being listed merely to flatter the powerful or to add to the prestige value of the authorship list. This convention can also cause confusion when comparing contributions across fields. For instance, a last author might be pre sumed by some professionals to have contributed the least to an article and by others to have backed the entire project. The convention followed most frequently in the addiction field is to list authors according to their relative contributions, with the first author assumed to be responsible for writing the article, corresponding with the journal edi tor, and making the most substantive contributions. The first author in such a system is sometimes called the corresponding author. In some cases a senior researcher who is not the first author is designated as corresponding author to facilitate the progress of the manuscript through the peer-review process. This practice is not acceptable if the main purpose is to take advantage of this researcher’s influence and prestige, rather than to reflect actual contributions to the manuscript. Although the convention is assumed to be based on the equitable distribu tion of authorship credits, the relative ordering of authors is often depend ent on the first author’s subjective judgment of others’ contributions. In the absence of conducting an inventory of contributions, effort, and follow through, it is likely that some contributors will receive more credit than they deserve, and others less, solely because of the ambiguity and arbitrariness of the process. Introduction As the number of authors continued to grow they outnumbered the patients by two to one or so. While PIs faxed memos to company headquarters the bees and the nurses took care of the orders. They’d signed up the patients, and followed them weekly heard their complaints, and kept casebooks so neatly. There were seniors from centres that enrolled two or three who threatened “foul play” if not on the marquee. But the juniors and helpers who worked into the night were simply “acknowledged” or left off outright. “Calm down” cried the seniors to the quivering drones there’s place for you all on the RPU clones. When the paper was finished and sent for review six authors didn’t know that the study was through. Oh the work was so hard, and the fights oh so bitter for the glory of publishing and grabbing the glitter. Imagine the wars when in six months or better The Editor’s response, “please make it a letter”. RPU=repeating publishable unit; PI=principal investigator RPU=repeating publishable unit; PI=principal investigator Reprinted from The Lancet, 348, HW Horowitz, NH Fiebach, SM Levitz, J Seibel, EH Smail, EE Telzak, GP Wormser, RB Nadelman, M Montecalvo, J Nowakowski, and J Raffall, “Ode to multiauthorship: A multicentre, prospective random poem, 1746, 1996, with permission from Elsevier. Box 11.1: Ode to multiauthorship: A multicentre, prospective random poem. 210 Publishing Addiction Science Conventions in Assigning Order of Authorship With the growth of multicenter clinical trials and other “big-science” col laborative projects, corporate authorship has also increased. This convention lists a team name as the author, with a footnote or acknowledgement describing the contributors and the corresponding author. One reason for this conven tion is to make citations and referencing more efficient in cases where there are large numbers of contributors. Corporate authorship might also help to avoid the difficulties associated with determining who contributed what to a Coin of the Realm 211 Coercion authorship is a gift authorship that is demanded rather than voluntarily awarded. Contributorship consists of listing the contributions of each person involved in the project, avoid ing the attribution of authorship entirely. Corporate authorship lists the name of a project as author, along with a separate acknowledgement describing the contributors and the corresponding author (as an alternative to long author lists in multi-authored reports). Corresponding author is often the first author listed on an article, assumed to be the main researcher and writer of the article and the person responsible for corresponding with the journal editor. In some cases the corresponding author is not listed first when the writing and corresponding functions are divided. Ghost authorship is the failure to include as co-author of a work a person who satisfies the criteria for authorship (e.g., a science writer employed by a drug company). Gift authorship awards authorship credit because of a person’s power or prestige rather than for substantial contribution to the work. Group authorship See “Corporate authorship.” Guarantor is the person who takes responsibility for the contents and integrity of the work as a whole. Honorary authorship See “gift authorship.” Mutual-admiration authorship occurs when two or more researchers agree to list each others’ names on their own articles despite the others’ minimal involvement. Mutual-support authorship See “mutual-admiration authorship.” Pressured authorship See “Coercion authorship.” Surprise authorship occurs when a researcher finds out after publication that his or her name appears on an article. 2 Publishing Addiction Science 212 multi-authored article, and how much credit each author should receive. Some journals require contributors to formally name at least one person in the mast head, however (e.g., Alexander Bloggins for the Addiction Research Group). Conventions in Assigning Order of Authorship When participating in multidisciplinary or international collaborations, dif fering authorship conventions must also be taken into account, as authorship criteria and authorship order can have significantly different connotations in different disciplines (Anderson et al., 2011). As noted previously, in some disci plines, the last author may indicate the person who contributed the least effort, whereas in others it might signify the senior author or laboratory head. Because of the problems associated with determining who merits authorship credit, one editor (Smith, 1997) proposed the concept of contributorship. This involves listing the contributions of each person involved in the project, and avoiding the attribution of authorship entirely. Although this convention has not been adopted by any journal in its pure form (probably because the prob lems it causes with referencing), some journals, such as the American Journal of Public Health, request that all authors list their contributions when an article is submitted and publish a summary as a footnote or acknowledgement (Ameri can Journal of Public Health Instructions for Authors at ajph.aphapublications. org/page/authors.html). In summary, a variety of conventions have been used to arrange the names of individual contributors in multi-authored articles. Some conventions are used more than others, with the main-author-first convention used most often. Other conventions (e.g., group authorship) tend to be used in special situa tions as the case demands. The purpose of these conventions, particularly more recent variants, is to assure that proper credit is assigned so that individual responsibility for a publication can be inferred by the reader. Box 11.2: Ghost authorship by the tobacco industry. Box 11.2: Ghost authorship by the tobacco industry. In the journal Science, Dr. Gerald P. Schatten was listed as a co- corresponding author and senior author of an article on a high- efficiency method for generating stem cells (University of Pittsburg, 2006). Soon after publication, allegations of scientific misconduct, including scientific fraud and data manipulation, on the part of Dr. Woo Suk Hwang, the lead author, were made public and ultimately the article was retracted. Although Dr. Schatten was absolved from par ticipating in any misconduct, he was culpable for research misbehavior. Dr. Schatten wrote much of the article but did not verify the authen ticity of the raw data and did not critically examine discrepancies that occurred through the drafting process. An investigative board ruled that Dr. Schatten assumed senior authorship to enhance his scientific reputation, improve opportunities for funding, and obtain financial benefit. The board also ruled that only a few of the 25 authors listed had actually read the article before submission. Publication Policies and Publication Misconduct Over the past 25 years, journal editors, research administrators, and funding agencies have devoted increasing attention to the ethical and practical issues of scientific authorship. Concern about authorship has been heightened by a number of events and situations that have at times compromised, and at other times embarrassed, the entire scientific enterprise (Box 11.2 and Box 11.3).hli i The most flagrant examples involve scientific misconduct. In a number of well-publicized cases (Broad & Wade, 1982), investigators have published sci entific articles that have been retracted because the data were fraudulent or the contents plagiarized from other sources. What is remarkable about many of these cases is that, in addition to the person directly involved in scientific mis conduct (e.g., John Darsee, who was the lead author on numerous fraudulent articles; Relman, 1983), there have typically been a number of co-authors who apparently had no idea that the senior author was fabricating data or copying others’ ideas. This implies that in some cases co-authors are not in a position Coin of the Realm 213 213 In 1983 and 1986, the International Advertising Association published pro-tobacco reports on tobacco advertising bans and smoking prev alence, with the work credited to Dr. J. J. Boddewyn of Baruch Col lege, The City University of New York (Davis, 2008). Supporters of the tobacco industry enthusiastically touted the reports, but a later review of publicly available tobacco industry documents paints a different pic ture. Not only were the reports ghost written by Paul Bingham, then an employee of British American Tobacco, but Dr. Boddewyn was also a paid consultant of the tobacco industry, and the research itself was highly flawed. The relationship between Mr. Bingham, British American Tobacco, Dr. Boddewyn, and the International Advertising Association was not disclosed in the reports or in later hearings in front of the U.S. Congress. In 1983 and 1986, the International Advertising Association published pro-tobacco reports on tobacco advertising bans and smoking prev alence, with the work credited to Dr. J. J. Boddewyn of Baruch Col lege, The City University of New York (Davis, 2008). Supporters of the tobacco industry enthusiastically touted the reports, but a later review of publicly available tobacco industry documents paints a different pic ture. Not only were the reports ghost written by Paul Bingham, then an employee of British American Tobacco, but Dr. Publication Policies and Publication Misconduct Boddewyn was also a paid consultant of the tobacco industry, and the research itself was highly flawed. The relationship between Mr. Bingham, British American Tobacco, Dr. Boddewyn, and the International Advertising Association was not disclosed in the reports or in later hearings in front of the U.S. Congress. Box 11.3: Gift authorship of a retracted article. Box 11.3: Gift authorship of a retracted article. to take public responsibility for the contents of a scientific report, which is now considered to be one of the main criteria for authorship credit. In reality, there is a significant amount of basic trust across a number of domains that authors must invest in each other when collaborating on a publication, no matter what 214 Publishing Addiction Science their authorship position. Basic domains include honesty regarding the origi nality of the origins of any writing contributions, open disclosure about any conflicts of interest (e.g., financial investment in a business that is dependent on research outcomes, personal relationships with potential reviewers), and being thorough and ethical in any data entry and statistical analyses. With the rise in publication pressures that authors face at their own institutions and funding agencies (e.g., having to produce a minimum number of publications per year to stay employed), it is important to address a range of ethical concerns in pub lishing. In its updated statement on authorship standards for submissions to biomedical journals, the International Committee of Medical Journal Editors (2013) indicates that authors should be able to identify the specific parts of an article that the other co-authors have been responsible for.i Extreme cases aside, the abuse of scientific authorship has been suspected in an even greater number of cases where the scientific misconduct is much more subtle. Examples include the addition of authors to curry favor, conferring co- authorship by virtue of status or power, rewarding students or junior faculty with co-authorship to advance their careers, and adding a prominent name to a list of co-authors to receive a more sympathetic editorial review. Related to these problems and to the ever-growing importance of “research productivity” are disturbing trends toward the proliferation of authorship credits attached to publications, a growth in the number of mediocre quality publications (“paper inflation”), and the multiplication of reports using the “least publishable unit” to maximize the output from a single study (Lafollette, 1992). In part to prevent these kinds of problems, many journal editors and other individuals in scientific publishing have promoted policies designed both to detect misconduct and prevent the more blatant forms of authorship abuse. Box 11.3: Gift authorship of a retracted article. These policies include publishing detailed descriptions of the criteria for sci entific authorship, requiring that all authors sign a statement of authorship responsibility, putting limits on the number of authors listed on the masthead, and requesting that co-authors provide a written explanation of their individ ual contributions to a publication. How does all of this apply to individual authors? Even if most authors in the addiction field have never encountered an instance of data fabrication or plagiarism, they are likely to encounter the more subtle forms of irresponsi ble authorship and publication misconduct, such as gift authorship and ghost authorship (Flanagin et al., 1998). Honorary or gift authorship consists of awarding authorship credit because of the person’s power and prestige or as “payment” for another kind of contribution rather than for time, effort, and substantive contributions to the work. An extreme example of this is sur prise authorship, where a researcher finds out that his or her name appears on an article only after publication (Anderson et al., 2011). When someone demands (and receives) an honorary authorship, it is sometimes called a coer cion authorship or pressured authorship (Claxton, 2005; Freeser, 2008). Closely related to gift authorship is mutual-admiration or mutual-support authorship, Coin of the Realm 215 in which two or more researchers agree to list each other as authors despite little involvement in each other’s articles, usually as a means to expand their individual publication histories (Claxton, 2005). Ghost authorship refers to the failure to include as co-authors those who satisfy the criteria for authorship (Sheikh, 2000). This happens most often in the publication of pharmaceutical company trials in which an industry-paid scientific writer drafts the article but is not listed as a co-author to avoid the perception of conflict of interest. It also occurs with funded students and research assistants (Newman & Jones, 2006) who might contribute substantively to a publication but do not receive credit because the contribution is considered “part of the job.” In the remainder of this chapter, we review guidelines that have been devel oped to deal with publication misconduct and then some practical steps that can be taken by individuals, project teams, centers, departments, and profes sional organizations to ensure responsible authorship. Formal Guidelines Except under exceptional circumstances, a student is listed as principal author on any multi- authored article that is substantially based on the student’s doctoral dis sertation. Faculty advisors discuss publication credit with students as early as feasible and throughout the research and publication process as appropriate. Box 11.4: Authorship guidelines proposed by the american psychological association. Source: Section 8.12, American Psychological Association (2010). Psychologists take responsibility and credit, including authorship credit, only for work they have actually performed or to which they have substantially contributed. Principal authorship and other publication credits accurately reflect the relative scientific or professional contribu tions of the individuals involved, regardless of their relative status. Mere possession of an institutional position, such as department chair, does not justify authorship credit. Minor contributions to the research or to the writing for publications are acknowledged appropriately, such as in footnotes or in an introductory statement. Except under exceptional circumstances, a student is listed as principal author on any multi- authored article that is substantially based on the student’s doctoral dis sertation. Faculty advisors discuss publication credit with students as early as feasible and throughout the research and publication process as appropriate. Box 11.4: Authorship guidelines proposed by the american psychological association. Source: Section 8.12, American Psychological Association (2010). and further revised in 2013 (see www.icmje.org). The International Commit tee of Medical Journal Editors now indicates that each author should meet the following criteria: (a) substantial contributions to the conception or design of the work or the acquisition, analysis, or interpretation of the data; (b) drafting the work or revising it critically for important intellectual content; (c) approval of the final version to be published; and (d) agreement to be accountable for all aspects of the work in ensuring the questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. In addition, the International Committee of Medical Journal Editors recommends that an author should have confidence in the contributions of their co-authors and be able to identify which parts of the work he or she was responsible for. Additional changes were made by the International Committee of Medical Journal Editors to deal with contributors who do not meet authorship crite ria, such as people who provide general supervision or administrative support for a research group, technical help, writing assistance, language editing, or proofreading. Formal Guidelines To develop a more coherent, equitable, and ethical set of guidelines for addic tion journals, various policies have been proposed in the scientific literature. These policies include the guidelines recommended by the Council of Science Editors (Biagioli et al., 1999), the Sigma Xi standards for responsible author ship (Jackson & Prados, 1983), the statement of the International Committee of Medical Journal Editors (2013), and a variety of proposals from individual commentators (e.g., Broad & Wade, 1982; Fine & Kurdek, 1993; Newman & Jones, 2006). Box 11.4 describes the general guidelines developed by the Amer ican Psychological Association (2010). These have been the subject of a consid erable amount of interpretation and discussion in the psychological literature, and some attempts have been made to develop operational definitions of the specific criteria. i Winston (1985) developed a system in which points are assigned for vari ous professional contributions to a scholarly publication, with research design and report writing earning the most points. A certain number of points must be earned to qualify for authorship credit, and the individual with the highest number is granted first authorship. i One of the most cited sources on authorship is the 1985 consensus statement of the International Committee of Medical Journal Editors (1985). The state ment indicated that only those in a position to take public responsibility for the work could claim authorship. Although this definition would preclude gift authorship and help to minimize ghost authorship, there were still problems with the definition of a “substantial” contribution (see Yank & Rennie, 1999) especially in situations in which collaborating investigators band together on a project to take advantage of expertise that is unlikely to be concentrated in one individual. These problems were corrected in a 2003 revision to this statement 6 Publishing Addiction Science 216 Psychologists take responsibility and credit, including authorship credit, only for work they have actually performed or to which they have substantially contributed. Principal authorship and other publication credits accurately reflect the relative scientific or professional contribu tions of the individuals involved, regardless of their relative status. Mere possession of an institutional position, such as department chair, does not justify authorship credit. Minor contributions to the research or to the writing for publications are acknowledged appropriately, such as in footnotes or in an introductory statement. Practical Steps to Determine Authorship The foregoing discussion of conventions, problems, and policies suggests that authorship of an article is foremost a social process that requires a considerable amount of discussion, negotiation, and influence. If there is a general percep tion that the procedures for attributing authorship credits are inadequate and ineffective (see Yank & Rennie, 1999), then it may be because the social nature of authorship has not been taken into account in the design of policies and procedures for responsible authorship. Most guidelines focus on individual accountability in relation to abstract ethical principles, with bureaucratic con trols and punitive sanctions emphasized instead of practical guidance about what to do at the level of the group where real influence and control are concen trated. In this section, we describe a model process to demonstrate how many of the helpful suggestions provided in the literature on scientific authorship can be implemented in a practical, systematic, and open way. The process is based on the assumption that, because the writing of a multi-authored article is a social process, the responsibility, accountability, and equitable distribution of credit reside in the group of individuals most responsible for conducting the research and writing the article. This process can easily be implemented by an external agency or even within an institution, department, or research center. It needs to be conducted in an open, democratic, and ethical way so that all col laborating investigators agree to accept the basic values of scientific integrity. i As in any group process, one or more individuals need to take a leadership role. There is general agreement in the scientific community that the person most closely associated with the project should take responsibility for drafting the arti cle and being first author. Exceptions to this rule are possible, such as when the investigator who conceived and directed a project cedes responsibility to a junior investigator who made special contributions and who is capable of carrying the written report to a successful conclusion. A crucial skill that should be taken into account in the choice of one or more leaders for a scientific publication is famili arity with the authorship issues described in this chapter. Formal Guidelines These individuals and their contributions should be listed in an acknowledgements section. To the extent that a listing of such persons could be interpreted as an endorsement of the data or conclusions, the International Committee of Medical Journal Editors concluded that all persons listed must provide written permission to be acknowledged. and further revised in 2013 (see www.icmje.org). The International Commit tee of Medical Journal Editors now indicates that each author should meet the following criteria: (a) substantial contributions to the conception or design of the work or the acquisition, analysis, or interpretation of the data; (b) drafting the work or revising it critically for important intellectual content; (c) approval of the final version to be published; and (d) agreement to be accountable for all aspects of the work in ensuring the questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. In addition, the International Committee of Medical Journal Editors recommends that an author should have confidence in the contributions of their co-authors and be able to identify which parts of the work he or she was responsible for. Additional changes were made by the International Committee of Medical Journal Editors to deal with contributors who do not meet authorship crite ria, such as people who provide general supervision or administrative support for a research group, technical help, writing assistance, language editing, or proofreading. These individuals and their contributions should be listed in an acknowledgements section. To the extent that a listing of such persons could be interpreted as an endorsement of the data or conclusions, the International Committee of Medical Journal Editors concluded that all persons listed must provide written permission to be acknowledged. Coin of the Realm 217 Coin of the Realm 217 Planning Stage The planning stage of the publication process begins when a scientific investi gation or other project (e.g., a review article) has advanced to the point where it is likely that a scientific article is appropriate or warranted. This decision is usually made by the project leader, who either takes direct responsibility for the direction of the publication or designates one or more individuals to initiate the publication planning process. The following tasks and activities are suggested. • One or more senior members of the research or writing team take responsi bility for developing an outline of the article, a timetable for the completion of the article, and a list of potential co-authors, based on actual contribu tions to date and expected contributions in the future. The outline is distrib uted to all prospective authors, with the understanding that authorship will depend on substantive contributions, as well as effort and follow through, as described in relevant policies and publications (including this chapter). • Plans are made for a periodic reassessment of the research team’s contri butions throughout the planning, drafting, and finalization stages. If it is found that previous expectations are not being met, then assignment of authorship credit may be modified, based on actual contributions at the time of publication completion. • Relevant policies and publications (including copies of this chapter) are dis tributed to prospective authors along with the outline. • A meeting is called to discuss the proposed publication and the distribu tion of responsibilities for its completion. Assignments are made for data analysis and writing sections of the first draft. A timeline of key tasks is distributed and discussed. Practical Steps to Determine Authorship If the person has had no formal training in research ethics, the articles cited in the reference section of this chapter should be reviewed, giving special attention to several key sources (e.g., Fine & Kurdek, 1993; International Committee of Medical Journal Editors, 2013).l To avoid conflict, misunderstandings, and publication misconduct, both the lead author and the group should follow generally accepted procedures that are characterized by openness and transparency and should decide as early as possible who will be listed as an author, the order of authorship, and the other contributors to mention in the acknowledgments (American Psychological Association, 2010). In the following paragraphs, we provide an outline for a model that can be modified to fit the needs of a project team.hi ii The model requires the completion of specific tasks at each of three stages in the publication process. As described below, periodic discussions about 8 Publishing Addiction Science 218 authorship and accountability should be conducted at the planning stage, the drafting stage, and the finalization stage of a publication. According to Lafollette (1992), “The issue is absolutely clear. Who did what and how much? Answering those questions early on—and continuing to ask them as projects change—can help to prevent disputes or embarrassment later” (p. 107). Drafting Stage After the first draft of an article is completed or as relevant sections are finished, the drafting author or authors circulate the article for comments. At this stage, potential authors must be reminded not only about their rights to possible authorship but also about their responsibilities.i A crucial task at this stage is to identify who qualifies for formal authorship credit according to generally accepted criteria for responsible authorship. One Coin of the Realm 219 way to accomplish this task is to ask all potential contributing authors (includ ing the lead author) to describe their contributions to the project. Box 11.5 provides a checklist of contributions that prospective authors could be asked to complete by the lead author in order to determine eligibility for authorship at this stage. Although this one was designed for original research reports and may not apply to all publication types (e.g., reviews), similar disclosure check lists have been found to be useful for determining authorship credit (Yank & Rennie, 1999). Once the checklists are completed, the lead author could call a meeting to discuss authorship and other matters related to the proposed publication. At the meeting, each person is asked to describe his or her contributions to the project to date. In such a setting, individuals often reveal contributions that others were not aware of and, in other cases, describe activities that might not be considered substantial in comparison with those of others. At this time, it is important to discuss generally accepted criteria for authorship, such as those listed in Box 11.5, to make sure that everyone agrees on the standards for deter mining who should be listed on the article and in what order the names should be arranged. To provide authority to the process, it could be advantageous to mention that most journals now require a similar process of asking authors to sign a statement attesting that they have met minimal criteria for authorship, and some journals (e.g., The Lancet, BMJ, American Journal of Public Health) require authors to describe their individual contributions, the text of which is published along with the article.fi One of the most difficult decisions in the assignment of authorship credit is the distinction between major (or substantial) and minor contributions. A major contribution usually involves the independent development or inter pretation of ideas that are crucial to the advancement of a scientific study or a scholarly article. Drafting Stage It may also involve the use of special skills to perform a complex task without which the project could not have been done, such as the application of a sophisticated statistical technique. The emphasis in these definitions is more on quality than quantity. All persons making major contri butions should receive authorship credit, provided that they also participate in the writing of the article and any revisions required by the editor. Such indi viduals should also be capable of taking public responsibility for both general and specific aspects of the publication, recognizing that opinions differ as to what this means. Although the checklist provided in Box 11.5 was compiled from a variety of sources, we borrowed heavily from Yank and Rennie (1999), who distinguished between “major” and “partial” contributions. In a content analysis of articles in which authors provided a description of their roles in the publication process, they also report the 10 most common author contribu tions. A major contribution meant that the contributor fulfilled a majority of the activities for a given category (examples below). A partial or minor con tribution referred to a more limited role, presumably in terms of time, effort, or substance. 220 Publishing Addiction Science 220 Instructions: Use the checklist to describe your contributions to the pro ject to date. Under each item you have checked, describe the nature of your contribution, the amount of effort you put into it (e.g., hours, days, months), and whether your contribution fulfilled all of the requirements for that task or some of the requirements (e.g., in collaboration with oth ers, you wrote part of the article or you collected part of the data). Drafting Stage • Were responsible for conception of the project (planning meetings, drafting of research proposal, etc.) • Were responsible for conception of the project (planning meetings, t • Were responsible for conception of drafting of research proposal, etc.) drafting of research proposal, etc.) • Reviewed the literature • Obtained funding or other resources • Assembled the project team • Coordinated study (5) by assigning responsibilities and tasks • Trained of personnel • Supervised personnel • Obtained human (or animal) subjects approvals • Designed the methodology or experimental design (2) • Advised on design or analysis (9) • Wrote the research protocol • Collected data (4), including follow-up data • Performed clinical analysis or management (6) • Performed randomization or matching • Performed statistical analysis of data (8) • Performed economic analysis of data • Managed data (10) • Provided technical services (coding questionnaires, laboratory anal yses (7), etc.) • Provided or recruited patients • Provided materials or facilitiesi • Presented and defended findings in a public forumt • Responded to reviewers’ comments • Were responsible for other activity or service (describe) Box 11.5: Checklist for conducting an inventory of major and minor contri butions to a scientific article. Box 11.5: Checklist for conducting an inventory of major and minor contri butions to a scientific article.h i Note: The numbers in parentheses refer to the top-10 overall categories of con tribution identified by Yank and Rennie (1999) in a content analysis of arti cles according to the most frequently mentioned contributions to authorship. Coin of the Realm 221 Examples of major contributions that fulfilled Yank and Rennie’s (1999) “lenient” interpretation of the International Committee of Medical Journal Editors (1991) authorship criteria were (a) conception of the idea for the study or article, (b) design of the study, (c) statistical analysis or interpreta tion of data, (d) laboratory analysis, (e) management or analysis of clinical aspects, and (f) performance of field work or epidemiology. Anyone who wrote or revised the article (even sections) fulfilled the second part of the criteria (i.e., drafted the article or revised it critically for important intel lectual content). In considering the relative importance of major contributions, we believe two additional factors should be taken into account by the project leader and team: effort and follow through. Effort pertains to the amount of time spent on the particular contribution. Finalization Stage Before an article is formally submitted to a journal, a corresponding author needs to be designated. This person is usually the first author, but sometimes it is also the senior project leader in cases in which the first author is inexpe rienced with publication submission. A prominent or senior co-author should never be designated as corresponding author solely to influence the review pro cess. If there is general agreement about the authorship order throughout the writing process, this order can be reviewed again at the final stage to determine whether preparation and revision altered the relative order of contributions enough to require changes. Authorship Disputes If attempts to resolve authorship status before writing or publishing a manu script are unsuccessful, four processes for authorship dispute resolution have been proposed: direct dialogue, mediation, peer panel, and a binding decision (National Institutes of Health, 2010). Direct dialogue requires the parties in a dispute to discuss their differences with each other in order to reach an agreea ble solution. If direct dialogue is unsuccessful, they may enter mediation, which uses a neutral, third-party mediator to assist in finding a resolution. Parties in dispute may also present their perspectives on authorship to a three-person peer panel and agree to abide by the panel’s decision. If the dispute remains unresolved, then a scientific director or person in a similar position may make a binding decision. Although these processes have been created by a U.S. insti tution, they are applicable to any research environment and can be modified to best suit the authors’ circumstances. Drafting Stage Follow through involves active participation at various stages throughout the project. For example, if a person has participated in a study in a minor way or has made a major contribution that involves minimal effort (e.g., the development of an idea for the study or a novel hypothesis) and/ or follow through, this does not necessarily entitle the individual to authorship if other persons have made greater contributions with respect to effort and fol low through. g Nonsubstantive considerations should not determine the order of authorship or whether to include an individual as an author. Examples of nonsubstantive factors include rank or status, need for publication credits to justify advance ment, involvement in the project as a consequence of routine duties for which the individual is paid (e.g., collecting laboratory samples), or ability to provide access to study participants. The person who is named as the principal investi gator of a project or a grant for administrative reasons might not even qualify for authorship under these circumstances if she or he has had no role in the design and conduct of a particular project (e.g., the secondary analysis of data collected for another purpose). Members of a research team also need to recognize that, in general, indi viduals will be expected to contribute to projects in a collegial fashion with out necessarily receiving credit in all project publications. And, as noted in Chapter 5, the group may want to give consideration to the special situation of students and postdoctoral fellows where different standards for a contribution may apply.fi Taking all of the above information into account, it should not be difficult in most cases to reach consensus about who qualifies for authorship and what the most equitable relative ranking of contributions should be. When contribu tions are discussed in an open forum in relation to generally accepted criteria and ethical principles, secondary (nonsubstantive) considerations tend to be difficult to defend, especially when there is a written record of each individual’s perceived contributions. If there are discrepancies between what an individual perceives to be his or her contributions and the perceptions of others, these dif ferences often can be resolved through open discussion. 222 Publishing Addiction Science 222 Publishing Addiction Science The authors thank Ian Stolerman for his helpful comments and suggestions. The authors thank Ian Stolerman for his helpful comments and suggestions. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. Conclusion Intellectual honesty is a fundamental ingredient of scientific integrity, and this extends to the need for complete accuracy and transparency in repre senting contributions to research reports and other scientific writing. The contributions of colleagues and collaborators need to be recognized in all sci entific publications, but authorship must be assumed or awarded only on the basis of substantive contributions to an article and the ability of its authors to take public responsibility for its contents or, at least, for major parts of the contents. Decisions regarding authorship should be seen as part of a process that begins with the development of a publication plan and ends with the final revision of an accepted article. In between, it is best to have all potential contributors to a publication participate in an open process of stating their perceived contributions to a given project in the context of generally accepted Coin of the Realm 223 Coin of the Realm 223 criteria for authorship. Such a process is likely to manage expectations and prevent publication misconduct as well as misunderstandings and conflicts. To the extent that authorship credit continues to be seen as the coin of the realm in addiction science, both sides of the coin (credit and responsibility) need to be valued. Authorship Credit Exercise Appendix A contains two case studies that describe sensitive and possibly contentious authorship credit scenarios. For each case, answer the questions at the end and then discuss your answers with colleagues or a mentor in order to apply the principles described in this chapter. Also review Chapters 5, 14, and 15 for additional information about resolving ethical dilemmas in rela tion to authorship. References and Additional Reading American Psychological Association Ethics Committee. (2010). Ethical prin ciples of psychologists and code of conduct including 2010 amendments. Retrieved from http://www.apa.org/ethics/code/principles.pdf. Anderson, M., Kot, F. C., Shaw, M. A., Lepkowski, C. C., & De Vries, R. G. (2011). Authorship diplomacy: Cross-national differences complicate allo cation of credit and responsibility. American Scientist, 99, 204–207. DOI: https://doi.org/10.1511/2011.90.204 Biagioli, M., Crane, J., Derish, P., Gruber, M., Rennie, D., & Horton, R. (1999). CSE Task force on authorship draft white paper. Retrieved from http://www. councilscienceeditors.org/resource-library/editorial-policies/cse-policies/ retreat-and-task-force-papers/authorship-task-force/cse-task-force-on- authorship/. Broad, W., & Wade, N. (1982). Betrayers of the truth: Fraud and deceit in the halls of science. New York, NY: Simon & Schuster. 24 Publishing Addiction Science 224 Claxton, L. D. (2005). Scientific authorship. Part 2: History, recurring issues, practices, and guidelines. Mutation Research, 589, 31–45. DOI: https://doi. org/10.1016/j.mrrev.2004.07.003 Davis, R. M. (2008). British American Tobacco ghost-wrote reports on tobacco advertising bans by the International Advertising Association and J J Boddewyn. Tobacco Control, 17, 211–214. DOI: https://doi.org/10.1136/ tc.2008.025148h Feeser, V. R., & Simon, J. R. (2008). The ethical assignment of authorship in sci entific publications: Issues and guidelines. Academic Emergency Medicine, 15, 963–969. DOI: https://doi.org/10.1111/j.1553-2712.2008.00239.xl Fine, M. A., & Kurdek, L. A. (1993). Reflections on determining authorship credit and authorship order on faculty-student collaborations. American Psycholo gist, 48, 1141–1147. DOI: https://doi.org/10.1037/0003-066X.48.11.1141 Flanagin, A., Carey, L. A., Fontanarosa, P. B., Phillips, S. G., Pace, B. P., Lun dberg, G. D., & Rennie, D. (1998). Prevalence of articles with honorary authors and ghost authors in peer-reviewed medical journals. JAMA, 280, 222–224. DOI: https://doi.org/10.1001/jama.280.3.222 Horowitz, H. W., Fiebach, N.H., Levitz, S. M., Seibel, J., Smail, E. H., Telzak, E. E., . . . Raffalli, J. (1996). Ode to multiauthorship: A multicentre, prospective random poem [Letter to the Editor]. The Lancet, 348, 1746. DOI: https:// doi.org/10.1016/S0140-6736(05)65883-7 g International Committee of Medical Journal Editors. (1985). Guidelines on authorship. British Medical Journal, 291, 722. DOI: https://doi.org/10.1136/ bmj.291.6497.722 International Committee of Medical Journal Editors. (1991). Uniform require ments for manuscripts submitted to biomedical journals. New Eng land Journal of Medicine, 324, 424–428. DOI: https://doi.org/10.1056/ NEJM199102073240624 International Committee of Medical Journal Editors. (2003). Uniform require ments for manuscripts submitted to biomedical journals: Writing and editing for biomedical publication. Retrieved from http://www.icmje.org/about- icmje/faqs/icmje-recommendations/. International Committee of Medical Journal Editors. (2013). Recommendations for the conduct, reporting, editing, and publication of scholarly work in medi cal journals. Retrieved from http://www.icmje.org/icmje-recommendations. pdf. Jackson, C. I., & Prados, J. Discussion Questions 1. What are the ethical implications and whose interests are involved? 1. What are the ethical implications and whose interests are involved? 2. What should Dr. Muck do about the manuscript and the request to add Dr. Camel as a co-author? 3. What should have been discussed among the collaborators before the raw data was made available? Multicentered Trial with Multiple Investigators Multicentered Trial with Multiple Investigators Dr. Joe Camel is an assistant professor at Small State University where he is the principal investigator of a large, multicenter trial to determine the effectiveness of a new nicotine inhaler at reducing cigarette use and nicotine cravings. The main findings of the study were positive and have already been published in the Journal of Reputable Results. To maximize use of the data collected, Dr. Camel has made the raw data available to each of his colleagues for secondary analyses. It was agreed on by the group that a brief outline of the analyses to be performed and a list of potential co-authors should be prepared by those requesting to use the data to ensure there are no duplicate analyses. The group also agreed to prepare com ments and critiques in response to data requests. Dr. Muck E. Muck, a professor at Ivy League University, informs the Small State group that his team would like to perform an analysis on the effect of alcohol use in nicotine-cessation therapy. In response, Dr. Camel insists on being listed as the last and corresponding author even though he will not contribute to the data analysis, interpretation of the results, or manuscript preparation. Dr. Camel tells Dr. Muck that, as principal investigator of the trial, he has the right to be listed as an author on all related publications, and because he made the data freely avail able to Dr. Muck, he will not supply the data unless he does so. References and Additional Reading W. (1983). Honor in science. American Scientist, 71, 462–464. Kennedy, M. S., Barnsteiner, J., & Daly, J. (2014). Honorary and ghost author ship in nursing publications. Journal of Nursing Scholarship, 46, 416–422. DOI: https://doi.org/10.1111/jnu.12093 LaFollette, M. C. (1992). Stealing into print: Fraud, plagiarism, and misconduct in scientific publishing. Berkeley, CA: University of California Press. Coin of the Realm 225 Mowatt, G., Shirran, L., Grimshaw, J. M., Rennie, D., Flanagin, A., Yank, V., . . ., Bero, L. A. (2002). Prevalence of honorary and ghost authorship in Cochrane reviews. JAMA, 287, 2769–2771. DOI: https://doi.org/10.1001/ jama.287.21.2769 National Institutes of Health. (2010). Processes for authorship dispute reso lution. Retrieved from https://oir.nih.gov/sourcebook/ethical-conduct/ responsible-conduct-research-training/processes-authorship-dispute- resolution. Newman, A., & Jones, R. (2006). Authorship of research papers: Ethical and professional issues for short-term researchers. Journal of Medical Ethics, 32, 420–423. DOI: https://doi.org/10.1136/jme.2005.012757f Relman, A. S. (1983). Lessons from the Darsee affair. [Editorial]. New Eng land Journal of Medicine, 308, 1415–1417. DOI: https://doi.org/10.1056/ NEJM198306093082311h Rennie, D., & Flanagin, A. (1994). The Second International Congress on Peer Review in Biomedical Publication. JAMA, 272, 91. DOI: https://doi. org/10.1001/jama.261.5.749 Rennie, D., Yank, V., & Emanuel, L. (1997). When authorship fails: A proposal to make contributors accountable. JAMA, 278, 579–585. DOI: https://doi. org/10.1001/jama.1997.03550070071041 g j Sheikh, A. (2000). Publication ethics and the research assessment exercise: Reflections on the troubled question of authorship. Journal of Medical Ethics, 26, 422–426. DOI: https://doi.org/10.1136/jme.26.6.422t Smith, R. (1997). Authorship: Time for a paradigm shift? [Editorial]. BMJ, 314, 992. DOI: https://doi.org/10.1136/bmj.314.7086.992 University of Pittsburgh. (2006). University of Pittsburgh summary investi gative report on allegations of possible scientific misconduct on the part of Gerald P. Schatten, Ph.D. Retrieved from https://ecommons.cornell.edu/ bitstream/handle/1813/11589/Gerald_Schatten_Final_Report_2.08. pdf?sequence=1&isAllowed=y.h y Wilcox, L. J. (1998). Authorship: The coin of the realm, the source of complaints. JAMA, 280, 216–217. DOI: https://doi.org/10.1001/ jama.280.3.216 Winston, R. B. (1985). A suggested procedure for determining order of author ship in research publications. Journal of Counseling & Development, 63, 515–518. DOI: https://doi.org/10.1002/j.1556-6676.1985.tb02749.x Wislar, J. S., Flanagin, A., Fontanarosa, P. B., & Deangelis, C. D. (2011). Honor ary and ghost authorship in high impact biomedical journals: a cross sec tional survey. BMJ, 343, d6128. DOI: https://doi.org/10.1136/bmj.d6128 y g j Yank, V., & Rennie, D. (1999). Disclosure of researcher contributions: A study of original research articles in The Lancet. Annals of Internal Medicine, 130, 661–670. DOI: https://doi.org/10.7326/0003-4819-130-8-199904200- 00013 226 Publishing Addiction Science Junior Investigators Sharing Authorship on Each Other’s Articles Dr. Allen Quidproquo and Dr. Miriam Scratchmyback are the only postdoctoral fellows at the National Center for Addiction Science. They have both been work ing to publish their dissertation results. Dr. Quidproquo’s research focuses on the association of genes with initiation of substance use, whereas Dr. Scratchmyback researches the role of visual cues in treatment and relapse. The two fellows agree that their research has little in common and rarely discuss research topics in the office. But, being the only postdoctoral fellows at their center, they often share Coin of the Realm 227 meals together, talk about their nonacademic lives, and have quickly become friends. During one meal, Dr. Quidproquo talks about the pressure he is under to pub lish as often as possible. He can only stretch his data so far and has only a hand ful of publications to his credit. Dr. Scratchmyback has already been included as an author on more than a dozen publications. Therefore, Dr. Quidproroquo asks Dr. Scratchmyback if he could be a co-author on her publications to bump up his publication numbers, and, in return, he will list Dr. Scratchmyback as a co-author on all of his publications. Dr. Quidproquo reasons that this arrangement would effectively double the amount of publications on his list and substantially add to Dr. Scratchmyback’s list as well. He reasons this would better position them for future funding opportunities, faculty positions, and other research awards. How to cite this book chapter: Stolerman, I and Pates, R. 2017. Preparing Manuscripts and Responding to Reviewers’ Reports: Inside the Editorial Black Box. In: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P. (eds.) Publishing Addiction Science: A Guide for the Perplexed, Pp. 229–244. London: Ubiquity Press. DOI: https://doi. org/10.5334/bbd.l. License: CC-BY 4.0. Discussion Questions 1. How should Dr. Scratchmyback respond to her friend’s request?i 1. How should Dr. Scratchmyback respond to her friend’s request? 2. What can Dr. Scratchmyback do to maintain her own scientific integrity and/or prevent his colleague from committing scientific misconduct? 3. To what extent does either fellow stand to gain or lose from this arrangement? 2. What can Dr. Scratchmyback do to maintain her own scientific integrity and/or prevent his colleague from committing scientific misconduct? 2. What can Dr. Scratchmyback do to maintain her own scientific integrity and/or prevent his colleague from committing scientific misconduct? i 3. To what extent does either fellow stand to gain or lose from this arrangement? CHAPTER 12 Triage: the First Selection The author’s quest to find a suitable publication outlet ends with a letter stating, “I am pleased to inform you that your manuscript is acceptable for publication. . . .” But the first step is to get the manuscript into the peer-review system. Yet hav ing your article peer reviewed is not the inevitable consequence of submitting to a peer-reviewed journal. Some journals state formally that they operate a sys tem of “triage,” whereby the editor or his or her assistants decide which submit ted articles will be entered into the peer-review process. In practice, it is likely that all journals have such a system to protect the profile of the journal and to avoid bothering authors and peer reviewers with a long and laborious evalua tion process when it is easy to predict a negative result (see Box 12.1). Thus, if something is received that clearly has no hope of acceptance, it may be rejected without review. Here, the difference between journals is quantitative rather than qualitative: In the journals of highest impact in science and medicine generally, including addiction research, it may be that more than half of the submissions are rejected at this stage. Some addiction journals will, however, accept almost all articles, or reject only 20% or 25%. For some information on acceptance rates of addiction journals, see Chapter 3 and its appendix.h j There are some aspects of manuscript preparation that are so easy that every one should get them right. To ensure your manuscript has the best chance of passing through triage, make sure you do all these things as set out in the instructions to authors that every journal provides. Follow all advice and rec ommendations exactly, format your submission precisely as requested, make sure that all sections are complete, and be sure that no tables, figures, or figure legends are missing. Check the reference list to ensure that all cited references are in it, and no others. Check the accuracy of each citation. Look in the journal to see exactly how references are styled. Then check them over again, after you have made the corrections, until no more errors can be found. This sort of work is tedious but does not need expensive resources, profound knowledge of the subject, or outstanding intellectual ability. Triage: the First Selection If the editor sees at a glance that you do not even get these straightforward, mechanical things right, he or she may well develop a jaundiced view about your capability to deal with more complex matters. Try to look at your own manuscript as an editor might. If you do not bother to do the easier things required of an author, the editor might reason ably conclude that you will not be able to do complex revisions either, and you may not be given the opportunity to revise and resubmit. Introduction This chapter describes how the peer-review process works and presents sugges tions to authors of manuscripts. It is based on the experiences of scientists and clinicians who have many years of experience as editors of prominent addic tion journals. The task of the editor is to publish manuscripts appropriate for the journal and to assist would-be authors in the production of suitable mate rial. Many of the problems facing authors writing for scholarly peer-reviewed journals in the addiction field are similar to those in other fields. Therefore, it is recommended that readers consult one or more of the full-length books that have already been published in this general area. Hundreds of books have been published, as can be seen by searching Amazon.com or PubMed for “scientific writing.” For example, a search of Amazon.com on December 1, 2014, pro duced 16,904 results, many of which were relevant. A short list of recent books is provided at the end of this chapter in Appendix A. Nearly all academic journals now work exclusively with computerized systems that allow for submitting manuscripts, sending articles to reviewers, responding to the reviewers’ comments, and making a decision on the manuscript (e.g., accept/minor changes/major changes/reject). The advantages of these systems are increased efficiency for the editorial staff and an easier submission role for the author. It also makes it easier to keep track of manuscripts. As a rule, nearly all the communications to and from the journal are now done electronically. 230 Publishing Addiction Science Communication with more Experienced Writers Would-be authors may seek the advice of more experienced colleagues at almost any stage of the publication process. When planning a publication, discussion Preparing Manuscripts and Responding to Reviewers’ Reports 231 • The submission is outside the scope of the journal (e.g., it is about a misused substance but it is not relevant in any discernible way to misuse of or dependence on it). • The manuscript type is not appropriate (e.g., a case report is submit ted to a journal that does not publish case reports). • It contains clear ethical problems such as apparent violation of cur rent generally accepted standards for the treatment of human or animal subjects. • The article is poorly organized. • The report is purely descriptive, has no hypotheses, or reaches no conclusions. • There are major methodological weaknesses. • The article appears to offer nothing new. • Instructions to authors are flagrantly ignored in some way not men tioned here. Box 12.1: Reasons for rejection by triage. Note: The editor has a duty to reviewers, as well as to authors, and tries not to waste reviewers’ time by requesting evaluations of work that has no chance of acceptance for one or more of the reasons above. • The submission is outside the scope of the journal (e.g., it is about a misused substance but it is not relevant in any discernible way to misuse of or dependence on it).h • The submission is outside the scope of the journal (e.g., it is about a misused substance but it is not relevant in any discernible way to misuse of or dependence on it).h • The manuscript type is not appropriate (e.g., a case report is submit ted to a journal that does not publish case reports). • It contains clear ethical problems such as apparent violation of cur rent generally accepted standards for the treatment of human or animal subjects.h • The article is poorly organized.h • The report is purely descriptive, has no hypotheses, or reaches no conclusions.h • There are major methodological weaknesses.hf h • The article appears to offer nothing new.l • The article appears to offer nothing new • Instructions to authors are flagrantly ignored in some way not men tioned here. Box 12.1: Reasons for rejection by triage.h Box 12.1: Reasons for rejection by triage.h j y g Note: The editor has a duty to reviewers, as well as to authors, and tries not to waste reviewers’ time by requesting evaluations of work that has no chance of acceptance for one or more of the reasons above. Note: The editor has a duty to reviewers, as well as to authors, and tries not to waste reviewers’ time by requesting evaluations of work that has no chance of acceptance for one or more of the reasons above. with colleagues after presenting the work at a seminar in the home institu tion may yield some tips as to the type of journal that may be interested in the study. Subsequently, during preparation of the manuscript, it may be appropri ate to seek the advice of local colleagues on technical aspects, such as statistical analyses. When a manuscript exists in a complete form, it is often immensely helpful to ask at least one person to read it and make comments and sugges tions. People are very often willing to help if authors make clear that they value an expert opinion on aspects such as coverage of the literature, clarity, style, language, and validity of conclusions. If there is no person in the author’s own institution, it is possible to approach outsiders and ask if they would be willing to comment. Both people whom you know personally and others who have published in the area are likely to feel flattered and pleased that you value their opinion and may well provide advice. The manuscript that cannot be improved has yet to be written, and even experienced authors often seek the opinions of colleagues because, after working on a manuscript for months through revision after revision, authors may find it difficult to spot the little problems that spring to the attention of a new reader. with colleagues after presenting the work at a seminar in the home institu tion may yield some tips as to the type of journal that may be interested in the study. Subsequently, during preparation of the manuscript, it may be appropri ate to seek the advice of local colleagues on technical aspects, such as statistical analyses. When a manuscript exists in a complete form, it is often immensely helpful to ask at least one person to read it and make comments and sugges tions. Writing in a Foreign Language It is an unavoidable fact that many authors have to write in a language other than their own, and conveying complex scientific ideas with clarity and preci sion can be a difficult task even in one’s native language. Authors may therefore find it worthwhile to seek the assistance of colleagues with more experience in writing in the chosen language and, if possible, enlist a native speaker of the language to correct the manuscript. If that is not possible, it may be neces sary to obtain the assistance of a professional translator to suggest corrections. Journals provide varying amounts of assistance in the correction of errors after accepting a manuscript for publication, but they cannot do anything to assist reviewers of poorly written manuscripts. A fuller consideration of language issues and language editing services may be found in Chapter 4. Box 12.1: Reasons for rejection by triage.h People are very often willing to help if authors make clear that they value an expert opinion on aspects such as coverage of the literature, clarity, style, language, and validity of conclusions. If there is no person in the author’s own institution, it is possible to approach outsiders and ask if they would be willing to comment. Both people whom you know personally and others who have published in the area are likely to feel flattered and pleased that you value their opinion and may well provide advice. The manuscript that cannot be improved has yet to be written, and even experienced authors often seek the opinions of colleagues because, after working on a manuscript for months through revision after revision, authors may find it difficult to spot the little problems that spring to the attention of a new reader. 232 Publishing Addiction Science The Peer-Review Process: Selection of Reviewers The next step for the editor is selection of reviewers who will advise him or her of the strengths and weaknesses of the work and recommend whether or not it should be accepted, reconsidered after revision, or rejected outright. To improve the manuscript, reviewers are also expected to make constructive suggestions in a report that can be sent to the authors. The criteria used for selecting reviewers are diverse, and probably few if any journals have tightly defined procedures. Box 12.2 shows the main criteria used by editors to identify reviewers. The number of reviewers for each article varies within and between journals, but most commonly there are two. The editors of some journals may work with only one reviewer, but this seems to be increasingly rare. Occasion ally, three or more reviewers are used, depending on the journal and the edi tor’s perception of the complexity and significance of the work. For example, multidisciplinary manuscripts may require more than two reviewers to ensure sufficient expertise. Similarly, if a study seems likely to have a major practical impact, for example on policy or treatment, the editor may wish to be especially certain that it is assessed thoroughly. If the two reviewers initially selected disa gree about the article, an editor may seek additional advice from a third person to reach a decision. For all reports, regardless of whether they are quantitative or qualitative, each journal has its own set of instructions for reviewers; journals differ with respect to the attributes of their “ideal” manuscripts. There will sometimes be a requirement for reviewers to complete a questionnaire as part of the review, with ratings of the manuscript according to criteria such as importance and likely impact on the field, as well as technical competence. The reviewers are usually also asked to make a recommendation on the fate of the manuscript and to justify it in confidential comments to the editor. Finally, reviewers are in all cases expected to produce a report that the editor will forward to the Preparing Manuscripts and Responding to Reviewers’ Reports 233 authors. The main purposes of this report are (a) to make suggestions enabling the author to improve the manuscript and (b) to list criticisms that the reviewer believes need to be addressed if the report is to be published. Criteria for Evaluation of Manuscripts If the journal has published its instructions to reviewers or put them on a web site, these instructions will give you an idea about the features at which both editors and reviewers will look. Many journals probably look for the same desirable features of highly rated studies.fi If a study is quantitative, the criteria include the use of a sufficiently large and suitably representative sample of the population under study, the presence of a high response rate among invited participants, the use of valid measures, the absence of procedural biases, minimal confounding of one independent variable with another, and the use of appropriate controls. Similarly, review ers will look for as full a description of the methods as available space allows, with reference to earlier publications that provide more detail and establish the validity of the methods and measuring instruments (where applicable). Results must be described in a clear and logical sequence, with all necessary informa tion presented. No more detail should be provided than can be covered in the discussion section. The discussion should bring out the importance of all the main findings and indicate how the work advances the state of knowledge and understanding in the relevant subfield. In addition, alternative interpretations of the data may be given, thus acknowledging limitations of the study. Review ers pay attention to all the preceding points—and to many others. In quantitative research, the data analysis section is prone to several prob lems. These include the following: • failing to deal adequately with confounding variables; • failing to deal adequately with confounding variables; • claiming to have shown something without performing a (statistical) test that supports it directly and unequivocally; • failing to control for multiple comparisons; and • drawing inappropriate conclusions from non-significant associations or differences: we probably all realize that lack of significance means only that we have failed to find an effect and does not prove that no effect exists, but we don’t always remember this in our enthusiasm to explain how our results fail to support the ideas of a scientist whose theory we dislike. The Peer-Review Process: Selection of Reviewers The report to the authors should not include specific recommendations for acceptance or rejec tion of the manuscript because that decision is the editor’s. Chapter 13 provides further advice on how to become a competent reviewer. Reviewers are asked to act according to ethical guidelines that are presented and discussed elsewhere in this book (see Chapters 14 and 15). The task of the editor is to reconcile sometimes conflicting reports from different reviewers and to make a personal judgment based on a variety of other considerations. The task is made more difficult if the reviews contain conflicting recommenda tions for publication. • Recognized expertise in the specific field of the manuscript as noted in the journal’s database of previous reviewers and authors. • Previous invitations to the reviewer that have resulted in thorough, well-written, polite reviews submitted in a timely manner.i p y • Record of recent publications in the field as determined by searches of databases such as MEDLINE and PsycINFO. The following individuals are typically excluded from consideration as reviewers: • Persons who are known to have a very close connection to the authors or to have a conflict of interest with the authors will be avoided. • People who are currently reviewing another manuscript for the same journal or who have reviewed one within a set period (e.g., three months) will be avoided.h • Those who work is excessively praised or criticized in the manu script to be assessed are avoided. Box 12.2: Some criteria editors use to identify reviewers for a particular manuscript. Box 12.2: Some criteria editors use to identify reviewers for a particular manuscript. Note: Some journals ask authors to suggest reviewers or to name persons they do not wish to have as reviewers. How to use these suggestions is the editor’s decision. Different bulleted points from those above will be used in combina tion to reach a decision on whom to invite, and there will inevitably be appre ciable variations between journals with respect to the use of these different methods of selection. 234 Publishing Addiction Science Preparing Manuscripts and Responding to Reviewers’ Reports 2 235 • Give clear criteria for the selection of data or subjects. Position the material carefully in the social and cultural space. For example, different genres of fiction represent different segments of the culture. if • Present a detailed account of where and when data were collected or which existing data sets were used. In studies based on fieldwork, describe the relation between the fieldworkers and the subjects and discuss the possi ble influence of the data collection on the phenomenon under study. Keep careful records of the data so that they can be provided for independent examination if necessary. • Clearly state how the analysis was done, with an indication of whether reli ability was assessed—for instance by replicating the analysis. • Describe any themes, concepts, and categories derived from the data. Divide the interpretation process into short steps, specifying rules of clas sification and interpretation. • Outline steps taken to guard against selectivity in the use of data; discuss exceptions and deviant cases. Ideally, the reader should be able to apply the same classifications, take the same analytic steps, and reach the same kind of results with another data set.i • Present data systematically so that quotations, field notes, and so on are easily identifiable.f i • Offer enough primary evidence to show a relation between evidence and conclusions, but avoid the presentation of too many illustrations; the focus should be on the most representative examples. Criteria for Evaluation of Manuscripts Authors developing reports of randomized controlled trials may wish to follow the CONSORT (Consolidated Standards of Reporting Trials) checklist, which includes 22 items considered essential to judge the reliability or relevance of the findings (presented in Appendix B to this chapter in slightly abbreviated form).h The criteria for the evaluation of qualitative reports vary depending on the type of data and methods of analysis (e.g., participant observation and eth nography, qualitative interviews, content analysis, textual analysis, discourse analysis, ethnography and conversation analysis). Chapter 8 provides more information about how to write and publish articles using qualitative methods. Most types of qualitative reports should do the following. Preparing Manuscripts and Responding to Reviewers’ Reports Common Problems with Manuscripts If the reviewers discover these weaknesses, they will consider them selves smart and are likely to make sure you know it; if you show that you are aware of the limitations and understand the implications, they will perceive you as smart and honest, which counts for a lot. Do not waste time and space discussing “trends” that are not statistically significant; if the effect is not there, its implications do not need discussing. Remember that there are more than enough “significant” effects that do not replicate and there is no need to create new myths. If you believe that a real and important difference was undetected because of a lack of statistical power, the study needs to be repeated; that may be a factor to discuss.f Somewhat different problems are associated with reviews and theoretical articles. If a review claims to be comprehensive, it should state the way the lit erature search was carried out and define the criteria used for including articles (see Chapter 9). Articles that do not claim to be integrative reviews but rather argue the case for a particular theoretical viewpoint or set of ideas are often less comprehensive. In such instances, authors often cite publications that support their own position in a rather uncritical manner, and they may refer to few or no articles that oppose it. Editors may then firmly but politely ask the author to state the assumptions made and ensure that the article clearly indicates any controversial issues. Alternatively, where the intention is to let a distinguished writer express a personal view based on his or her selective citation of the lit erature, it should be made clear that a case is being made for a theory and that a balanced assessment of the state of the field is not being attempted. i Finally, remember you are writing a scholarly article and not running a cam paign! Do not enter into politics and polemics. For example, if your main find ing is that a widely used intervention is less favorable than another that lacks some sort of official approval for general use in your country, make the case for its relative merits and, if appropriate, argue for a policy change. But do not abuse the politicians and do not keep repeating the argument in more and more florid and emphatic language. Common Problems with Manuscripts All parts of a manuscript are open to criticism from the title onward. The first requirement for gaining the confidence of an editor or a reviewer is to describe the findings objectively and in a sober style without the use of hyperbolic lan guage. If your data are good, they will speak for themselves. It is always better for the reader to find that the results themselves are stronger than you claim. i Every “data not shown” statement may raise reviewers’ suspicions that the authors are trying to hide something. If there really is not enough space to show important data graphically or in tabular form, then give some examples of the more important of such results in the text (with means, standard errors, or other indicators of variance and numbers of subjects, if it is a quantitative study).hfi The discussion section is the most difficult part of a manuscript to write, and it often shows. Sometimes the opening paragraph is only a summary of the results, which is not satisfactory. One approach is to decide which are the main new findings, mention only them, and summarize two or three important conclusions that follow from them. It is also common to find that the discus sion does not focus on the aims as stated in the introduction and sometimes Publishing Addiction Science 236 discusses issues on which no background was given. Such a failure to place findings in the context of previous knowledge means that the case for publi cation is not made. Reviewers and editors want to know what is new, what is confirmatory, and what fails to confirm previous findings. Instead, authors may attempt to extract too much from their data by trying to address too many dif ferent issues. The effect of this error is to dilute strong conclusions with weakly supported ones, giving an overall unfavorable impression leading to rejection.h The discussion should also consider alternative interpretations of the study and acknowledge major limitations. These may arise from methodological weaknesses or unexpected findings that could not be pursued to a firm con clusion because of practical limitations, such as the project period coming to an end or a financial constraint (these nonscientific reasons do not need to be stated). The Editor’s Decision The much-anticipated response from the editor finally arrives, together with the statements from the reviewers. The editor will often need to reach a deci sion based on the balance between innovation and quality of work. The per fect manuscript would have important new ideas with far-reaching importance backed up by sound data obtained by means of thoroughly validated methods. In reality, such manuscripts are seen only rarely, if ever, and the editor and the reviewers have to make judgments. If the approach to a problem is highly novel or the study is a potential stimulus for further valuable work, a manuscript may be accepted with data that are less than wholly convincing. On the other hand, if there is not very much that is really new but the study is the first one to address a particular methodological weakness of previous work, then clear data of high quality will probably be essential.hl g y y The reviewers’ reports and recommendations inevitably influence the edi tor’s decision, but they are not the sole determining factors. Editors may study a manuscript in varying amounts of detail and may have concerns that are not reflected in reviewers’ reports. These concerns may relate to any of the range of issues that the reviewers also address but may especially relate to the appro priateness of the subject matter for the journal, whether there are any ethical problems, and whether the importance of the work is sufficient to justify pub lication in their journal rather than in a publication of lesser status that may be struggling to fill its pages. Studies may be technically competent and pre sented well but may be unimportant because they merely confirm well-known facts or focus on apparently trivial issues. When the reports of reviewers are in agreement with each other, the editor will most frequently accept the recom mendations made. It is a brave editor indeed who overturns the opinions of two independent experts—reviewers may soon stop assisting an editor who consistently ignores the advice given. When reviewers disagree, the editor may seek to sort out the matter by studying the manuscript and coming down in support of one or other reviewer; this is the ideal method if the editor can reach a clear view because he or she can reach a decision quickly without wasting another expert’s valuable time. Common Problems with Manuscripts Political battles are not won in the pages of aca demic journals. Preparing Manuscripts and Responding to Reviewers’ Reports 237 The Editor’s Decision However, sometimes reviewers reach opposing conclusions on the basis of equally well-argued cases, and then the editor may feel it is essential to obtain advice from a third person. This is especially likely to occur if the work is outside the editor’s main area of expertise.i When a third reviewer reaches a definite view supporting one or the other of the earlier reviewers, then the way forward is clear; but this does not always happen. If the first reviewer supports publication strongly and the second reviewer recommends rejection, the third reviewer quite often says the manu script is weak but may reach publication standard after major revision; in such cases the contribution of the third reviewer may swing the decision one way or the other depending on the journal’s needs at the time. If the journal is trying to 8 Publishing Addiction Science 238 raise the standard of published items, such marginal manuscripts will probably be rejected, whereas if the study is in a field that is under-represented in the journal, the editor may wish to include it. An editor may also seek to publish the article in a shorter form that reflects its lesser merits.hh l The abusive reviewer is a particular annoyance to editors. The most com monly identified, although happily quite rare, form of abuse occurs when a reviewer attempts a review of the author instead of the manuscript. It is one thing, and perfectly acceptable, to state that an argument is constructed poorly and is unconvincing, that it is presented badly, or that it does not take account of previous knowledge; it is quite another thing to assert that the author is stu pid, careless, or ignorant. Editors have a duty to alter or remove such inap propriate remarks from a reviewer’s report so that unnecessary distress is not caused and the author will be encouraged to improve the manuscript. If the reviewer is young and inexperienced, the editor may also explain the prob lem with the report, whereas a senior person will more likely not be invited to review again. Additional advice for inexperienced reviewers may be found in Chapter 13 of this book. A particularly difficult situation arises if the review process generates suspi cion that the author has engaged in scientific misconduct or another form of unethical behavior. The Editor’s Decision Such misconduct may be either minor or major in nature, and the editor typically has available a range of sanctions to apply. These may include refusing to consider further work from the author, reporting the mat ter to the author’s institution or employer, and publishing a statement in the journal to alert the scientific community to the issue. The availability of a code of practice by which editors can abide in such circumstances is very helpful (see The Farmington Consensus for the ethical practice guidelines developed by the International Society of Addiction Journal Editors). Editors are also wary of trying to resolve contentious ethical issues; they often do not have the resources to conduct a full investigation. Equally important, they cannot simply brush the matter aside by refusing to publish suspect material but must take reason able steps to ensure that appropriate action is taken. These and other related issues are discussed in greater detail in Chapters 14 and 15, which deal with ethical considerations in scientific publishing. Responding to Reviewers’ Reports: General Rules of Conduct Authors who regularly achieve immediate acceptance of a manuscript as sub mitted are rare indeed. Revisions are almost always required before accept ance, and in many cases a final decision cannot be reached until the revised version has been assessed. Therefore, the way in which authors respond to the reports of reviewers and to the editor can have a major influence on the outcome. If editors invite resubmission, they expect to receive the manuscript back again. Preparing Manuscripts and Responding to Reviewers’ Reports 239 Preparing Manuscripts and Responding to Reviewers’ Reports 239 An invitation to resubmit is not a half-hearted and cowardly way of say ing the work is unpublishable but rather an implicit suggestion that the editor remains interested in the article and that it is likely to be accepted if the author is responsive to the questions and recommendations of the reviewers. In such cases, it is nearly always worth resubmitting unless there is some clear and una voidable requirement with which you cannot possibly comply. The remainder of this section offers guidance for authors on how to navigate through this maze successfully. The overriding aim of the response is to engender trust among editors and reviewers. Authors should never claim to have made changes that in fact they have not done. If the cover letter says all requested changes have been made and an editor or reviewer checks two or three points at random and finds noth ing much has changed, he or she may reject the manuscript without looking carefully at the rest of it. If you have made major changes by rewriting whole sections of the manuscript, state that is the case and identify the sections. Alter natively, if just a few words needed to be inserted or deleted, make clear which words were changed so that reviewers can see what has been done. If you were asked to shorten something, you should almost always do so and perhaps state by how much (i.e., by how many words or pages). Do not try to fool the editor by printing the new version in smaller type or by other stylistic changes. Be polite, even if you feel that the reviewers have not understood your intentions. When you have been through all the points of criticism, you should have an idea of the changes you think are appropriate. The Cover Letter: Make Life Easier for the Editor Once you complete the changes to the manuscript, write a detailed reply to the reviewers. It is worth spending a significant amount of time getting your reply to reviewers as near to perfect as you can. Sometimes constructing the letter takes as long as revising the article, but it will not take as long as botching the job and then being obliged to reformat the manuscript for another journal to start the whole process over again. Nevertheless, it is best to keep this reply as short as possible. Typical successful replies will be in the range of one to three single-spaced pages. If the reviewer makes a point in just three lines and you need a page to rebut it, it is likely you have not gotten straight to the heart of the matter, and your reply will probably not be convincing. It is best to write the minimum needed to refute the criticism. If the reviewer cannot understand a point that you made in the manuscript, it may be because he or she is lacking in intellectual capacity (as we often think when we encounter such comments on our own work). However, if one person does not follow what you have written, the same may apply to others. Review ers are all published research workers and are often the very people whom you might hope would read your article; if a reviewer cannot understand your point, try to analyze your text to see how the misunderstanding may have arisen. Then make changes to ensure it will not happen again. Do remember that if a reviewer asks a question, other readers may want to know the answer to it too. The answer should therefore usually be contained in the revised manuscript and not in the cover letter. Save the reviewers’ time and they will love you; do not answer a question in the letter and then refer review ers to a section in the manuscript that they have to read over and over to check if it is really there! y If possible, reply in numbered sections that correspond with the reviewers’ numbered points. Explain the revisions you made to deal with most of the criti cisms, and also explain why you did not deal with the rest. Describe briefly each change you made, referring to the relevant page or paragraph in the revised manuscript. Responding to Reviewers’ Reports: General Rules of Conduct Will they be enough?t If after reading the reports you have concluded that none of the recommen dations is worth accepting and you do not want to make any changes, it is com mon sense to take a break from the job and look at it again on another day! It is simply not realistic to expect editors and reviewers to accept that none of the changes they request and the criticisms they make is well founded. Reviewers spend anything from an hour to a full day preparing their reports. If you dis miss this effort out of hand, you will get nothing published. You must therefore aim to make changes to deal with as many as possible of the points raised and, preferably, with a clear majority of them. Occasionally authors may feel that an editor’s decision to reject their manu script was unnecessary because the criticisms made could be answered through revisions. In such cases, in which there was no other clear reason given for rejection, authors may wish to seek approval to resubmit. For example, there may be no criticism of the conduct of the study or analysis of the data, but the reviewer may feel that the interpretation is so seriously flawed that the conclusions are not supported by the data. The manuscript might therefore be publishable if the authors are willing to revise their conclusions. Resubmission after rejection should be preceded by a carefully considered letter to the editor explaining why you believe that you can deal with the criticisms made. The editor will then decide whether to alter the previous negative decision and may agree to consider a revised version. Seek approval before resubmitting because Publishing Addiction Science 240 if a rejected manuscript is resubmitted without prior agreement, it is very likely that the editor will refuse to consider it. Some journals have stated their appeals procedure whereas others deal with appeals on an ad hoc basis. The Cover Letter: Make Life Easier for the Editor Try not to respond in a combative, overly assertive style. If there are major and important changes recommended that you are sure are wrong, then present a concise, logically argued rebuttal. If there are minor changes requested that you feel do not really improve matters, do them anyway because it helps a lot if you can truthfully claim to have dealt with the majority of points. At all stages, remember that although reviewers and editors may appear to be distant, self-opinionated, and arrogant, they are also human beings with their Preparing Manuscripts and Responding to Reviewers’ Reports 241 own feelings, emotions, and problems. If you want acceptances, make life easy for them by writing clearly, and do not antagonize them with criticisms or gra tuitous insults, however unwise and misguided you think the reviewers may be. It is also worth making the changes via a tracking system in a different color (such as red) on the manuscript to show clearly where changes, additions, and deletions have been made. It is sensible to maximize and stress the points that you agree with that the reviewers wrote and to acknowledge their contribution when they have made suggestions that improve the manuscript. Do not build minor disagreements into major issues. You probably need to make only minor changes to accom modate them and then mention the changes in the cover letter and should not waste time arguing and or risk offending the reviewer in the process. However, it is not necessary or appropriate to minimize disagreements to the point of dishonesty; they should be dealt with by logical rebuttal in the cover letter and, sometimes, by acknowledging and discussing the point in the manuscript.fi Perhaps the most difficult case occurs when you feel that a reviewer shows a bias towards a theoretical approach that differs from yours, and therefore undervalues the work. Here you can explain in the cover letter that there are different approaches to the problem (state what these are), that yours is equally valid, that there is a genuine difference of opinion and that you have a different but scientifically legitimate point of view. However, this strategy is probably unwise unless you have a strong case and there is no other way to deal with the issue. The Cover Letter: Make Life Easier for the Editor In the end, the editor will have to decide and what one person per ceives as objective and unbiased looks very different from another viewpoint. At the end of the day, the editor wants to have articles to publish. The number of acceptances rather than of rejections is therefore the mark of success and of an editor’s job well done. Authors, editors, reviewers, and publishers must all work together to ensure the production of a journal of high quality that achieves its intended objectives. Appendix A: General Publications on Scientific and Medical Publishing This is a very short selection from the huge number of publications. Many addi tional works may be found by searching biomedical databases such as PubMed or on-line booksellers.h Albert, T. (2000). The A-Z of Medical Writing. London, England: BMJ Books. Albert, T. (2000). The A-Z of Medical Writing. London, England: BMJ Books. American Psychological Association. (2010). Publication Manual of the Ameri can Psychological Association (6th ed). Washington, DC: Author. American Psychological Association. (2010). Publication Manual of the Ameri can Psychological Association (6th ed). Washington, DC: Author. y g g British Medical Association. http://bma.org.uk/about-the-bma/bma-library/ library-guide/reference-styles This is a useful website established by the British Medical Association, giving general guidance on resources for peo ple publishing in the biomedical field. i Hofmann, A. K. (2013). Scientific Writing and Communication: Papers, Propos als, and Presentations. New York, NY: Oxford University Press. Huth, E. J. (1990). How to Write and Publish Papers in the Medical Sciences (2nd ed). New York, NY: Williams & Wilkins. Iverson, C. (Ed.). (1998). American Medical Association Manual of Style: A Guide for Authors and Editors (9th ed.). New York, NY: Williams & Wilkins.i Katz, M. J. (2009). From Research to Manuscript: A Guide to Scientific Writing (2nd ed.). New York, NY: Springer. McInerney, D. M. (2002). Publishing Your Psychology Research: A Guide to Writing for Journals in Psychology and Related Fields. Thousand Oaks, CA: Sage.h Moher, D., Schulz, K. F., Altman, D. G., & for the CONSORT Group. (2001). The CONSORT statement: Revised recommendations for improving the quality of reports of parallel-group randomised trials. The Lancet, 357, 1191–1194.i gh Peat, J., Elliott, E., Baur, L., & Keena, V. (2002). Scientific Writing: Easy When You Know How. London, England: BMJ Books. Richardson, P. (Ed.). (2002). A Guide to Medical Publishing and Writing: Your Questions Answered. London, England: Quay Books.h Strunk, W., Jr., & White, E. B. (1999). The Elements of Style (4th ed.). New York, NY: Longman. Acknowledgements We thank Klaus Mäkelä and Kerstin Stenius for assistance with drafting the material about qualitative research. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. 242 2 Publishing Addiction Science Appendix B: Checklist of Items to Include When Reporting a Randomized Trial Blinding Whether participants, th interventions, and those a were aware of group assign Statistical analysis Statistical methods used to mary outcome; methods f such as subgroup analyses a RESULTSl Appendix B: Checklist of Items to Include When Reporting a Randomized Trial This section consists of a slightly shortened version of the checklist from Moher et al. (2001). TITLE AND ABSTRACT How participants were allocated to interventions (e.g., “random allocation,” “randomized,” or “ran domly assigned”). TITLE AND ABSTRACT How participants were allocated to interventions (e.g., “random allocation,” “randomized,” or “ran domly assigned”). Preparing Manuscripts and Responding to Reviewers’ Reports 243 INTRODUCTION Scientific background and explanation of rationale METHODS Participants Eligibility criteria for participants and the setting and locations where the data were collected. Interventions Precise details of the interventions intended fo each group and how and when they were actuall administered. Objectives Subjective objectives and hypotheses. Outcomes Clearly defined primary and secondary outcom measures and, when applicable, any methods use to enhance the quality of measurements (e.g., mul tiple observations, training of assessors). Sample size How sample size was determined and, when appli cable, explanation of any interim analyses and stop ping rules. Randomization Method used to generate the random allocatio sequence, including details of any restriction (e.g blocking, stratification). Allocation concealment Method used to implement the random allocatio sequence (e.g., numbered containers or central tel ephone), clarifying whether the sequence was con cealed until interventions were assigned. Implementation Who generated the allocation sequence, wh enrolled participants, and who assigned partici pants to their groups. Blinding Whether participants, those administering th interventions, and those assessing the outcome were aware of group assignment. Statistical analysis Statistical methods used to compare groups for pri mary outcome; methods for additional analyses such as subgroup analyses and adjusted analyses. RESULTS Participant flow: Flow of participants through each stage (a dia gram is strongly recommended) Specifically fo INTRODUCTION Scientific background and e METHODS Participants Eligibility criteria for parti and locations where the dat Interventions Precise details of the inte each group and how and w administered. Objectives Subjective objectives and hy Outcomes Clearly defined primary a measures and, when applic to enhance the quality of m tiple observations, training Sample size How sample size was deter cable, explanation of any int ping rules. Randomization Method used to generate sequence, including details blocking, stratification). Allocation concealment Method used to implemen sequence (e.g., numbered c ephone), clarifying whethe cealed until interventions w Implementation Who generated the allo enrolled participants, and pants to their groups. INTRODUCTION Eligibility criteria for participants and the settings and locations where the data were collected. Eligibility criteria for participants and the settings and locations where the data were collected. Precise details of the interventions intended for each group and how and when they were actually administered. Subjective objectives and hypotheses.i Clearly defined primary and secondary outcome measures and, when applicable, any methods used to enhance the quality of measurements (e.g., mul tiple observations, training of assessors). How sample size was determined and, when appli cable, explanation of any interim analyses and stop ping rules. Method used to generate the random allocation sequence, including details of any restriction (e.g., blocking, stratification). i Method used to implement the random allocation sequence (e.g., numbered containers or central tel ephone), clarifying whether the sequence was con cealed until interventions were assigned. Who generated the allocation sequence, who enrolled participants, and who assigned partici pants to their groups. Whether participants, those administering the interventions, and those assessing the outcomes were aware of group assignment. Statistical methods used to compare groups for pri mary outcome; methods for additional analyses, such as subgroup analyses and adjusted analyses. RESULTS Participant flow: RESULTS Participant flow: Flow of participants through each stage (a dia gram is strongly recommended). Specifically, for each group, report the numbers of participants who were randomly assigned, received intended treatment, completed the study protocol, and were analyzed for the primary outcome. Describe pro tocol deviations from study as planned, together with reasons.i Recruitment: Dates defining the periods of recruitment and follow-up. 244 Publishing Addiction Science Baseline demographic and clinical characteristics of each group. Number of participants (denominator) in each group included in each analysis and whether the analysis was by “intention to treat.” State the results in absolute numbers when feasible (e.g., 10/20, not 50%). For each primary and secondary outcome, a sum mary of results for each group, and the estimated effect size and its precision (e.g., 95%confidence interval). Address multiplicity by reporting any other analy ses performed, including subgroup analyses and adjusted analyses, indicating those prespecified and those exploratory.f All important adverse events or side effects in each intervention group. How to cite this book chapter: Balster, R L. 2017. Reviewing Manuscripts for Scientific Journals. In: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P. (eds.) Publishing Addiction Science: A Guide for the Perplexed, Pp. 245–263. London: Ubiquity Press. DOI: https://doi.org/10.5334/bbd.m. License: CC-BY 4.0. DISCUSSION Interpretation Interpretation of the results, taking into account study hypotheses, sources of potential bias or imprecision, and the dangers associated with multiplicity of analyses and outcomes.i External validity of the trial findings. External validity of the trial findings. i verall evidence General interpretation of the results in the context of cur rent evidence. i ce General interpretation of the results in the context of cur rent evidence. Source: Moher, D., Schulz, K. F., & Altman, D.G., for the CONSORT Group. (2001). The CONSORT statement: Revised recommendations for improving the quality of reports of parallel-group randomised trials. The Lancet, 357, 1191–1194. CHAPTER 13 Reviewing Manuscripts for Scientific Journals Robert L. Balster Introduction One of the main moral principles of virtually all major religions and cultures is the ethic of reciprocity, sometimes known as the Golden Rule: Treat others as you would like to be treated. Show mutual respect. This Golden Rule is also a fundamental principle of the process of peer review, including the review of manuscripts submitted to professional journals. If you have been asked to review someone’s article, it is very likely the case that you are an author yourself and will have been subjected to the same peer-review process. Keeping in mind how you expect your own journal submissions should be reviewed, you could readily derive from that experience nearly all of the advice I will be offering you in your role as a reviewer. Goal The goal of this chapter is to provide rather specific principles and suggestions on how to be a competent reviewer. Most editors of peer-reviewed journals see the peer-review process similarly, even if some of their specific journal policies differ. It is those commonalities that I will address here, approaching the topic from my various roles as former editor-in-chief of Drug and Alcohol Depend ence, member of several editorial boards for other journals, and reviewer for Publishing Addiction Science 246 many others. Interested readers may also want to consult prior publications on peer review in general (Moghissi, Love & Straja, 2013; Godlee & Jefferson, 2003) and on peer review of journal submissions specifically (Girden & Kabacoff, 2010; Smart, Maisonneuve & Polderman, 2013; Hames, 2007). Chapters 7, 8, 9, and 12 in this book also provide information that is relevant to journal reviewing. I should mention that reviewers of journal submissions are sometimes referred to as referees. I am not aware of any distinction between being a reviewer and a referee. I have always preferred the term reviewer because referee conjures images of a sporting contest with winners and losers. When you are a good reviewer, eve ryone wins—authors, editors, and the scientific community—because reviewing is fundamentally a constructive process. Yes, reviewing involves making judg ments and recommendations, but reviewers are not the decision makers in the process. This responsibility falls to the editor. Thus, I will use the words reviewing and reviewer throughout but just as a matter of personal preference. It seems likely that the words used for the reviewer function in non-English languages have nuances of their own to consider, but that is a matter for another discourse by someone with greater language skills than I. Brief Overview of the Journal Review Process Editor decides if further review is needed; if so, the process recommences at Step 4. Box 13.1: Steps in the Journal Review Process. 1. Editor develop a reviewer database. 1. Editor develop a reviewer database. 2. Authors submit manuscripts to the journal. 2. Authors submit manuscripts to the journal. 3. Editor(s) make an initial assessment to decide if the article is suitable for the journal and if peer review is warranted. 4. Editor selects reviewers and invites them to review. 5. Editor monitors the timeliness of peer review and sends remind ers or invites new reviewers if necessary. 6. Reviewers complete the review and provide recommendations and comments to the editor and comments to the authors. 7. Editor makes decision to accept the submission, asks authors for a revision, or rejects the submission. 8. Most journals notify reviewers of the editor’s decision. 9. If required, authors revise submissions and return to the editor. 10. Editor decides if further review is needed; if so, the process recommences at Step 4. Brief Overview of the Journal Review Process If a journal declares itself to be a “peer-reviewed” journal, this normally means that all articles that are eventually published in that journal have been reviewed. Peer review implies review by outside reviewers as well as the editorial staff. Of course, journals differ in their application of the peer-review process, and it is common for journals to publish editorials, commentaries, book reviews, and similar content without peer review, reserving that form of assessment for research reports and critical reviews. Before moving on to a more detailed discussion of journal reviewing, I want to give a brief overview of the process so readers can appreciate the steps involved. Box 13.1 outlines the basic steps of the review process used by most journals, keeping in mind that editorial structure differs among journals. For most journals today, submissions are made using an interactive Internet-based system, whereby all articles are processed in a centralized editorial office, or at least as email attachments. The editor then decides whether to edit the article or assign it to some kind of associate or assistant editor. Some journals may have more than one submission office or site, depending on where the author comes from or the general topic of the article. Some journals may have more than one editor look at the submission before assigning it to reviewers, whereas others may have an editorial team that considers the recommendations of the review ers. To simplify this discussion, I will assume there is one “decision editor” who assigns reviewers and makes decisions. I will refer to this individual as the edi tor, regardless of the specific editorial title assigned by the journal. Reviewing Manuscripts for Scientific Journals 247 1. Editor develop a reviewer database. 2. Authors submit manuscripts to the journal. 3. Editor(s) make an initial assessment to decide if the article is suitable for the journal and if peer review is warranted. 4. Editor selects reviewers and invites them to review. 5. Editor monitors the timeliness of peer review and sends remind ers or invites new reviewers if necessary. 6. Reviewers complete the review and provide recommendations and comments to the editor and comments to the authors. 7. Editor makes decision to accept the submission, asks authors for a revision, or rejects the submission. 8. Most journals notify reviewers of the editor’s decision. 9. If required, authors revise submissions and return to the editor. 10. Box 13.1: Steps in the Journal Review Process. Box 13.1: Steps in the Journal Review Process. The first step in the peer-review process is the assignment of reviewers. In the next section, I will discuss reviewer databases and how editors select and invite reviewers (Steps 1 and 4). All journals have a procedure for monitor ing the review process after reviewers have been assigned (Step 5). This has become easier with computer-based systems that notify the editorial team when assigned reviewers decline the invitation to review, when reviews are completed, and when they are late. At some point, the editor stops the review process and decides whether to accept the manuscript for publication (Steps 6 and 7). Editors carefully con sider the recommendations of the reviewers and their comments on the article, but ultimately editors themselves must make the decision based on both the reviews and their own assessment of the submission. Editors usually inform reviewers of the decision (Step 8), and often share the comments of all the reviewers with each other, which I believe is a good practice to strengthen the review process.h There are three basic options open to the editor. First, the editor may accept the submission for publication as submitted. For many journals, it is rare to accept a first submission of an article without asking for any revisions at all, but it does happen. Second, if the submission seems to the editor to be potentially publishable, he or she will ask the authors to make revisions (Step 9). Most journals divide revisions into minor and major categories. Minor revisions do not change the article very much, and the resubmission usually does not require additional 8 Publishing Addiction Science 248 outside reviews. Major revisions typically require significant changes to the article, such as the collection of additional data, a change in the way the exist ing data are analyzed or presented, or even changes to some of the conclusions of the report. Manuscripts with major revisions are often returned to the same peer reviewers for their comments and recommendations, although at times an editor may send the manuscript to different reviewers (Step 10).h f The third option open to editors is rejection of the submission. Box 13.1: Steps in the Journal Review Process. Some submis sions may be rejected without peer review (Step 3), such as when the topic is not appropriate for the journal readership or the form of article (e.g., case report, book review) is not one published by the journal. Editors may also decide that the article’s methods or other characteristics give it little or no chance of receiv ing a positive recommendation in the peer-review process. Giving immediate feedback on such articles may be in the authors’ best interest, because it allows them to submit elsewhere without delay. Rejection without review also saves the time of busy reviewers, who are more useful to the journal when reviewing manuscripts with a greater chance of success. Most often, editors base their rejections on negative recommendations in the peer-review process. In such cases, authors usually receive comments from the editor or reviewers that describe some of the weaknesses of their submission. Ideally, authors consider these comments and revise the article before they send it to another journal. One of the newer developments in journal publishing is the cascading of journal submissions within publishing consortia of individual journals (Bar roga, 2013). With the author’s permission, editors can forward a rejected sub mission and the completed reviews of an article to another journal within the consortium where the article may have a better chance of final acceptance. This saves reviewer time and can expedite final publication because the review pro cess does not need to start all over again. A typical consortium includes jour nals within a publishing company. How Do Editors Select Reviewers? As we turn to the principle of reciprocity, who do we, as authors, want assigned as reviewers of our article? To be honest, we probably prefer reviewers who are known to be favorably impressed with our work. But minimally, we want reviewers who are knowledgeable about our field of study and who will be fair in the review process. This is what editors want too; they want competent review ers with specialized knowledge of the potential advantages and pitfalls of vari ous research approaches. They also want reviewers who are fair and unbiased, without conflicts of interest. Finally, editors want reviewers who complete their work on time and who write constructive comments about the submission.h The process of selecting appropriate reviewers usually involves the use of a database. At a minimum, such a database includes email addresses by which to contact reviewersIn the case of a large, multidisciplinary journal, editors also Reviewing Manuscripts for Scientific Journals 249 need some means of matching reviewers with submissions. Smaller, more spe cialized journals often rely mainly on their normal editorial advisory boards for reviews, but ad hoc reviewers carry most of the load for larger journals. In addition to up-to-date contact information, a typical reviewer database includes some means of identifying their reviewers’ areas of expertise. For example, the database may assign to each reviewer one or more keywords or classifications (e.g., molecular genetics, pharmacotherapy, prevention, policy). Commercial editorial software systems are particularly adept at matching the keywords assigned to both submissions and reviewers to provide editors with a list of knowledgeable reviewers. These programs also tell editors if reviewers are currently assigned other manuscripts, the date of their last review, and the number of reviews they have done lately. It is also possible to see if reviewers have defaulted on prior assignments or how long they have taken to complete their prior reviews. Some systems even allow editors to rate reviewers to help them remember who provided useful suggestions and constructive and timely comments in previous reviews.h There are several ways to get one’s name added to a journal’s database. Per haps the most common way is by publishing an article in that journal. Edi tors typically prefer reviewers who themselves have published several articles as senior or corresponding author. How Do Editors Select Reviewers? If a suitable reviewer is not already in the database, many editors do a quick author search using PubMed or other search tool to see if potential reviewers have other publications, and the editorial soft ware often facilitates this search. Sometimes authors suggest reviewers for their manuscripts. If the editor agrees with the suggestion and the reviewer is not in the database, the reviewer will be added. Editors may ask their editorial advi sory boards to suggest new reviewers for the database, and most editors are also pleased to receive self-nominations. Finally, an editor sometimes receives submissions for which no suitable reviewer is available. In such cases, editors use their knowledge of the field or use authors cited in the submission who are clearly doing related work to add new reviewers to the database. y g Selecting the best possible reviewer for a submission is one of the most impor tant responsibilities of an editor. In my experience, taking time at this step to ensure a good match between reviewer and submission will often save time— and requests for more reviews—later on. Reviewers, too, prefer to assess arti cles in areas in which they feel qualified. Each editor goes about the matching process in a different way. In addition to seeking reviewers with expertise in the area, editors also may seek to balance their selection of reviewers for a single submission along several dimensions. For example, editors may seek a methodo logical balance, in which a reviewer with specific knowledge of a data-analytic approach complements another with knowledge of the content area of the work. It is often good to pair a senior scientist with a less-experienced reviewer because this can serve to train the junior person who subsequently sees the comments of the senior reviewer. Sometimes the editor needs a reviewer known to be unbi ased in a particularly controversial area. Editors usually like to have reviewers Publishing Addiction Science 250 who provide a broad perspective on the work to ensure geographical, cultural, or gender balance. In general, only two reviewers are needed for each submission, but there are times when more than two are invited. If the review process becomes delayed because of problems obtaining timely reviews, editors may add a reviewer they know to be particularly reliable. These and many other subtle factors make the reviewer-selection process a challenging one. How Do Editors Select Reviewers? In addition to selecting knowledgeable and reliable reviewers, editors do their best to avoid actual or perceived conflicts of interest (COIs) and bias. As I will discuss later, editors cannot know about every possible COI or bias that reviewers may have; therefore, they rely on reviewers to tell them. There are some relatively straightforward methods editors use to try to avoid appar ent COIs. They generally exclude as potential reviewers scientific colleagues close to the author, such as reviewers who have co-authored in recent years with any of the authors of the submission or persons known to be part of the same research team. It is usually wise not to assign reviewers from the same institution as the authors, but, in the case of authors based at large, multi campus institutions, such precautions may not always be necessary. Because editors are usually experts themselves, they may be aware of longstanding disagreements or controversies and take care to select unbiased reviewers. It is impossible to eliminate potential bias completely, but editors do the best they can.hf The actual process of inviting reviewers differs among journals. Some edi tors send a copy of the submission and reviewing instructions directly to the reviewer. Other editors first invite possible reviewers, usually by sending the abstract by email and asking them first to agree to do the review by a cer tain deadline. If the reviewers agree, they are then assigned the review and provided with access to the full article, review forms, and instructions. One advantage to this invitation process is that potential reviewers can identify COIs they may have or indicate that they lack expertise in the area of study. Some editors will invite several reviewers and then assign only the first two who agree. Computer technology and the use of email have automated some of these steps, making the process faster. It is now possible to receive a sub mission, invite reviewers, assign them, and get the review process started all in one day. Editorial Decision Making The most obvious reason to seek reviews of journal submissions is to help the editor make a decision about the article’s acceptability for publication in that journal. One should always remember that reviewers only make recommenda tions; it is the editor who chooses whether to follow those recommendations. This fact often frustrates reviewers, who may feel that the editor ignored their advice. A consideration of some of the factors that affect the editor’s decisions can relieve some of that frustration. Editors must make decisions based on all of their reviewers’ input, and sometimes different reviewers give conflicting recommendations. The editor may feel that a problem identified by a reviewer could be addressed in a revision or that the author may have good arguments for why the problem is not an important consideration. Indeed, the editor may read the article and disagree with the reviewer’s interpretation. An editor may feel that the importance of the article mitigates some problems identified by the reviewers. On the other hand, reviewers may not find any fault with an article that the editor decides carries little impact or belongs in a different journal. Most journals receive more submissions than they can publish and thus editors must choose among potentially publishable articles with no major flaws. All of these factors and others go into the editorial decision-making process. Editors who choose not to follow a reviewer’s recommendations may still consider the review excellent. Reviewers who are provided access to the editor’s decision letter, or to the comments of the other reviewer, can usually glean the basis for the editor’s decision. Explaining the Basis for Rejection Nearly all journals ask reviewers to provide comments and suggestions on the submission, which are then sent to the author with the decision letter. When reviewers recommend rejection of a manuscript, they should provide the basis for that recommendation in their comments to the authors. Not only does this give authors the feedback they deserve on their submissions, but it also helps the authors to revise their articles for submission elsewhere and to improve their work in general. Why Have Peer Review? Before further discussion of how to be a good reviewer, I will explain why we use peer review and why someone would want to be a peer reviewer. Peer review has four primary objectives: (a) advise the editorial decision-making process, (b) justify rejections, (c) improve the quality of acceptable manuscripts, and (d) identify instances of ethical or scientific misconduct. Reviewing Manuscripts for Scientific Journals 251 Improving the Quality of Published Articles Certainly, one of the most important reasons for obtaining constructive com ments during the review process is to make the articles that are ultimately pub lished the best they can possibly be. I have seen modest articles transformed during the review process into far better and more important contributions to the field. I received many statements of appreciation by authors for the Publishing Addiction Science 252 improvements in their manuscripts brought about by peer review. Of course, not all authors appreciate constructive criticism or relish doing the extra work required to revise their submissions to satisfy reviewers, but I believe that most authors value expert criticism of their work. I suppose that some contributions could be weakened when authors follow reviewer advice, but I cannot recall a specific example where I know this occurred. It is the responsibility of the editor to make judgments about major changes reviewers ask authors to make and to tell authors in a cover letter if the editor does not agree with a reviewer’s suggestions. Editors should also carefully consider counter arguments made by authors who prefer not to make some recommended changes.i g What is the scientific evidence that peer review improves the quality of scien tific publication? Although this chapter is not the place to have a thorough discus sion of this topic, it may interest the reader to know that there has been relatively little research on this topic, and the work that has been done does not provide a clear answer to the question (Fletcher & Fletcher, 2003; Overbeake & Wagner, 2003; Jefferson et al., 2007). Nor is there much empirical research on various approaches to peer review (e.g., open vs. blinded review). Criticisms have been published of the peer-review process for both journal publications and research grant applications (e.g., Smith, 2006). Nonetheless, editors in general see articles improve through the peer-review process, but perhaps they have a bias, and peer review is the only system we have for quality control in journal publication. Identify Areas of Ethical or Scientific Misconduct Most journals request that reviewers comment on any research subject- protection issues or other ethical concerns they detect in a submission. Reviewers’ careful examination of the data may reveal inconsistencies between reported methods and the ways in which data are presented or analyzed or may uncover highly unlikely data sets (e.g., with no variability) that may lead reviewers to suspect errors in data reporting or outright data fabrication. Reviewers may know of concurrent submissions by the same authors of the same manuscript with the same data to two or more journals or prior publica tions. It is important for reviewers to communicate their concerns about pos sible ethical or scientific-misconduct problems to the editor. Typically, this is done in confidential comments to the editor, who then must investigate these concerns. Reviewers do not need proof of these types of problems, just a rea sonable basis for concern. Fulfill Your Professional Responsibility The peer-review process is a simple application of the Golden Rule. You need people to review your articles, and therefore you should review those of others. A system of all authors and no reviewers is doomed. Thus, it is your profes sional responsibility to be part of the process, both as an author and reviewer. I do not subscribe to the view that senior scientists can be excused from the peer-review process because they “did their duty” earlier in their careers. If you write, you should review. Indeed, the perspective of experienced scholars can be particularly important. Improve Your Understanding of the Peer-Review Process Younger scholars particularly need to learn the peer-review process, because much of their career success will depend on it. Many scientific mentors include in their training a gradual exposure to doing peer reviews. A good strategy is for a mentor to ask a junior colleague to prepare a review of a manuscript that has been assigned to the mentor. The mentor should explain fully the confiden tiality issues surrounding the review process when asking a junior colleague for help. The novice reviewer then returns the review to the mentor who modifies it as needed and gives feedback to the trainee. The mentor then submits the review to the journal. In such instances of guided peer review, mentors should tell the editor the name of the junior colleague who helped with the review. Mentors might even recommend that the junior colleague be added to the reviewer database once they feel the colleague is ready to do independent work. Why be a Journal Reviewer? In the following, I expand on seven of the main reasons researchers review manuscripts for scientific journals. But keep in mind that the most important Reviewing Manuscripts for Scientific Journals 253 Reviewing Manuscripts for Scientific Journals 253 reason many people do it is this: They enjoy it. Nonetheless, thinking critically about science, staying informed of the latest advances, and making a contribu tion to health are what attracted many of us to science in the first place. Review ing is a scholarly, creative, focused, important activity that is capable of being completed with a few hours of work. What could be better? Learn More about Research in the Field Reviewing a scientific article can give a reviewer a much better understanding of the work than does just reading it because of the critical thinking involved in doing a review. In addition, you will probably be asked to review articles that you would not normally read because they are a little outside your specific area of work. The comments and suggestions of the editor and other reviewer(s) are sometimes more interesting and important to you than the article you reviewed. Some of the best scientific writing I have encountered has taken the form of reviews. After all, reviewers are experts in the field who are asked to summarize the salient strengths and weaknesses of a scientific study or new hypothesis in a few paragraphs. Improve Your Critical Thinking The review of other people’s work improves your critical thinking about your own work. Good reviewers attempt to articulate both the strengths and weak nesses of a particular scientific approach. You may be using a similar approach in your own work without thinking about it critically as often as you should. Perhaps you are considering the use of methods similar to those in the article you are reviewing. Thinking about some of the article’s weaknesses can lead you to improve the approach. 254 Publishing Addiction Science One of the most useful ways to evaluate your own critical thinking is to see what the other reviewer(s) and editors say about the work. I strongly believe that journals should make all comments to authors and decision letters available to reviewers on an anonymous basis, although not all journals do. When another reviewer or the editor identifies a serious flaw in experimental design or data analysis that you missed, it can be both instructive and a little embarrassing. Improve Your Own Writing and Data Presentation Reviewing a manuscript (and comments from other reviewers and the editor) gives you new insights into how to improve your own writing, presentation, and data analyses. By seeing how authors make revisions and respond to reviewer comments, you can improve your own approach to revising articles. Review ers often advise authors on how articles can be shortened and more sharply focused. As reviewers help authors focus their writing, they likewise learn to do this with their own articles. In addition, proofreading other manuscripts for errors improves the proofreading of your own writing. Fulfill Service Obligations for Promotion As scientists, our work is constantly being evaluated. This includes assessment for promotion and/or tenure. For most scholars, service to the profession is one of the areas where we are evaluated and judged. As mentioned above, journal reviewing is an important service that should be acknowledged and rewarded. I know that reviewers typically report their journal reviewing activities on their curricula vitae and include them in regular activity reports. Simply being invited to review a manuscript is evidence that you are known in the field and that an editor has some confidence in your expertise. Many journals regularly publish lists of recent reviewers and some use additional incentives (such as small gifts or reduced costs for the publisher’s books and journals) to reward good reviewers. How to be a Good Reviewer Much of the advice in this section stems from the goals of the review process, as described above. Simply stated, a good reviewer helps the editor achieve the goals of peer review. Another guiding principle is our friend the Golden Rule. You should be the kind of reviewer that you would want to review your work. It is always important for reviewers to try to take an author’s perspec tive and to remember that publishing is vitally important to authors. Some times an author needs one more publication to ensure a promotion or to receive a favorable review on a grant application. Authors may be performing research in a highly competitive area where having an article accepted for publication is crucial evidence of their precedence. Reviewers should easily be able to imagine an author’s response to a careless review or one that is delayed by months. Reviewers often offer excuses to the editor for late or cur sory reviews, but the author feels the delay—and the curt treatment—even more keenly than the editor. I suggest placing the following aphorism on your desk as you participate in the review process: Review others as you would like to be reviewed. Below, I present what could be viewed as one former editor’s advice on being a good reviewer. I have ordered this section essentially in the order of steps in the review process as presented in Box 13.1. Build Relationships with Journals The databases that include you as a reviewer generally will also include you as an author. Persons who are regular authors and reviewers for a journal and who are successful in both these roles build a relationship with a journal, becom ing good “journal citizens.” The editorial team comes to know who you are, appreciate better your areas of expertise, and develop confidence in your work. Your good relationship with a journal may result in your being asked to join its editorial advisory board or take on editing roles yourself. Reviewing Manuscripts for Scientific Journals 255 Reviewing Manuscripts for Scientific Journals 255 Reviewing Manuscripts for Scientific Journals 255 Respond Promptly to Invitations to Review As I mentioned above, many journals now use email or fax to invite review ers for a submission. The invitation typically includes only basic information about the article and an abstract. The worst thing you can do with an invitation is ignore it. It takes only a minute or two to decide if you are able to accept the Publishing Addiction Science 256 invitation and then notify the editor. If you want to do the review but cannot complete it by the deadline, contact the editor and see if a later deadline is acceptable. If you decide you cannot accept the invitation, you can help the editor with some suggestions for other reviewers, but the main thing is to tell the editor promptly. Failure to reply puts the editor in the difficult situation of deciding how long to wait until contacting other potential reviewers and puts the review process behind schedule. To avoid delays with this invitation step, some editors invite several qualified reviewers and hope that the required num ber will agree promptly. Reviewers decline invitations all the time. Editors are used to this. If you are too busy at the moment, have other review assignments to complete, do not feel competent to review the article, or have a COI, editors will understand, espe cially if you regularly agree to write reviews for that journal. If you will never agree to do a review for that journal, it is best to tell the editor so you can be removed from the reviewer database. Maintain Confidentiality Submissions to journals are confidential information. Reviewers should scru pulously respect the secrecy of the information, including even the existence of the submission. Reviewers who solicit input on a review from a colleague or trainee must first inform the colleague or trainee about the confidentiality of the information. The reviewer is responsible for any disclosures by his or her consultants.h The most unethical use of information in manuscripts under review occurs when the reviewer uses the information to facilitate directly the reviewer’s own work, for example by using a new methodology before it has been published or by citing the work in his or her own publication or grant application. Failure to maintain confidentiality and misuse of information obtained in the review process is scientific misconduct.f i I strongly advise reviewers not to contact author(s) with questions or offers to negotiate some changes in the manuscript or for any other reasons related to the submission. This applies even if the journal uses an unblinded review process, whereby the authors know the identities of their reviewers. Such com munications usually go badly for both reviewer and author, who can end up arguing about the article, and they improperly exclude the editor from the decision-making process, perhaps concealing from him or her important aspects of the review process. It also can damage important scientific relation ships among the parties involved. Complete the Review on Time Editors give reviewers a fixed period within which to return a review recom mendation. Reviewers who are late ultimately disrespect the author. When a reviewer accepts an invitation to review with a specified deadline, there is lit tle excuse for tardiness. If you know you are going to be late with a review, notify the editor, who can decide whether to wait for your review or invite someone else. One of the most disagreeable aspects of being a journal editor is the need to remind reviewers, often several times, of late reviews. Computer ized reviewer databases keep track of how long it takes reviewers to complete reviews so that chronically late reviewers can be removed from the database. Notify Editor of Any Potential COIs or Previous Reviews You Did of the Article If you have an obvious COI, you should decline the invitation to review. Often reviewers are uncertain if they should declare what appears to be a slight COI, such as a small collaboration with an author or a collaboration that occurred many years ago. In these cases, it is best to tell the editor about your concerns, who can then decide if it represents a real conflict. Putting such ambiguous conflicts on the record can often lessen an editor’s concern. On the other hand, if you feel there might be a conflict that might affect your ability to give a fair and unbiased assessment, then you probably are in conflict and should not accept the invitation to be a reviewer.t More often than one might expect, reviewers are invited to review articles they have already reviewed and rejected for other journals. Many reviewers are in more than one reviewer database, and these journals match reviewers to submissions in much the same way. Some reviewers prefer to decline the second invitation, often stating that they do not want to place the author in double jeopardy. In such instances, I recommend asking the editor what to do. I personally had no problem with reviewers assessing the same manuscript for two different journals. If the new article is identical to the one reviewed earlier and the reviewer feels that the same recommendation and comments are in order, then he or she should submit them again. But if the author has made improvements before resubmitting to the new journal, the recommendation should address this improved manuscript. Authors should be advised that they take a risk if they submit a rejected article to another journal without address ing the concerns raised with the initial submission, because their manuscript may be assigned to the same reviewer. Reviewing Manuscripts for Scientific Journals 257 Reviewing Manuscripts for Scientific Journals 257 Make a Publication Recommendation Completing a review requires at least two steps: making a publication recom mendation and writing comments to the authors (and editor, when needed). Both are important, but I begin with some advice about making recommen dations. Read the reviewer instructions carefully to learn on what basis the Publishing Addiction Science 258 journal wants you to make a recommendation. How does the editor want you to balance technical merit versus importance to the field, etc.? Most journals ask for your overall recommendation based on the review criteria specified. Although journals differ on this, they usually want one of four possible deci sions: accept, minor revision, major revision, or rejection. Because many jour nals receive more technically acceptable articles than they can publish, editors want recommendations that also consider the importance of the information and whether it covers new ground or applies a novel perspective. In my experi ence, reviewers are least comfortable with judging the importance of submis sions, but as experts in the area, they may be in the best position to make this judgment. Complete Questionnaires Most journals ask reviewers to complete questionnaires, which may include items about COIs, ethics, technical merit, significance, language usage, or other matters. Some of these items (e.g., COI and research subject protection) may be crucial to the review process. It should not take you long to complete the questionnaire, and the information you provide will help the editor. Provide Confidential Comments to the Editor Nearly all journals give reviewers the option of providing comments to the edi tor. These comments are confidential and not shared with the author or other reviewer(s). There is no need to reproduce your comments to the author, but do include things you believe the editor should know besides your comments to the author. The following are some of the matters you would want to bring to the confidential attention of the editor. Identify COIs not previously reported. If you have some relationship to the authors or have some financial or personal interest in the work you are reviewing, you should tell the editor. The editor will take this information into account when making decisions based on your recom mendations. If the editor believes the COI precludes you from being a reviewer, your review may not be considered in making a decision and your comments to authors may not be sent on. Situations like this are rare, but it is better to tell the editor too much than too little.h Identify your areas of expertise. There may be some aspects of the arti cle about which you do not feel competent to provide a review. As I mentioned before, you may have been invited to complement another reviewer who lacks expertise in your area. Tell the editor if there are parts of the article for which another expert is needed. It is far better to place statements about your areas of expertise in the comments to the editor than in your comments to the author.i List concerns about ethics or scientific misconduct. As I have already mentioned, you need not be certain of ethical problems to report your suspicion. If you have sufficient cause for question, tell the editor. fi Provide other comments. Reviewers may have additional information or some options the editor should consider that are not appropriate to send to the authors. Reviewers should feel free to help editors in any way they can to make the right decision and seek changes in a submission. 259 Reviewing Manuscripts for Scientific Journals Reviewing Manuscripts for Scientific Journals Examples include when a reviewer is familiar with a controversy sur rounding the reported research or when there is excessive overlap with other reports coming from the same research group. Provide Comments for Authors Here again, the Golden Rule tells us how to act. As an author, you would undoubtedly want to know the basis for an editor’s decision; so, too, do the authors of the articles you review. Comments to authors are a crucially impor tant part of the review process and should be written for the edification of both the author and the editor. There is no standard length for these comments. In my experience, one to two pages is usually sufficient, but if the key basis for your recommendation can be stated in a paragraph or two, that is fine. Some reviewers do fairly detailed page-by-page suggestions for improvements; these are much appreciated by editors if they are constructive. Below are guidelines for writing a good review. State the article’s main strengths and weaknesses. Start the comments to authors with your views of the main strengths and weaknesses of the manuscript. In general, after reading this section the editor should understand the basis for your final recommendation (which you should not include in the comments to authors). Make your statement of weak nesses as constructive as possible and suggest possible avenues by which the author might address the problems in a revision. Weaknesses that are inherent in the design and execution of the study cannot be fixed in a revision; therefore, you can expend less effort in telling authors how they should have done their study. Even if you recommend rejection, you should provide constructive suggestions for improving the article in the event that the editor gives the author an opportunity to address the weaknesses. Many reviewers organize the comments to authors by describing the strengths and weaknesses of each of the sections of the article (e.g., introduction, methods, results, and discussion). Try to balance technical merit with scientific significance. This bal ance has been the subject of debate in the grant-review process for many Publishing Addiction Science 260 years, with many believing that technically competent but scientifically unimportant or uncreative proposals have an advantage, although they may lack significance. It is important for reviewers of journal publica tions to identify particularly important or creative articles and, con versely, to indicate if in their judgment an article represents only a minor advance in the field.i i Provide specific suggestions for improvements. Provide Comments for Authors Detailed suggestions for improving the writing in the article, the figures, or the tables are very helpful to both the editor and the author. Identify paragraphs or sen tences that are unclear, point out areas where information is missing, and explain how the writing can be clearer. If you have trouble under standing the article, there is a good possibility that other readers will as well. Reviewers familiar with the journal’s formatting requirements can point out departures for correction. Reviewers also can identify relevant publications that should have been cited by the author.t It is important for reviewers to appreciate that journal space is often limited and that articles should be as short as they can be while still covering the necessary material. Reviewers can be especially helpful in pointing out how to shorten articles, perhaps by eliminating tables or figures or summarizing data in text. Introductions are often longer than they need be: Point out nonessential background information that can be removed. Similarly, discussions can be too speculative or focused on minor aspects of the study results. Chapter 12 provides advice on writ ing articles for addiction journals; reviewers can offer similar advice in their comments to authors. Reviewers are often exceptionally generous with their help, especially to inexperienced authors. I have been truly impressed and am grateful to the many reviewers who see the peer- review process as a mentoring opportunity.i g y Having advised reviewers to provide both general and specific sugges tions for improving manuscripts, I would not want authors to see this as a rationale for submitting rough drafts or articles that have not been proofread so they can get reviewer feedback. Submission of unpolished drafts shows a lack of respect for the reviewers and editors and is not a good application of the Golden Rule. Authors should submit articles that they would be happy to review. Reviewers often become exasper ated when reviewing sloppy submissions—it colours their recommen dations and discourages them from providing detailed suggestions. Authors should submit their very best work. Comment on language issues. Chapter 4 discusses the problems faced by authors having to submit their articles to English-language journals when English is not their first language. Reviewers cannot be expected to correct language problems. Provide Comments for Authors When assigned an article obviously writ ten by a non-native English speaker, reviewers should do their best to focus on the science being presented and simply point out areas where Reviewing Manuscripts for Scientific Journals 261 language usage needs to be improved. There are various means by which authors and editors can handle this problem; therefore, reviewers should not be biased in their recommendations based on poor English language usage. Instead of addressing language issues in peer review, some journal publishers provide language assistance, or the editor can request that the author seek help from a native English speaker or a lan guage editing service if the article is likely to be accepted. Avoid unconstructive comments. Some things do not belong in com ments to authors. Foremost are pejorative comments about authors or the work. Reviewers should strive to be constructive at all times. Editors may remove overly personal criticisms or other material that insults the authors, their institution, or their geographical location. Jokes and witty remarks also do not belong in comments to authors. Editors may appreciate them in comments to the editor, but for authors this is serious business. They may perceive such remarks as a lack of serious intent by the reviewer. Unless the journal uses unblinded reviews, reviewers should not reveal their identity in the comments to authors. Personal pronouns can provide clues to the reviewer’s identity (“We did a study that showed. . .”). Often, reviewers pro vide an author with several of the reviewers’ own publications that they feel should have been cited, leading the author to suspect who the reviewers are. Reviewing Revisions Most of what I have said about reviewing applies to reviewing revisions of man uscripts. I feel that reviewers have a special obligation to agree to review revised articles that they reviewed in an earlier version. Reviewers should evaluate how the authors addressed the weaknesses they identified in the earlier version. If authors have failed to address the concerns successfully, it should be stated in this subsequent review. On the other hand, if the authors were successful in their revisions or, alternately, convinced you that your concerns were unwar ranted, this should also be stated in your comments. In any case, you should check to make sure the authors actually made the changes in the manuscript. Addressing problems only in a cover letter is not constructive. g p y It is also helpful for reviewers to look at the concerns of the other reviewer(s) and see how the authors addressed them. If you disagree with the other reviewer(s) and agree with the authors’ explanation and defense of their origi nal article, help out the editor by discussing these issues. If you feel that another reviewer was way off base, it is probably best to discuss your concerns in the comments to editor rather than sharing your views with the authors. g y Sometimes when authors clarify their methods or data analyses, you see that the submission is even weaker than you initially thought. If that is the case, Publishing Addiction Science 262 state your new observations clearly, and make your recommendation accord ingly. Sometimes in reading a revision of an article, reviewers will identify weaknesses they missed in the first review. It is fine to voice these new concerns in your written review, but ideally you should have identified these problems in your earlier review. Depending on the scope of additional changes you and the editor request in a second revision, the editor may return the same article to you a third time for your recommendation. What Do Editors Do with Peer Reviews? After reviews of a manuscript are completed, the editor evaluates the article and the reviews and makes a decision. As I mentioned, with some journals this decision-making process may involve multiple editors and/or members of the editorial advisory board; others may involve one editor’s decision. Many factors go into making a decision on a submission. The primary factor is the recom mendations of the reviewers, but other factors include the editor’s assessment of the importance of the article and how well it fits with the journal’s area of coverage, additional concerns the editor may have that were not identified by the reviewers, the likelihood that the submission can be successfully revised to address its weaknesses, and the overall rejection rate for the journal.f Addiction journals differ greatly in their rejection rates with some able to publish much less than half of the submitted articles. Journal editors far prefer the opportunity to select only the best articles for their journal over not having enough acceptable submissions to fulfill their page budget. Journals with high rejection rates usually have higher impact factors because they choose only top-quality articles, and better and more important articles tend to be cited heavily. It is important for reviewers to understand this dynamic to appreciate why their reviews are so important to both the authors and the journals. Summary and Conclusions I have tried to provide background and helpful advice on reviewing articles for scientific journals. I provided an overview of the peer-review process, reasons for having peer review, why scholars would want to be peer reviewers, and tips on how to be a good reviewer. I have done this primarily from the point of view of a former journal editor but have emphasized authors’ points of view as well. I encouraged you to apply the Golden Rule and be the type of reviewer you would want to review your submissions to journals. If you take an author’s point of view, it will greatly help you be a good reviewer. Peer review is a very important part of the scientific process. Your work as reviewers is greatly appre ciated and necessary. I hope this chapter helps you improve your reviewing skills. Reviewing Manuscripts for Scientific Journals 26 Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. SECTION 4 Ethics Matter References and Further Reading Barroga, E. F. (2013). Cascading peer review for open access publishing. Euro pean Science Editing, 39(4), 90–91.hf Fletcher, R. H., & Fletcher, S. W. (2003). The effectiveness of journal peer review. In T. Jefferson & F. Godlee (Eds.), Peer review in health sciences, 2nd edition (pp. 62–75). London, England: BMJ Books.f Girden, E. R., & Kabacoff, R. I. (2010). Evaluating Research Articles, 3rd edition. Thousand Oaks, CA: Sage Publications.f h Godlee, F., & Jefferson, T. (Eds). (2003). Peer review in health sciences, 2cd edi tion. London: BMJ Books.i Hames, I. (2007). Peer review and manuscript management in scientific journals: Guidelines for good practice. Hoboken NJ: Wiley-Blackwell.ff Jefferson, T., Rudin, M., Folse, S. B., & Davidoff, F. (2007). Editorial peer review for improving the quality of reports in biomedical studies. Cochrane Database of Systematic Reviews. DOI: https://doi.org/10.1002/14651858.MR000016. pub3 Jefferson, T., & Godlee, F. (Eds.). (2003). Peer review in health scheinces, 2cd edition. London: BMJ Books.i Moghissi, A. A., Love, B. R., & Straja, S. R. (2013). Peer Review and Scientific Assessment. Alexandria, VA: Institute for Regulatory Science.h Overbeke, J., & Wagner, E. (2003). The state of evidence: What we know and what we don’t know about journal peer review. In F. Godlee and T. Jefferson (Eds.), Peer review in health sciences (2nd ed.). London, England: BMJ Books. Smart, P., Maisonneuve, H., & Polderman, A. (Eds.). (2013). Science Editor’s Handbook, 2cd edition. Cornwall, UK: European Associatoin of Science Editors (especially Section 4).l Smith, R. (2006). Peer review: A flawed process at the heart of science and jour nals. Journal of the Royal Society of Medicine, 99, 178–82. SECTION 4 How to cite this book chapter: Babor, T F, McGovern, T and Robaina, K. 2017. Dante’s Inferno: Seven Deadly Sins in Scientific Publishing and How to Avoid Them. In: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P. (eds.) Publishing Addiction Science: A Guide for the Perplexed, Pp. 267–298. London: Ubiquity Press. DOI: https://doi. org/10.5334/bbd.n. License: CC-BY 4.0. Ethics Matter CHAPTER 14 Dante’s Inferno: Seven Deadly Sins in Scientific Publishing and How to Avoid Them Thomas F. Babor, Thomas McGovern and Katherine Robaina Thomas F. Babor, Thomas McGovern and Katherine Robaina “Relinquish all hope, ye who enter here.” Dante Alighieri, The Divine Comedy, Inferno, Canto III, 9 Some 700 years ago, Dante Alighieri (1265–1321) wrote an epic poem about a man’s journey through the afterworlds of hell, purgatory, and heaven. In his Divine Comedy, he catalogued the vices and virtues of people who had passed into those spiritual domains, in part to provide a valuable insight to us, the living. Dante described hell as a very unhappy and inhospitable place that had nine different levels ranging from the blazing inferno of the eternally damned to a rather benign area, called the First Circle, which was reserved for worthy individuals who were born before the world was redeemed and therefore could not enter the gates of heaven (Alighieri, 1947). Within this general metaphor, this chapter will take the reader on an educa tional journey through the various levels of scientific misconduct, from unin tentional but questionable research practices, such as citation bias, to serious scientific fraud, such as the fabrication of data. Our purpose is not to scare the fear of God into the gentle hearts of our readers. Rather, like Dante on his jour ney through the netherworld, we too should see the mortal consequences of scientific misconduct so that we can learn how to avoid them. Table 14.1 shows the seven types of misconduct this chapter explores. In addition to describing Publishing Addiction Science 268 these various “sins” and the people who commit them, we also discuss their relative seriousness, the punishments that can result, and how to prevent these kinds of problems before they arise. In Chapter 15, we discuss the same issues within a framework of ethical decision making, using case studies to illustrate each topic.hii p The first issue is carelessness, exemplified by unconscious or conscious cita tion bias, misrepresenting the accomplishments or findings of others, and neglecting to reference findings that an informed reader would need to know to interpret the author’s conclusions. In its most benign form, this problem con sists of a failure to read and understand the articles one cites. A more serious offence is the distortion of others’ work so that their ideas or findings support a preconceived point of view that the author is trying to advance. Carelessness can also be manifested in poor management or inaccurate presentation of data. Thomas F. Babor, Thomas McGovern and Katherine Robaina The second ethical issue is dual and redundant publication, which occurs when two or more articles share any of the same data without full cross-referencing. The third issue we consider is unfair or irresponsible authorship. According to standard Ethical Practice Guidelines published by the International Society of Addiction Journal Editors (ISAJE) and similar guidelines of other organiza tions (e.g., Committee on Publication Ethics (COPE)), all persons named as The first issue is carelessness, exemplified by unconscious or conscious cita tion bias, misrepresenting the accomplishments or findings of others, and neglecting to reference findings that an informed reader would need to know to interpret the author’s conclusions. In its most benign form, this problem con sists of a failure to read and understand the articles one cites. A more serious offence is the distortion of others’ work so that their ideas or findings support a preconceived point of view that the author is trying to advance. Carelessness can also be manifested in poor management or inaccurate presentation of data.h Sin Exampes Punishments 1 Carelessness Citation bias, understatement, negligence Request for correction, letter to editor 2 Redundant and duplicate publication Same tables or literature reported without noting prior source, same article published in different journals Rejection of manuscript, copyright infringement, retraction 3 Unfair authorship credit Failure to include eligible authors, inclusion of honorary authors, use of ghost writer Angry colleagues, complaints to editor or employer 4 Undeclared conflict of interest Failure to cite funding source Letter to editor, public apology 5 Human/animal subject violations No ethical approval Rejection of manuscript, notification of employer 6 Plagiarism Reproducing others’ work or ideas as one’s own Retraction of manuscript, notification of employer 7 Scientific fraud Fabrication or falsification of data, misappropriation of others’ ideas or plans given in confidence Retraction of manuscript, notification of employer, publication ban Table 14.1: The seven deadly sins and punishments of those who engage in publishing misconduct. Dante’s Inferno 269 Dante’s Inferno 269 269 authors should have made a major contribution to the work, not just a token contribution. Failure to declare a conflict of interest (COI) is the fourth ethical issue con sidered in this chapter. A COI is a situation or relationship in which profes sional, personal, or financial considerations could be seen by a fair-minded person as potentially in conflict with the researcher’s or author’s independence of judgment. Thomas F. Babor, Thomas McGovern and Katherine Robaina The fifth ethical violation is the failure to conform to minimum standards of protection for animal subjects or human research participants. The latter includes confidentiality of patient records and other data, informed consent, and proper explanation of the risks of research participation. Abiding by stand ards set by national and institutional boards for the protection of animal or human subjects is an important aspect of research under this rubric. Plagiarism is the sixth issue. Plagiarism literally means the act of “literary theft” by using or closely imitating the language and thoughts of another author as if they were one’s own.hiih The final level is scientific fraud. This form of misconduct consists of the deliberate fabrication of data or the alteration of findings to make a study more credible and acceptable for publication. A meta-analysis of survey studies conducted by research scientists and their student trainees representing a range of disciplines indicates that up to 33.7% admit to engaging in questionable research practices, and 0.3%–4.9% have fab ricated or falsified data. Misconduct is reported more frequently by medical and pharmacological researchers than those from other disciplines (Fanelli, 2009). How prevalent are these various ethical problems among addiction sci entists? ISAJE conducted an informal survey of its members to learn about the kinds of ethical misbehavior of most concern to journal editors (Stenius & Babor, 2003). Duplicate publication in various forms and inappropriate cita tions were the most common problems encountered by journal editors in their routine processing of manuscripts. A substantial number of journals had expe rienced at least some of the more serious forms of scientific misconduct, such as plagiarism and failure to declare COI. Authorship problems were also noted quite often. Although most problems were considered infrequent occurrences by the editors, it is likely that these issues are often hidden from the eyes of busy editors and reviewers. For example, editors and reviewers are unlikely to detect scientific fraud in the normal editorial process because data fabrication can be easily hidden in lab records and computer files that are inaccessible during the review process. Skilled reviewers are more likely to detect plagiarism and cita tion bias, but there is a general suspicion that the cases of identified and prov able misconduct are the tip of an iceberg. Negligent Carelessness and Citation Bias The first Deadly Sin described in Table 14.1 refers to minor forms of negligent carelessness and citation bias that are likely to mislead readers and distort the value of scientific research. Perhaps the most benign and most prevalent form of ethical impropriety, negligent carelessness, is characterized by such deficien cies as a failure to adequately review the literature on a topic, lack of candor or completeness in describing one’s research methods, or presentation of data that are based on faulty statistical analyses. A related problem occurs when an author cites articles taken from other reports or from published abstracts with out having read the primary sources.i A more serious form of carelessness in scientific writing is citation bias. One form of this bias is the selective citation of only those articles that support a particular point of view, ignoring or understating the importance of articles that contradict that viewpoint. For example, a study of all therapeutic intervention studies included in meta-analyses published between January 2008 and March 2010 in the Cochrane database found that studies with statistically significant findings were cited twice as often as nonsignificant studies (Jannot et al., 2013). A citation bias favoring significant results is also evidenced in the psychiatric literature (Nieminen et al., 2007). Within the addiction field, Etter and Stapleton (2009) found that randomized controlled trials for nicotine replacement therapy that included positive and statistically significant results were more often cited than articles that did not (N = 41 vs. 17, p < .001). In addition, a meta-analysis of 42 studies reporting smoking among people with schizophrenia found that the actual average prevalence of smoking among this population is 62%, as opposed to the 80%–90% rate frequently reported. The analysis also found that, for every 10% increase in prevalence reported in a study, there was a 28% increase in the likelihood of that study being cited. These higher rates were also inaccurately reported in publically available information and by the media (Chapman et al., 2009). The intention to deceive others may not be operative in all or even most cases, but this does not make this practice any less unacceptable. Another form of citation bias is selective citation to enhance one’s reputation, epitomized by self-citation. We discuss these issues in Chapter 10 in terms of various deviations from ideal citation practice. Negligent Carelessness and Citation Bias A case analysis of these practices in Chapter 15 further illustrates the ethical dimensions of such transgressions. Thomas F. Babor, Thomas McGovern and Katherine Robaina In the following sections of this chapter, each of these ethical improprieties is discussed in terms of its relative importance, possible consequences, and strate gies for avoidance. Table 14.2 provides definitions of the various types of ethical problems discussed in the chapter. 270 Publishing Addiction Science 270 Citation bias A form of carelessness that ranges from a rather benign failure to read the articles one is citing to distorting the meaning of others’ work. Copyright The legal right granted to an author, publisher, or distributor to exclusive publication, production, sale, or distribution of a scientific work. Divided publication Information from a single research study is divided for publication in two or more articles. Also called “salami science.” Duplicate publication Re-publication of the same article in two places without clear reference to the other publication. Fabrication Presenting data in a research report that have not been obtained in the manner or by the methods described in the report. Fractionally divided publication Reporting in a single article only a fraction of the data that have been or will be reported in their entirety in another article. Ghost authorship A published article fails to acknowledge the original writers’ contributions. Guest authorship A researcher is invited to add his or her name to a study or publication without fulfilling authorship criteria. Misappropriation Illicitly presenting or using in one’s own name an original research idea, plan, or finding disclosed in confidence. Misrepresentation (falsification) of findings Altering or presenting original findings in a way that distorts the result in a scientifically unjustified way or by omitting results or data pertinent to conclusions. Partial repetitive publication Repeatedly publishing parts of the same information in modified form. Plagiarism Presenting someone else’s manuscript, article, text, or idea as one’s own. Redundant/repetitive publication Publishing the same information two or more times (e.g., in journal articles and book chapters). Self-plagiarism Copying and presenting one’s own text or article without properly attributing its original source. Unethical authorship Authorship which violates the principle that all persons named as authors should have made a major contribu tion to the work reported and be prepared to take public responsibility for it. Similar to guest authorship. Table 14 2: Definitions of terms referring to various forms of scientific Dante’s Inferno 271 Dante’s Inferno 271 Consequences If the effect of these practices is to mislead or misinform the reader, then they are considered a form of scientific misconduct, even if they only occur at the drafting stage when they are often detected by observant colleagues or review ers who are likely to request a more balanced literature review or the correc tion of obvious mistakes. In some cases, an editor may reject an otherwise Publishing Addiction Science 272 acceptable manuscript if reviewers raise questions about the author’s objectiv ity or intellectual sloppiness. The consequences could be more serious if care lessness or citation bias is detected only after the article is published. If readers of a published article detect a statistical mistake, a clear bias in the formulation of a research question, or the selective reporting of the literature, they may write letters to the editor pointing out the problem. Editors in turn may ask for corrections to the text or the data analyses, which are subsequently published as a special note to readers. Beyond these embarrassing consequences, failure to cite relevant studies and bias in the interpretation of previous research is likely to create a negative impression of the author among his or her colleagues. The institution with which the author is affiliated may experience criticism and damage of reputation. Furthermore, if articles showing favorable results with large effect sizes are cited more often, readers can be misled into thinking treat ments or interventions are more effective than they really are. This may affect the health of individuals and the way services are organized for the public, or it could have other policy implications. Figure 14.1 provides an illustration of how citation bias could have adverse policy and clinical implications. Citation bias favoring studies with higher prevalence rates For every 10% increase in a study's reported smoking prevalence rate, there is a 28% increase in the likelihood of it being cited. Media reports 80%–90% of people with schizophrenia smoke (compared with a prevalence rate closer to 62%). Public and clinicians are mislead, beleiving that most schizophrenics smoke and that smoking is linked to their disease. Possible adverse policy and clinical implications (i.e., no resources allocated to help them quit) Figure 14.1: Citation bias and its potential consequences. Source: Chapman et al. (2009). Prevention As discussed in Chapters 7, 8, and 10, the best way to avoid these problems is to follow appropriate citation practices, conduct a thorough review of the literature (by searching for positive as well as negative outcomes), read all of the articles you cite, present research findings accurately, and interpret them objectively. Locating unpublished studies and/or outcomes may also help to reduce bias. Authors who collaborate on multi-authored articles have a special responsibility to read all drafts of a manuscript with extreme care to make sure these problems are detected during the early stages of the publication process. Even when several authors divide responsibility for writing different sections of a research report, authors should always check each other’s work. Consequences Citation bias favoring studies with higher prevalence rates For every 10% increase in a study's reported smoking prevalence rate, there is a 28% increase in the likelihood of it being cited. Media reports 80%–90% of people with schizophrenia smoke (compared with a prevalence rate closer to 62%). Public and clinicians are mislead, beleiving that most schizophrenics smoke and that smoking is linked to their disease. Possible adverse policy and clinical implications (i.e., no resources allocated to help them quit) Figure 14.1: Citation bias and its potential consequences. Source: Chapman et al. (2009). Dante’s Inferno 273 Dante’s Inferno 273 Duplicate and Redundant Publication Authors wishing to reach the widest possible audience, or a variety of specific audiences, may seek to report a single definable body of research in more than one article, in repeated reports of the same work, in fractional reports, or in reports in more than one language (Huth, 1986). But there are also less noble motives for duplicate and redundant publication, including the desire for mul tiple publications to enhance one’s reputation. Redundant publication occurs when two or more articles share the same data without full cross-reference (COPE, 1999). Duplicate (or dual) publish ing, according to the International Committee of Medical Journal Editors (ICMJE, 2013, p. 8), refers to the “publication of a paper that overlaps substan tially with one already published without, clear, visible reference to the previ ous publication.” In general, journal editors expect authors to ensure that no significant part of the submitted material has been published previously and that the article is not concurrently being considered by another journal. Meta- analyses (Choi et al., 2014; Gotzsche, 1989) indicate that repetitive publishing practices have become a serious problem, and the evidence suggests that this is true across nations, accounting for 18.1% of all retractions of articles published on PubMed between 2008 and 2012 (Amos, 2014). In Finland and China, the rate of retractions for duplicate publication is much higher (37.5% and 29.4%, respectively) (Amos, 2014). Therefore, many journals now require authors to state in writing whether the data have been previously reported in part or in whole (ICMJE, 2013).f As indicated in Table 14.2, a number of different terms have been used to describe this phenomenon. Although there are some important differences among prior, duplicate, repetitive, fragmented, and redundant publication, they are all part of a common problem. Duplicate and redundant publication and their variants consume valuable resources that otherwise might be devoted to other authors who are publishing original data or ideas. Because of limited 274 Publishing Addiction Science 274 journal space, the publication of one person’s article means that another’s article will be rejected. If there are questions about the extent of the overlap between two articles, editors and reviewers need to take extra time to review several publications to determine the extent of redundancy and whether it violates any copyright agreements. Duplicate and Redundant Publication Regardless of whether the repetition occurs with data or ideas (e.g., repetitive review articles), the information from duplicated sources is sometimes inad vertently cited in a way that implies that the findings or conclusions are inde pendent of each other, when in fact they are based on the same source. Without full disclosure in the original sources, authors of subsequent meta-analyses and review articles based on these source articles may come to biased conclusions because the effect of a given finding is multiplied or distorted. Self-Plagiarism Self-Plagiarism A special case of redundant publication is “self-plagiarism,” a topic on which relatively little has been written. According to Griffin (1991), this occurs when an author re-uses text from his or her own previously published article in a way that fails to give proper acknowledgement to its source and its owner. By owner, we mean the person who or organization that owns the copyright (see Table14.2 for definition), which is often the publisher of the previous version of the borrowed text, not the original author. This problem typically occurs when authors re-use text from a literature review or the methods section of an article either without changing the wording or by quoting the original text. Unlike the re-use or re-publication of original data, self-plagiarism is some thing that is more the result of laziness than dishonesty. It can also be a form of self-aggrandizement. Instances of Acceptable Secondary Publication As Huth (1986) has noted, some types of repetitive publication are legitimate and should not be considered scientific misconduct. This is particularly the case in the publications associated with large data sets that involve multiple investigators across many sites. Often, the collaborating investigators have included measures related to a particular hypothesis or methodology, which could and should be reported in separate articles even though the article pre sents the same subjects, methods, procedures, and even some of the same data as other articles. Such publications may be intended to highlight the relevance of particular clinical findings for a particular audience, especially if they have been first published in a technical journal that did not permit the reporting of particular findings or the discussion of clinical implications. Articles presented at scientific conferences or meetings but not published in full may also be sub mitted to journals for publishing. In such cases provide an explanatory letter along with copies of related materials (ICMJE, 2013).i g It is also acceptable to re-publish ideas, data, or review findings when journal editors or book editors request that a popular author write a topical review or commentary for their publication, as long as the author tells the editor about previously published material and cites all relevant reports in the commis sioned article.t Another possibly acceptable variant is publication of the same article, often in its entirety, in two languages when the editors of both journals agree to it and when the translated version cites the original version as the primary publication (which cannot be submitted simultaneously). Submitting the same article to two journals may also be justifiable if the two journals are in very different disciplines and the publication is intended for different groups of readers, the authors have received permission from the editors of both journals, the title indicates it is a secondary publication of a primary publication, and the reviewers’ comments bring about considerable changes to the manuscript (ICMJE, 2013). Dante’s Inferno 275 Dante’s Inferno 275 Box 14.1: Case study: Impact of duplicate publication on meta-analysis. widespread self-plagiarism, the editor may reject the article. Nevertheless, the more that authors re-use text without proper quotation or attribution, the more they risk adverse consequences from editors and publishers, ranging from a reprimand to legal action for copyright violation. Consequences Furthermore, the duplicated randomized controlled trials reported greater efficacy than nonduplicated studies. If duplicated data were included in the meta-analysis, the efficacy of ondansetron would be overestimated by 23% (Tramèr et al., 1997). Box 14.1: Case study: Impact of duplicate publication on meta-analysis. Consequences If a duplicate publication constitutes a copyright infringement, it may result in a reprimand for the author, a retraction of the article, or an apology to the journal editors and the publishers involved. Editors, likely embarrassed by the need to publish a retraction, have adopted policies and regulations to pre vent this questionable research practice. Recommendations for the Conduct, Reporting, Editing, and Publication of Scholarly Work in Medical Journals (pre viously known as Requirements for Manuscripts Submitted to Biomedical Jour nals), which is endorsed by over 500 medical journals, cautions the following: “Authors who attempt duplicate publication without such notification should expect at least prompt rejection of the submitted manuscript. If the editor was not aware of the violations and the article has already been published, then the article might warrant retraction with or without the author’s explanation or approval” (ICMJE, 2013, p. 9). Furthermore, redundant articles may mislead researchers because of the duplicate counting of subjects in a meta-analysis, as illustrated in the case described in Box 14.1 (Tramèr et al., 1997). And when instances of scientific misconduct like this are reported to the public, they diminish the reputation of scientists and their work. In general, an author is not allowed to re-use previ ously published material when the rights have been assigned to the publisher, which occurs in most instances of scientific journal publications. Reprinting more than one or two sentences verbatim without proper attribution may con stitute a violation of copyright and could result in legal sanctions, although this rarely occurs in cases of minor copyright violations.h The negative consequences of self-plagiarism may be less obvious and edi tors are unlikely to consider small amounts (the BMJ uses a baseline of 10%) of “borrowing” to be a major problem, but if an observant reviewer detects 276 Publishing Addiction Science Investigators conducting a meta-analysis of randomized controlled trials for the medication ondansetron found that 17% were duplicate publications that had not been cross-referenced, resulting in a 28% duplication of patient data. Furthermore, the duplicated randomized controlled trials reported greater efficacy than nonduplicated studies. If duplicated data were included in the meta-analysis, the efficacy of ondansetron would be overestimated by 23% (Tramèr et al., 1997). Investigators conducting a meta-analysis of randomized controlled trials for the medication ondansetron found that 17% were duplicate publications that had not been cross-referenced, resulting in a 28% duplication of patient data. Prevention Authors of overlapping articles would be seriously remiss in failing to cite their previously published work (see Jerrells, 2001, for a discussion of this problem) or submitting the same article to two different journals while intending the piece to be recognized as two original articles. The fault in this sin does not insomuch lie with the duplicate publication itself but with the author’s intent to deceive. When there is any possibility of repetitive publication, authors must notify editors to explain the connection between the current article and its pre decessors. Ideally, the author should submit all related publications to the edi tor along with an explanation of the potential overlap and the reasons for the new report. Second, all versions of related articles must contain appropriate citations and complete references to the related articles so that readers and edi tors can evaluate the implications of the repetition and overlap. This includes citing illustrations or tables reprinted or adapted from other journals. When publishing an article in two different journals, each publication should clearly state, “This paper is also published as ‘Title of paper’ in the Title of Journal, Vol x(x), pp. x.” (Bretag & Mahmud, 2009, p. 194), or secondary publication should refer to the primary one in the title (ICMJE, 2013). A survey on redundant publishing (Yank & Barnes, 2003) found that both editors and authors believe that journals do not do enough to expose, condemn, and penalize this publishing sin. Authors also felt that that redundant publica tions occur because the practice is not condemned by academic leaders and because authors do not understand how redundant reporting distorts the aggre gation of data (i.e., meta-analyses). Therefore, editors, authors, and academic Dante’s Inferno 277 Dante’s Inferno 277 leaders should clarify and enforce mutually acceptable standards on redundant publication (e.g., Recommendations for the Conduct, Reporting, Editing, and Publication of Scholarly Work in Medical Journals, Section III.D.2 and IV.B).f f y Regarding self-plagiarism, set off short quotations from a previously pub lished article in quotation marks, and cite the original version. Permission must be requested from the publisher or other copyright holder when large sections are reproduced. Prevention When there is a need to repeat the information contained in a previously published literature review or a methods section, the best solution is to change some of the wording in each sentence and to refer the reader to relevant sources for previously published material (e.g., “As discussed in our previous report [give author names and year of publication],” etc.). It has also been suggested (Bretag & Mahmud, 2009) that authors could use text-matching software to ensure that they have appropriately described all previously pub lished work; however, it should be noted that there has been some concern over potential ethical and legal issues surrounding the software (McKeever, 2006). Unfair Authorship Authorship of a scientific report refers not only to the writing of a manu script but also to the origin of a writing project, any experimentation or other research connected with it, and the substantive kinds of work that led up to it. According to the ISAJE Ethical Practice Guidelines (www.isaje.net) and other codes (COPE, 1999; ICMJE, 2013), all persons named as authors should have made a major contribution to the work reported and be prepared to take public responsibility for its contents (in proportion to the credit they claim on the author list). An editorial (Huth, 1982, p. 613) in the Annals of Internal Medicine defines relevant terms as follows: Responsibility means the ability and willingness to defend the content of the paper if it is challenged by readers. Public means that authors are willing to carry out this responsibility in a published defense, such as a signed letter to the editor; private defense in private correspondence would not reach the scientific public. i Content means not simply packages of data but also the conceptual framework on which they are hung; the justification for a study or clini cal observations; the basis for the study design; methods for collection of valid data; the analysis and interpretation of the data; and the logic that led to the conclusions. The ICMJE’s (2013) four criteria for authorship are also relevant in this con text: (a) substantial contributions, (b) drafting the work or revising it criti cally, (c) final approval of the version to be published, and (d) agreement to be accountable. 278 8 Publishing Addiction Science There are a number of ways in which authorship decisions can result in ethi cal improprieties. First, some persons who have made significant contributions to an article may not receive sufficient credit or may receive no credit at all. This occurs when an article is drafted without the knowledge or consent of someone who made a substantive contribution earlier in the process. It also occurs when a decision to list the order of contributions is not made fairly with the full agreement of the co-authors, as when a major contributor is listed after a minor contributor to enhance the ego or career of the minor contributor. Unfair Authorship Another instance of inappropriate credit occurs when a co-author, such as a science writer, is not listed because the research group might be embarrassed to admit that someone else wrote the article, such as a science writer hired by a drug company to expedite the publication of favorable findings. This is called ghost authorship because the real author’s identity is unknown to those who read the article. Ghost writers are used by drug companies (Moffatt & Elliott, 2007) and were used by the tobacco industry (see Box 14.2) (Davis, 2008). By contrast, guest authorship occurs when articles are prepared by hired writers but published under the names of academics or scientists who allow themselves to be listed (sometimes for a payment or other incentives) without satisfying authorship criteria (Stern & Lemmens, 2011). The concern with this unethical practice lies with COIs and the potential for bias, as evidenced by ghostwrit ten articles on hormone replacement therapy, Vioxx (an anti-inflammatory drug that was withdrawn amid safety fears) and Fen (a popular diet drug withdrawn for safety reasons). A less serious form of ghost writing can occur when researchers, who are either too busy or poor writers, employ professional science writers to draft manuscripts of original research. For the purposes of this discussion, we concentrate on the former definition. An analysis of tobacco industry documents and transcripts of tobacco litigation testimony showed that British American Tobacco ghost-wrote the International Advertising Association (IAA) report titled “Tobacco Advertising Bans and Consumption in 16 Countries,” originally pub lished in 1983 and again as a revision in 1986. J.J. Boddewyn, a mar keting professor, served as “guest” editor of the reports. The reports concluded that tobacco advertising bans did not result in a reduction of tobacco use. These reports were then publicized in print materials, media campaigns, and legislative hearings during the 1980s and later. The Tobacco Institute, the major trade association representing the major U.S. cigarette manufacturers at the time, helped arrange for Bod dewyn to present the findings to the U.S. Congress and the media. Box 14.2: Case study: Ghost writing by the tobacco industry. Dante’s Inferno 279 A second type of authorship problem arises when some persons are listed as co-authors even though they made no substantive contribution to the article or the research. Unfair Authorship A common example is the practice of listing the head of a depart ment or a research center director, often at the end of the author list, a custom known as gratuitous, honorary, or gift authorship. Again, in the light of this practice, one must question the ethical climate in research settings that allows such behavior to exist. Ethical guidelines, appropriately crafted and imple mented, might deter such transgressions. Between these two extremes, there are a number of related infractions, such as the failure to give proper recognition to a person’s contribution by listing him or her inappropriately low in the author list, or the tendency to award co- authorship for minor contributions based on personal or political considera tions. A more complete discussion of authorship issues is provided in Chapters 5 and 11, which also describe procedures to minimize ethical and interpersonal problems related to authorship credits. Our purpose here is to discuss the seri ousness and consequences of this type of misconduct and to summarize the steps that can be taken to prevent its occurrence. Undeclared Conflict of Interest When a gift or gesture of any size is bestowed, it imposes on the recipient a sense of indebtedness. The obligation to directly reciprocate, whether or not the recipient is conscious of it, tends to influence behavior (Katz et al., 2003).i A COI is a situation or relationship in which professional, personal, or finan cial considerations compromise, or could be seen by a fair-minded person as potentially compromising independence of judgment (ISAJE, 1997). This problem has become exacerbated by closer relationships between government and industry (e.g., Bonner & Gilmore, 2012), industry-civil society partner ships, and cuts to government funding which encourage the procurement of industry sponsors. Prevention How can authors best deal with ethical issues related to authorship? As noted in Chapter 11, we advise early agreement on the precise roles of the contributors and collaborators and on matters of authorship and publication. The ICMJE (2013) has attempted to control unfair authorship practices by requiring that journals ask detailed questions about each author’s contributions. The lead author should periodically review the status of authorship credits within a designated working group by having open discussions of substantive contributions with all prospec tive collaborators. To avoid disputes, lead authors should distribute and discuss authorship guidelines with all potential collaborators on a manuscript. Those who may have been listed as an “honorary author” should instead be mentioned in the acknowledgments and have their contributions specified. An open dis cussion of authorship should be on the academic agenda of research centers. Involving an institutional ethics committee in drawing up institutional guide lines might also be helpful. Open and ongoing conversation about these issues, combined with institutional policies, is the best way to avoid problems. Consequences Authorship credits may be one of the most contentious issues in scientific publishing. At the level of collaborating research groups, the consequences range from hurt feelings to formal complaints made to a scientist’s unit direc tor or institutional authority. In between these extremes, there are likely to be recriminations, perceptions of unfairness, and poisoned working relation ships, which could damage the reputations of some of the parties involved. In the case of ghost writing, the funder (for example a drug manufacturer) obtains the credibility and prestige attached to the guest author, which may translate into distorted perceptions of the evidence base and affect public health. When instances of unfair authorship credit are detected, the editor’s response could range from the rejection of a pending manuscript to the call for a correction to a published article. Some journals (e.g., PLoS Medicine), call for a formal retraction if unacknowledged ghostwriting is discovered after publication and reporting of authors’ misconduct to institutions, in addition to banning the guest author from future submission (PLoS Medicine Editors, 2009). But these questionable research practices rarely come to the attention of editors unless there is a case of scientific fraud, where co-authors might claim that they were not sufficiently involved in the writing of the article to detect the fabrication in the first place. Some have called for academic sanctioning (Moffatt & Elliott, 2007), and because ghostwritten articles have been used in litigation to support drug companies’ claims, others (Stern & Lemmens, 2011) argue that a guest author’s claim for credit of an article written by someone else constitutes legal fraud. 280 Publishing Addiction Science Real, Apparent and Potential COIs Real (or actual) COIs should first be distinguished from “apparent” and “poten tial” conflict situations (See Table 14.3), as a COI only indicates the potential for bias, not the likelihood. A real COI means that the author, or the administrative unit with which the author has an employment relationship, has a financial or other interest that could unduly influence the author’s position with respect to the subject matter being considered. An apparent COI exists when an interest would not necessarily influence the author but could prompt others to question the author’s objectivity. Sometimes a conflict may exist, but the link is not so clear, as was the case with a young investigator who failed to declare funding Dante’s Inferno 281 Real or actual COI A direct conflict exists between professional judgment/ objectivity and private interests Apparent COI It appears or could be perceived that competing interests are improperly influencing the professional’s judgment, whether or not that is actually the case Unapparent COI A conflict may exist, but the link is unclear Potential COI Private interests are not but could come into direct conflict with professional judgment Table 14.3: Conflict of interest (COI) situations. Table 14.3: Conflict of interest (COI) situations. from the Institute for Research on Pathological Gambling. When contacted by the journal about her failed declaration, the researcher reported that she had no idea that the Institute’s funding came from the gambling industry. Unapparent COIs such as these occur when sponsorship is provided through an industry- funded social aspects organization or another third party, or when the recipient of the funding is unaware of the funding source. A potential COI involves a situation that may develop into a real COI. One’s perception of COI is just as important as COI itself, as even paid travel, honoraria, or other relationships can subconsciously “create strong disposi tions or obligations to reciprocate” (Mauss, 1967). As explained by Katz et al. (2003) “When a gift or gesture of any size is bestowed, it imposes on the recipi ent a sense of indebtedness. The obligation to directly reciprocate, whether or not the recipient is conscious of it, tends to influence behavior.” This means that one does not necessarily need to have a financial interest in the outcome of one’s research to constitute having a COI.i COIs can be financial, personal, political, or academic. Real, Apparent and Potential COIs Financial interests can include employment, research funding, stock or share ownership, payment for lectures or travel, consultancies, or company support for staff (COPE, 1999). These kinds of conflict are most often discussed in ethics codes and reports on research integrity because they are easier to document and quantify. Per sonal conflicts might include a vendetta against another researcher whom the author dislikes. Political conflicts exist when researchers distort their findings or interpretation to conform to a specific political idea or ideology. Academic conflicts include the attempt to validate “pet” theories that support one’s own ideas. These kinds of conflict are difficult to detect, but authors should never theless consider them when evaluating their own work. Authors in the past received little guidance in evaluating and responding appropriately to issues of regarding COIs. The existence of compliance offices in research settings is helpful, but these institutions themselves will not solve the problem. Research ers and research groups need appropriate training about the ethical dimensions involved as well as about opportunities for ongoing dialogue and conversation (Institute of Medicine, 2002). 2 Publishing Addiction Science 282 One way to determine whether a COI exists is to ask the following ques tion: If the situation or relationship were revealed to the editor or the reader only after the article was published, would it make a reasonable person feel misled or deceived? COI is not in itself wrongdoing. However, scientific mis conduct does occur when there is a failure to declare real or potential conflicts to an editor, one’s co-authors, and the readers of an article, to the extent that potential conflicts are very important in the evaluation of any piece of scien tific work. As discussed in more detail in Chapter 16, the potential for COI in the addiction field is enhanced by any relationship or funding connected to the tobacco industry, the alcohol beverage industry, for-profit health care systems, private hospitals, the pharmaceutical industry, or “social aspect organizations” that receive their primary support from industry sources. For example, in the search for medications that may be used to treat tobacco, alco hol, or illicit drug dependence, scientists involved in research on a particular product may have financial ties with companies that have a business interest in that product.h The alcohol and tobacco industries have also funded researchers to conduct policy studies or policy-related program evaluations. Box 14.3: Organizations receiving industry support.* This list is not exhaustive. • Foundation for Alcohol Research (formerly ABMRF) • Institut de Recherches Scintifiques sur les Boissons (IREB) • National Center for Responsible Gaming (NCRG) • Center for Consumer Freedom (CCF) • European Foundation for Alcohol Research (ERAB) • International Alliance for Responsible Drinking (IARD, formally ICAP) • Alcohol Information Partnership Real, Apparent and Potential COIs Sometimes the industry funds studies directly; other times, it funds studies indirectly through social aspect organizations, think tanks, or other third par ties that receive support from industry sources (see Box 14.3 for a list of these organizations). In addition to research funding, industry ties can include paid consultancies, conference presentations, stockholding, advisory board mem bership, or patent holding. Two major questions regarding the need for COI policies and precautions are whether industry funding affects the quality and eventual publication of research and whether the effect is deleterious. Bias toward “positive” results may exist even among articles that disclose financial ties to industry (Cho, 1998). For example, pharmaceutical industry–supported medication studies are sig nificantly more likely to report “positive” findings (i.e., that the manufacturer- associated medication is better than the placebo) than non–industry-funded • Foundation for Alcohol Research (formerly ABMRF) • Institut de Recherches Scintifiques sur les Boissons (IREB) • National Center for Responsible Gaming (NCRG) • Center for Consumer Freedom (CCF) • European Foundation for Alcohol Research (ERAB) • International Alliance for Responsible Drinking (IARD, formally ICAP) • Alcohol Information Partnership Box 14.3: Organizations receiving industry support. This list is not exhaustive. Dante’s Inferno 283 Dante’s Inferno 283 studies (Stelfox et al., 1998). Several examples of such biases have been observed in the addiction field. One analysis found that industry-supported studies were more likely than non–industry-funded studies to conclude that secondhand smoke has no health effects (Lambe et al., 2002). In reviewing all randomized controlled trials on nicotine replacement therapy included in the Cochrane database, Etter et al. (2007) found that industry-supported trials were more likely to produce statistically significant results when compared with independ ent trials. Researchers (Cataldo et al., 2010) conducting a meta-analysis on the link between smoking and Alzheimer’s disease found that, in tobacco-industry- affiliated studies, smoking was associated with a significantly decreased risk for Alzheimer’s disease, whereas those with no industry affiliation demonstrated a significant increased risk. Such instances of COI could be made worse by publi cation bias, in which industry-favorable studies are more likely to get published than are unfavorable ones.hl There are several possible mechanisms to explain how conflicts, especially those connected with industry ties, may lead to publication bias (see Cho, 1998). Real, Apparent and Potential COIs One is suppression of publication, whereby negative findings are not published because either the author fears loss of funding from industry spon sors or the industry itself imposes restrictions on publication. Another mecha nism is self-selection or industry selection of researchers who are more likely to get positive results. Even when grants are awarded by industry-funded organi zations that convene expert review panels, the panel members themselves may be influenced by receipt of honoraria, travel funds and invitations to speak at industry-supported conferences. A third possibility is industry control of the research agenda, so that funding is only provided for topics that are not likely to threaten an industry’s financial interests. A final possibility is that even when the funding source has no influence on the findings, researchers compromise their own credibility by being associated with industries that have a vested interest in the outcomes of the research. From the literature reviewed in this section, we conclude that industry fund ing can affect the nature, quality, and credibility of research, and the effect is likely to be deleterious. Consequences The existence of a COI does not mean that the conflict will result in adverse consequences. However, people with a conflict often fail to realize the extent to which the conflict has affected their judgment, because this can occur sub consciously. Another consequence of having competing financial interests is the possible limitation of publication options. Although most journals do not ban publication of articles because of their authors’ financial interests, some journals have now begun to prohibit authors of editorials and review articles from publishing if the author has a substantial financial interest in the product Publishing Addiction Science 284 discussed in the editorial or review (Relman, 1990). This policy does not apply to authors of scientific reports that present original data. i Undeclared COIs, when detected, may have serious consequences, such as the rejection of a pending article, the retraction of a published article, or the author’s need to publish an apology. A more subtle effect of real or apparent COI is the perception by one’s scientific colleagues that one’s scientific work is biased because of a personal or financial interest. Industry relationships can also threaten the integrity of the author’s host institution itself. p Note: Adapted from Pew Charitable Trusts (2013). Prevention Researchers must first be made aware of the ethical issues that arise when exploiting COIs. Many schools are requiring ethics classes that include edu cation on COIs, and many academic institutions and medical centers have adopted rules governing financial support for faculty activities. These rules describe when faculty must disclose particular interests and when they must divest themselves of particular financial interests. In 2013, an expert Task Force convened by the Pew Charitable Trusts published COI best practice recom mendations for academic medical centers, which can be read in Table 14.4. COI committees, when they operate as part of ethical review committees, are a part of institutional compliance oversight and hold promise in this respect. COI area Best practice recommendation Disclosing COIs Required to disclose all industry relationships that relate to academic activities in teaching, research, patient care, and institutional service. Acceptance of gifts and meals Prohibited Industry-funded speaking Prohibited Industry-sponsored fellowships Clinical training: prohibited Research training: permitted COI curriculum Required Consulting and advising relationships Marketing: prohibited Scientific activities: permitted Industry support of accredited continuing medical education Should not be supported Ghostwriting and honorary authorships Prohibited Table 14.4: Recommended best practices in medical conflict of interest (COI) policies. Note: Adapted from Pew Charitable Trusts (2013). Dante’s Inferno 285 Authors should pay close attention to the guidelines issued by these commit tees. As noted in Chapter 16, the scientific community has issued warnings about the advisability of accepting any funding from the tobacco and alcohol industries and has suggested rigorous adherence to voluntary ethical codes when such funding is accepted. According to Loue (2000), the best way to avoid problems associated with potential COI is self-elimination from participation in potentially conflicting activities. Short-term consulting arrangements with the tobacco, alcohol, and pharmaceutical industries are often not worth the questions the researcher must face about his or her objectivity. Arrangements with industry can be par ticularly problematic when the researcher is asked to sign a restrictive con tract regarding the ownership of data, the sponsor’s control of the data, and the investigator’s right to publish them. Even when these guidelines have been followed appropriately, however, authors should declare to the editor any real, potential, or apparent COI with respect to their involvement in a particular publication. Prevention Authors should declare conflicts between (a) commercial entities and authors personally and (b) com mercial entities and the administrative unit with which the authors have an employment relationship. Authors should also declare sources of funding for a study, review, or other publication in a way that can be clearly understood by the reader, even if the journal does not require authors to do so. A footnote or an acknowledgment is the most appropriate mechanism. Describe funding sources in sufficient detail so that an average reader can recognize potential COIs. If a funding source is a social aspect organization with an ambiguous name such as The Alcohol and Health Fund, the reader should be informed that, for example, the organization is supported by a group of beer companies. Disclosure alone will not necessarily eliminate publication bias. Research ers who are serious about avoiding even the appearance of COI are advised to dilute the conflicting relationship by getting funding from both industry and nonindustry sources and by refusing to sign industry agreements that do not guarantee the researcher’s right to publish the results regardless of the study’s outcome. Other management strategies include avoiding additional financial ties that are not absolutely necessary to the pursuit of the research, such as the acceptance of advisory board memberships, stock options, or consulting fees from companies sponsoring research (Cho et al., 2002). Human/Animal Subjects Violations Addiction research involving human and animal subjects has been conducted for over a century. During this period, regulations governing human and ani mal experimentation have developed into a very complex set of procedures that are typically governed by appointed committees located at institutions involved Publishing Addiction Science 286 in biomedical research. These procedures include ethical review of research protocols, safety monitoring of animals and human research participants, and informed consent requirements for human participants. These procedures were developed out of concern for the rights of research participants follow ing a series of well-publicized medical experiments in which human subjects were exposed to harmful agents or had effective treatments withheld without their knowledge or consent (Loue, 2000). It has now become customary, if not mandatory, to submit proposed research for independent review by an ethical research committee to determine its ethical acceptability from the perspective of the local community and the researcher’s institution (Federman et al., 2003). y Such boards focus primarily on the protection of research participants by assuring that the study’s procedures minimize risks of unwarranted harm to participants. Although regulations regarding types of study requiring ethical approval vary across the world, formal international standards developed to guide experimentation involving human participants have been put forth in the 1964 Declaration of Helsinki, which states that in medical research involv ing human participants, the well-being of the individual research subject takes precedence over all other interests. In particular, the 1975 and 1983 revisions emphasized the importance of voluntary informed consent to participate in research (Loue, 2000). Additional ethical issues may also have to be considered for certain types of research involving individuals who are substance dependent. Does drug or alcohol dependence in combination with other factors limit capacity to give informed consent? What other factors—intoxication, withdrawal, chronic recidivism? Do the criteria for dependence imply impaired decision making? If someone is using drugs despite reoccurring problems and does not seek treat ment, should he or she be categorized as not exhibiting concern for his or her welfare and therefore incapable of providing informed consent? p p g Genetic research raises similar if not even more challenging ethical issues. Genetic research in relation to addiction exposes subjects, their families, and the broader social community to additional risks (Chapman et al., 2012). Risks to subjects include the loss of privacy and the loss of control over sensitive personal information. Consequences Failure to follow recommended or required journal procedures regarding protection of human and animal research subjects could have several impor tant consequences. Although most journals do not ban publication of articles because they have not been submitted for ethical review, some journals now require authors to state whether their research conforms to the minimum standards outlined in the Declaration of Helsinki, a set of ethical principles regarding human experimentation developed by the World Medical Associa tion. In particular, social and behavioral research such as survey studies and research involving archival records may not require stringent informed consent procedures. However, it would be an error to rely on this perception. Surveys, on occasion, have resulted in significant harm to individuals and to institu tions. It is safer to submit all research for institutional review and to let the committee decide whether the researcher is exempt or not. Failure to obtain ethical approvals or informed consent from research participants may lead an editor to question the purpose and value of the research and could result in a decision not to send the manuscript out for review or, when the failure is detected during peer review, to decline the manuscript. Another consequence could be the notification of an official from the author’s institution. Human/Animal Subjects Violations Financial remuneration for research participation may increase the use of drugs or alcohol if adequate precautions are not taken. Incentive payments to parents to encourage them to enroll their children in genetic studies are unacceptable because of the risk of coercing children to par ticipate. Editors and authors have a duty to make sure that published research is subject to rigorous ethical review. Nevertheless, in some cases, particularly the social sciences, there is the per ception that ethical review has gone too far in its attempts to minimize risks that may not be present. As explained by Mäkelä (2006): (a) social research is generally much less invasive than medical research; (b) its impact on research participants involves different casual chains; (c) social research design tends to be more open ended; and (d) in social research, the context of the relationship Dante’s Inferno 287 between researcher and participant is closer to that of a journalist and a minis ter rather than that of a doctor and patient. between researcher and participant is closer to that of a journalist and a minis ter rather than that of a doctor and patient. Prevention It is always wise to mention both in the cover letter to the editor and in the text of a submitted manuscript that the researchers have followed appropriate ethical review procedures. If there are any questions regarding the applicabil ity of human subjects requirements, these should be raised with the editor in the cover letter or in a telephone call or email message before submission of a manuscript. Often these questions can be resolved by consulting the journal’s website or instructions to authors. The ICMJE (1991, p. 339) has provided the following guidance regarding ethical issues: When reporting experiments on human subjects, indicate whether the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional or regional) and with the Helsinki Declaration of 1975, as revised in 1983. Do not use patients’ names, initials, or hospital numbers, espe cially in illustrative material. When reporting experiments on animals, indicate whether the institution’s or the National Research Council’s Publishing Addiction Science 288 guide for, or any national law on, the care and use of laboratory ani mals, was followed. Scientific journals also have an important role to play in the protection of human and animal research subjects. Journals are responsible for the dissemi nation of research findings. They “are obligated to publish research that meets high ethical standards . . . for which the authors have attested to their compli ance with regulatory and ethical standards” (Federman et al., 2003, p. 205). A number of journals have implemented policies requiring authors to certify compliance with informed consent procedures, and ISAJE (1997) subscribes to these policies. Note: Adapted from COPE (2011). Plagiarism Plagiarism refers to both the theft of intellectual property, such as ideas and images, and the copying of unattributed textual material. Plagiarism ranges from the unreferenced use of others’ published and unpublished ideas, includ ing research grant applications, to submission under “new” authorship of a complete article, sometimes in a different language. It can also include copying of another’s work verbatim or nearly verbatim in a way that misleads the ordi nary reader about the author’s own contribution. Table 14.5 provides examples of instances that can be constituted as “clear plagiarism,” such as copying an entire article, as well as less serious forms like the “minor copying” of a string of words (COPE, 2011). It may occur at any stage of planning, research, writing, or publication. It applies to both print and electronic versions of a publication. The Office of Research Integrity, an office within the U.S. Department of Health and Human Services that monitors investigation of research misconduct, considers plagia rism to include both the theft or misappropriation of intellectual property and the substantial unattributed textual copying of another’s work, such as sen tences, paragraphs or even entire manuscripts, in a way that misleads the ordi nary reader regarding the contribution of the author. Least serious Most serious Extent Few words Whole paragraph Whole article Originality Commonly used Used by small number of authors Original idea Referencing Full and accurate referencing Not referenced Intent Unintentional deception Intentional deception Table 14.5: Features of plagiarism identified by the Committee on Publication Ethics. Note: Adapted from COPE (2011). Least serious Most serious Extent Few words Whole paragraph Whole article Originality Commonly used Used by small number of authors Original idea Referencing Full and accurate referencing Not referenced Intent Unintentional deception Intentional deception Table 14.5: Features of plagiarism identified by the Committee on Publication Ethics. Note: Adapted from COPE (2011). Dante’s Inferno 289 Consequences Developments in text-matching software (e.g., CrossCheck, eTBLAST) have made detecting instances of plagiarism much easier. The consequences of pla giarism can be serious, ranging from an editor’s reprimand to a formal hearing and loss of employment after an allegation is reported to the author’s insti tutional officials. Box 14.4: Guidelines for avoiding plagiarism. Source: Roig (2013). Plagiarism The US Office of Research Integrity generally does not pur sue the limited use of identical or nearly identical phrases that, for example, describe a commonly used methodology or previous research, because these are not considered to be substantially misleading to the reader or of great sig nificance. Journal editors can be unrelenting and at times unforgiving if they detect instances of plagiarism. The typical approach is first to request a writ ten explanation from the author soon after the plagiarism has been discov ered. Most often, these instances are discovered by knowledgeable and vigilant reviewers or by readers who sometimes report that their own words, sentences, paragraphs, or articles have been misappropriated. If the author’s explanation is credible and the amount of copying is small, the consequences may be noth ing more than a letter of reprimand and possibly the rejection of the manu script. More extensive types of plagiarism may result not only in the rejection of the manuscript, but also in the publication of a correction or retraction if the Box 14.4: Guidelines for avoiding plagiarism. Source: Roig (2013). 1. Cite idea sources and identify the contributions of others with out exception, even when paraphrasing or summarizing. 2. Use quotation marks for any verbatim text taken from another author. 3. Clarify for readers which ideas are the author’s own and which are derived from another source. 4. Be familiar with copyright law. 5. Paraphrasing and summarizing requires authors to produce the same meaning using their own words. 6. Paraphrasing and summarizing requires authors to possess a comprehensive understanding of the material. 7. Refer to the primary literature, as opposed to a secondary source. 8. Always double check citations and reference section. 9. If uncertain as to whether an idea or fact is common knowledge, cite the original source. 10. Do not partake in ghostwriting. Box 14.4: Guidelines for avoiding plagiarism. Source: Roig (2013). 1. Cite idea sources and identify the contributions of others with out exception, even when paraphrasing or summarizing. 2. Use quotation marks for any verbatim text taken from another author. 3. Clarify for readers which ideas are the author’s own and which are derived from another source. 4. Be familiar with copyright law. 5. Paraphrasing and summarizing requires authors to produce the same meaning using their own words. 6. Paraphrasing and summarizing requires authors to possess a comprehensive understanding of the material. 7. Plagiarism Refer to the primary literature, as opposed to a secondary source. 8. Always double check citations and reference section. 9. If uncertain as to whether an idea or fact is common knowledge, cite the original source. 10. Do not partake in ghostwriting. 1. Cite idea sources and identify the contributions of others with out exception, even when paraphrasing or summarizing. 2. Use quotation marks for any verbatim text taken from another author. 3. Clarify for readers which ideas are the author’s own and which are derived from another source. 4. Be familiar with copyright law. 5. Paraphrasing and summarizing requires authors to produce the same meaning using their own words. 6. Paraphrasing and summarizing requires authors to possess a comprehensive understanding of the material. 7. Refer to the primary literature, as opposed to a secondary source. 9. If uncertain as to whether an idea or fact is common knowledge, cite the original source. 10. Do not partake in ghostwriting. Source: Roig (2013). Publishing Addiction Science 290 material has already been published, and authors may be banned from submit ting to the journal in the future (COPE, 2011). Studies indicate that retractions for this deadly sin are increasing in recent years, accounting for 9.8%–17.0% of retractions (Fang et al., 2012). More importantly, such matters may then be referred to the author’s institutional employer, who typically will have responsibility for dealing with allegations of scientific misconduct. This is discussed in more detail in the next section. Although failure to attribute the original source of a sentence or paragraph may constitute a copyright infringement and could result in civil proceedings, such cases are rarely prosecuted. Prevention The US Office for Research Integrity offers 26 Guidelines on Avoiding Plagia rism (Roig, 2013), which focus on disclosing all sources through appropriate citation or quotation conventions (see Box 14.4 for relevant guidelines). If the author plans to use a large amount of other people’s written or illustrative mate rial, he or she must seek permission to reprint the material (COPE, 1999). Legal definitions may vary from country to country regarding plagiarism, copyright, and intellectual property rights. The author should review these with the editor when there is any question (Roig, 2013). A more common problem that may result in an embarrassing revelation is the unintentional copying of small amounts of textual material or the borrow ing of others’ ideas or concepts without appropriate attribution. These cases are usually the result of negligence, sloppiness, or laziness, as when an author fails to use quotation marks or paraphrases someone else’s ideas without stating the source. In these instances, the best prevention method is the careful documen tation of all source documents in the course of note taking and the develop ment of writing habits that allow ample time to prepare a manuscript. Authors can ensure they have appropriately cited their work using text-matching soft ware recommended by the Office of Research Integrity. Other Types of Scientific Fraud According to various ethical authorities (e.g., Committee on Publication Eth ics, 2011), scientific fraud is manifested in the following forms: • fabrication or falsification of data, that is, presenting data in a research report that have not been obtained in the manner or by the methods described in the report or altering or presenting original findings in a way that distorts the result in a scientifically unjustified way, or by omitting results or data pertinent to conclusions; Dante’s Inferno 291 291 • plagiarism, that is, presenting someone else’s manuscript, article, or text as one’s own; • plagiarism, that is, presenting someone else’s manuscript, article, or text as one’s own; • misappropriation, that is, illicitly presenting or using in one’s own name an original research idea, plan, or finding disclosed in confidence; and • misappropriation, that is, illicitly presenting or using in one’s own name an original research idea, plan, or finding disclosed in confidence; and • noncompliance with legislative and/or regulatory requirements. ii • noncompliance with legislative and/or regulatory requirements. Although the terms fraud and misconduct are often used interchangeably, it is important to note that fraud implies intentional deception. Fraud can occur in the course of proposing, conducting, or reporting research. It is most often detected at the time of publication, primarily because reviewers, editors, and read ers of scientific articles are very critical and skeptical by nature and profession. In the course of this chapter, and in other parts of this book (see Chapters 5, 10, and 11), we have described several of the less serious instances of scientific In December 2003, the Court of Justice of the Canton of Geneva gave its sentence in an (in)famous case of scientific fraud. A Swedish profes sor at The University of Geneva and formerly of Gothenburg University had charged two tobacco activists with libel after they accused him of ‘unprecendented scientific fraud’ concerning the risks of passive smok ing. The court dismissed the case, stating that “Geneva has indeed been the platform of a scientific fraud without precedent in the sense that. Box 14.5: An example of scientific fraud from the tobacco field. Sources: Domstol i Geneve slår fast svenskt vetenskapsfusk (Court in Geneva gives sentence on Swedish scientific fraud). Svenska Dagbladet, 16.12.2003 www.prevention.ch/rypr151203.htm, accessed 11 June 2004. Other Types of Scientific Fraud Professor Ragnar Rylander has acted in his capacity of associate profes sor at the University, taking advantage of its influence and reputation and not hesitating to put science at the service of money, in disregard of the mission entrusted to this public institution.” According to the court, for thirty years the professor had had a close but secret relationship with Philip Morris, which included substantial financial rewards. Thus he lied when he stated to The European Journal of Public Health that he had never had contact with Philip Morris. In his research on passive smoking and in several conferences on the topic he questioned the risks connected with passive smoking. According to the Court, the professor “did not hesitate to deceive the general public in order to show himself favorable to the tobacco company.” In particular, the Court reported as apparently fraudulent a study on respiratory diseases in children in which he altered the database so that no link could be made between passive smoking and the frequency of respiratory infections. 2 Publishing Addiction Science 292 misconduct, such as the selective interpretation of others’ findings, inappro priate citation practices, unfair authorship practices, selective reporting of data, or use of inappropriate statistics. The problem with these questionable research practices and the more serious forms of fraud (e.g., data fabrication) is the damage it does to the scientific enterprise, to the extent that it misleads other scientists and establishes a false record that may be misinterpreted by the public, policymakers, or clinicians. Box 14.5 provides an example of scientific fraud from the field of addiction research. Consequences As is the case with the previous publishing sins, fraud also distorts research findings and can erode the public’s trust in research (Gupta, 2013). Consequences Journal editors, funding agencies, and academic institutions take allegations of scientific misconduct seriously, especially those institutions that depend on public support for their research. Typically, an editor who receives informa tion about possible fraud or who suspects it during the course of a manuscript review has a limited number of options, starting with the notification of the author. Many scientific and academic institutions have procedures to deal with allegations of fraud and misconduct; therefore, an editor can begin by passing the allegation and the author’s response to an appropriate institutional official or review committee for further action if the allegation seems credible. In general, the process begins with a preliminary investigation, followed by a more formal inquiry if the allegation has sufficient substance or importance. In such cases, the withdrawal or rejection of the manuscript, or the publication of a correction in the case of an already published article, is the least of the author’s worries. Fraud can lead to disciplinary action, banishment from advisory committee or review boards, and the re-review and possible retraction of previously published articles. As is the case with the previous publishing sins, fraud also distorts research findings and can erode the public’s trust in research (Gupta, 2013). Journal editors, funding agencies, and academic institutions take allegations of scientific misconduct seriously, especially those institutions that depend on public support for their research. Typically, an editor who receives informa tion about possible fraud or who suspects it during the course of a manuscript review has a limited number of options, starting with the notification of the author. Many scientific and academic institutions have procedures to deal with allegations of fraud and misconduct; therefore, an editor can begin by passing the allegation and the author’s response to an appropriate institutional official or review committee for further action if the allegation seems credible. In general, the process begins with a preliminary investigation, followed by a more formal inquiry if the allegation has sufficient substance or importance. In such cases, the withdrawal or rejection of the manuscript, or the publication of a correction in the case of an already published article, is the least of the author’s worries. Fraud can lead to disciplinary action, banishment from advisory committee or review boards, and the re-review and possible retraction of previously published articles. Prevention There can be no substitute for careful mentoring and training of scientists in the prevention of scientific misconduct. Most scientists have such high respect for the values of science that they would never deliberately fabricate data or mislead their colleagues about the data they have collected or its interpretation. Milder forms of scientific misconduct may result from ignorance, so that delib erate exposure to ethical training may help individual scientists avoid these kinds of problems. Researchers are encouraged to review the resources listed in Table 14.6. Because scientists typically work in groups along with research sup port staff, the best way to prevent fraud is to check the data as well as colleagues’ work carefully at every stage in the process of conducting a research project and preparing a scientific report. BMJ goes so far as to require investigators to submit full data sets to accompany trials that are published in that journal. Dante’s Inferno 293 1. Code of conduct for social science research UNESCO [undated] http://www.unesco.org/new/fileadmin/MULTIMEDIA/HQ/SHS/pdf/Soc_Sci_Code. pdf. 2. Guidelines for research ethics in the social sciences, law and the humanities The National Committee for Research Ethics in the Social Sciences and the Humani ties (NESH), 2006 https://graduateschool.nd.edu/assets/21765/guidelinesresearchethicsinthesocials cienceslawhumanities.pdf 3. The concordat to support research integrity Universities U.K., 2012 http://www.universitiesuk.ac.uk/highereducation/Documents/2012/TheConcordat ToSupportResearchIntegrity.pdf 4. European code of conduct for research integrity European Science Foundation (ESF) and ALLEA (All European Academies), 2011 http://www.esf.org/fileadmin/Public_documents/Publications/Code_Conduct_ ResearchIntegrity.pdf 5. Singapore statement on research integrity 2nd World Conference on Research Integrity, 2010 http://www.singaporestatement.org/statement.html 6. Teaching the responsible conduct of research in humans (RCRH) Koreman, S. G., Office of Research Integrity, 2006 http://ori.hhs.gov/education/products/ucla/default.htm 7. Declaration of Helsinki—Ethical principles for medical research involving human subjects World Medical Association, 1964 http://www.wma.net/en/30publications/10policies/b3/index.html Table 14.6: Resources. Finally, we encourage readers to come forward with good-faith allegations of scientific misconduct and remind readers about protections for whistleblowers, for example, those endorsed by the US Office of Research Integrity, Department of Health and Human Services (2014) in the Whistleblower’s Bill of Rights. Conclusion Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. Conclusion At various times in its short history, addiction research has had its credibility damaged because of ethical breaches in its research and publication practices. Today the field is experiencing an even greater crisis in values, caused by increas ing pressure to publish, COIs, and ethical committee restrictions on research 4 Publishing Addiction Science 294 (Babor, 2009). Furthermore, questionable research practices may be implicitly encouraged by publication practices that focus on significant findings.h ii This situation has been exacerbated by researchers and organizational enti ties such as journals and professional societies not having a consistent frame work of ethical standards and ethical decision making that can protect authors, the scientific community, and the public from the ethical problems that arise in research and scientific writing. A practical, case-based approach with appropri ate ethical analysis, designed to address the realities of research and publishing, follows in Chapters 15 and 16. In most countries, the general public rates biomedical and social scientists highly in terms of their occupational prestige and credibility. When scientific misconduct is detected and publicized, scientists violate this trust and science loses public support. By following the preventive measures described in this chapter, researchers can avoid most of the major and minor ethical dilemmas associated with scientific misconduct. But the obligation of ethical conduct in reporting research in journal publications does not rest with the authors alone. The Institute of Medicine (2002) report affirms what this chapter espouses in terms of the integrity of individual authors (researchers) by advocating “above all a commitment to intellectual honesty and personal responsibility for one’s actions and to a range of practices that characterize the responsible conduct of research” (p. 5). This report also notes that individuals can only flourish in institutions that “establish and continuously monitor structures, processes, policies and procedures [that support] integrity in the conduct of research and use this quality improvement” (Institute of Medicine, 2002, p. 5). There is no one strategy that can be relied on to fully overcome questionable research prac tices or instances of serious research misconduct. Therefore, a multipronged approach is required by researchers, academics, journal editors, peer review ers, funders, ethics committees, and regulatory authorities. Such an approach would not only go a long way in preventing the Seven Deadly Sins, it would also remove the need for punishments meted out in the Circles of Hell. References Alighieri, D. (1947). The Divine Comedy (translated by Laurence Binyon in The Portable Dante, The Viking Portable Library #32). New York, NY: Viking Press. Dante’s Inferno 295 Dante’s Inferno 295 295 Amos, K. A. (2014). The ethics of scholarly publishing: Exploring differences in plagiarism and duplicate publication across nations. Journal of the Medical Library Association, 102, 87–91. Babor, T. F. (2009). Alcohol research and the alcoholic beverage industry: Issues, concerns, and conflicts of interest. 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Retrieved from http://www.esf.org/fileadmin/Public_documents/Publications/Code_ Conduct_ResearchIntegrity.pdf. Fanelli, D. (2009). How many scientists fabricate and falsify research? A sys tematic review and meta-analysis of survey data. PLoS ONE, 4(5), e5738. Fang, F. C., Steen, R. G., & Casadevall, A. (2012). Misconduct accounts for the majority of retracted scientific publications. Proceedings of the National Academy of Sciences of the United States of America, 109, 17028–17033. Federman, D. D., Hanna, K. E., & Rodriguez, L. L. (Eds.). (2003). Responsible research: A systems approach to protecting research participants. Washington, DC: National Academies Press.fi Griffin G. C. (1991). Don’t plagiarize—even yourself! Postgraduate Medicine, 89, 15–16. Gøtzsche, P. C. (1989). Multiple publication of reports of drug trials. European journal of clinical pharmacology, 36(5), 429-432. Gupta, A. (2013). Fraud and misconduct in clinical research: A concern. 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Jannot, A., Agoritsas, T., Gayet-Ageron, A., & Perneger, T. V. (2013). Citation bias favoring statistically significant studies was present in medical research. Journal of Clinical Epidemiology, 66, 296–301. Jerrells, T. R. (2001). References Duplicative publication of data and other ethical issues in publishing of scientific findings. Journal of Substance Abuse Treatment, 20, 111–113. Dante’s Inferno 297 Dante’s Inferno 297 297 Katz, D., Caplan, A. L., & Merz, J. F. (2003). All gifts large and small: Toward an understanding of the ethics of pharmaceutical industry gift-giving. American Journal of Bioethics, 3, 39–46. Koreman, S. G. (2006). Teaching the responsible conduct of research in humans (RCRH). Office of Research Integrity. Retrieved from http://ori.hhs.gov/ education/products/ucla/default.htm. Lambe, M., Hallhagen, E., & Boethius, G. (2002). Cyniskt spel inom tobak sindustrin. 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National Committee for Research Ethics in the Social Sciences and the Humani ties (NESH). (2006). Guidelines for research ethics in the social sciences, law and the humanities. Retrieved from https://graduateschool.nd.edu/ assets/21765/guidelinesresearchethicsinthesocialscienceslawhumanities.pdf. Nieminen, P., Rucker, G., Miettunen, J., Carpenter, J., & Schumacher, M. (2007). Statistically significant papers in psychiatry were cited more often than oth ers. Journal of Clinical Epidemiology, 60, 939–946.fi Office of Research Integrity, Department of Health and Human Services. (2014). Whistleblower’s Bill of Rights. Retrieved from http://ori.hhs.gov/ Whistleblower-Rights. Pew Charitable Trusts. (2013). Task force recommendations on relationships with industry. Retrieved from http://www.pewhealth.org/reports-analysis/ data-visualizations/task-force-recommendations-on-relationships-with- industry-85899525795. PLoS Medicine Editors. (2009). 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The Road to Paradise: Moral Reasoning in Addiction Publishing Thomas McGovern, Thomas F. Babor and Kerstin Stenius With the gesture of a guide, whose goal’s in sight, She spoke: “We from the greatest body move, Emerging in the heaven that is pure light; Light of the understanding, full of love, Love of the true good, full of joy within, Joy that transcends all the heart conceiveth of.” Dante Alighieri (1947), Paradiso, Canto XXIX, 37–42 Dante Alighieri (1947), Paradiso, Canto XXIX, 37–42 References New England Journal of Medicine, 338, 101–106. Stenius, K., & Babor, T. F. (2003). Ethical problems and ethical guidelines of addiction journals: A report to the ISAJE membership. Unpublished manu script, International Society of Addiction Journal Editors. Stern, S., & Lemmens, T. (2011). Legal remedies for medical ghostwriting: Imposing fraud liability on guest authors of ghostwritten articles. PLoS Med, 8(8), e1001070. Tramèr, M. R., Reynolds, D. J., Moore, R. A., & McQuay, H. J. (1997). Impact of covert duplicate publication on meta-analysis: A case study. BMJ, 315, 635–640. Yank, V., & Barnes, D. (2003). Consensus and contention regarding redun dant publications in clinical research: Cross-sectional survey of editors and authors. Journal of Medical Ethics, 29, 109–114. World Medical Association. (1964). Declaration of Helsinki – Ethical Prin ciples for Medical Research Involving Human Subjects. Retrieved from http://www.wma.net/en/30publications/10policies/b3/. CHAPTER 15 Introduction The descent into the various levels of the Inferno, described in Chapter 14, resulting from the capital sins (vices) associated with varying degrees of sci entific misconduct, is replaced in this chapter by a description of an ascent into Paradise, a realm of enlightened ethical conduct and decision making. This transformation is achieved by the practice of virtue and by adherence to ethical principles and moral reasoning. The ascent into the heavenly spheres is achieved by those virtuous ones who exemplify fortitude, prudence, justice, and temperance in the overarching context of faith, hope, and love. “Being good” (character ethics) exemplifies this state, and this is a quality found in society, Publishing Addiction Science 300 institutions, and individuals. Moral reasoning and ethical reflection inculcate attitudes that promote good behavior, the moral stance of “being good.”l Reflection and conversation are at the heart of ethical dialogue in any setting. The road to the unethical publication pitfalls described in the previous chapter is paved with good intentions. Perhaps “good conversation” is equally a culprit, as discussion about ethical issues in research does not seem to have influenced actual behavior. Kass (2002), a seasoned veteran in the field of bioethics, com plained that “in bioethics at the present, the action is mostly talk” (p. 57). In our search for the best solution to ethical problems, we have lost sight of the original goal of ethics: to improve the quality of our behavior. How then can we provide meaningful direction for researchers wishing to avoid the seven deadly sins pertaining to scientific writing and fraudulent research and at the same time devise a virtuous path leading to responsible research? Let us abandon ethics as good conversation, an approach advocated by many respected authorities (Brody, 1990; Glaser, 1994), and concentrate exclusively on practical recommendations to guide the behavior of authors and research ers. The Greeks, in their wisdom, saw virtue—the quality of being good in any human endeavor—as the condition of being poised between the two extremes (vices) of any given situation. In proposing an approach to ethical issues in addiction research and publishing, we embrace the advice of the Greeks, which accounts for both talk and action in fashioning practical responses to moral questions. Introduction In pursuing a path that leads to ethical behavior guided by moral reason ing, we find guidance in the initiatives promoted by the International Society of Addiction Journal Editors (ISAJE) and by the Committee on Publication Ethics (COPE). ISAJE was organized in 1997 and has authored the Farming ton Consensus (1997) and Ethical Practice Guidelines in Addiction Publish ing: A Model for Authors, Journal Editors and Other Partners (ISAJE Ethics Group, 2002). Since its own inception in 1997, COPE has published guide lines, policy statements, and more than 500 case reports that provide guid ance in promoting ethical standards in all aspects of publication while at the same time addressing the pitfalls of unethical behavior and the associated vices described in Chapter 14. Table 15.1 describes the types of the cases cov ered by COPE in its ethical analysis of moral problems associated with over all research, including addiction research. The data show that a wide variety of ethical problems have precipitated inquiries for ethical analysis, especially with regard to questionable and unethical research. Most of these issues have already been discussed in Chapter 14. COPE also provides a variety of pol icy statements and guidelines that address major issues in publication from the perspective of publishers and editors and issues resulting from the case reports described above.l We begin with some reflection about ethics as the human endeavor in addic tion research. This approach to ethics is characterized by twin goals: “to be good” and “to do good.” Being good is at the heart of “virtue” or “character” The Road to Paradise 301 Type of case Frequency Percentage Questionable and unethical research 167 21.1% Redundant and duplicate publication 113 14.3 Data sloppiness, fabrication, etc. 105 13.3 Misconduct/questionable behavior 99 12.5 Correction of literature 82 10.3 Conflict of interest 61 7.7 Plagiarism 55 7.0 Miscellaneous 54 6.8 Peer review 54 6.8 TOTAL 790 100% Table 15.1: Frequencies, percentages, and types of ethical cases covered by COPE. Source: Frequency data obtained from COPE website: http://publicationethics. org/cases, accessed June 21, 2015. ethics and espouses qualities such as integrity, honesty, and compassion for others. Doing good is the basis for principle-based ethics, such as autonomy, beneficence, and justice (see Table 15.2 for definitions of italicized ethical terms). The Ethical Challenge Scientific issues in the addiction field, as elsewhere, embrace three ethical realms: the individual, the institution, and society (Glaser, 1994). In affording respect to each of these realms, researchers honor what Kass (2002) describes as “the rich broth of our social, civil, cultural, and spiritual life together and of the ways in which it seasons us without our knowledge” (p. 65). The well-being of the individual, of institutions, and of society as a whole is at stake in assessing the ethical issues that arise in addiction research, as illustrated by recent stud ies of corporate social responsibility programs, research on chronic drug users, and the use of animals involved in addiction research (Casswell, 2013; Fisher, 2011; Lynch et al., 2010; Miller et al., 2011). Consider the following scenario as an invitation to apply our discussion up to this point to the realities of a pos sible research publication situation: A university research team wishes to examine drug use in a poor, disad vantaged minority neighborhood with an identifiable ethnic population. The intent of the study is to test a new treatment for addiction that holds great promise for society as a whole. The political climate in which the research is conducted is one that is willing to provide research support for biological and social research but is not prepared to address the deeper societal issues underlying drug problems. In addition, the community in which the research is to be conducted sees drug use as both a matter of choice and best controlled through stringent and oppressive legal meas ures. Furthermore, the larger community views the minority drug-using group with suspicion and distrust. The individuals who will constitute the research population are disadvantaged, have little education, and are a vulnerable population that can be easily exploited in a research endeavor. Can such research be conducted in a manner that meets appropriate scientific standards? The answer is yes: Many measures can be taken to assure its appro priateness. For instance, researchers can offer guarantees that ensure respect for the dignity of the research participants. Researchers can also safeguard the vul nerability of the individuals involved, together with the community as a whole, by meeting the standards of ethical review committees and other governmental and institutional regulations on research. At first sight, the ethical and scientific standards for responsible research seem to be met at the individual level. Introduction ii We combine both goals in the ethical discussion in this chapter and bring them to bear on two basic questions that are interwoven in every research enterprise: (a) Can we do it (the technical or research question), and (b) should we do it (the ethical or moral question)? Both questions, individually and col lectively, are challenging: They hold us to equally rigorous scientific and ethical standards in all of our research and publishing undertakings. The materials produced by COPE are also an invaluable resource in this undertaking. Autonomy Respect people’s choices, and do not obstruct their actions unless those actions are harmful to others. Beneficence Do good: Provide competent and compassionate care and maximize benefits to individuals, institutions, society. Nonmaleficence Do no harm: Minimize risks to individuals, institutions, society. Justice Give each person his or her due. Fairness Avoid discrimination and exploitation. Stewardship Use resources efficiently and justly. Table 15.2: Key ethical principles used in moral reasoning and decision making. Autonomy Respect people’s choices, and do not obstruct their actions unless those actions are harmful to others. Beneficence Do good: Provide competent and compassionate care and maximize benefits to individuals, institutions, society. Nonmaleficence Do no harm: Minimize risks to individuals, institutions, society. Justice Give each person his or her due. Fairness Avoid discrimination and exploitation. Stewardship Use resources efficiently and justly. Table 15.2: Key ethical principles used in moral reasoning and decision making. 302 Publishing Addiction Science The Ethical Challenge But what of the larger community and societal implications of this research? How will the individuals involved be treated by the larger com munity if the study shows a high prevalence of drug dependence in the pop ulation? Conceivably, an increase in discrimination and oppression might occur (McGovern, 1998), a result researchers would want to avoid. Another consideration centers on who shall benefit from the favorable outcomes of The Road to Paradise 303 the research: the individuals in the poor neighborhood or the more privi leged members of society? Balancing individual, institutional, and societal concerns can lead to a better understanding of the risks and benefits of research in such situations. Whether or not such research should be under taken determines whether or not it is published. A helpful perspective on research and publishing as a whole, as well as the case under consideration, can be found by applying the theories and principles associated with “doing good.” In analyzing the proposed scenario, a utilitarian approach might seek to maximize the good and minimize the harm. In its most simplistic application, utilitarianism is based on the maxim that the end justi fies the means. One could argue from this perspective that the benefit accru ing to the majority of the population outweighs the harm to the individual research participants. A duty-driven or deontological approach would counter by arguing that humans can never be used as a means to an end, that their basic dignity must be valued as an end in itself. Two very different responses to the legitimacy of the research and of its subsequent publication thus result from invoking the utilitarian and the deontological positions. One must always be skeptical of research and publications that are justified on the basis of utility or expediency. Grave harm can be inflicted on minority populations and on persons unable to adequately protect their basic dignity (Elwood, 1994). Such a caveat needs to be heeded by authors and editors alike. In following the road to ethical paradise in research and in publication, then, it is helpful to remember a number of principles derived from ethical theory. Autonomy, or respect for persons, obliges the researcher in our scenario and those who oversee research to respect the dignity of those involved in the research project. Toward a Problem-Solving Approach The first step in the development of an effective problem-solving approach to ethical dilemmas in addiction publishing is to create a code of professional practice for use by research organizations and scientific journals. Such a code now exists in the form of the ethical practice guidelines developed by ISAJE (ISAJE Ethics Group, 2002). The ISAJE guidelines articulate values and define the boundaries of appropriate and inappropriate conduct in addiction research. As such, they provide a moral compass authors can use to guide ethical deci sion making. One should also note the very significant contributions of COPE in providing moral direction in our research undertakings. However, the most enlightened and practical direction might be found in the comprehensive analysis of actual situations, especially if they can be considered paradigm cases. This approach finds expression in casuistry, with its ancient roots in moral philosophy and in theology, which provides a consistent focus on individual moral behavior (Jonsen & Toulmin, 1988). It values broad consen sus, the development of maxims based on practical wisdom, and the acceptance of probable certitude as the ultimate outcome. Casuistry is attractive because it most closely resembles how we approach moral issues in day-to-day living. Brody (1990) argues that if we examine any ethical situation in research or pub lishing from every possible angle, we will be able to arrive at a consensus and, in doing so, cover all the various ethical approaches, including theory and princi ples. The case reports and ethical analysis provided by COPE, which have been previously referenced, are also an invaluable resource in this respect. Another necessary step toward ethical decision making is to learn how to apply these codes in a practical way. To this end, the main part of this chapter is devoted to the analysis of a set of case studies. These cases are presented in the form of short vignettes that describe a situation or problem, followed by an analysis of the ethical principles involved and the appropriate course of action to be taken by the author. The vignettes have a touch of humor in their presentation, intended as a relief from the doom and gloom of traditional moral analysis. We have also organ ized the incidents depicted in the vignettes according to the following topics: 1. citation bias: a selective reporting of the literature; 2. The Ethical Challenge This is guaranteed by safeguarding privacy and confidential ity and by receiving informed consent—with special attention given to assure that research participants fully understand the risks and benefits involved in the study. Likewise, the principle of nonmaleficence—that is, doing no harm to individuals, communities, and society as whole—is of the utmost importance. Conducting research in a competent and compassionate fashion is embod ied in the principle of beneficence. Although often criticized as the basis for a paternalistic approach, this principle is indispensable in addressing the needs of vulnerable individuals and vulnerable communities, as in the scenario under consideration.h The ethical principle of justice guarantees persons their due and guards against discrimination. We would invoke this principle to ensure that we do not expose the research population to undue risks for the benefit of another population. Fairness, as a guiding principle, is difficult to invoke in a society overzealous in its defense of individualism and autonomy, without equal atten tion to the common good (see Ross et al., 1993, pp. 17–28, for discussion of these principles). Finally, stewardship demands that investigators use resources responsibly and efficiently. 304 Publishing Addiction Science Toward a Problem-Solving Approach redundant publication: when two or more articles share any of the same data or text without full cross-referencing; 3. unethical authorship: all persons named as authors should have made a major contribution to a publication and be prepared to take public responsibility for its contents;l 4. undeclared conflict of interest; l 5. failure to conform to minimal standards of protection for animal or human subjects; j 6. plagiarism: unreferenced use of others’ published and unpublished ideas; andi 6. plagiarism: unreferenced use of others’ published and unpublished ideas; and 7 scientific fraud 7. scientific fraud. 7. scientific fraud. The Road to Paradise 305 The analyses provide guides to action, rather than definitive decisions, by deriving conclusions about the most appropriate course of action from sound (and, for the most part, universal) ethical principles such as auton omy, beneficence, justice, honesty, conscientious refusal, stewardship, and nonmaleficence. The analyses provide guides to action, rather than definitive decisions, by deriving conclusions about the most appropriate course of action from sound (and, for the most part, universal) ethical principles such as auton omy, beneficence, justice, honesty, conscientious refusal, stewardship, and nonmaleficence. A Synthetic Model for the Analysis of Ethical Dilemmas In their book, Critical Incidents: Ethical Issues in the Prevention and Treatment of Addiction, White and Popovits (2001) describe a synthetic model for ethical decision making that borrows from the major traditions and ethical principles described above. The goal is not to provide definitive answers to difficult ethical choices but rather to stimulate thinking about ethical complexity and to suggest options for an ethical course of action. The model involves the application of three questions: 1. Whose interests are involved, and who can be harmed? Stating this ques tion in another way, who are the potential winners and losers? In the situations described in this chapter, the main parties likely to be involved are the authors of a particular journal article, the editor of the journal, the author’s co-workers, the institution with which the author is affiliated, the professional community of addiction researchers, and society at large. By reviewing the interests and vulnerabilities of these different stakehold ers, it becomes possible to identify areas of conflicting interest, where the benefits to one party must be balanced against the harm that could be done to another party or institution.i 2. What universal or culturally specific values apply to this situation, and what course of action is suggested by these values? According to White and Pop ovits (2001), this question requires one to explore how widely held ethical values (defined in Table 15.2) can be applied to guide the best course of action in a particular situation. The identification of values that may be in conflict (e.g., honesty vs. loyalty) is an important part of this process, leading to a resolution of the conflict by choosing the higher value. White and Popovits indicate that “the higher value is often determined by the degree of good to be achieved or the degree of harm to be avoided [as] identified through the first question” (p. 27). ii 3. What standards of law, professional propriety, organizational policy, or historical practice apply to this situation? The third step in this process involves the review of established standards of professional conduct, which prescribe or proscribe certain actions for the situation in ques tion. These standards include legal mandates (e.g., copyright laws), pro fessional practice standards, human-subjects requirements, and institu tional policies. ii 3. What standards of law, professional propriety, organizational policy, or historical practice apply to this situation? Box 15.1: Checklist for analysis of critical incidents. Adapted from White and Popovits (2001). y Adapted from White and Popovits (2001). Box 15.1: Checklist for analysis of critical incidents. Case Studies In this section, we present seven case studies, each dealing with an important ethical dilemma. Following each case are a series of discussion questions that draw attention to the moral reasoning issues covered. After considering these questions, the reader should follow the outline shown in Box 15.1, which pro vides further guidance about how to resolve a particular dilemma. Then com pare your responses with the ethical analysis that follows each case, which is conducted according to the moral reasoning procedures proposed by White and Popovits (2001). A further source of case discussion and ethical analysis is found in the materials published by COPE, which provides ready access to case materials, including ethical analysis, under the following headings: authorship, conflict of interest, consent for publication, contributorship, data, editorial independence, funding/sponsorship, miscellaneous (books, social media, legal issues), misconduct/questionable behavior, mistakes, peer review, and plagia rism. In the discussion of the cases that occur in this chapter, ethical opinions from COPE are included in the ethical analysis of the cases we have chosen. In this section, we present seven case studies, each dealing with an important ethical dilemma. Following each case are a series of discussion questions that draw attention to the moral reasoning issues covered. After considering these questions, the reader should follow the outline shown in Box 15.1, which pro vides further guidance about how to resolve a particular dilemma. Then com pare your responses with the ethical analysis that follows each case, which is conducted according to the moral reasoning procedures proposed by White and Popovits (2001). A further source of case discussion and ethical analysis is found in the materials published by COPE, which provides ready access to case materials, including ethical analysis, under the following headings: authorship, conflict of interest, consent for publication, contributorship, data, editorial independence, funding/sponsorship, miscellaneous (books, social media, legal issues), misconduct/questionable behavior, mistakes, peer review, and plagia rism. In the discussion of the cases that occur in this chapter, ethical opinions from COPE are included in the ethical analysis of the cases we have chosen. A Synthetic Model for the Analysis of Ethical Dilemmas The third step in this process involves the review of established standards of professional conduct, which prescribe or proscribe certain actions for the situation in ques tion. These standards include legal mandates (e.g., copyright laws), pro fessional practice standards, human-subjects requirements, and institu tional policies. ii 3. What standards of law, professional propriety, organizational policy, or historical practice apply to this situation? The third step in this process involves the review of established standards of professional conduct, which prescribe or proscribe certain actions for the situation in ques tion. These standards include legal mandates (e.g., copyright laws), pro fessional practice standards, human-subjects requirements, and institu tional policies. 06 Publishing Addiction Science 306 Incident/situation_____________________ 1. Whose interests are involved; who can be harmed, how serious is the potential harm? Which interests, if any, are in conflict? significant moderate minimal/none Your own interests Co-workers Research participants Your institution Professional field or science Society 2. Application of universal values. Check all that apply to your case. ____ Autonomy (freedom over one’s own destiny) ____ Beneficence (do good, help others) ____ Nonmaleficence (do not hurt anyone) ____ Justice (be fair, distribute by merit) ____ Obedience (obey legal and ethically permissible directives) ____ Conscientious refusal (disobey illegal or unethical directives) ____ Gratitude (pass good along to others) ____ Competence (be knowledgeable and skilled) ____ Stewardship (use resources wisely) ____ Honesty and candor (tell the truth) ____ Fidelity (keep your promises) ____ Loyalty (do not abandon) ____ Diligence (work hard) ____ Discretion (respect confidence and privacy) ____ Self-improvement (be the best that you can be) ____ Restitution (make amends to persons injured) ____ Self-interest (protect yourself) ____ Other culture-specific values 3. What laws, standards, policies, practice guidelines, and historical practices should guide us in this situation? 1. Whose interests are involved; who can be harmed, how serious is the potential harm? Which interests, if any, are in conflict? 3. What laws, standards, policies, practice guidelines, and historical practices should guide us in this situation? The Road to Paradise 307 Discussion Questions Discussion Questions 1. What could Mr. Lective and Prof. Dorphin have done to avoid this situa tion? 1. What could Mr. Lective and Prof. Dorphin have done to avoid this situa tion? 2. Who is responsible for the selective reporting of the literature, the first author (Mr. Lective), the second author (Prof. Dorphin), or both? 2. Who is responsible for the selective reporting of the literature, the first author (Mr. Lective), the second author (Prof. Dorphin), or both? 3. Whose interests are involved, and what ethical principles apply to this case? 3. Whose interests are involved, and what ethical principles apply to this case? Case 1. Selective Reporting of the Literature Although your study does not seem to contain any fatal flaws, I have decided Publishing Addiction Science 308 not to accept the article because of the reviewer’s criticism that the background, rationale, hypotheses, and discussion are all in need of major revision, and the level of scholarship reflected in the article’s introduction suggests that the authors are either unfamiliar with recent research on the topic or are being unusually biased in their reporting of the background to their study.” not to accept the article because of the reviewer’s criticism that the background, rationale, hypotheses, and discussion are all in need of major revision, and the level of scholarship reflected in the article’s introduction suggests that the authors are either unfamiliar with recent research on the topic or are being unusually biased in their reporting of the background to their study.” Case 1. Selective Reporting of the Literature Mr. C. Lective is a graduate student in clinical psychology at Orgone University who has just finished his doctoral dissertation under the direction of his men tor, the prominent clinical psychologist Prof. Ann Dorphin. The dissertation topic was based on Prof. Dorphin’s Theory of Addiction Reflection, which proposes that drug users’ brainwaves give off an aura of escaping endogenous opiates that can be captured by perceptive therapists and recycled to form a therapeutic alliance. After several promising quasi-experimental studies and case reports of Addiction Reflection therapy, all published by Prof. Dorphin or her students, two independ ent randomized trials produced negative results. A review article was then pub lished questioning the validity of the theory as well as the unorthodox research methods used at Orgone University. Consistent with previous studies at Orgone University, Mr. Lective’s dissertation has produced positive but unimpressive results in support of the theory. Prof. Dorphin strongly suggests that the results be published and collaborates in the drafting of an article that recommends that Addiction Reflection therapy be adopted widely in routine clinical practice. The article is submitted to a small psychotherapy journal. After receiving the reviews, the editor of the journal writes the following letter to Mr. Lective:hi “I have now received two reviews of your manuscript. The first reviewer liked the article and has few recommendations for revision. The second reviewer, how ever, notes that your literature review fails to describe recent studies of Addiction Reflection therapy, including a highly critical review article, and thereby presents an inaccurate and misleading characterization of the current status of the theory. Ethical Analysis The responsibility for providing a complete account of the literature and research pertaining to Addiction Reflection therapy rests with both authors, with Prof. Dorphin shouldering most of the responsibility because of her supervisory position. Selective reporting of the literature to support a par ticular point of view is a significant ethical infraction. It clearly deviates from accepted standards of citation, as described in Chapter 10. Using the White– Popovits grid (see Box 15.3) for the analysis of critical incidents as a guide, this ethical violation has significant moral implications for the authors, their institution, the addiction field, and society as a whole. The reprimand that the authors received from the editor, together with the rejection of the manuscript and the accompanying professional embarrassment, is minor inconvenience compared with the greater harm that might have resulted from the publication of their work. Consider how their faulty research might have harmed the well- being of clients being treated by service providers who, in good faith, followed the researchers’ clinical recommendations. The authors’ actions, probably motivated by self-interest, violated the ethical principles of nonmaleficence and justice. There is a clear mandate to “do no harm” enshrined in the principle of nonmaleficence. Mr. Lective and Prof. Dorphin’s lack of honesty in espousal of self-interest has the potential to endanger the well-being of all clients and institutions involved with the new therapy. In addition, the principle of justice (fairness) becomes relevant when one consid ers the fruitless expenditure of scarce resources on a futile mode of treatment. In addition, Prof. Dorphin is clearly in a position to violate the student’s auton omy (self-determination) by bringing undue pressure on him to publish his research in a manner supportive of her original theory. This form of coercion, The Road to Paradise 309 which is clearly unethical, is often ignored in research situations, with conse quences for everyone involved when this is uncovered. Much of the harm, real and potential, involved in this situation could have been avoided by following the established standards of citation practice—that is, to present all sides of the related literature, as described in Chapter 10. COPE provides further insight into the ethical issues raised by this case in their discussion of the potential fab rication of data in primary studies included in articles for publication (http:// publicationethics.org/cases; Case number 14-01 2014). Discussion Questions 1. What should Dr. Science and her co-investigators have done with the reporting of the survey findings? 1. What should Dr. Science and her co-investigators have done with the reporting of the survey findings? i 2. What, if anything, should they have told the editor at the time they sub mitted the manuscript? i 2. What, if anything, should they have told the editor at the time they sub mitted the manuscript? 3. Whose interests are involved, and what ethical principles apply to this case? 3. Whose interests are involved, and what ethical principles apply to this case? Case 2. Redundant Publication A junior faculty member, Dr. Salame Science, is approaching tenure review at a large university that places great emphasis on the number of first-authored pub lications as the main criterion for promotion. Dr. Science, who has been working with three other investigators on a large collaborative survey study, suggests that the investigators report their findings separately for each of 16 drugs, thereby giv ing each of the investigators four first-authored publications. Dr. Science develops a template in which the literature review, methods, and statistical analyses are virtually the same for each article, with only the name of the drug being changed for the 16 articles. When one of the articles dealing with a new rave drug is sub mitted to a journal for review, the authors fail to advise the editor of the other 15 articles under review at different journals, and do not cite any of these articles in their report. Moreover, the co-authors all sign an ethical statement required by the journal indicating that the article has not been published in whole or in part by another journal and is not under consideration by another journal. Ethical Analysis As noted in Chapter 14, a place in Hell is reserved for those guilty of promot ing their own self-interest in the practice of redundant publication, in viola tion of accepted ethical norms. Dr. Science and her three collaborators find themselves in this unholy situation by submitting material that is (partially) 0 Publishing Addiction Science 310 under consideration by another journal and by using verbatim material with out quotation marks or attribution. By signing the journal’s ethical statement, they have blatantly lied about the existence of the other articles and their rela tionship to the rave drug study.h Thus, however inadvertent it initially appears, the deception involved in fail ing to disclose the relationship between the articles has serious ethical impli cations. Referencing again the White–Popovits analysis grid (see Box 15.1), several types of harm can result at professional, clinical, and societal levels. First, if all 16 articles were in fact published (as opposed to one or two com prehensive articles), the authors would deny as many as 15 competing and per haps equally worthy authors of the opportunity to publish in the same journals, because many journals have limited space and must reject a high proportion of submitted articles. Second, the task of reviewing and processing these redun dant articles creates unnecessary work for reviewers and editors, most of whom volunteer their time as a service to the peer-review system. Whether the possi ble harm rises to the level of significant in the White–Popovits grid is debatable; it is certainly moderate, in terms of harm inflicted by any standard of ethical analysis. Clearly, the authors’ actions have violated the standards of honesty, candor, fidelity, and diligence. The decision of the authors to lie in their ethical declaration attacks the basic trust that undergirds the scientific enterprise and has the capacity to inflict the type of “irreparable damage to scientific investiga tors, editors, and the community” described in Chapter 14. By following established standards for citing the interrelationships involved in their collaborative studies, and by responding honestly to the statement required by journal editors and publishers, the authors could have avoided both the ethical and legal censure resulting from their deception and dishonesty. A case report from COPE (number 06-22 2006) provides further insights into the ethical issues created by redundant publications (http://publicationonethics. org/cases/). 1. Whose interests are involved, and what ethical principles apply to this case? 1. Whose interests are involved, and what ethical principles apply to this case? 2. What should Dr. Doogood do about the suggestion to add the name of the scientific director of NARC? i 3. What should Dr. Doogood do about the suggestion to add the name of the research assistant? 3. What should Dr. Doogood do about the suggestion to add the name of the research assistant? Case 3. Authorship Credits Dr. Mary Doogood is a postdoctoral fellow at the prestigious National Addiction Research Collaborative (NARC). She is conducting research on prescription-drug addiction under the direction of her mentor, Dr. Arthur Stringalong. After a pre liminary analysis of the findings, Dr. Stringalong (who helped design the study, secure grant funding, and analyze the data) suggests that they prepare an article for submission to the Journal of Irreproducible Results.iit When Dr. Doogood finishes the first draft, Dr. Stringalong insists on two addi tions to the list of authors: (a) the scientific director of NARC, who had nothing to do with the study or the writing of the manuscript, and (b) the research assis tant who conducted the interviews, entered the data, and did a literature search but who otherwise had little involvement in the study design, data analyses, The Road to Paradise 311 interpretation of findings, or drafting of the manuscript. Dr. Stringalong tells Dr. Doogood that with the NARC director as last author, the article would have a better chance of being accepted by the Journal of Irreproducible Results. He also suggests that the research assistant, Ms. Day Tamanager, deserves to be listed as a reward for her hard work; a publication credit will help her application for admis sion to graduate school. Discussion Questions Ethical Analysis One could argue that this situation has significant ethical implications for Drs. Doogood and Stringalong on an individual basis and moderate implica tions for the scientific director and the research assistant. Dr. Stringalong vio lates Dr. Doogood’s autonomy as first author by insisting on the addition of the extra names, although he would not violate her autonomy if he merely suggested it. Dr. Stringalong’s insistence is all the more egregious because of the implica tions of the duress deriving from his position of authority. There are also issues of doing no harm and of fairness, understood as distribution of credit accord ing to merit. Ms. Tamanger, the research assistant, may have some claim to co- authorship from a fairness perspective but does not really meet the criteria for authorship described in Chapter 11 of this book. Of course, Dr. Doogood could include both in the acknowledgment section without violation of the rule of appropriate attribution-of-authorship credit. Should the names be included as co-authors, an argument could be made that the profession, the field, and soci ety could be moderately damaged. Dr. Stringalong might counter, from a utilitarian viewpoint, that using the scientific director’s name to assure the publication of the data would work toward the betterment of individuals and society and, thereby, outweigh the harm involved by including the additional author. He might likewise remind us that names are regularly added to lists of authors without being seen as a major ethical violation.h The counter-argument points to the damage, certainly moderate and possi bly significant, inflicted on the field by the violations of honesty, equity, fidelity, and loyalty involved in this practice of gift authorship. It is clearly contrary to Publishing Addiction Science 312 the practice guidelines endorsed by journal editors over the past several dec ades. In summary, the issues raised in this case involve ethical violations at the individual, institutional, and societal levels and therefore cannot be justified. Case 4. Undeclared Conflict of Interest Dr. Boyam I. Greedy was asked by the editor of the Journal of Neuropsychop harmacoepidemiology (NPPE), Dr. Tom Naïve, to submit a review article on the subject of anti-dipsotropic medications. Dr. Naïve based his invitation on Dr. Greedy’s expertise in the pharmacological treatment of craving and his widely cited articles on a new anti-craving drug called Payola. Dr. Greedy prepared the review and submitted it to the journal editor. In the article, Dr. Greedy cited both published and unpublished reports to support his contentions that: • anti-craving drugs like Payola reduce drug craving and substance abuse; • a large multi-center clinical trial of Payola is currently underway by the man ufacturer, Chemical Therapeutics, Inc.; and h • methods to deliver Payola via patch technology have been developed. Because the Journal of NPPE has no formal policy, Dr. Greedy was not asked to declare any real or apparent conflicts of interest. In addition, in the acknowledge ments section of the article, Dr. Greedy included pertinent information about the people who helped him prepare the article. But neither his communications with the editor nor the acknowledgements section revealed the following information: • Dr. Greedy holds U.S. Patent 6,375,999 on “Methods and Devices for Trans dermal Delivery of Payola.”ih • Dr. Greedy holds U.S. Patent 6,375,999 on “Methods and Devices for Trans dermal Delivery of Payola.”ih • Dr. Greedy is a member of the scientific advisory board of Chemical Thera peutics, Inc., and as such received an option to purchase 7,000 shares of stock at 5 cents per share. When the projected initial public offering of shares by Chemical Therapeutics, Inc., occurs in the near future at the corporation’s esti mated share price of $25.00 per share, Dr. Greedy’s equity will be valued at $175,000. • Dr. Greedy received substantial consulting payments from Chemical Thera peutics, including first-class airfare to numerous international meetings, where he spoke about his research on Payola. Ethical Analysis Dr. Greedy has many personal, professional, and financial interests embed ded in the promotion of Payola. His ability to influence a wider public and to advance the acceptance of the new drug is closely tied to the publication of his review article. A real conflict of interest exists and a host of ethical concerns arise at the individual, institutional, and societal levels. At the outset, it is important to establish the stakeholders—that is, those who are likely to benefit or lose from the publication of a review article that fails to acknowledge the author’s financial stake in Payola’s development. First, the author stands to profit in many ways from the publication of the review, although the extent of this benefit depends partly on the prestige of the journal and its influence on readers. Second, patients experiencing addiction stand to gain if knowledge of the efficacy of the new medication becomes widespread following the article’s publication. In his defense, Dr. Greedy might say that the promotion of the new product was the province of the advertising arm of Chemical Therapeutics, Inc., and that neither he nor the company would benefit unduly from the publication of the review article itself. He might even add that his ownership of the patent and his financial ties to the company were matters of public record and these activities are perfectly legal and ethical (even in academic circles) in his role an entrepreneur-scientist. He made his decision to publish his findings solely out of respect for the editor, Dr. Naïve. If the journal had a disclosure policy about conflict of interest, he would have had the option of either complying with it or declining the invitation to publish his data. Another important set of stakeholders in this case includes the journal itself, its editor, and the publisher. An objective bystander might question the profes sional and ethical judgment of the editor, Dr. Naïve, in inviting Dr. Greedy to submit an article without first consulting the editorial board. Here Dr. Naïve has failed in his fiduciary responsibilities to the author, the publisher, the journal, and its readers. Even if Dr. Greedy’s review were fair, balanced, and critical, deserving of the broadest possible dissemination, the integrity of both the journal and the field are nonetheless called into question by Dr. Naïve’s lack of responsibility. The absence of a conflict-of-interest disclosure policy excuses neither the editor nor the author. Discussion Questions 1. What ethical issues could arise in this convergence between Dr. Greedy’s role as a scientist writing a review article and his connections with the drug company, Chemical Therapeutics, Inc.? 1. What ethical issues could arise in this convergence between Dr. Greedy’s role as a scientist writing a review article and his connections with the drug company, Chemical Therapeutics, Inc.? The Road to Paradise 313 313 2. To what extent does Dr. Greedy stand to gain financially by gratuitously promoting his patented Payola patch?i 2. To what extent does Dr. Greedy stand to gain financially by gratuitously promoting his patented Payola patch?i 3. To what extent does Dr. Greedy stand to gain financially from the interest that his positive assessment of Payola might generate for Chemical Thera peutics, Inc., in advance of a public stock offering?l f 4. What are the real or apparent conflicts of interest in this case? l 5. What are Dr. Greedy’s ethical obligations in this case? Ethical Analysis In a like vein, neither Dr. Greedy nor 314 Publishing Addiction Science Dr. Naïve should claim that the possible good resulting from the publication of the review article outweighs the harm done. One could further argue that if this practice of nondisclosure became widely accepted, irreparable harm could result for patients, the publishing field, and society as a whole.h i This case gives us pause when we acknowledge a certain reluctance on the part of the entire scientific community—in its individual, academic, and research components—to provide full disclosure. The relationship among research, industry, and publishing outlets is a necessary one, but ethical stand ards are needed to manage conflicts between self-interest and concern for the common good. g COPE, in many of its case reviews and related publications, emphasizes the importance of addressing conflict of interest as an ongoing issue of ethical con cern in the publication of research. In a case titled “Multiple failure to declare a relevant conflict of interest” (case number 07-33 2007), it provides excellent guidance on how to deal with situations like this. Ethical Analysis In this case, it is appropriate to emphasize the vulnerability of persons with addictions in all aspects of their well-being, including treatment and research, and the intensification of such vulnerability in particular environments, such as correctional facilities. Such concerns are central to Dr. Ploit’s research, which describes the response of parolees to an innovative treatment program. Even though the way in which participants were originally assigned to the new treat ment arose out of limited resources, ethical review is very important to make sure that coercion was not a factor. These questions arise in the presence or absence of a research protocol.h In this case, it is appropriate to emphasize the vulnerability of persons with addictions in all aspects of their well-being, including treatment and research, and the intensification of such vulnerability in particular environments, such as correctional facilities. Such concerns are central to Dr. Ploit’s research, which describes the response of parolees to an innovative treatment program. Even though the way in which participants were originally assigned to the new treat ment arose out of limited resources, ethical review is very important to make sure that coercion was not a factor. These questions arise in the presence or absence of a research protocol.h The question of ethical approval, requested by the editor as a condition for accepting this piece for review, is an important one. Ethical review gives some assurance that the research itself meets basic ethical standards and also includes the expectation to provide oversight of the ongoing research in terms of partici pant well-being in a research environment. The ethical review board, if it had been involved in the discussion of this research, could have decided that the research enjoyed exempt status under the rubric of quality assurance and chart review. On the other hand, it may have required full compliance with all the requirements of a regular research protocol. In addressing a journal’s ethical concerns about compliance with ethical review committees or other supervi sory bodies, the nature of Dr. Ploit’s work changes when it becomes research. The editorial board could reasonably restrict Dr. Ploit’s research to data gath ered subsequent to approval.i Compliance with regulatory bodies generally satisfies legal requirements in research undertakings and guarantees that basic ethical standards are in place. Discussion Questions 1. Why did the editor require Dr. Ploit to submit documentation that he had met ethical review requirements for the study? 2. What is the function of institutional and editorial requirements regarding the treatment of human participants? 3. Do compliance standards in themselves assure ethical behavior in research? Case 5. Human Subjects Requirements Dr. X. Ploit, a clinical psychologist working at the Department of Parole, hears about a dataset consisting of clinical records, demographic information, and rear rest data for parolees (i.e., convicted criminals who are released to the commu nity under close supervision) who were exposed to a new substance use disorder treatment program. Because the program could not accommodate all parolees, only people being released from prison on alternate weeks were assigned to the program. The others received no treatment. When Dr. Ploit learns of this “natural experiment,” he concludes that the data could comprise a very valuable contribu tion to the literature, because the parolees were, in effect, randomly assigned to treatment and control conditions and were not pre-selected for participation in a research project. Because of his lack of ethical training, Dr. Ploit is unaware of the need to obtain ethical review board approval to access these kinds of records for research purposes, even though he has legitimate access to the same records because of his clinical responsibilities. Thus, he obtains the names of the selected paroled prisoners, looks up their remand records, and conducts a statistical analy sis. The analysis reveals that the parolees who were exposed to treatment were significantly less likely to return to prison for parole violations associated with alcohol and other drug use. Dr. Ploit writes up the results and submits them to the Journal of Drug Criminalization. When he submits the article, Dr. Ploit is asked to sign a form stating that the study had received all necessary human subjects approvals by an ethical review board. Although Dr. Ploit feels conflicted about signing the statement, he decides to lie about his failure to seek ethical approval, reasoning that (a) the results do not identify individual prisoners and (b) the ethical review board would probably have given him permission to access the data anyway. Dr. Ploit also hesitates to The Road to Paradise 315 seek post hoc permission from the ethical review board at this point, because they might now deny permission. He reasons that the value of the findings for society and the prisoners far outweighs his minor ethical transgression. Ethical Analysis The regulatory research bodies share with journal editors a concern for the promotion of good and the avoidance of harm at the individual, insti tutional, and societal levels. The author has a fiduciary relationship with the 316 Publishing Addiction Science 316 ethical review board and with the editor, and all parties are mutually depend ent on each other acting in good faith and in compliance with a commonly accepted ethical framework that promotes the common good. Compliance standards in and of themselves guarantee minimum protection for stakehold ers in research undertakings; ethical standards often espouse a higher degree of care.h The ethical dimensions involved with the protection of human subjects have societal, institutional, and individual implications. This has been discussed in the ethical analysis of this case, and further insight into this analysis is provided by the COPE publication on inadequate assurance of human research ethics for questionnaires, case number 12-33 2012 (http://publicationonethics.org/ cases/, retrieved June 3, 2015). Discussion Questions 1. How could the students have avoided the reprimand of the journal editor and the possible censure of their chair and university? p y 2. What harm, real or potential, could result from the students’ action? 3. Could the students claim that they were unfamiliar with the ethical rules of publishing? If they were unfamiliar, whose obligation was it to inform them? 3. Could the students claim that they were unfamiliar with the ethical rules of publishing? If they were unfamiliar, whose obligation was it to inform them? Case 6. Plagiarism Wilhelm Reicht and Ena G. Orgone are new doctoral students working on a project at the University of Freudberg that explores the impact of the therapist– patient relationship in psychoanalytic treatment for female abusers of prescribed psychotropics. Reading the background literature, they find a very good article by Professor Eve N. Id in one of the big U.S.-based psychoanalytic journals. In the article, Dr. Id explores how the angle of the analyst’s sofa can influence the level of subconsciousness that the patient is able to reach in therapy. The article estab lishes the so-called Divanaltitude theory.h The two ambitious students decide to submit an article to the Bayerische Zeitschrift für Psychoanalytische Alkoholstudien to demonstrate that they are on the cutting edge of current research. Their article, written in German, pre sents the Divanaltitude theory along with some findings from a small, local survey that the students conducted to learn what alcohol and other drug therapists think about the design of sofas in therapeutic settings. Reicht and Orgone inform the editor that they consider their text to be an overview and not a piece of original research. The editor, who is not familiar with the Divanaltitude theory, sends the text to a referee. The referee’s critique comes back after two weeks. She has discovered that the introduction is a direct translation of Professor Id’s abstract. Several subtitles and the structure of the first part of the article are identical to Dr. Id’s. That the authors have one reference to Dr. Id’s article in the second paragraph of the text is obviously not enough; the referee considers this to be a case of plagiarism.h The editor subsequently sends a letter to the young authors stating that he can not accept the article for publication because large sections of the text are identical to an already published article. He states that their submission breaches interna tionally accepted ethical rules of publishing and demands an explanation. The editor also informs the authors that he will send a copy of his letter to the head of their department at Freudberg. The Road to Paradise 317 1. Was it ethical for Dr. Stein to use the one-tailed test? 2. How should Stein respond to the editor? 1. Was it ethical for Dr. Stein to use the one-tailed test? 2. How should Stein respond to the editor? 2. How should Stein respond to the editor? Ethical Analysis The students’ plagiarism has important implications, with the possibility of harm for the students themselves, the original author, the research institution, the addiction field, and for society as a whole. The students, according to the White–Popovits grid, exposed themselves to the risk of possible dismissal from their doctoral program as punishment for their violation of accepted ethical norms. It is conceivable, however, that they acted out of ignorance and that they had not received appropriate ethics training from their professors or their institution. Had the individual professors and the institution been remiss in providing appropriate direction for the students, then the institution and its representatives would be as culpable as the students.ht p p The actions of the students obviously involved a form of theft where Dr. Id’s work is concerned, but any damage to her reputation will be moderate or mini mal according to the White–Popovits scale. Their transgressions also present the possibility of injuring the professional field and society as a whole, especially if such actions were to become commonplace in the publishing field. Accord ing to the White–Popovits scheme of universal values, the students violated the values of justice, honesty, and diligence in their failure to acknowledge the work of the original author. They acted out of self-interest, with lack of regard for established ethical and professional guidelines. They might be accused of violating the original researcher’s autonomy by denying her the opportunity to control her own work through appropriate citations. If the students failed to receive appropriate ethical formation and direction from their institute, then the administrators and professors at the institute would be in violation of the principles of beneficence and nonmaleficence. Institutions have a moral responsibility to provide an environment in which integrity and honesty are an essential part of their research undertaking (Institute of Medicine, 2002). Stewardship also enters into the equation because, from a societal perspective, institutions have a social responsibility to use resources wisely.h The need to address plagiarism in its many forms, including self-plagiarism, is central in maintaining the integrity of research publication, with ongoing attention to the ethical dimensions addressed in the analysis of this case. In examining a report of possible self-plagiarism, COPE case number 14-10 2014 8 Publishing Addiction Science 318 provides further insight into this important issue (http://publicationonethics. org/cases/, retrieved on June 3, 2015). Case 7: Scientific Fraud—“Data Trimming” Dr. Frank N. Stein is a junior faculty member in the Department of Anatomical Protuberances at a large Transylvanian medical school. His latest research pro ject deals with the effects of brain transplants on addiction careers. Preliminary analysis of the data on the first 10 transplants shows an interesting trend, but the p value is just shy of statistical significance. Dr. Stein’s statistician, Mr. Igor Num bers, suggests they conduct a few more transplants to increase statistical power and then add an equal number of cases to the control group (without the ben efit of random assignment). Igor also suggests they conduct a one-tailed test to get a more favorable alpha level and drop some of the covariates to increase the degrees of freedom. After Dr. Stein and Mr. Numbers have made all these protocol changes, they submit their article for publication as a true random assignment study with significant differences between groups. One of the reviewers questions the use of a one-tailed test, suggesting that the authors include more covariates in their analyses and asks the editor to obtain more detailed information from the authors (Dr. Stein and Mr. Numbers) about the way they assembled their samples. Dr. Stein’s institution has granted appropriate approval for the research. In addition, the research enjoys societal approval through funding that provides appropriate resources for good scientific work. Ethical Analysis The stakeholders are the recipients, the scientists, the medical school, and society as a whole. The good espoused by Dr. Frank N. Stein’s research is the enhancement of the addiction field through the advancement of knowl edge about the effects of brain transplants. Whether to continue this research depends on outcome studies, largely dependent on the findings of Dr. Stein and Mr. Numbers, who are convinced that the changes in their statistical analy sis are minor and ethical. They feel that the continuation of their work will confer immense benefits on all involved and especially people with addictive disorders. Their decision to use the new statistical analyses, together with their justification of this approach in their response to the review process, shows The Road to Paradise 319 they believe the end justifies the means. After all, this is a new cutting-edge enquiry where data trimming on a minor scale may be considered no more than a minor peccadillo.h The researchers, despite their idealism and good intentions, are blind to the implications of honesty, stewardship, and fairness in their decisions. Their dis honesty impinges on the well-being and safety of the recipients of brain trans plants. In addition, they are not good stewards of the funds that supported this research. Furthermore, their unethical use of funds constitutes disservice to the other, unfunded scientists whose requests for funding are based on honest and responsible findings. i Our tongue-in-cheek response to this fanciful scenario uncovers many ethi cal pitfalls resulting from what might appear prima facie as minor adjustments in one’s statistical approach. Rigorous honesty must inform the research itself, and authors must be candid with editors about methods and outcomes. The relationship between the two parties is a fiduciary one, and the engendered trust touches the basic integrity of scientific publishing. Using the White–Pop ovits grid, one could award this case a perfect score of “significant” on all the interests and vulnerability items.i Fraud, as we have identified in the ethical analysis of this scenario, is the most egregious violation of professional integrity in research undertakings. COPE, in its analysis of case number 14-05 2014, again provides excellent insights into the implications of fraud in research situations (http://publicationethics. org/case/fraud-or-sloppiness-submitted-manuscript). Distinguishing between fraud and sloppiness is difficult to determine, and this case analysis is helpful in this respect. Conclusion The intent of this chapter was to illustrate an ethical framework that provides practical guidance for investigators in publishing responsible and trustworthy research. Central to this understanding is a high degree of trust, as demon strated in the case analyses. A fiduciary relationship is at the heart of the assur ance whereby researchers address the well-being of individuals, institutions, and the overall common good.t In a climate of self-interest, often nurtured by a high regard for an exaggerated form of individualism (which is inimical to the common good), it is difficult to develop a consistent appreciation of the place of trust in research undertakings, as is the case elsewhere in society (Institute of Medicine, 2002). Societal safe guards need to be in place, as envisaged by ethical review committees and other regulatory agencies, to ensure that the trust that individuals, institutions, and society afford to research is well placed and respected. Research communities and regulatory agencies need to establish the highest level of collaboration as a first step in creating and maintaining a climate of trust. 320 Publishing Addiction Science Regulatory agencies in and of themselves cannot ensure ethical behavior in research or publishing, both of which have trust as their foundation. Other forces are in play, such as virtue or character considerations. Individuals, insti tutions, and publishing enterprises should ideally encompass qualities such as integrity, fairness, and trust in their undertakings evaluating the presence or absence of virtue in larger bodies is not easy. It is difficult to determine if an institution is virtuous based on an analysis of the goodness of the institution where the research occurs. Other forces are equally important, such as virtue or character considerations involving individuals and institutions in the research and publishing enterprises. Inserting virtue ethics by encompassing qualities such as integrity, fairness, and trust is not an easy task. Equally difficult is the infusion of like qualities into the culture of institutions where research occurs. Many centuries ago, in his dialogue with Socrates, Plato wrestled with this problem as recounted in his work, Meno: “Can you tell me Socrates, is virtue something that can be taught? Or does it come by practice? Or is it neither teaching nor practice that gives it to a man, but natural aptitude or something else?” (translation by Thompson, 1980). Conclusion In fashioning a character-based ethic to guide the behavior of researchers and authors, traditional wisdom might prompt one to respond “all of the above” in answer to Plato’s questions.h The “something else” to which Plato alludes is intriguing and invites com ment as a concluding thought for this chapter. Perhaps Plato was hinting, for our present-day edification, that the fullest ethical analysis of persisting con temporary issues in research and publication, along the lines of the case stud ies in this chapter, is that “something else.” Ongoing conversation about actual issues is the best assurance that an ethical climate will inform research ethics and promote responsible publishing behavior. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. References Alighieri, D. (1947). The Divine Comedy (translated by Laurence Binyon in The Portable Dante, The Viking Portable Library #32). New York, NY: Viking Press. Brody, H. (1990). Applied ethics: Don’t change the subject. In B. Hoffmas ter, B. Freedman, and F. Raser (Eds.), Clinical ethics: Theory and practice (pp. 183–200). Clifton, NJ: Human Press. The Road to Paradise 321 Casswell, S. (2013). Why do we not see the corporate interests of the alcohol industry as clearly as we see those of the tobacco industry? Addiction, 108, 680–685. DOI: https://doi.org/10.1111/add.12011h Elwood, W. (1994). Rhetoric in the War on Drugs: The triumph and tragedy of public relations. Westport, CT: Praeger. Fisher, C. B. (2011). Addiction research ethics and the Belmont Principles: Do drug users have a different moral voice? Substance Use & Misuse, 46, 728–741. DOI: https://doi.org/10.3109/10826084.2010.528125 g Farmington Consensus. (1997). Addiction, 92, 1617–1618. DOI: https://doi. org/10.1080/09652149736332h Glaser, J. W. (1994). Three realms of ethics: Individual, institutional, societal. Kansas City, MO: Sheed and Ward.i Institute of Medicine. (2002). Integrity in scientific research: Creating an envi ronment that promotes responsible conduct. Washington, DC: National Academy of Sciences Press. ISAJE Ethics Group. (2002). Ethical practice guidelines in addiction publishing: A model for authors, journal editors and other partners. London, England: International Society of Addiction Journal Editors. Retrieved from: www. isaje.net. Jonsen, A. R., & Toulmin, S. (1988). The abuse of casuistry: A history of moral reasoning. Berkeley, CA: University of California Press.h Kass, L. R. (2002). Life, liberty and the defense of dignity: The challenge of bioeth ics. San Francisco, CA: Encounter Books. Lynch, W. J., Nicholson, K. L., Dance, M. E., Morgan, R. W., & Foley, P. L. (2010). Animal models of substance abuse and addiction: Implications for science, animal welfare, and society. Comparative Medicine, 60, 177–188.l McGovern, T. F. (1998). Vulnerability: Reflection on its ethical implications for the protection of participants in SAMSHA programs. Ethics and Behaviour, 8, 293–304. Miller, P. G., de Groot, F., McKenzie, S., & Droste, N. (2011). Vested interests in addiction research and policy. Alcohol industry use of social aspect pub lic relations organizations against preventative health measures. Addiction, 106, 1560–1567. Ross, J. W., Glaser, J. W., Rasinski-Gregory, D., McIver Gibson, J., & Bayley, C. (1993). Health care ethics committees: The next generation. Chicago, IL: America Hospital Publishing.hh Thompson, E. S. (Ed.). (1980). The Meno of Plato. New York, NY: Garland Publishing. White, W. L., & Popovits, R. How to cite this book chapter: Miller, P, Babor, T F, McGovern, T, Obot, I and Bühringer, G. 2017. Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies, and Other Funding Agencies: Holy Grail or Poisoned Chalice? In: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P. (eds.) Publishing Addiction Science: A Guide for the Perplexed, Pp. 323–352. London: Ubiquity Press. DOI: https://doi. org/10.5334/bbd.p. License: CC-BY 4.0. References M. (2001). Critical incidents: Ethical issues in the prevention and treatment of addiction (2nd ed.). Bloomington, IL: Light house Institute. CHAPTER 16 Relationships with the Alcoholic- Beverage Industry, Pharmaceutical Companies, and Other Funding Agencies: Holy Grail or Poisoned Chalice? Peter Miller, Thomas F. Babor, Thomas McGovern, Isidore Obot and Gerhard Bühringer Peter Miller, Thomas F. Babor, Thomas McGovern, Isidore Obot and Gerhard Bühringer Introduction The ethical dimensions of the relationships among researchers, research organ izations, journal editors, and the various industries that profit from addictive substances and behaviors are complicated and extensive. They embrace the indi vidual, institutional, and societal dimensions of ethical reflection. In a way, this chapter is a case study on a grand scale that calls for profound ethical analysis. The forces and interests involved are of necessity interwoven, and researchers are dependent on many funding sources as a mainstay for their research. These will be covered in detail as the chapter unfolds. At the heart of the ethical con versation is an issue of trust for individuals and institutions. Ultimately, there are no simple guidelines to help an investigator decide which funding sources to accept or reject. However, it is vital that researchers go through an ethical assessment to consider the issues involved. In this chapter, we will explore the ways in which different interest groups have influenced the research process before demonstrating the use of the PERIL (purpose, extent, relevant harm, identifiers, link) analysis (Adams, 2007), an ethical decision-making framework 4 Publishing Addiction Science 324 developed specifically to address ethical decision-making. We will extend this previous work to challenge even this framework by asking whether it is simply enough just to question the intentions of vested interests in their funding of research. We will close by stressing the importance of understanding corporate political activity in the context of how vested interests are capable of undermin ing evidence-based policy at local, state, national, and international levels. A high proportion of an active researcher’s workload is spent applying for grant income. Successful receipt of grant monies is seen as an independ ent measure of a scientist’s worth to the field. But the successful awarding of research money can occasionally be a “poisoned chalice” because of the prob lems engendered by an association with a funding agency. Such problems include having commercial or other vested interests set the research agenda, determine the way in which research is conducted, or define when and where research is published. Contracts that might seem reasonable when the cash is being waved under one’s nose may prevent entire studies from being pub lished or, even worse, result in selective publication that does not portray the actual findings accurately. These types of experiences can devastate individual researchers, both personally and professionally. Introduction From the outset, we want to emphasize that individual researchers cannot deal with these issues alone but need support from senior colleagues, their institutions, professional associa tions, and academic journals. A Growing Concern In a climate of self-interest, often nurtured by a high regard for an exag gerated form of individualism (which is inimical to the common good), it is difficult to develop a consistent appreciation of the place of trust in research undertakings, as is the case elsewhere in society. (McGovern et al., Chapter 15). (McGovern et al., Chapter 15). Concerns about the integrity of the evidence base of addiction science have been raised in a number of forums recently (e.g., Adams, 2007; Babor & Robaina, 2013; Hall, 2006a; Miller, 2013; Miller et al., 2006; Stenius & Babor, 2010). Many of the authors expressing these concerns have reminded us that, although safe guards such as ethical review committees and other regulatory agencies are in place, ensuring the integrity of the evidence is an ongoing task that requires an awareness of new players (e.g., energy-drink producers) seeking to influ ence the evidence base, as well as awareness of new technologies for doing so (Hall, 2006a), such as paid contributions to edited books that look scholarly but often have a hidden political agenda. On the other hand, there have been strong developments in the study of such industries and the way in which they use research to muddy the waters of evidence and influence the political process (Hawkins & Holden, 2014; Savell et al., 2014). This will be discussed later in the chapter in regard to assessing the purpose of industry-funded research. Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies 325 Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies 325 Miller et al. (2006) highlighted the influence that major funding bodies (e.g., pharmaceutical companies and governmental departments) can have on research findings and the information-dissemination process. This was con sidered important from two angles: (a) keeping true to the ideal of science and (b) adhering to the ethical principle of beneficence (Chapter 15). Maintaining the ideal of science was seen as essential for the field, in terms not only of sus taining public trust (as mentioned above) but also of ensuring that the field moves toward the most-effective interventions available. A Growing Concern Adhering to the ideal of beneficence (the obligation to maximize possible benefits and minimize possible harms) was viewed as equally important when considering whether research (which may be censored, be partially reported, or go unpublished) could truly be said to be in the best interests of the research participants.h The debate within academic journals and subsequent commentaries has added substantially to our knowledge of how funding bodies influence research both directly and indirectly (Adams, 2007; Ashcroft, 2006; Babor, 2006; Babor & Miller, 2014; Hall, 2006a, 2006b; Hough & Turnbull, 2006; Khoshnood, 2006; Lenton & Midford, 2006). The observations collected from various authori ties and presented in Box 16.1 highlight some of the main issues and point to “Because . . . research may adversely affect the reputations of govern ments and government departments, ‘project management’ has become an increasingly central part of contractual arrangements between researchers and funders” (Hall, 2006b, p. 240). “[I]n the current funding climate, universities and research centres have incentives not to adhere rigorously to these norms” (Ashcroft, 2006, p. 238). “In recent years almost all [Australian] state and federal funded drug edu cation research has been commissioned according to funder specifications, rather than being investigator driven” (Lenton & Midford, 2006, p. 244). “Certainly, too, government departments set research agendas—and specify research methodologies to suit their own interests, rather than to contribute in a disinterested way to the body of knowledge that relates to policy issues. Government departments do not intentionally commission research that will embarrass their ministers” (Hough & Turnbull, 2006, p. 242). “Senior academic researchers should be prepared to ‘out’ funding bodies for bad behaviour. Researchers with seniority and the protection afforded by tenure should be prepared to protect junior researchers and advocate for an unencumbered right to publish research results” (Hall, 2006b, p. 240). Box 16.1: Observations about research funding from different commentators. “Because . . . research may adversely affect the reputations of govern ments and government departments, ‘project management’ has become an increasingly central part of contractual arrangements between researchers and funders” (Hall, 2006b, p. 240). “[I]n the current funding climate, universities and research centres have incentives not to adhere rigorously to these norms” (Ashcroft, 2006, p. 238). “In recent years almost all [Australian] state and federal funded drug edu cation research has been commissioned according to funder specifications, rather than being investigator driven” (Lenton & Midford, 2006, p. 244). Types of Adverse Influence Miller et al. (2006) identified five major avenues through which funding bod ies can regulate research in an adverse way: (a) direct censorship (where mate rial is edited or dissemination is interfered with), (b) limiting access to data (either affecting some point or to be used as coercion for favorable interpre tation), (c) ongoing funding insecurity (attaching conditions to subsequent funding if previous findings have been awkward or unwelcome), (d) using under-qualified or easily-influenced researchers (which allows funders to con trol the quality of investigation being carried out, even before the research has commenced), and (e) setting research agendas or dilution (whereby decisions are based on the political, financial, or ideological interests of the funder). For example, pharmaceutical companies overemphasize studies that examine the efficacy of pharmacotherapeutic solutions to drug-related problems, which could make the evidence base appear to be overly favorable for such an inter vention (Wagner & Steinzor, 2007). Other authors (e.g., Gruning et al., 2006; Kassirer, 2005) have provided similar, although slightly different, descriptions of the ways in which interest groups have influenced health policy and scien tific research (Box 16.2). A Growing Concern “Certainly, too, government departments set research agendas—and specify research methodologies to suit their own interests, rather than to contribute in a disinterested way to the body of knowledge that relates to policy issues. Government departments do not intentionally commission research that will embarrass their ministers” (Hough & Turnbull, 2006, p. 242). “Senior academic researchers should be prepared to ‘out’ funding bodies for bad behaviour. Researchers with seniority and the protection afforded by tenure should be prepared to protect junior researchers and advocate for an unencumbered right to publish research results” (Hall, 2006b, p. 240). Box 16.1: Observations about research funding from different commentators. Publishing Addiction Science 326 the fact that influences on the research process go far beyond industry-related funding bodies alone. The Tobacco Industry The best known example of the way a funding body can act to undermine research integrity and muddy the waters surrounding a topic of public health interest is the concerted campaign by the tobacco industry first to deny the links between smoking and lung cancer and then more recently to support pro grams that attribute responsibility to the individual smoker rather than to the tobacco companies. Investigations into tobacco companies continue to identify new ways in which the industry seeks to encourage smoking and at the same time divest itself of responsibility for the subsequent health costs (Drope et al., 2004; Iida & Proctor, 2004; King, 2006; Muggli et al., 2004; Ong & Glantz, 2000). There are numerous examples of how tobacco companies have acted to undermine or adulterate health initiatives. The tobacco industry has been found to influence research using every one of the techniques discussed earlier (e.g., Hirshhorn et al., 2001; King, 2006). According to one authority, “perhaps research grants coming from tobacco companies should carry their own Surgeon General’s warning. Caution: Tobacco industry sponsorship may be hazardous to the pub lic’s health” (Parascandola, 2005, p. 549). Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies 327 Gruning and colleagues (2006) identified five ways in which the tobacco industry in Germany distorted science: Gruning and colleagues (2006) identified five ways in which the tobacco industry in Germany distorted science: • Suppression, through actions such as closing the German Indus try Research Institute (which it funded) when its head published results unfavorable to the industry and having subsequent scientists in its employment guarantee that unfavorable results would not be published; • Dilution, through selective funding of research and the recruit ment of scientists who had doubts about the adverse health effects of smoking or whose previous work had found no links, as well as funding research projects designed to find no association between smoking and disease (e.g., Wander & Malone, 2006); • Distraction, by selecting and supporting a large number of “con founder studies,” which are research projects aimed to distract attention from smoking by investigating other potential causes of smoking-related diseases; • Concealment, using third-party scientists whose connection to the industry was hidden to increase the credibility and impact of the studies published; and • Manipulation, the vetting of articles and presentations by the indus try before publication or presentation. Box 16.2: The tobacco industry in Germany. Source: Gruning et al. (2006). Box 16.2: The tobacco industry in Germany. Source: Gruning et al. (2006). Box 16.2: The tobacco industry in Germany. Source: Gruning et al. (2006). Rubin’s deposition in 2000 during a case brought by a health insurance company against Philip Morris for the costs connected to tobacco smoking. The line of questioning begins with questions of financial payments received by Dr. Rubin. It then continues to explore Dr. Rubin’s opinions about the qualifications of members of the Scientific Advisory Board who were senior executives of RJR Tobacco Company, and the practice of having grant applications screened initially by industry lawyers before they were submitted for scientific review. Given the outcome of the trial, Dr. Rubin’s testimony provides a good example of how financial COIs may influence the opinions of scientists who serve as expert witnesses. Box 16.3 provides excerpts taken from Dr. Rubin’s deposition in 2000 during a case brought by a health insurance company against Philip Morris for the costs connected to tobacco smoking. The line of questioning begins with questions of financial payments received by Dr. Rubin. It then continues to explore Dr. Rubin’s opinions about the qualifications of members of the Scientific Advisory Board who were senior executives of RJR Tobacco Company, and the practice of having grant applications screened initially by industry lawyers before they were submitted for scientific review. Given the outcome of the trial, Dr. Rubin’s testimony provides a good example of how financial COIs may influence the opinions of scientists who serve as expert witnesses. Box 16.2: The tobacco industry in Germany. Source: Gruning et al. (2006). One example of this is the tobacco industry’s support of scientific research and their use of academics as expert witnesses in court cases. As many senior researchers in the addiction field are occasionally asked to serve as expert wit nesses for a defendant or a plaintiff, it is instructive to examine cases where such testimony could have implications for public health, especially when it proves to be wrong. Can direct payment of a scientist bias that person’s opin ions and even sworn testimony in a court case? Until 1998, most of the tobacco industry funding for research on nicotine and tobacco came through Council for Tobacco Research (CTR) and the Center for Indoor Air Research (CIAR). These two organizations were established and maintained by funding from the tobacco industry. They played a central role in the lawsuits brought against the tobacco industry in the 1990’s, when it was found that industry-funded research contradicted the conclusions of inde pendent scientists (Shick and Glantz, 2007). A US judge presiding over two state cases described CTR as “nothing but a hoax created for public relations Publishing Addiction Science 328 purposes with no intention of seeking the truth or publishing it.” (Janson, 1988). The Master Settlement Agreement (MSA) in 1998 dissolved the CTR and CIAR, as they were implicated in a conspiracy of massive fraud. Tobacco companies also agreed to pay $206 billion over the first twenty-five years of the agreement to compensate the States for taxpayer money spent for health-care costs connected to tobacco-related illness. In a series of court cases and depositions, then Professor Emmanuel Rubin testified that the research conducted by the CTR was of high scientific quality and that its scientific review adhered to widely recognized scientific standards. For example, in 2000 testimony for Philip Morris Inc. (p. 29) he stated: “In my opinion the Council for Tobacco Research was an affective (sic), efficient, generous and thoroughly honest organization that provided funds for excellent biomedical research. It acted in an independent fashion that was no different from other agencies that provided grants. I think that the research that was funded by CTR contributed signifi cantly to understanding the issues of tobacco and health. And, for that reason, I have no objections to funding by the CTR.” Box 16.3 provides excerpts taken from Dr. Source: Blue Cross and Blue Shield of New Jersey, et al., Plaintiffs, vs. Philip Morris, Incorporated, et al., Defendants. Case no. 98 CIV 3287 (JBW) Video taped deposition of Emanuel Rubin, M.D., April 12, 2000, Bates Number: 522994762-522994916. pp 47; 110-111. Available at http://industrydocuments. library.ucsf.edu/tobacco/docs/yqnk008347. Box 16.3: Excerpts from Dr. Emmanuel Rubin’s Testimony in Blue Cross and Blue Shield of New Jersey vs. Philip Morris, Inc.f The Alcohol Industry Using terms of justification such as “corporate social responsibility” and “part nerships with the public health community,” the alcoholic-beverage industry (mainly large producers, trade associations, and “social-aspects” organizations) funds a variety of “scientific” activities that involve or overlap with the work of independent scientists using techniques that range from efforts to influence public perceptions of research to the direct commissioning of research that is consistent with their public-relations priorities (Babor & Robaina, 2013).h There are at least three organizations funded predominantly by alcohol- industry sources for the primary purpose of conducting scientific research on alcohol: the European Research Advisory Board, the ABMRF/The Foun dation for Alcohol Research, and the Institut de Recherches Scientifiques sur Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies 329 329 Q. It appears to me that you’ve given deposition testimony in six smok ing and health litigations and have given trial testimony in one. . . . . . Can you give me an estimate on how much money you have been com pensated for performing as an expert witness in the various tobacco and health litigations in which you have done so? Q. It appears to me that you’ve given deposition testimony in six smok ing and health litigations and have given trial testimony in one. . . . . . Can you give me an estimate on how much money you have been com pensated for performing as an expert witness in the various tobacco and health litigations in which you have done so? A. I haven’t kept records and that is for, you know, all of this time. I’m not in business, but I’d estimate all of those things, $500,000, $600,000. Q. Over a six year period? A. Yes. A. Yes. Q. Dr. Rubin, you just testified that it would not be proper for the presi dent of CTR to send grant applications to CTR’s lawyers for legal review solely on the basis of the fact that the research-called for could implicate cigarette smoking as a cause of human disease, correct? If you were shown evidence that that, in fact, did happen, would that change any of the expert opinions that you’ve expressed in your expert report? A. Well, I’d like to know the circumstances… Q. Did your opinions change if you were shown evidence to indicate that this was a continuing, regular practice, at CTR? A. Box 16.3: Excerpts from Dr. Emmanuel Rubin’s Testimony in Blue Cross and Blue Shield of New Jersey vs. Philip Morris, Inc. Source: Blue Cross and Blue Shield of New Jersey, et al., Plaintiffs, vs. Philip Morris, Incorporated, et al., Defendants. Case no. 98 CIV 3287 (JBW) Video taped deposition of Emanuel Rubin, M.D., April 12, 2000, Bates Number: 522994762-522994916. pp 47; 110-111. Available at http://industrydocuments. library.ucsf.edu/tobacco/docs/yqnk008347. The Alcohol Industry You would have to show me the evidence. A. You would have to show me the evidence. A. You would have to show me the evidence. *Q. refers to questions asked by attorneys for Blue Cross and Blue Shield of New Jersey (Plaintiffs). A. refers to answers provided by Dr. Rubin, expert witness for Philip Morris, Inc. Publishing Addiction Science 330 les Boissons. Although some consider the operations of these organizations as a model of the way industry should contribute to alcohol science, questions have been raised about the way they operate and their influence on the scien tific process (Babor & Robaina, 2013). For example, the Institut de Recherches Scientifiques sur les Boissons commissions its own studies in addition to funding investigator-initiated projects, thereby increasing the possibility that industry-favorable topics are promoted. It has also been suggested that a scien tist’s objectivity might be compromised by receipt of the honoraria and travel funds involved, as well as through the opportunities to fraternize with industry executives at international meetings. Each of these organizations also funds research on industry-favorable topics such as the health benefits of moderate drinking, which then are used as a part of the marketing strategies by the wine and beer industries or as reasons why regulation and taxation should not be imposed on the alcohol industry (Stenius & Babor, 2010). In addition to indirect support of research through third-party organiza tions, there have been several instances in which individual alcohol produc ers or industry-supported social-aspects/public-relations organizations provide direct support to university-based scientists engaged in alcohol research. The most-notable examples include the Ernest Gallo Clinic and Research Center established by the Gallo Winery at the University of California to study basic neuroscience and the effects of alcohol on the brain; Anheuser-Busch’s support of social norms research at seven U.S. universities; and a research center on youth binge drinking funded by Diageo Ireland, part of Diageo PLC, the world’s largest producer and distributor of alcohol (Babor, 2006; Babor et al., 1996). Little is known about the internal marketing research conducted by the alco hol industry and contract research organizations because the information is not shared with the public, the scientific community, or public health profes sionals. The Alcohol Industry In the case of tobacco, previously secret internal industry documents have revealed that independent analysis of research on sensory perception was used to inform product design for targeted segments of the cigarette market, including young adults (e.g., Carpenter et al., 2005), and there is evidence that the alcohol industry does similar research (Babor, 2009). Contract research requires the services of social and behavioral scientists; therefore, it may pose ethical problems to the extent that such research could facilitate the marketing of products (e.g., alcopops) that are misused by vulnerable populations.h These kinds of funding initiatives not only have the potential for competing interests, but they may also affect the objectivity of independent scientists and the integrity of science. At best, the scientific activities supported by the alcohol industry provide financial support and small consulting fees for basic and behav ioral scientists engaged in alcohol research. At worst, they confuse public discus sion of health issues and policy options, raise questions about the objectivity of industry-supported alcohol scientists, and provide industry with a convenient way to demonstrate “corporate responsibility” in its attempts to avoid taxation and regulation (see Box 16.4 for further examples of industry activities). Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies 33 331 ICAP is an industry-funded, social-aspects/public-relations organiza tion located in Washington, D.C., USA. It was founded in 1995 by a consortium of alcohol companies, including MillerCoors, which at that time was part of tobacco giant Phillip Morris. According to an article on the early history of ICAP (Jernigan, 2012), MillerCoors’s primary interests in the creation of ICAP were purely commercial, that is, to aid their planned international expansion by managing worldwide issues and thereby assisting their sales and marketing group in an increasingly competitive marketplace. ICAP is an industry-funded, social-aspects/public-relations organiza tion located in Washington, D.C., USA. It was founded in 1995 by a consortium of alcohol companies, including MillerCoors, which at that time was part of tobacco giant Phillip Morris. According to an article on the early history of ICAP (Jernigan, 2012), MillerCoors’s primary interests in the creation of ICAP were purely commercial, that is, to aid their planned international expansion by managing worldwide issues and thereby assisting their sales and marketing group in an increasingly competitive marketplace. Box 16.4: The research pedigree of the International Center for Alcohol Policies (ICAP), now called the International Alliance for Responsible Drinking (IARD). The Alcohol Industry Despite ICAP’s original mission to promote understanding of the role of alcohol in society and help reduce the abuse of alcohol worldwide, there is strong evidence that ICAP has evolved primarily into an industry public- relations organization dedicated to the advancement of industry-favora ble alcohol policies (Anderson & Rutherford, 2002; Babor & Robaina, 2013; Bakke & Endal, 2010; Foxcroft, 2005; Jernigan, 2012; McCreanor et al., 2000; Room, 2005). For example, ICAP sponsored conferences and governmental consultations in a number of African countries in which industry-invited representatives helped governmental officials draft national policy plans for their countries. In one analysis of this initia tive (Bakke & Endal, 2010), the national plans—ostensibly designed to fit the specific needs of four different African countries—were found to be virtually identical, with all documents originating from the MS Word document of a senior executive of SABMiller, one of the ICAP’s funders. There is also evidence that ICAP-supported research is of poor quality and is biased in favor of industry positions supporting alcohol educa tion over more-effective alcohol policies (Babor & Xuan, 2004). ICAP also pays scientists to edit and write chapters for commissioned books that have been criticized for their bias toward industry-favorable posi tions on alcohol policy (Caetano, 2008; Stimson, et al., 2006). Any pretense of ICAP’s objectivity and independence was abandoned in 2014 with their announced merger with the Global Alcohol Pro ducers Group, a major industry lobby organization. With this merger, ICAP was renamed the International Alliance for Responsible Drink ing (IARD). Since its inception in 2005, the Global Alcohol Producers Group has spent more than USD$1.15 million on lobbying the World Health Organization (OpenSecrets.org, 2015), taking positions that seem to be diametrically opposed to those recommended by the inter national public health community. Box 16.4: The research pedigree of the International Center for Alcohol Policies (ICAP), now called the International Alliance for Responsible Drinking (IARD). 332 Publishing Addiction Science The Pharmaceutical Industry The pharmaceutical industry has become more interested in the discovery and evaluation of medications that can be used for the treatment of addiction, including opiate-substitution therapies and nicotine-replacement therapies. As such, pharmaceutical companies represent a different type of research funder from those, such as the tobacco industry, who sell dangerous consumables. The pharmaceutical industry commissions and funds legitimate research that has genuine benefit for the treatment of substance-related disorders. However, this industry also produces psychotropic substances like analgesics, hypnotics and sedatives. They are helpful treatment options when adequately prescribed but there is also increasing concern about prescribed and over-the-counter non- medical use of these substances, caused by aggressive marketing and inade quate prescriptions by primary care doctors. Examples include the dramatic increase of prescribed opioid analgesics in Canada and the United States, lead ing to severe negative health consequences and premature death (Fischer et al., 2011; Fischer et al., 2013), or the fact that in many western countries the number of substance use disorders for these classes of drugs is as high as the number of alcohol use disorders (e.g. for Germany: Kraus et al., 2013)i g y Pharmaceutical companies are as profit driven as the tobacco and alcohol industries and have demonstrated a willingness to engage in such activities as suppression, through delayed or nonpublication of null or negative findings, and dilution, through the selective funding of certain types of research (Kas sirer, 2005). There is also evidence that some industry-supported research is biased (Brennan et al., 2006; Kassirer, 2005; Singer, 2008). In an interesting case study that combines pharmaceutical companies and tobacco, Etter et al. (2007) assessed whether the source of funding affected the results of trials of nicotine- replacement therapy for smoking cessation. They found that, compared with independent trials, industry-supported trials were more likely to produce sta tistically significant results and larger odds ratios. i In general, it has been found that researchers who report a financial compet ing interest are more likely to present positive findings (Friedman & Richter, 2004). Such behavior has not been documented within the addictions field, although medications used by many addicted patients for other complaints such as depression and anxiety have been the subject of controversial research practices. The Gambling Industry Problem gambling has been strongly linked to a range of personal and social problems (Gupta & Derevensky, 1998). The opportunities for addiction sci entists to receive funding from gambling-industry sources have increased Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies 333 significantly over the last decade, raising a number of ethical and organiza tional risks similar to those associated with accepting funding from other dan gerous consumption industries (Adams, 2007). g p As in the case of relationships with the tobacco and alcohol industries, rela tionships with social-aspects/public-relations organizations have been used to mitigate potential negative associations with gambling problems and to give the impression either that the activity leads to public good or that they have at least attempted to rectify potential harm (Adams & Rossen, 2006). In countries such as Australia and New Zealand, a governmental or quasi-governmental agency has been created to manage voluntary funds in a way that appears independent of the source. Adams and Rossen point out that the major problem with such arrangements “is the perception that donor organizations should still retain a significant say in how the money is used” (p. 11). This culture leads to uncritical acceptance of gambling-industry perspectives and misrepresents the industry’s willingness to trade profits for public health. This has meant in the past that industry officials were “consistently instrumental in ensuring that activities that might threaten the consumption of gambling were unlikely to receive signifi cant funding (this particularly applied to research, health advocacy, and public health initiatives)” (Adams & Rossen, 2006, p. 12). This may explain why there have been few studies of the role of the gambling industry in the promotion of gambling behavior and pathological gambling. It has been proposed that government-mandated contributions provide an alternative option to support research and provide a way to mollify criticism. In this arrangement, governments enact legislation that requires gambling pro viders to allocate a portion of their net income to projects, including research, with a community purpose. The major difficulty with this arrangement is the risk of increasing financial dependency, leading scientists to avoid criticiz ing gambling interests (Adams & Rossen, 2006). Likewise, the responsibility of governments to regulate gambling and prevent gambling problems may be compromised by the possibility that governments have themselves become “addicted” to the tax revenues derived from gambling. Governmental Agencies Albert Einstein (1934) once said that the “pursuit of scientific truth, detached from the practical interests of everyday life, ought to be treated as sacred by every government, and it is in the highest interests of all that honest servants of truth should be left in peace.” Einstein’s plea, directed at the fascist government of Mussolini, has been honored by most govern ment funding agencies, but there are many cases in which the interests of government are prioritized over scientific pursuit of truth. In a situation similar to that of the pharmaceutical companies, national and international 334 Publishing Addiction Science 334 governmental bodies fund many valuable research studies. However, as seen in earlier examples, research has sometimes been used to achieve political or financial goals, such as supporting current budget allocations, protecting policy makers who have made bad decisions, or undermining more-effective strategies because they are unpopular and politically risky. Miller et al. (2006) identified two examples in which governmental funders acted to distort research findings in Australia and the United Kingdom, particularly regarding more-controversial activities such as needle and syringe programs. Similar observations have been made about the diffi culty in obtaining funding for research into the effectiveness of needle and syringe programs and other forms of harm reduction in the United States (Pollak, 2007; Small & Drucker, 2006;). Other Interest Groups Funding bodies are not the only groups to control research findings. For instance, Hall (2006b) identified the possibility of drug-user groups and socially conservative members of ethics committees prioritizing their own interests at the expense of the integrity of the research. Members of ethics committees hold very powerful positions when it comes to rejecting, delaying, or modifying research proposals. Although most declare financial competing interests, ideo logical positions are different, and indeed many would not identify strongly held beliefs as being competing interests. For example, individual members of ethics committees who are strongly attached to abstinence-only programs may block or delay research into controlled-drinking interventions in the belief that they cannot be morally justifiable.h Funding bodies are not the only groups to control research findings. For instance, Hall (2006b) identified the possibility of drug-user groups and socially conservative members of ethics committees prioritizing their own interests at the expense of the integrity of the research. Members of ethics committees hold very powerful positions when it comes to rejecting, delaying, or modifying research proposals. Although most declare financial competing interests, ideo logical positions are different, and indeed many would not identify strongly held beliefs as being competing interests. For example, individual members of ethics committees who are strongly attached to abstinence-only programs may block or delay research into controlled-drinking interventions in the belief that they cannot be morally justifiable.h i There is also substantial room for competing interests inherent in the current peer-review framework (Hall, 2006a). With increasing competition over scarce resources, editors or reviewers may thwart the publication of research arti cles that counter their own theories or may thwart the publication of findings of their major competitors for funding. Although some journals have begun to publish ethical statements for editors, similar statements for reviewers of articles and funding applications may soon be required. Similarly, we should not forget that most researchers have their own pet theories, which can result in skewed research findings, particularly when those theories align with the interests of others such as professional societies, governments, or industry bod ies. As noted in Chapter 14, these kinds of competing interests are difficult to detect, but they should nevertheless be considered by authors when evaluating their own work.l Other social groups that might seek to influence research include profes sional associations, fellowship groups, religious organizations, and even service providers. Other Funding Agencies Increasingly, charitable organizations such as the Robert Wood Johnson Foun dation in the United States, the Joseph Rowntree Foundation in the United Kingdom, and the Millennium Trust in Australia have taken on agenda-setting roles that include research. Although most do not have profit imperatives akin to those seen in the tobacco, alcohol, and pharmaceutical industries, some nonetheless have their own agendas, and only a worthy few use transparent peer review. For example, the Wates Foundation in the United Kingdom has previously funded only research that supports abstinence-only approaches. Nepotism and personal competing interests can also come into play when trus tees back projects supported by their friends or projects in which they are per sonally involved. This lack of peer review and external accountability means that such organizations may end up skewing the evidence base by supporting research into only certain types of intervention. Although some of this might be balanced by different foundations having different interests, the reality is that these funders have the potential to, at times, favor ideologically and politi cally simple and popular interventions. For example, although a small num ber of trusts, such as the Soros Foundation, have funded research into harm reduction and drug-policy reform, there are many more foundations that will fund only abstinence-based programs or programs aimed at abstinence, such as education programs. Although there are many reasons for this, most revolve around trustees not being knowledgeable about the available evidence and the ory. In addition, many trustees and directors are politically aware individuals who are in the public spotlight. They may be reluctant to become associated with politically sensitive topics. All of this means that researchers should be aware of the possible consequences of applying for funding from such organi zations, because even limited research might contribute to the overall publica tion bias in the field. Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies 335 Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies 335 Other Interest Groups Professional associations (e.g., medical societies) have traditionally sought to maintain or increase their influence regarding any number of areas of knowledge and practice (Willis, 1989). Each discipline produces its own lit erature base. The size and complexity of this literature base helps to determine differential power structures within treatment settings. In the alcohol and drug sector, medicine and psychiatry (with the support of the pharmaceutical indus try) dominate the literature base, resulting in the medical model (and pharma cotherapies) having the strongest evidence base. In a different type of influence, some fellowship groups may influence research findings through nonparticipa tion (e.g., Wilton & DeVerteuil, 2006). Service providers are also not disinterested parties. Almost all (with a few notable exceptions) derive their income (and some of their raison d’etre) from treating addiction. This has substantial implications for the politics of treat ment and the vested interests many people bring to the research enterprise. The political and economic weight of mantras such as “treatment works” bear little 6 Publishing Addiction Science 336 relation to the complex evidence base and far more to the pragmatic needs of governments and service providers. Although many service providers use the discourse of charitable objectives, they are invested both financially and existen tially in the perceived success of the treatment they provide. This raises substan tial ethical issues when conducting program-evaluation research in treatment settings, especially if the evaluation is funded by the service provider or its fund ing body. Ethical considerations such as the true reporting of findings (even when negative), full editorial control of research projects, and the assurance of adequate dissemination should be negotiated before research commences. Such issues require that researchers, reviewers, and journal editors within the field apply a strong critical gaze to research and encourage an ethos of independence, even when such independence may not be economically prudent. Funding Issues in the Developing World All of the examples discussed thus far describe the situation in the developed world. However, the issues facing researchers in the developing world are likely to be even more complicated and are much less likely to be documented. As do their counterparts in the more-developed parts of the world, researchers in developing countries face many challenges in their work. In both environ ments, success is tied to the availability of resources and the overall intellec tual climate (Adair, 1995). Significant achievements as a scholar in a university or research institute require the ability to attract funding for research and to publish research findings, preferably in journals of high repute. Although the expectations from employers and the public might be the same, both activities are not always easy to execute by scholars in poor countries in which there are virtually no local resources for research. y When asked about the major problems encountered in their work, research ers and service providers affiliated with drug-demand–reduction organizations in Nigeria not surprisingly identified lack of funding as the leading challenge (Obot, 2004). Indeed, it is a rare country in Africa and other low-income parts of the world in which one can find consistent and near-sufficient outlay for scientific research on any topic, including addiction and other public health issues. This is especially the case for researchers in countries that constitute the “bottom billion” (Collier, 2007) or countries often described as least devel oped. In addition, competing for scarce resources with colleagues who are in resource-rich countries is often an impossible challenge. For the enterprising researcher, the response to this dearth of local funding opportunities is to con duct self-sponsored research (with all the limitations that this entails) or seek support from less-competitive external sources. This situation provides a good opportunity for organizations with ideological positions to propagate their interests and for others with economic interests to gain a foothold through financial support for research and training in these countries. Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies 337 Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies 337 This is a potential source of danger for research in many developing coun tries and one that has not received sufficient attention. Funding Issues in the Developing World Although there has been active discussion about unfair distribution of benefits of international research, especially coming from concerns about the ethical dimensions of clinical trials in developing countries (e.g., Bhutta, 2002), the exploitation that is implicit in some sources of funding for research in developing countries deserves greater scrutiny. Exploitation is more likely to occur in situations in which there is little understanding of competing interests, low economic capacity, limited infrastructure, and lack of ethical oversight—all of which are conditions that characterize many low-income countries. In the field of alcohol research, developing countries are experiencing a growing interest by representatives of the alcoholic-beverage industry mas querading as social-aspects organizations and seeking partnerships with researchers and policy makers. Usually the amount of money involved is a fraction of what would be spent for similar efforts in western countries, but it goes a long way for the scholar to whom such support is a lifeline, ena bling research and the publication of a book with an international imprint. In Africa, for example, the International Center for Alcohol Policies (ICAP; Box 16.3 above) has provided support for data collection, write-up, and pub lication of work with the potential of influencing local alcohol policy (e.g., Haworth & Simpson, 2004). For the funding organization, association with (usually) a high-profile academic or policy expert in a developing country validates their professed selfless motives. This can be a particularly pernicious strategy, because the developing-country scholar who has been co-opted by the alcohol, tobacco, or pharmaceutical industry might be the same scholar on whom government depends for advice when needed. It is not always lack of financial resources that drives the accommodation to untested imported theories and practices. Sometimes it is lack of knowledge, or even naïveté. A researcher in a developing country might find it difficult to suspect the motives of a funding agency that is acceptable to that country’s government and one that is supported or led by internationally recognized aca demics or professionals. To guard against establishing or sustaining relation ships with funding agencies that might lead to bad science or bad policy, it is important for researchers in developing countries to be more skeptical of easy money by questioning its source and the motives of its providers. Funding Issues in the Developing World That is easier to do today than it might have been 10 years ago, because most of the time all the information that is needed to decide whether to take the money can be found on the Internet. Competing Interests: What are They, Why are They Important Competing Interests: What are They, Why are They Important As suggested by the examples reviewed above, funding sources can influence scientific integrity in a variety of ways, ranging from subtle bias in the way 8 Publishing Addiction Science 338 research findings are presented to outright distortion of the research agenda or the scientific literature. One way to approach the ethical implications of many of the issues raised in this chapter is through the concept of competing interests. Competing interests can be financial, personal, ideological, political, and academic. A competing interest does not in itself constitute wrongdoing; rather, it acknowledges that the researcher has an interest that may be put above the integrity of the research being conducted. It is only the failure to declare real or potential competing interests to an editor, one’s co-authors, and the readers of an article that constitutes scientific misconduct. Potential competing interests are very important when it comes to the ability of the reader to assess the validity of any piece of scientific work. As noted above and in Chapter 14, competing interests may take many forms. For example, the issue of ideologi cal bias has been raised as a possible competing interest in medical research. A series of articles and responses about prayer as medicine has raised substantial concerns about the interface between faith and science (Clarke, 2007; Jantos & Kiat, 2007). It has been suggested that “for the benefit of a secular readership, in articles concerning religion and medicine in the Journal, the Editor should require the authors’ religious position to be stated under ‘competing interests’” (Clarke, 2007, p. 422). How to Avoid Competing Interests and Other Threats to Scientific Integrity and Academic Freedom Just as there are many forms of competing interests, so too are there many dif ferent ways to avoid or reduce undue influence, although many commenta tors believe that none of the possible options is entirely satisfactory or risk free (Adams & Rossen, 2006). By far the most commonly proposed way to avoid or ameliorate competing interests is through communication with one’s peers, particularly when done alongside ethics-awareness exercises (e.g., White & Popovits, 2001). Adams (2007) recommends that individuals, organizations, and others involved with interested parties engage in processes that raise ethi cal consciousness in conjunction with transparent regulatory frameworks that ensure accountability and independence from organizations and governmental and professional associations. This kind of communication and awareness rais ing has begun to occur at a number of levels. g g Recently, the institutions responsible for the production and dissemination of research (i.e., journals, professional societies, and academic institutions) have taken some important initiatives. Academic journals have increasingly begun to enact competing interest strategies including (a) requiring author state ments that declare funding source, which are then published with the article; (b) a positive statement that all authors had complete control over the research process; (c) reviewer and editor statements similar to those of authors; and (d) prior registration with an approved clinical-trials register as a prerequisite Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies 339 for publication. Journal editors have also begun to look at strategies for assess ing publication bias within their journals and at a more general level. Some journals have used their editorial pages to name and shame parties that behave inappropriately (e.g., Edwards et al., 2005) and to educate the scientific com munity about the need for competing interest policies (Babor & Miller, 2014). Professional associations have begun to draw up guidelines regarding the behavior of acceptable funding bodies, competing interests, and related issues. For example, the Federation of American Societies for Experimental Biology (2007) has issued a call to the scientific community to adopt more-consist ent policies and practices for disclosing and managing financial relationships between academia and industry in biomedical research. Box 16.5: Summary of the International Network on Brief Interventions for Alcohol and Other Drugs (INEBRIA) Position Statement on the Alcohol Industry. How to Avoid Competing Interests and Other Threats to Scientific Integrity and Academic Freedom The Federation of American Societies for Experimental Biology Toolkit (Federation of Ameri can Societies for Experimental Biology) consists of a set of model guidelines that speaks specifically to institutions that develop and enforce policies for their investigators, editors who develop disclosure policies for authors, and scientific and professional societies that have a role in promoting professional ethics. Similarly, the RESPECT Code of Practice (Dench et al., 2004) is a vol untary code of practice regarding the conduct of socioeconomic research. The proposed guidelines are a synthesis of several professional and ethical codes of practice designed to protect researchers from unprofessional or unethi cal demands. In one of the most thorough policy statements on the subject of competing interests, the International Network on Brief Interventions for Alcohol and Other Drugs issued a position statement that is summarized in Box 16.5. (1) INEBRIA believes that the commercial activities of the alcohol industry pose a conflict of interest of such magnitude that any form of engagement with the alcohol industry may influence its independence, objectivity, integrity, and credibility internationally. (2) All individuals wishing to present at an INEBRIA meeting will be required to complete a conflict-of-interest declaration for the work being presented. (3) Members of the coordinating committee will sign a conflict-of- interest declaration and may not have worked with or received funding from the alcohol industry, directly or indirectly, in the five years before their election date or during their term of office. (1) INEBRIA believes that the commercial activities of the alcohol industry pose a conflict of interest of such magnitude that any form of engagement with the alcohol industry may influence its independence, objectivity, integrity, and credibility internationally. j y g y y y (2) All individuals wishing to present at an INEBRIA meeting will be required to complete a conflict-of-interest declaration for the work being presented.l (3) Members of the coordinating committee will sign a conflict-of- interest declaration and may not have worked with or received funding from the alcohol industry, directly or indirectly, in the five years before their election date or during their term of office. 340 Publishing Addiction Science Institutions such as universities and research centers have developed policies regarding acceptable funding bodies, and some scrutinize research contracts for possible competing interests. How to Avoid Competing Interests and Other Threats to Scientific Integrity and Academic Freedom A growing number of universities (e.g., Kings College London) have refused to accept funding from the tobacco industry, and some research centers have developed their own internal policies (Box 16.6). Deakin University (Australia) now prohibits the receipt of research funding from the tobacco and gambling industries, as well as social, health or epidemiological research funded by the alcohol industry. There is also scope for institutional ethics review boards to assess the appropriateness of funder– researcher relationships. Questions regarding such relationships are now incor porated in the Australian National Ethics Application Form (www.nhmrc.gov. au/health-ethics/human-research-ethics-committees-hrecs/hrec-forms/neaf- national-ethics-application-for). Such responses are designed to support indi vidual researchers in the decision-making process and provide more-reliable and consistent approaches to this complex issue (Babor & McGovern, 2007; Miller et al., 2006). However, resolving these issues remains in large part the responsibility of individual authors, many of whom have a limited ability to understand or act upon the complex ethical, political, clinical, and scientific issues surround ing the initiatives coming from a particular funding source. Fortunately, most addiction scientists have chosen to eliminate themselves from participation in activities with obvious competing interests, such as consulting arrangements with the tobacco and alcohol industries and restrictions from funding sources that prevent them from retaining ownership of data and the investigator’s right to publish it (Babor & McGovern, 2007). Nevertheless, what is needed is a more-systemic set of procedures that allows individuals to conduct a risk analysis of different funding opportunities. Decision-Making Approaches Several approaches have been suggested to guide decision making by inde pendent scientists when they consider collaboration with the alcoholic-bever age industry and other dangerous consumption industries (Babor, 2009; Babor & McGovern, 2007; Stenius & Babor, 2010). Decisions regarding collaboration with bodies that may seek to influence research can range from a “hands-off” position to full collaboration. Adopting a hands-off position, in which mem bers of the scientific community and their organizational sponsors refuse to engage in communication or collaboration with industry representatives, is based on the assumption that commercial interests are incompatible with the values and aims of public health in general and with health-related scientific research in particular. Some have argued that the main effect of industry’s recent cooperation with scientists and public health professionals has been to improve their corporate image with the public and with governmental policy Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies 341 Dealing with Possible Competing Interests Related to the Financing of Our Research Projects The proportion of industry research funding within the financial budget of the institute has been very low since the foundation of the IFT Institut für Therapieforschung in 1973. But caution is needed, because this part of research support is provided by organizations and companies that produce or distribute psychoactive substances (e.g., alcohol or phar maceutical industry) or are active in the gambling business (includ ing gambling companies licensed or owned by the German States) and because of the internationally known incidents of scientific misconduct. The IFT does not reject funding of research by commercial institutions in principle but is aware of the particular responsibility in this area. In times of short or even declining public research funding and direct demands of the public to cooperate with industry and to expand com mercial third-party funds for research, it is hardly possible to abandon such sources of funding in principle. The institute has in this context the following rules: • Research requests to conduct a study on a given research question will be accepted only if (a) the research question is formulated glob ally and is undirected (e.g., the extent of drug abuse in the popula tion) and not biased (e.g., the study is not expected to demonstrate that a certain medicine bears no risk for the population), (b) the research question is scientifically relevant, and (c) the free and unre stricted further design of the study is guaranteed. • A further precondition for accepting funding by industry sources is the guaranteed independent formulation of the research objectives, hypotheses, and study methodology, and the unrestricted statistical analysis, interpretation, and publication of results. The funds have to be granted to the IFT as unrestricted educational grants or donations. • We do not accept funding of research projects by the tobacco indus try (reasons: evidence of long-lasting, one-sided, and unacceptable manipulation of scientists and scientific results). i • A single funding source must not contribute to more than 10% of the annual budget, and all industry funds should not exceed 20%. It is notable that these limits have never been reached: The average contribution is about 2%, and it has never exceeded 5% in the past. • All results will be published. • Lectures given in the context of industry organizations are acces sible via the website of the IFT. Dealing with Possible Competing Interests Related to the Financing of Our Research Projects • Lectures given in the context of industry organizations are acces sible via the website of the IFT. (Box continued on next page) (Box continued on next page) 342 Publishing Addiction Science Box 16.6: One research institution’s guidelines on acceptable research funding. Source: Institut für Therapieforschung, München, Germany (www.ift.de). makers, rather than to promote science (Babor & Robaina, 2013; Gmel et al., 2003; McCreanor et al., 2000; Munro, 2004).h makers, rather than to promote science (Babor & Robaina, 2013; Gmel et al., 2003; McCreanor et al., 2000; Munro, 2004).h The other end of the spectrum is to engage in dialogue with industry repre sentatives, accept industry funding for research, and participate as “partners” in industry-funded scientific activities such as the publication of books (e.g., Stimson et al., 2006). A third approach is based on the growing number of case studies, ethical reviews, and documentary information now available with respect to industries that have an important stake in products that affect public health (Brennan et al., 2006; Hirshhorn et al., 2001; Rampton & Stauber, 2002; Rundall, 1998). This approach avoids categorical recommendations to either allow or discour age relationships between science and industry in favor of a more-nuanced set of guidelines that outlines conditions of cooperation between science and industry (Adams, 2007). Funding in the “Gray Area” between Public and Commercial Organizations Examples are charitable organizations, (nonprofit) health insurance companies, and industry associations. In most cases, these organiza tions are accountable to the public or the commercial sector. The IFT applies in each case the same rules as for commercial organizations. PERIL Adams’ (2007) PERIL framework (purpose, extent, relevant harm, identifiers, link) provides a structured means of evaluating individual situations from an ethical perspective. Depending on circumstances, each of the five PERIL sub continuums is influenced in varying ways by the different domains of risk. Purpose refers to the degree to which purposes are divergent between funder and recipient. For example, if the primary purpose of the recipient is the advancement of public good, receiving funds from dangerous consump tion industries such as tobacco, alcohol, and gambling companies will prob ably conflict with this purpose. Similarly, the risk is mitigated partially if the funder has a clear public-good role. For example, the provincial government of Ontario runs a state monopoly on liquor distribution, the profits from which they invest in a broad range of research (Adams, 2007). Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies 343 Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies 343 Extent is the degree to which the recipient relies on this source of funding. As the proportion of income increases, it becomes more difficult to separate one’s research from expectations associated with the source. For example, a young investigator may find an award from an industry-sponsored organization is the sole source of salary support, which could create pressure to obtain industry- favorable results to ensure the continuation of funding. Relevant harm is the degree of harm associated with this form of consump tion. The level of harm generated by different forms of consumption varies. Lower potency products, such as lottery tickets or low-alcohol beer, are on the whole less likely to lead to problems than more-potent products, such as elec tronic gambling machines or alcoholic energy drinks. Funders are unlikely to contribute anonymously, because for them the point of the exercise is often to be identified, to form a visible association with pub lic-good activities for the purposes of positive branding. This in turn can be used for political or commercial purposes. The extent of visible association can be reduced by moving away from high-profile advertisements (such as media releases of findings) to more-discrete acknowledgements on plaques or at the end of publications. Through reputational risk, this strategy indirectly discour ages engaging in industry-supported research.h The more direct the link is between funder and researcher, the stronger the influence and the more visible the association are. PERIL For example, direct funding by a tobacco company involves more exposure than receiving the funding via an independent intermediary agency, such as a foundation or governmental funding body. As long as there are no major competing interests for the inter mediary agency, the separation reduces the likelihood that recipients will feel obligations, even coercion, for their activities to comply with the interests of the donor. The overall extent of moral jeopardy ranges from very high levels, as indicated by high ratings on all five subcontinuums, to very low levels, as indicated by consistently low ratings. Decisions regarding future industry rela tionships are made accordingly. Boxes 16.7 and 16.8 provide two case studies to illustrate how a PERIL analysis can be applied to specific funding opportunities. Is Industry Funding of Research the only Peril that Matters? A new genre of policy analysis suggests that vested interests use research to achieve their ultimate goals of profit maximization (Babor & Robaina, 2013). In their illuminating series of articles, Hawkins, McCambridge and col leagues highlight the way in which the alcohol industry uses both industry- funded research and their relationships with researchers to demonstrate their credibility and good intentions (Hawkins & Holden, 2014; Hawkins et al., 2012; McCambrige et al., 2013; Hawkins & Holden, 2014). These public-relations activities are commonly hidden in the rhetoric of corporate social responsibility, which is particularly important to recognize when considering the long-term 344 Publishing Addiction Science relationships between the alcohol industry and most politicians and the way in which these relationships are formed. Although politicians might read a newspaper article about new alcohol trends, they are easily calmed when their likeable industry representative, who knows their kids’ names and the schools they go to, assures them that there is no need to worry because the industry — often through one of its front bodies such as Drinkwise (Australia), Drinka ware (United Kingdom), EURAB (Europe), or the ABMRF/The Foundation for Alcohol Research (United States) — is working with a group of respected researchers to deal with the issue. Hawkins and Holden (2014) demonstrated convincingly just how effective this strategy is, especially when it is combined with the very long-term engagement approach that the alcohol industry adopts with politicians from all sides of the political fence. It is even more effective when they are able to suggest that the industry has actually funded the research into this important issue and that they have found it not to be so important or that the interventions they recommend are effective and much more palatable politically than “nanny state” interventions, such as raising taxes or restricting trading hours (Miller et al., 2011). In the end, whether or not other elements of the PERIL analysis such as repu tational risk or extent of funding are of concern, the overriding consideration in the strategic funding of research by the alcohol industry is their ability to use those relationships to gain a place at the discussion table regarding policy at the state, federal, and global levels. A university-based school of medicine distributes an email announc ing to all faculty and staff the availability of a new research funding opportunity. independent research and a review of the literature on tobacco-industry tactics. independent research and a review of the literature on tobacco-industry tactics. Is Industry Funding of Research the only Peril that Matters? The announcement reads: “Please see the link below for an available funding opportunity from the Philip Morris External Research Foundation The website invited scientists to submit funding proposals to Philip Morris’s independent, peer-reviewed, external research pro gram, which is willing to support research on the disease mechanisms and health endpoints of tobacco smoking and smoke exposure. The pro gram’s scientific advisory board members are listed on one of the pages of the request for applications, an impressive-looking group of academ ics, including department chairs, distinguished professors, and even the President of the Hungarian Academy of Sciences. This announcement raises a number of questions about the moral hazards of industry spon sorship of scientific research. Assume you are a tobacco researcher at a large academic medical center whose dissertation was recently completed on a topic related to the announcement. Should you apply for the funds? A PERIL analysis along the lines recommended in the Adams article would require some Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies 345 Box 16.7: PERIL analysis of a funding opportunity from Phillip Morris. PERIL Analysis Is the purpose of your academic institution (e.g., “excellent medical care through research and education”) consistent with the stated pur pose of Phillip Morris (i.e., to sell cigarettes to adults, without taking any responsibility for the millions of adolescents who become addicted before they can legally purchase tobacco products)? If your institution is in any way devoted to health, the answer is that the purposes are incom patible. In addition, some have pointed to the anti-scientific record of Phillip Morris. The reason Phillip Morris’s research foundation is now called “external” is that the company was ordered to disband a prior organization that was found by a U.S. court to be biased in the way it awarded grants to scientists. What about the extent of the funding? Is it sufficient to compromise the independence of an academic medical center with a large portfolio of research grants and contracts? It probably is not, but for individual investigators it could create a dependence on tobacco money when other sources of funding become more scarce. Is there relevant harm associated with Phillip Morris’s continued mar keting of tobacco products? The evidence is incontrovertible. Will the recipient of the funds be identified with the funder so that Phil lip Morris might benefit from its support of university-based scientists? And could funded scientists eventually be exposed to reputational risk if their names were associated with Phillip Morris? The answer is a pos sible yes to both questions. Finally, is the nature of the link between recipient and donor direct or indirect? In this case it is indirect; therefore, it may not involve a major competing interest, and there are no limitations on publication imposed by the funder. In summary, the analysis indicates that there are incompatible institu tional interests, a potential for developing dependence on an industry funding source, relevant harms to the public if tobacco sales continue as more research is conducted, a potential for future reputational risk, and a possible political benefit for Phillip Morris. Box 16.7: PERIL analysis of a funding opportunity from Phillip Morris. 6 Publishing Addiction Science 346 A residential rehabilitation charity approaches you to collaborate in an application to fund doctoral research into the long-term effectiveness of its project. The charity reports that it has been involved in research previously and has found it beneficial. The methodology is discussed and agreed. PERIL Analysis The application is designed to go to a governmental funding body that provides matching funds for collaborations between commu nity organizations and universities. The charity expresses concern about the confidentiality of its service users and requests that “We would, however, want the research findings to be kept confidential except in so far as they are needed to fulfill the requirements for the degree.” Subse quent investigation shows that, although the charity refers to a strong research pedigree, findings have been published only in non-peer- reviewed trade magazines or internal reports. PERIL Analysis Is the purpose of your academic institution (e.g., excellent medical care through research and education) consistent with the stated purpose of the charity? At first glance it would appear that the charity has the laudable goal of assessing its effectiveness through independent research. However, its desire to control dissemination (presumably in case of unfavorable findings) and its previous track record of publishing only in non-peer- reviewed journals would suggest that its goal might not be excellence. What about the extent of the funding? In this example, this is unlikely to be a major factor because the amount involved would be comparatively small. Is there relevant harm? There is a chance of some harm in this case if the findings are unfavorable and the charity chooses not to disseminate the report. In this situation, the charity is clearly providing ineffective treatment and using resources that might be better used elsewhere. In addition, it may be skewing the knowledge base through omission of negative findings. There is also a significant issue that the researchers and university will be identified with the evaluation. It is within the interest of the charity to point to the fact that the research was independently conducted. Finally, is the nature of the link between recipient and donor direct or indirect? In this case it is indirect; therefore, it may not involve a major competing interest, and there are no limitations on publication imposed by the funder. In this case, it would be possible for the researchers or the Relationships with the Alcoholic-Beverage Industry, Pharmaceutical Companies 347 university to insist that the charity remove its right to control release of the data. If that were done, the PERIL analysis would suggest that the funding is worth pursuing. Box 16.8: PERIL analysis of a funding opportunity limited by conditions imposed by a collaborating organization. Conclusion Every individual, discipline, and funding organization brings its own agenda to the research process. The practical and ethical conundrums associated with research funding are becoming increasingly complex in a context in which research plays a greater role in the regulation and marketing of potentially addictive products. The examples reviewed in this chapter suggest that addic tion scientists should be vigilant and critically reflective about the funding they accept from any source, particularly in relation to the ultimate purpose of such funding. This is even more so the case when there are restrictions on the design, interpretation, and publication of the resulting data. Thus, researchers should always be very wary about accepting research funding directly from dangerous consumption industries, their trade associations, and public-relations organi zations. Consulting arrangements wherein scientists are paid by parties with a clear competing interest to critique the work of other scientists can constitute a serious financial competing interest that is unlikely to benefit either science or the investigator. Acceptance of fees for writing book chapters, preparing background reports, attending industry-organized conferences, and writing letters to the editor should be prefaced by careful consideration of the follow ing questions: (1) To what extent is the scientific activity designed to promote the commer cial interests of a particular industry? (2) Will the funding source be acknowledged? (3) How could this research or my institution’s relationship with this com pany be used to undermine the implementation of effective policy? Addiction scientists also need to be careful that their objectivity and independ ence are not compromised by fraternizing with industry executives as well as paid travel to meeting sites and consulting fees (Wagner & Steinzor, 2007). Investigators in particular need to be attentive to the possibility that industry funding in many health areas is being contested on both ethical and scientific grounds (Foxcroft, 2005; King, 2006; Brennan et al., 2006). Finally, researchers 8 Publishing Addiction Science 348 should examine all funding sources using a framework such as the PERIL anal ysis, which allows the individual scientist and his or her institution to review relevant information about the motives of the funding source and the uses of the research that will be conducted. 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Spaces of sobriety/sites of power: Exam ining social model alcohol recovery programs as therapeutic landscapes. Social Science & Medicine, 63, 649–661. SECTION 5 How to cite this book chapter: Babor, T F, Stenius, K and O’Reilly, J. 2017. Addiction Publishing and the Meaning of [Scientific] Life. In: Babor, T F, Stenius, K, Pates, R, Miovský, M, O’Reilly, J and Candon, P. (eds.) Publishing Addiction Science: A Guide for the Perplexed, Pp. 355–364. London: Ubiquity Press. DOI: https://doi.org/10.5334/bbd.q. License: CC-BY 4.0. How to cite this book chapter: Addiction Publishing and the Meaning of [Scientific] Life Thomas F. Babor, Kerstin Stenius and Jean O’Reilly Conclusion CHAPTER 17 The Meaning of Science A seminal article by Ilkka Niiniluoto (2002), professor of philosophy at Helsinki University, traces the history of science through the various mile stones in the search for knowledge from the time of the ancient Greeks to the present time.hi The first milestone, which is the legacy of Aristotle, lies above all in the organ ized description of how we come to know the world and its generally accepted laws (“why” knowledge). A second phase in the history of science came with Galileo’s search for regularities in how the world changes (“how” knowledge). Compared with these steps, the third one is more complicated. A much later advance in the development of science began at the end of the 19th century when Charles Pierce introduced the notion of fallibility, which claimed that human beings constantly make mistakes in their search for knowledge and that all claims about the real world should be questioned. “This is true also of research, even if the scientific method of the research community, at least in the long run, is the most reliable way to produce and motivate conceptions of the world” (Niiniluoto, 2002, p. 32, authors’ translation).h Niiniluoto talks about science as a self-correcting process. The modern sci entific community has its own quality-assessment system (e.g., the peer review process), scientific claims are public, and all parties in the scientific community have the right to discuss, criticize, or refute those claims. According to Niini luoto, contemporary science is characterized by objectivity (gaining as true a picture of the object studied as possible), a critical attitude (research should be public and open for critical discussion in the research community), autonomy (the scientific community operates independently of religious, political, eco nomic, personal, and social influences), and progressivity (science creatively seeks new solutions and builds on old ones). Introduction The global scientific discipline of addiction studies that has developed during the past half century would be impossible without the infrastructure of the publishing enterprise. At the core of this infrastructure lie the peer-reviewed scientific article and the expanding network of journals that publish such arti cles. Throughout this book, we have focused on publishing scientific articles in peer-reviewed journals because this is a key part of the meaning of scientific life. Publishing allows the scientist to communicate findings, ideas, and opin ions within a forum representing the scientific community. In this final chapter, we will explore this theme in relation to addiction science, which for many highly trained researchers throughout the world has become a career commit ment that is not only personally rewarding but also beneficial to society. In brief, our argument is as follows: Science is meaningless unless it is com municated. Publication communicates scientific findings, and it is also the hallmark of a productive scientific career. Scientific integrity is another core feature of a successful career, and it must be nurtured by individuals, groups, and institutions, including scientific journals. To the extent that science consti tutes a universal language, there is a special need to foster addiction careers in low- and middle-income countries. 356 Publishing Addiction Science The Meaning of Science Arguing further that science is a social institution, Niiniluoto refers to Merton’s (1973) four imperatives for the ethos of science: (a) universalism (the truth of claims shall be judged on impersonal grounds irrespective of the race, nationality, class, or personal characteristics of the person who presents them), (b) communism (scientific findings result from social cooperation and should be common property), (c) disinterestedness (scientists present and analyze scientific knowledge without considering the career or prestige of the researcher), and (d) organized skepticism (scientists assess scientific results on the bases of empirical and theoretical criteria).i According to Niiniluoto, Merton’s principles have been criticized as deficient, insufficient, and inconsistent with the everyday life of research in the contem porary world. “Big science,” increasing competition for personal repute, and the inequitable concentration of resources have eroded the ethos of science, as has the use of science in war and commercial production, which has produced a form of applied science that is businesslike and breaches the “communism” principle of common ownership of intellectual property. Niiniluoto argues, Addiction Publishing and the Meaning of [Scientific] Life 357 though, that this activity is not really scientia and should be viewed as some thing other than academic research. In addiction research, the increasing competition for research positions and financial resources can foster the temptation to neglect ethical rules as well as the ethos of science. Career considerations can orient one’s research to what is popular or fundable rather than toward what is interesting or important. The growth in private research funding may lead to secrecy instead of the open exchange of new ideas and research results, and may lead to new priorities that favor business interests rather than the public good. If we accept Niiniluoto’s assertions, we can understand why good publica tion practices, of the type described in this book, are crucial for science and the search for meaning in scientific life. Good publication practices represent the principles that should guide the quest for truth and, at the same time, dem onstrate how to become a respected member of the scientific community. If science is to be used properly in the search for meaning as well as the basis for the betterment of humankind, there needs to be free access to the enormous reservoir of scientific knowledge in the world. The Meaning of Science That knowledge not only needs to be readily available, but it must also be recorded in a way that is understand able, useable, and certifiably scrutinized for error and bias. This is the role of journals and the responsibility of their authors. As noted by LaFollette (1992), a journal serves as the arbitrator of the authenticity and legitimacy of knowledge. It provides a historical record of a particular area of knowledge and confers implicit certification on authors for the originality of their work. Careers in Addiction Science Publishing with scientific integrity is for many the sine qua non of a produc tive scientific career in addiction science. The remarkable growth of addiction science worldwide (Babor, 1993, 2002; see also Chapter 2) coincides with the development of a variety of career options for those interested in basic, clinical, or social research. Research societies, subspecialties within professional organ izations, and research centers have proliferated in many parts of the world, as has the availability of addiction specialty journals (see Chapter 3). There is growing evidence that a career in addiction science has become a viable and rewarding way to spend one’s professional life (Edwards, 1991, 2002). As noted in Chapter 3, journals and the process of scientific publication serve the inter ests of career advancement and provide a vehicle for scholarly achievement. Indeed, the easiest way to understand a scientist’s career is to review the pub lications proudly listed in his or her curriculum vitae. When one looks at the seminal thinkers and scientists in the field, published works constitute the main record of their professional lives. Boxes 17.1–17.2 provide examples of how productive and influential addiction researchers reflect on their research and scientific communications. 8 Publishing Addiction Science 358 Born in Antwerp, Belgium, in 1931, Charles S. Lieber received his medical degree in 1955. Soon thereafter, he moved to the United States and obtained senior research appointments at Harvard Medical School, Cornell Medical College, and Mount Sinai Medical School. His research focused on alcohol abuse and its biological components, including the mechanisms underlying the development of alcoholic cirrhosis of the liver (Edwards, 2002). In the following, he describes one of the discov eries that changed the course of biological research on alcohol: “There seemed to be an adaptive system which helps us survive in mod ern society because it is relatively non-specific and detoxifies foreign compounds even when the body has never been exposed to them before. When we observed a similar morphological response after alcohol, I pos tulated that alcohol may therefore also be a substrate for this system. This hypothesis led to the discovery of the microsomal ethanol oxidizing sys tem (MEOS) as a new pathway of ethanol metabolism” (Edwards, 2002, p. 19). Box 17.1: Charles S. Lieber, M.d. (1931–2009). Born in Antwerp, Belgium, in 1931, Charles S. Lieber received his medical degree in 1955. Careers in Addiction Science Soon thereafter, he moved to the United States and obtained senior research appointments at Harvard Medical School, Cornell Medical College, and Mount Sinai Medical School. His research focused on alcohol abuse and its biological components, including the mechanisms underlying the development of alcoholic cirrhosis of the liver (Edwards, 2002). In the following, he describes one of the discov eries that changed the course of biological research on alcohol: “There seemed to be an adaptive system which helps us survive in mod ern society because it is relatively non-specific and detoxifies foreign compounds even when the body has never been exposed to them before. When we observed a similar morphological response after alcohol, I pos tulated that alcohol may therefore also be a substrate for this system. This hypothesis led to the discovery of the microsomal ethanol oxidizing sys tem (MEOS) as a new pathway of ethanol metabolism” (Edwards, 2002, p. 19). Box 17.1: Charles S. Lieber, M.d. (1931–2009). In 1972, Martha Sanchez-Craig took a position as director of a halfway house for homeless alcoholics at the Addiction Research Foundation in Toronto, Canada. Five years later, she became a senior scientist at the Clinical Institute of the Addiction Research Foundation. Here, her research centered on brief interventions for people with alcohol- and other drug-related problems (Edwards, 2002). Despite her extensive publication career, she cautions about the “publish or perish” mentality: “. . . one of the senior people, who was conducting experiments with small numbers of non-human subjects, said ‘I don’t have much regard for any scientist who doesn’t publish at least six papers a year in peer-reviewed journals’’. I was very worried about that. I met colleagues who would get depressed or seriously worried if they couldn’t publish a paper every month. I began to think that there are a lot of people here who like to do science that looks good, and only a few who like to do good science” (Edwards, 2002, p. 124). Box 17.2: Martha Sanchez-Craig, Ph.d. In 1972, Martha Sanchez-Craig took a position as director of a halfway house for homeless alcoholics at the Addiction Research Foundation in Toronto, Canada. Five years later, she became a senior scientist at the Clinical Institute of the Addiction Research Foundation. Here, her research centered on brief interventions for people with alcohol- and other drug-related problems (Edwards, 2002). Box 17.2: Martha Sanchez-Craig, Ph.d. Individual Responsibility Research can be a solitary endeavor, involving late nights spent in your study or laboratory, preparations to defend a thesis or to question someone else’s disser tation, and standing alone on a podium to present a scientific article. In many cases, it is impossible, at least without considerable effort, for an outsider to know whether a researcher has conducted his research ethically. All researchers are thus responsible for guarding the integrity of the public trust in research. But research is also a highly social enterprise, which introduces its own ethi cal concerns. Much scientific research is now conducted via teams of investiga tors and support staff that share responsibility for the completion of a project and the publication of a scientific report. In this context, individual responsi bility sometimes becomes diluted and ambiguous in relation to ethical mat ters. The research world is also very hierarchical. Younger researchers are like apprentices being trained by their masters, economically dependent on them for positions and promotions. These differential power relations can further dilute ethical responsibility. Despite these threats to research integrity, addiction scientists must adhere to the ideal of the polis of the ancient Greeks, whereby every free man (we will have to ignore the gender discrimination of the time) was an equal, with simi lar responsibilities to decide matters of importance and civil rights to support those responsibilities. Similarly, every researcher must accept his or her personal responsibility for creating a more transparent and ethical addiction research community, which includes young investigators and senior researchers alike, as well as editors of journals and peer reviewers. Everyone, for example, has a responsibility to use citations in a fair and informative way (Chapter 10), to ensure the proper assignment of authorship credits (Chapter 11), and to adhere to ethical rules (Chapters 14, 15 and 16). When all researchers view themselves as equals in the republic of science, they will create the best foundation for crea tive discussions, which in turn will lead to progress in research. Careers in Addiction Science Despite her extensive publication career, she cautions about the “publish or perish” mentality: “. . . one of the senior people, who was conducting experiments with small numbers of non-human subjects, said ‘I don’t have much regard for any scientist who doesn’t publish at least six papers a year in peer-reviewed journals’’. I was very worried about that. I met colleagues who would get depressed or seriously worried if they couldn’t publish a paper every month. I began to think that there are a lot of people here who like to do science that looks good, and only a few who like to do good science” (Edwards, 2002, p. 124). Box 17.2: Martha Sanchez-Craig, Ph.d. Addiction Publishing and the Meaning of [Scientific] Life 359 Addiction Publishing and the Meaning of [Scientific] Life 359 Box 17.3: Mustapha Soueif, Ph.d. Also helpful to scientific integrity are more formal structures, such as policies for the ethical conduct of research (Chapter 15) and procedures for the deter mination of authorship credits (Chapter 11). In recent years addiction journals have emerged from their relatively obscure and modest origins to take a lead ership role in the prevention of scientific misconduct. The ethical principles for authors included in this book represent the consensus of editors who are members of the International Society of Addiction Journal Editors. Integrity in scientific publishing can be enhanced only by education, vigilance, clear poli cies, and institutional norms that put science first. Creating Good Institutions In many instances, exhortations to individual responsibility are not enough to guarantee scientific integrity. Good institutions must support creative research milieus with sound ethical principles. Informal structures, such as open com munication within departments (not only about research but also about ethical problems), the reading and critiquing of each other’s work, democratic deci sion making, and cooperation on multidisciplinary projects all emanate from participatory norms and strong leadership. In Boxes 17.3–17.4, two influen tial addiction researchers reflect on the social and institutional aspects of their research and scientific communications. 60 Publishing Addiction Science 360 Mustapha I. Soueif was born in 1924. He completed his graduate studies in psychology at University of Cairo, Egypt. In addition to teaching psy chology at the University of Cairo, he also worked for the World Health Organization (Edwards (1991). Here, he describes the challenges of publishing in different languages and the conflicts between having a national commitment and an international vision: “It is a long time now that I have been living with this double identity; on the one hand I feel a world-citizen, on the other I belong to Egypt. This complex ‘consciousness’ or oscillating began in the late fifties when I was carrying out my first piece of clinical research in Egypt (at Abbassia Psy chiatric Hospital) while keeping an eye on getting it published abroad. This was the paper on ‘Testing for organicity in Egyptian psychiatric patients’. It was accepted for publication in Acta Psychologica (in Amsterdam). That was the first step towards establishing my reference group, defined in this case as a group of international scientists who would judge the worth of my research on its objective merits. My international identity, however, was definitely promoted through my contact with the WHO in Geneva. In 1966 I was approached by the WHO people to prepare a paper for publication in the UN Bulletin on Narcotics reporting on our work on ‘Hashish Con sumption in Egypt’ which has been under way since 1957. This I did, and the paper was published in 1967. In 1970 I was invited to participate in a ‘Scientific group’ meeting to be held at WHO headquarters. The recognition my work received there was deeply gratifying” (Edwards, 1991, p. 436). Awareness of Global Inequality Addiction is a global concern, and the concepts of universalism and autonomy suggest that knowledge gained from research should be shared Addiction Publishing and the Meaning of [Scientific] Life 3 Kettil Edmund Bruun received his doctoral training in sociology from the University of Helsinki. He is perhaps best known for his influential book, Alcohol Control Policies in Public Health Perspective, published in 1975 under the auspices of the World Health Organization. Some times called the “purple book” (owing to its cover in the English-lan guage version), the publication gained wide attention for its basic tenet: “changes in the overall consumption of alcoholic beverages have a bearing on the health of the people in any society. Alcohol control measures can be used to limit consumption: thus, control of alcohol availability becomes a public health issue” (Bruun et al., 1975, p. 90; see also Edwards, 1991, and Room, 1986). In the following, Bruun describes with characteristic modesty the process that gave rise to the book: “The background was that I had to rethink my ideas of alcohol control in the light of the Finnish experience in 1968/69 when controls had been sud denly relaxed with dramatic increase in consumption and harmful effects. My own liberal views on alcohol policies had received a blow. Then I was confronted in the European Office with international issues. I thought that I had to reconsider my position and that probably the best way to do it was to try to have a group which could develop a perspective beyond the specific sit uation in Finland. The situation was fortunate because many of the relevant questions had by then been focused for research. The group which emerged from my invitation did a marvelous job” (Edwards, 1991, pp. 371–372). Box 17.4: Kettil Edmund Bruun, Ph.d. (1924–1985). throughout the world. Unfortunately, resources for both research and sci entific communications are limited in many parts of the world, and research conducted in the more-resourced countries often follows parochial national interests. Moreover, the dominance of English as the de facto language of sci ence comes at a price for the majority of the world, in which other languages predominate. Addiction researchers in the English-speaking and the more-developed countries have a special obligation to conduct and present their research, whenever possible, in a way that benefits the rest of humankind. Conclusion: The Meaning of Scientific Life In Chapter 1, we referred to the medieval philosopher Maimonides and his Guide for the Perplexed. This was perhaps not a very modest analogy. We do not want to suggest that this book—or any book for that matter—can remove all confusion and provide a researcher with the guidance needed to have a suc cessful career in addiction science. Rather, we hope the information in this book will lead its readers to the agora of science, a community square or com mon ground on which open and democratic discussions can take place among equals about the difficult problems all researchers, novices and career profes sionals alike, encounter in their everyday work.fii One of those difficult problems is the meaning of scientific life itself. It is a question perhaps secondary to the broader question of life’s meaning in general, but it is nevertheless worth asking if we want to make our own lives meaningful as addiction scientists. Various spiritual, religious, and philosophical traditions from the East, West, North, and South have contributed to this profound line of questioning. Despite their important insights, biologist Edward O. Wilson (2014) believes philosophy is ill-equipped to tackle the meaning of existence. Wilson con cludes that, by default, the task of explaining meaning necessarily falls to sci ence itself. Among the disciplines that he favors in determining meaning are evolutionary biology and neuroscience. To those we would add the behavioral, social, and population sciences, which may help us understand how addiction is the antithesis of harmony with the natural world and how modern civiliza tion seems designed to make that harmony difficult for many to achieve. And we should not defer entirely to science when meaning can surely be derived from religion, literature and other areas of knowledge.l g g In the most spiritual and reflective period of his life, Leo Tolstoy (1886) wrote a novella called The Death of Ivan Ilyich, which tells the story of the last days of a high-court judge in 19th-century Russia. It is at its core a philosophical commentary on the meaning of life as revealed in the interactions one has with family, work colleagues, and people encountered in day-to-day living at all social levels. What are the lessons for us, the living? Awareness of Global Inequality The peer- review process should be open to scientists from all languages and nationali ties, as should the editorial boards of the journals serving as the gatekeepers for scientific truth. Language and culture should not limit publication in addiction science. Not only is this a question of fairness, but it also speaks to the cross-cultural generalizability of scientific findings and the need to dis cover universal truths. 362 Publishing Addiction Science Addiction Publishing and the Meaning of [Scientific] Life 363 Addiction Publishing and the Meaning of [Scientific] Life 363 Addiction Publishing and the Meaning of [Scientific] Life 363 Presenter addresses the question this way: “Well, it’s nothing very special. Try to be nice to people, avoid eating fat, read a good book every now and then, get some walking in, and try and live together in peace and harmony with people of all creeds and nations.” Or, as comedian Groucho Marx observed, “If you’re not having fun, you’re doing something wrong.”i Ultimately, the meaning of scientific life is a question you will have to answer yourself. Even if a single answer to the question may elude you, that elusiveness is no great tragedy. More important is the search itself and the insights you gain as you realize that addiction science is a wonderful way to add benefit to society and depth to your own understanding of human nature. And finally, it is a way to have fun. Please visit the website of the International Society of Addiction Jour nal Editors (ISAJE) at www.isaje.net to access supplementary materials related to this chapter. Materials include additional reading, exercises, examples, PowerPoint presentations, videos, and e-learning lessons. Conclusion: The Meaning of Scientific Life One lesson is that if our lives are intimately invested in addiction science, this would be a good time to take inventory of what we have accomplished and what remains to be done. Have we avoided meaningless writing projects that lead to publications that nobody reads or values? Have we worked amicably with colleagues, supported their ideas, and given credit where it is due? Have we considered the plight of the alcoholic and the drug addict; the families who lose children to drunk drivers; and the evidence-based policies that could prevent drunk driving, fetal alcohol spectrum disorder, and underage drinking? Beyond literature, science, and philosophy, perhaps the answer lies elsewhere. At the end of the Monty Python film, aptly called The Meaning of Life, the Lady Room, R. (1986). Kettil Bruun, 1924–1985: An appreciation. The Drinking and Drug Practices Surveyor, 21, 1, 42–49. Tolstoy, L. (1886). The Death of Ivan Ilyich. Russia. Wilson, E. O. (2014). The meaning of human existence. New York, NY: Liveright Publishing Corporation. Room, R. (1986). Kettil Bruun, 1924–1985: An appreciation. The Drinking and Drug Practices Surveyor, 21, 1, 42–49.h h Wilson, E. O. (2014). The meaning of human existence. New York, NY: Liveright Publishing Corporation. g y Tolstoy, L. (1886). The Death of Ivan Ilyich. Russia.h Drug Practices Surveyor, 21, 1, 42–49. Tolstoy, L. (1886). The Death of Ivan Ilyich. Russia. Wilson, E. O. (2014). The meaning of human existence. New York, NY: Liveright Publishing Corporation. References Babor, T. F. (2002). In their own words: Conversations about the evolution of a specialist field. In G. Edwards (Ed.). Addiction: Evolution of a specialist field (pp. 383–389). Oxford, England: Blackwell Science. Babor, T. F. (1993). Megatrends and dead ends: Alcohol research in global per spective. Alcohol Health and Research World, 17, 177–186. Bruun, K., Edwards, G., & Lumio, M., Mäkelä, K., Pan, L., Popham, R. E., . . ., Österberg, E. (1975). Alcohol control policies in public health perspective. Helsinki, Finland: Finnish Foundation for Alcohol Studies, Vol. 25.li Edwards, G. (Ed.). (1991). Addictions: Personal influences and scientific move ments. New Brunswick, NJ: Transaction Publishers.i Edwards, G. (Ed.). (2002). Addiction: Evolution of a specialist field. Oxford, England: Blackwell Science. LaFollette, M. C. (1992). Stealing into print: Fraud, plagiarism, and misconduct in scientific publishing. Berkeley, CA: University of California Press.hh i Merton, R. (1973). The sociology of science: Theoretical and empirical investiga tions. Chicago, IL: University of Chicago Press.h Niiniluoto, I. (2002). Tieteen Tunnuspiirteet [The characteristics of science]. In S. Karjalainen, V. Launis, R. Pelkonen, & J. Pietarinen (Eds.), Tutkijan Eet tiset Valinnat [The ethical choices of the researcher], (pp. 30–41). Tampere, Finland: Gaudeamus. 364 Publishing Addiction Science h Publishing Corporation. Index acknowledgement 84, 117, 130, 150, 171, 196, 210–12, 223, 241, 275 appropriate 147 footnoted 93 acknowledgement function 192–93 acknowledgements section 216, 312 acknowledgments 217, 280, 285 addiction field 4–5, 18, 22, 24–25, 27, 30, 34, 46, 49, 51, 54, 71–74, 104–5, 282–83, 317–18 Addiction Journal Editors 5, 8, 31, 33, 38, 79, 85, 104, 110, 165, 263, 268, 294, 298, 300 addiction journals 48, 50, 71–75, 78, 82, 104, 107, 148, 156, 163–64, 167, 169–70, 229–30, 260, 262 indexed 74 peer-reviewed 38 specialized 46 addiction libraries 22, 33 A A abstracting services 24, 47, 49, 52, 54–55, 57, 58 abstracts 24, 38, 42, 57–58, 64–65, 67, 69, 139, 164, 176, 179, 196–97 conference 177 published 271 structured 142 academic journals 12, 22, 75, 124, 229, 236, 324–25, 338 open-access 65 peer-reviewed 106 acceptance 28, 39–42, 58–59, 83, 89, 104, 138, 230, 233, 238, 241, 285, 304, 313 chances of 47, 58–59, 81, 231 final 248 rapid 53, 104 acceptance letters 52 acceptance rates 5, 46, 58, 104, 127, 137, 230 i 27, 30, 34, 46, 49, 51, 54, Addiction Journal Editors 5, 8, 31, 33, 38, 79, 85, 104, 110, 165, 263, 268, 294, 298, 300 open-access 65 j 78, 82, 104, 107, 148, 156, 163–64, 167, 169–70, 229–30, 260, 262 specialized 46 addiction libraries 22, 33 Publishing Addiction Science 366 advancement 135, 219, 221, 318, 331, 342 academic 53 scientific 97 tenure-track 89 advertising bans 213, 224, 278, 295 advisor 96, 100–101, 113–14 major 116–17 advisor agreement 101 advisory board memberships 282, 285 advisory boards 76, 249, 254, 262 scientific 312, 328 Africa 16, 31, 83, 99, 336–37 African Journal of Drug and Alcohol Studies 39, 74, 83, 170 African research 75 Alberta guidelines 93 alcohol 8–15, 17, 19–22, 24, 26–28, 31–34, 38–40, 46–47, 62–63, addiction literature 46, 57 abstracted 14 international 22 addiction medicine 21, 33 addiction professionals 29, 73, 120 addiction psychiatrists 21, 27 addiction publishing 4, 6–7, 38, 72, 84, 133, 299–300, 304, 321, 355 addiction literature 46, 57 abstracted 14 international 22 addiction medicine 21, 33 addiction professionals 29, 73, 120 addiction psychiatrists 21, 27 addiction publishing 4, 6–7, 38, 72, 84, 133, 299–300, 304, 321, 355 331, 342 , academic 53 scientific 97 tenure-track 89 advertising bans 213, 224, 278, 295 advisor 96, 100–101, 113–14 major 116–17 advisor 96, 100–101, 113–14 major 116–17 321, 355 advisor agreement 101 advisory board memberships 282, 285 addiction research 7–8, 11–13, addiction research 7–8, 11–13, addiction research 7–8, 11–13, 16–18, 20–21, 27–28, 33, 37, 47, 50, 73–74, 82–83, 292–93, 300, 302, 304 16–18, 20–21, 27–28, 33, 37, advisory boards 76, 249, 254, 262 scientific 312, 328 Africa 16, 31, 83, 99, 336–37 African Journal of Drug and Alcohol Studies 39, 74, 83, 170 African research 75 Alberta guidelines 93 47, 50, 73–74, 82–83, 292–93, advisory boards 76, 249, 254, 262 scientific 312, 328 300, 302, 304 addiction research centers 11–12, 16–18, 31, 33 addiction research centers 11–12, 16–18, 31, 33 addiction researchers 50, 74, 83, 305, 357, 359, 361 Alberta guidelines 93 Addiction Research alcohol 8–15, 17, 19–22, 24, 26–28, Foundation 11–12, 358 addiction science 3–5, 9–14, 16–18, 31–34, 38–40, 46–47, 62–63, 73–74, 103–4, 119, 170, 330–31, 358 31–34, 38–40, 46–47, 62–63, 73–74, 103–4, 119, 170, 22, 24–27, 29–30, 49, 76–77, 79, 135–37, 144–45, 147, 184, 355, 361–63 22, 24–27, 29–30, 49, 76–77, 22, 24–27, 29–30, 49, 76–77, 79, 135–37, 144–45, 147, 184, 355, 361–63 79, 135–37, 144–45, 147, 184, 330–31, 358 alcohol abuse 11, 16, 18, 24, 33–34, 170, 331, 358 career in 32, 109, 135, 357, 362 Addiction Science & Clinical Practice 58 addiction science workforce 27 addiction scientists 9, 27, 29, 71, 74, 85, 136, 269, 332, 340, 347, 359, 362 career in 32, 109, 135, 357, 362 Addiction Science & Clinical Practice 58 career in 32, 109, 135, 357, 362 Addiction Science & Clinical Practice 58 addiction science workforce 27 addiction scientists 9, 27, 29, 71, 74, 85, 136, 269, 332, 340, 347, 359, 362 Alcohol and Alcohol Problems Science Database 24, 151 alcohol dependence 18, 32, 46, 51, 170, 245, 286 addiction science workforce 27 addiction scientists 9, 27, 29, 71, alcohol education 331, 349 Alcoholics Anonymous 136, 171 alcohol industry 285, 321, 328, 330, 332–33, 339–40, 343–44, 348–50 352 alcohol education 331, 349 Alcoholics Anonymous 136, 171 alcohol industry 285, 321, 328, 330, 332–33, 339–40, 343–44, 74, 85, 136, 269, 332, 340, 347, addiction societies 21–22, 31, 33 addiction specialty journals 4–5, addiction societies 21–22, 31, 33 addiction specialty journals 4–5, 11–13, 38, 46–47, 50, 57, 74, 127, 357 dd d addiction specialty journals 4–5, 330, 332–33, 339–40, 343–44, 11–13, 38, 46–47, 50, 57, 74, 348–50, 352 127, 357 alcohol industry funding 28 Alcoholism Treatment Quarterly 170 alcohol research 11, 32, 58, 62, 85, 119, 170, 282, 295, 328, 330, 337, 344, 348, 363 Alcohol Studies 11–12, 26, 33, 56, 74, 83, 86, 170–71, 363 alcohol use disorders 13, 184, addiction studies 10–11, 25–26, 32, 49–50, 56, 185, 307, 355 addiction studies 10–11, 25–26, 32, 49–50, 56, 185, 307, 355 , , , , addiction treatment 17–18, 21, 33, 131, 305, 321, 332, 352 Addictive Behaviour 33 Addictologie 20, 49, 56 ad hoc reviewers 249 Adicciones 20 Adiktologie 42, 131, 188 advanced search option 66, 69, 152 , , , , addiction treatment 17–18, 21, 33, 131, 305, 321, 332, 352 Addictive Behaviour 33 Addictologie 20, 49, 56 ad hoc reviewers 249 Adicciones 20 Adiktologie 42, 131, 188 advanced search option 66, 69, 152 addiction treatment 17–18, 21, 33, 131, 305, 321, 332, 352 119, 170, 282, 295, 328, 330, Addictive Behaviour 33 Addictologie 20, 49, 56 ad hoc reviewers 249 Adicciones 20 337, 344, 348, 363 Alcohol Studies 11–12, 26, 33, 56, 74, 83, 86, 170–71, 363 alcohol use disorders 13, 184, 188, 332 Adiktologie 42, 131, 188 advanced search option 66, 69, 152 Alcoologie 20, 49, 56 Index 367 Index 367 American Psychological Association 56, 68–69, 92, 110, 138, 148, 150, 208, 215–17, 242 American Psychological Association 56, 68–69, 92, 110, 138, 148, 150, 208, 215–17, 242 105–6, 205, 334, 340, 344, 349–50, 352 author, corresponding 14, 210–11, 222, 226, 249 author, corresponding 14, 210–11, 222, 226, 249 222, 226, 249 analysis adjusted 243–44 appropriate 156 content 219–20, 234 laboratory 116, 220–21 multi-variate 149 power sample 126 qualitative 157, 159, 161, 172 quantitative 175, 180 reference 63 author fees 5 authorship 90–94, 97, 101, 103, 112–14, 116–18, 207–8, 210–12, 215, 217–25, 270, 277, 280, 307, 311 assigning 91 assumed senior 213 attribution of 211–12 coercion 211, 214 corporate 208, 210–11 early-career 90 gift 211, 213–15, 279, 311 guest 270, 278 honorary 208, 211, 214, 284 honorary and ghost 225 irresponsible 214, 268, 296 mutual-admiration 211 mutual-support 211, 214 pressured 211, 214 responsible 208, 215, 217–18 unethical 270, 304 unfair 7, 268, 277 reference 63 animal experimentation 285, 321 animal experimentation 285, 321 d b bl h Apparent COI, 250, 280–81, 284–85 Appendices 145 article fees 53l Article Influence Score 200 articles commissioned 274 duplicate 178 fabricated 54 first 7, 129, 193 ghostwritten 278–79, 298 multi-authored 6, 212, 216, 217, 273 rejected 129, 256 reviewing 262 revising 254 theoretical 48, 236 top-cited 61 assessment 108, 137, 180, 201, 20 246–47, 255 duplicate 178 p responsible 208, 215, 217–18 unethical 270, 304 unfair 7, 268, 277 first 7, 129, 193 i ghostwritten 278–79, 298 multi-authored 6, 212, 216, authorship assignment 92, 97, 218–19, 359 p g 218–19, 359 p g 218–19, 359 rejected 129, 256 reviewing 262 authorship contribution 94, 97 authorship contribution 94, 97 authorship conventions 212 authorship credits 6, 91–93, 103–4, authorship conventions 212 authorship conventions 212 authorship credits 6, 91–93, 103–4, theoretical 48, 236 top-cited 61 111–12, 116–17, 147, 207, 210, 212–16, 219, 223–24, 279–80, 310, 360 111–12, 116–17, 147, 207, 210, 212–16, 219, 223–24, 279–80, assessment 108, 137, 180, 201, 203 246–47, 255 246–47, 255 310, 360 authorship criteria 92, 103, 212, audiences authorship criteria 92, 103, 212, 216, 221, 270, 278 Authorship Disputes 222 authorship guidelines 93,111, 216, 280 authorship order 89, 97, 101, 109, 111, 114, 212, 217, 221–22, 224–25 authorship problems 208, 269, 279 216, 221, 270, 278 appropriate 85 Authorship Disputes 222 global 68 g important 56 authorship guidelines 93,111, p intended 47, 54, 58, 96–97, 198i g 216, 280 scientific 106, 136 authorship order 89, 97, 101, 109, 111, 114, 212, 217, 221–22, 224–25 authorship problems 208, 269, 279 i Australasian Professional Society 20 Australasian Professional 368 Publishing Addiction Science 368 B Beall’s list of predatory publishers 53, 55, 58, 110 beneficence 301, 305–6, 325 principles of 303, 317 bias 184, 186, 195, 198–200, 203, 250, 252, 271–73, 278, 280, 282–83, 285, 331, 337, 339 ideological 338 methodologic 203 minimising 162 potential 131, 244, 250 procedural 234 bibliography 153 bibliometric indicators 63, 204 author-based 201 biomedical journals 204, 214, 224, 275, 296 high impact 225 randomized 77, 180, 204 co-authors 89, 100–101, 108, 117, 208, 211–12, 214–16, 226–27, 278–79, 282, 309, 311, 338 potential 97, 108, 218, 226 co-authorship 114, 214, 311 Cochrane Database 140, 263, 271, 283 Cochrane Reviews 208, 225 COIs 250, 256, 258–59, 269, 278, 280–85, 293 financial 328 possible 250 real 280–81 collaboration 10, 17, 30, 73, 90, 125, 129, 220, 256, 319, 340, 346 cross-national 106 faculty-student 111, 224 international 12, 31, 212 commentaries 7, 246, 274, 325 comments 7, 115, 145–46, 203, 218, 220, 226, 229, 231, 240, 247–49, 251, 254, 256, 258–61 confidential 232, 252, 258 competing interests 281, 330, 335, 337–40, 349 financial 332, 335, 347 major 343, 345–46 personal 334 possible 338, 340–41 potential 338 complaints 209, 225, 268, 332 formal 279 B 280–85, 293 high impact 225 C career 18, 29, 32, 84, 91, 95, 109, career 18, 29, 32, 84, 91, 95, 109, 116, 121, 135, 214, 253, 278, 355–58, 362 116, 121, 135, 214, 253, 278, 355–58, 362 i competing interests 281, 330, 335, academic 95 professional 6, 27, 29–30, 33, 131 academic 95 professional 6, 27, 29–30, 33, 131 337–40, 349 financial 332, 335, 347 major 343, 345–46 personal 334 possible 338, 340–41 potential 338 complaints 209, 225, 268, 332 formal 279 case reports 48, 122, 139, 231, 248, 300, 307, 310 case reports 48, 122, 139, 231, 248, 300, 307, 310 case studies 48, 107, 122, 158, 223, 268, 275, 278, 298, 304, 307, 323, 332, 342–43, 351 centers 16–17, 23, 26, 31, 33, 52, 91, 215, 282, 302, 327 citation analysis 55, 63 citation bias 77, 184, 267–72, 295–96, 304 conscious 268 citation procedures 61 citations 55, 60–61, 64, 67–68, 76, 165, 168, 177, 181, 187, 191– 203, 205, 209–10, 289, 296 p complaints 209, 225, 268, 332 formal 279 compliance 68, 281, 288, 315–16 confidentiality 257, 269, 303, 346 conflicts 117–18, 178, 214–15, 217, 256, 268–69, 280–81, 283, 285, 295, 297, 305–7, 313–14, 285, 295, 297, 305–7, 313–14, 285, 295, 297, 305–7, 313–14, citation procedures 61 citation procedures 61 348–49, 351 348–49, 351 citations 55, 60–61, 64, 67–68, 76, 165, 168, 177, 181, 187, 191– 203, 205, 209–10, 289, 296 appropriate 276, 290, 317 clinical trials 194, 210, 312, 337 addressing 314 apparent 312–13 direct 281 ethical 128 addressing 314 g apparent 312–13 apparent 3 Index 369 Index 369 copyright holder 277 copyright infringement 268, 275, 290 copyrighting dissertation materials 98 copyright laws 289, 305 CORK Database 66, 151 Correction of literature 301 corrections 199, 230, 232, 260, 268, 271–72, 279, 289, 292 Correspondence 87 cover-letter content 103 criteria editors use 233 critiques 137, 146, 226, 347 fair 145 referee’s 316 CrossCheck 289 Cross Ref 64 financial 28 potential 184, 282 real 256, 313 relevant 314 undeclared 280, 304, 312 financial 28 potential 184, 282 real 256, 313 relevant 314 undeclared 280, 304, 312 potential 184, 282 real 256, 313 relevant 314 undeclared 280, 304, 312 Consolidated Standards 139, 180, 234 CONSORT, 139, 180, 189, 234, 242, 244 Statement 143, 242, 244 Contemporary Drug Problems 170 contributions 114, 135, 137, 147, 150, 191, 193, 200–201, 210– 12, 214–16, 219–22, 252–53, 278, 280, 288–89 actual 210, 218 advisor’s 114 author’s 280 important 129, 251 individual 214, 219 intellectual 103 minor 216, 219–20, 279 original 114, 142 partial 219 substantial 97, 210–11, 215–16, 277 substantive 210, 214, 218, 222, 278–80 contributors 145, 210–12, 216–17, 219, 225, 280 individual 212 major 278 minor 278 potential 222 contributorship 211–12, 307 COPE Report 295, 301 copyediting 52, 81,146 copying 212, 270, 288–89 minor 288 substantial unattributed textual 288 unintentional 290 copyright 24, 270, 275–76, 290 copyright agreements 274 copyright laws 289, 305 Consolidated Standards 139, 180, 234 , 180, 234 CONSORT, 139, 180, 189, 234, 242, 244 Statement 143, 242, 244 Contemporary Drug Problems 170 contributions 114, 135, 137, 147, 150, 191, 193, 200–201, 210– 12, 214–16, 219–22, 252–53, 278, 280, 288–89 actual 210, 218 advisor’s 114 author’s 280 important 129, 251 individual 214, 219 intellectual 103 minor 216, 219–20, 279 original 114, 142 partial 219 substantial 97, 210–11, 215–16, 277 substantive 210, 214, 218, 222, 278–80 contributors 145, 210–12, 216–17, 219, 225, 280 individual 212 major 278 minor 278 potential 222 contributorship 211–12, 307 COPE Report 295, 301 copyediting 52, 81,146 copying 212, 270, 288–89 minor 288 substantial unattributed textual 288 unintentional 290 copyright 24, 270, 275–76, 290 copyright agreements 274 CONSORT, 139, 180, 189, 234, 242, 244 CONSORT, 139, 180, 189, 234, 271–72, 279, 289, 292 242, 244 Statement 143, 242, 244 Contemporary Drug Problems 170 contributions 114, 135, 137, 147, 150, 191, 193, 200–201, 210– 12, 214–16, 219–22, 252–53, 278 280 288 89 150, 191, 193, 200–201, 210– 12, 214–16, 219–22, 252–53, 278, 280, 288–89 CrossCheck 289 Cross Ref 64 D D data analyses 97, 105, 138, 164, 218, 226, 254, 261, 272, 310 data analysis section 234 databases 14, 22–24, 56–57, 60–61, 64, 66–70, 75, 85, 151–53, 176–78, 183, 185–86, 248–49, 254, 257 scientific 150–51 searchable 67, 178–79 data fabrication 214, 252, 269, 292 decision letters 251, 254 design 122, 140, 142, 162, 179, 216–17, 220–21, 259, 310, 316, 341, 347 appropriate 120 cross-sectional 180 experimental 220, 254 observational study 180 developing countries 19, 72, 76–77, 83, 86, 105, 107, 119, 336–37, 348 disciplinary journals 38, 46–47, 50, 169 disclosure 219, 225, 257, 274, 285, 314 data analyses 97, 105, 138, 164, 218, data analyses 97, 105, 138, 164, 218, y 226, 254, 261, 272, 310 y 226, 254, 261, 272, 310 data analysis section 234 databases 14, 22–24, 56–57, 60–61, original 114, 142 64, 66–70, 75, 85, 151–53, 176–78, 183, 185–86, 248–49, design 122, 140, 142, 162, 179, 216–17, 220–21, 259, 310, 316, 341, 347 appropriate 120 cross-sectional 180 experimental 220, 254 observational study 180 copying 212, 270, 288–89 minor 288 developing countries 19, 72, 76–77, 83, 86, 105, 107, 119, 336–37, 348 336–37, 348 disciplinary journals 38, 46–47, 50, 169 disclosure 219, 225, 257, 274, 285, 314 disclosure 219, 225, 257, 274, 285, 314 Publishing Addiction Science 370 Eigenfactor 61, 200, 203, 205 e-learning lessons 8, 31, 62, 85, 110, 130, 150, 165, 171, 187, 202, 223, 241, 263, 294 disclosure policies 313, 339 disputes 92, 218, 222, 280 resolving authorship 7 dissertation 64, 90, 94–99, 107–10, 112, 114, 117–18, 143, 147, 151, 177, 344, 359 article-based 89 final 96 graduate student’s 92, 117 monograph-style 89, 96 dissertation abstracts 24, 98 DOI, 32–34, 37, 62–64, 85–89, 110, 112, 115, 131, 135, 188, 191, 203, 223–25, 267, 321–23 DORA (Declaration on Research Eigenfactor 61, 200, 203, 205 e-learning lessons 8, 31, 62, 85, 110, 130, 150, 165, 171, 187, 202, 223, 241, 263, 294 223, 241, 263, 294 112, 114, 117–18, 143, 147, electronic databases 56–57, 75, 176, 197 main 24 endnotes 138, 153, 177–78 proprietary 153 English abstracts 58 English editing services 81 English grammar and syntax 53, 81 English language 75–76 English-language abstracts 49, 74 English-language journals 6, 38, 48–49, 58, 60, 71–72, 74–75, 78–80, 83, 108, 169–70, 200, 260 international 80 open-access 12 Equator Network 139 ethical analysis 300, 304, 307–11, 313, 315–20, 323 appropriate 294 ethical codes 82, 285, 339 ethical conduct 8, 294, 360 ethical dilemmas 6, 90, 304–5, 347 resolving 223 151, 177, 344, 359 article-based 89i final 96 DOI, 32–34, 37, 62–64, 85–89, 110, 112, 115, 131, 135, 188, 191, 203, 223–25, 267, 321–23 English grammar and syntax 53, 81 DORA (Declaration on Research Assessment) 200–201, 203 DORA (Declaration on Research Assessment) 200–201, 203 Drug and Alcohol Dependence 46, 51, 170, 245 Drug and Alcohol Review 46, 131, 170 DrugScope 24, 65, 66 duplicate publication 273, 275 English-language journals 6, 38, 48–49, 58, 60, 71–72, 74–75, 78–80, 83, 108, 169–70, Drug and Alcohol Dependence 46, 51, 170, 245 200, 260 200, 260 international 80 open-access 12 Equator Network 139 ethical analysis 300, 304, 307–11, 313, 315–20, 323 appropriate 294 ethical codes 82, 285, 339 ethical conduct 8, 294, 360 ethical dilemmas 6, 90, 304–5, 347 resolving 223 international 80 Drug and Alcohol Review 46, 131, 170 open-access 12 DrugScope 24, 65, 66 duplicate publication 273, 275 E e-books 73, 148 EBSCO, 151, 176 EBSCO platforms 152 edited books 324 editing 81, 108, 143, 145, 224, 275, 277, 296 editing services 82 editorial 32, 65, 70, 76, 85–86, 199, 204, 225, 246, 249–51, 254, 277, 283–84, 295–97, 349 normal 249 editorial boards 53–54, 65, 76, 78, 80, 87, 245, 313, 315, 361 editors 49–55, 77–82, 142–48, 167–68, 229–33, 235–42, 245–62, 268–69, 274–77, 285–87, 290–92, 307–10, 312–13, 315–16, 338–39 editor’s decision 233, 237, 239, 247, 251, 259, 262 E appropriate 294 ethical codes 82, 285, 339 ethical conduct 8, 294, 360 ethical dilemmas 6, 90, 304–5, 347 resolving 223 Ethical guidance for genetic research on addiction 295 ethical issues 7, 164, 208, 210, 269, 271, 278, 280, 284, 286, 296, 300, 302, 305, 309–10, 312, 321, 336, 352 g g on addiction 295 rial 32, 65, 70, 76, 85–86, 199, 204, 225, 246, 249–51, 254, 277, 283–84, 295–97, 349 mal 249 204, 225, 246, 249–51, 254, 277, 283–84, 295–97, 349 300, 302, 305, 309–10, 312, 321, 336, 352 normal 249 normal 249 Ethical practice guidelines in addiction publishing 300, 321 Ethical practice guidelines in addiction publishing 300, 321 ethical principles 221, 287, 299, 301, 303–5, 308–9, 311, 325, 359–60 ethical problems 7, 163, 237, 258, 269, 294, 298, 300, 330, 359 ethical review board 314–16 ethical review committees 284, 302, 315, 319, 324 Ethical practice guidelines in addiction Ethical practice guidelines in addiction publishing 300, 321 ethical principles 221, 287, 299, 301, 303–5, 308–9, 311, 325, 359–60 ethical problems 7, 163, 237, 258, 269, 294, 298, 300, 330, 359 ethical review board 314–16 ethical review committees 284, 302, 315, 319, 324 editorial boards 53–54, 65, 76, 78, publishing 300, 321 ethical principles 221, 287, 299, editors 49–55, 77–82, 142–48, 167–68, 229–33, 235–42, 245–62, 268–69, 274–77, 285–87, 290–92, 307–10, 312–13, 315–16, 338–39 359–60 359–60 315, 319, 324 Index 371 Index 371 ethical rules 164, 317, 357, 359 ethical standards 287–88, 294, 300–301, 314, 316 ethical violations 269, 308, 311, 312 ethics 104, 245, 258–59, 295–96, 299–301, 321, 348 character-based 320 principle-based 301 professional 339 ethics-awareness exercises 338 ethics committees 125, 294, 335 institutional 280 ETOH, 14, 24, 151 European Addiction Research 170 European Association of Science Editors 85 F funders 279, 294, 325–26, 334, 340, 342–43, 345–46 funder specifications 325 funding 6–7, 27, 67, 75, 115, 117, 213, 220, 280–83, 280, 285, 310, 318–19, 326–28, 330, 332–37, 340–47 pharmaceutical company 295, 349 funding bodies 325–26, 335–36, 339–40, 350–51 governmental 343, 346 industry-related 326 funding opportunities 28, 227, 340, 343, 345, 347 available 344 local 336 new research 344 ethical rules 164, 317, 357, 359 ethical standards 287–88, 294, 300–301, 314, 316 ethical violations 269, 308, 311, 312 ethics 104, 245, 258–59, 295–96, 299–301, 321, 348 character-based 320 principle-based 301 professional 339 ethics-awareness exercises 338 ethics committees 125, 294, 335 institutional 280 ETOH, 14, 24, 151 European Addiction Research 170 European Association of Science Editors 85 F funders 279, 294, 325–26, 334, 340, 342–43, 345–46 funder specifications 325 funding 6–7, 27, 67, 75, 115, 117, 213, 220, 280–83, 280, 285, 310, 318–19, 326–28, 330, 332–37, 340–47 pharmaceutical company 295, 349 funding bodies 325–26, 335–36, 339–40, 350–51 governmental 343, 346 industry-related 326 funding opportunities 28, 227, 340, 343, 345, 347 available 344 local 336 new research 344 ethical rules 164, 317, 357, 359 ethical standards 287–88, 294, 300–301, 314, 316 ethical violations 269, 308, 311, 312 ethics 104, 245, 258–59, 295–96, 299–301, 321, 348 character-based 320 principle-based 301 professional 339 ethics-awareness exercises 338 ethics committees 125, 294, 335 institutional 280 ETOH, 14, 24, 151 European Addiction Research 170 European Association of Science Editors 85 funders 279, 294, 325–26, 334, 340, 342–43, 345–46i 342–43, 345–46 funder specifications 325 pi funding 6–7, 27, 67, 75, 115 ethical violations 269, 308, 311, 312 ethics 104, 245, 258–59, 295–96, 213, 220, 280–83, 280, 285, 213, 220, 280–83, 280, 285, 299–301, 321, 348 310, 318–19, 326–28, 330, 310, 318–19, 326–28, 330, 332–37, 340–47 332–37, 340–47 332–37, 340–47 pharmaceutical company 295, 349 pharmaceutical company 295, y funding bodies 325–26, 335–36, funding bodies 325–26, 335–36, 339–40, 350–51 governmental 343, 346 industry-related 326 funding opportunities 28, 227, 340, 343, 345, 347 available 344 local 336 new research 344 ethics-awareness exercises 338 ethics committees 125, 294, 335 343, 345, 347 available 344 local 336 Editors 85 F G G fabrication 267–68, 270, 279, 290, 301 deliberate 269 potential 309 falsification 268, 270, 290 fabrication 267–68, 270, 279, 290, 301 deliberate 269 potential 309 falsification 268, 270, 290 gambling 13–15, 34, 38, 104, 143, gambling companies 341–42 gambling industries 14, 28, 281, p falsification 268, 270, 290 falsification 268, 270, 290 g 332–33, 340 Farmington Consensus 54, 63, 104, 111, 238, 296, 300, 321 332–33, 340 Gender Equality 138, 188 gender issues 181 genetic research 17, 286, 295 ghost authorship 208, 211, feedback 78, 113, 129, 145, 248, 251, 253 helpful 106 negative 129 213–15, 224–25, 270, 278, 213–15, 224–25, 270, 278, 284, 289, 297 284, 289, 297 ghostwritten journal articles 297 gifts 255, 280–81, 284, 296–97 Google Scholar 57–58, 61, 67, 99, 102 150 284, 289, 297 ghostwritten journal articles 297 gifts 255, 280–81, 284, 296–97 Google Scholar 57–58, 61, 67, 99, discounted 54 nominal 98 102, 150 government documents 151, 196 graduate students 6, 89–90, 92, 94, government documents 151, 196 graduate students 6, 89–90, 92, 94, 97, 100–101, 111–12, 115–18, 136, 307 graduate studies 85, 112, 360 group authorship 211–12 guidelines 6, 8, 92–93, 100–101, graduate studies 85, 112, 360 group authorship 211–12 guidelines 6, 8, 92–93, 100–101, graduate studies 85, 112, 360 group authorship 211–12 guidelines 6, 8, 92–93, 100–101, 112–18, 138–39, 141, 175, 188 89 215 224 285 289 112–18, 138–39, 141, 175, fraud liability 298 fraudulent research 212, 291, 300 188–89, 215, 224, 285, 289–90, 297, 339 fraudulent research 212, 291, 300 Publishing Addiction Science 372 indexing databases 58, 69 indexing services 24, 39–41, 46, 48, 55, 57–58, 61, 64, 70, 104, 151 index terms 67, 152 industry 7, 28, 280, 282–83, 285, 297, 314, 323–24, 326–27, 330, 332, 339–42, 344, 351 l h l b 7 282 295 departmental 92, 103 disciplinary 97 ethical 53, 233, 279, 298 institutional 96, 280 intellectual-property 93 indexing databases 58, 69 g 55, 57–58, 61, 64, 70, 104, 151 g 55, 57–58, 61, 64, 70, 104, 151 index terms 67, 152 industry 7, 28, 280, 282–83, 285, industry 7, 28, 280, 282–83, 285, 297, 314, 323–24, 326–27, 330, 332, 339–42, 344, 351 H 332, 339–42, 344, 351 332, 339–42, 344, 351 Harm Reduction Journal 88, 170, 352 Harm Reduction Journal 88, 170, 352 health sciences 65, 69, 75, 101, 151, 263 health services research 27 high-impact journals 59, 75, 199 h-index 61, 69, 201, 204 historical research 123 history 32, 34, 46, 51, 60, 119, 169, 184, 204, 215, 224, 321, 356 early 331 short 293 Holy Grail 7, 323 honesty 214, 301, 305–6, 308, 310–11, 317, 319 intellectual 222, 294 honoraria 281, 283, 330 human subjects 7, 269, 286–87, 304, 316 involving 293 human subjects requirements 287, 314 Harm Reduction Journal 88, 170, 352 alcohol beverage 7, 282, 295, 328, 340 health sciences 65, 69, 75, 101, 151, 263 dangerous consumption 333, 340, dangerous consumption 333, 340, 342, 347–48 drinks 348, 351 pharmaceutical 104, 282, 285, 296, 332, 334–35, 337, 341 342, 347–48 342, 347 48 drinks 348, 351 pharmaceutical 104, 282, 285, 296 332 334 35 337 341 drinks 348, 351 industry-funded research 283, 324, 327, 343 industry-funded research 283, 324, 327, 343 industry funding 28, 282–83, 342–43, 347 industry funding 28, 282–83, 342–43, 347 information science 56, 68, 131, 204 Holy Grail 7, 323 Information Specialists 70 informed consent 269, 286–88, 303 infrastructure 5, 10, 12, 22, 25, Information Specialists 70 informed consent 269, 286–88, 303 infrastructure 5, 10, 12, 22, 25, 27, 355 addiction research 31i Institute for Scientific Information i (ISI) 60, 65, 87 Institute of Medicine 281, 294, 296, 317, 319, 321 Institute of Medicine 281, 294, 296, 317, 319, 321 integrity 47, 51–53, 55, 211, 216, integrity 47, 51–53, 55, 211, 216, 294, 296, 313, 317, 320–21, 324, 335, 338–39, 348–49, 359–60 academic 49 basic 319 professional 319 ll l h J journal articles 24, 58, 64, 66, 92, 94, 106, 108, 135, 148, 151, 170, 197, 270 junior investigators 16, 93, 94, 105, 136, 217 junior investigators 16, 93, 94, 105, 136, 217 journal articles 24, 58, 64, 66, 92, 94, 106, 108, 135, 148, 151, 170, 197, 270 accessing 108 content of scientific 24, 57 peer-reviewed 90 journal choice 37 51 198 I 294, 296, 313, 317, 320–21, ICAP (International Center for Alcohol Policies) 331, 337, 349, 351 324, 335, 338–39, 348–49, 359–60 academic 49 basic 319 ICMJE (International Committee of Medical Journal Editors) 208, 214–17, 224, 273–77, 280, 287, 296 professional 319 intellectual property rights 290 l l intellectual property rights 290 intellectual property rights 290 international journals 32, 48–49, intellectual property rights 290 international journals 32, 48–49, international journals 32, 48–49, 73, 76, 82–83, 111, 127, 165, 73, 76, 82–83, 111, 127, 165, impact factors 46–47, 49, 54, 59–61, 75, 86, 104, 107, 127, 198–200, 202–4 348, 350 investigators 17, 75, 79, 84, 212, 217, 275, 285, 292, 309, 319, 323, 325, 339–40, 347 collaborating 217, 274 investigators 17, 75, 79, 84, 212, investigators 17, 75, 79, 84, 212, 217, 275, 285, 292, 309, 319, 323, 325, 339–40, 347 collaborating 217, 274 indexing 42, 52, 54, 65, 73–74, 98, 152 217, 275, 285, 292, 309, 319, indexing 42, 52, 54, 65, 73–74, 98, 152 journal’s 47, 55 323, 325, 339–40, 347 collaborating 217, 274 journal’s 47, 55 Index 373 Index 373 early 94 established 4, 8 multiple 226, 274 new 104 novice 90 principal 115–18, 209, 221, 226 scientific 310 early 94 established 4, 8 multiple 226, 274 new 104 novice 90 principal 115–18, 209, 221, 226 scientific 310 j , , , , 73–75, 77–78, 80–83, 103–4, 127–31, 136–38, 140–42, 195– 201, 203–8, 229–34, 245–59 discipline-oriented 13 electronic 37, 70 high-impact-factor 127, 296 indexed 74, 77 non-indexed 177 non-peer-reviewed 346 open access 12, 54, 58, 65 pay-per-page 47 peer-review 118 questionable 53–54 regional 60, 70, 72, 76 127–31, 136–38, 140–42, 195– 201, 203–8, 229–34, 245–59 discipline-oriented 13 electronic 37, 70 high-impact-factor 127, 296 indexed 74, 77 non-indexed 177 non-peer-reviewed 346 open access 12, 54, 58, 65 pay-per-page 47 peer-review 118 questionable 53–54 regional 60, 70, 72, 76 high-impact-factor 127, 296 indexed 74, 77 non-indexed 177 non-peer-reviewed 346 open access 12, 54, 58, 65 pay-per-page 47 peer-review 118 questionable 53–54 regional 60, 70, 72, 76 high impact factor 127, 296 indexed 74, 77 non-indexed 177 non-peer-reviewed 346 open access 12, 54, 58, 65 pay-per-page 47 peer-review 118 questionable 53–54 regional 60, 70, 72, 76 ISAJE (International Society of Addiction Journal Editors) 5, 31, 33, 51–52, 54, 78–79, 83, 85, 104, 106, 268–69, 294, 296, 300, 320–21 ISAJE member journals 46 ISAJE member journals 46 ISAJE-PARINT Online Mentoring Scheme 111 ISAJE-PARINT Online Mentoring Scheme 111 Scheme 111 journal submissions 245–46, 248, 251, 257 junior investigators 16, 93, 94, 105, 136, 217 K accessing 108 content of scientific 24, 57 peer-reviewed 90 keywords 57–58, 66–67, 152, 176, 185, 249 knowledge 4, 22, 24, 29, 116, 125–26, 172–73, 234, 236, 238, 249, 313, 325, 335, 356–57 applied 30 basic 9, 29 certifiable 22 common 289 fundamental 10 scientific 30, 72, 77, 356–57 specialized 4, 71, 248 journal choice 37, 51, 198 Journal Citation Reports 54, 60, 200, 204 journal impact factors 6, 59, 63, 199–202, 204 Journal of Addictions Nursing 170 Journal of Addictions Nursing 170 Journal of Alcohol and Drug Education 170 Journal of Drug Education 170 l f Journal of Alcohol and Drug Education 170 Journal of Drug Education 170 Journal of Drug Issues 165, 170, 172 Journal of Drug Policy 350 L language editing 81, 216 language editing services 232, 261 language issues 232, 260, 261 language problems 81 language questions 86 languages 42–50, 57, 60, 67, 69, 72–73, 75–76, 78–79, 81–83, 86–87, 178, 231–32, 269, 273–74, 360–61 L L language editing 81, 216 language editing services 232, 261 language issues 232, 260, 261 language problems 81 language questions 86 languages 42–50, 57, 60, 67, 69, 72–73, 75–76, 78–79, 81–83, 86–87, 178, 231–32, 269, 273–74, 360–61 L language editing 81, 216 language editing services 232, 261 language issues 232, 260, 261 language problems 81 language questions 86 languages 42–50, 57, 60, 67, 69, 72–73, 75–76, 78–79, 81–83, 86–87, 178, 231–32, 269, 273–74, 360–61 Journal of Ethnicity in Substance Abuse 170 f y Abuse 170 Journal of Gambling Issues 170, 348 Journal of Gambling Studies 350 Journal of Groups in Addiction 111 Journal of Studies on Alcohol and Drugs 12, 41, 46, 170 Journal of Substance Abuse Treatment 34, 46, 170, 296 Journal of Substance Use 131, 170 Journal of Studies on Alcohol and Drugs 12, 41, 46, 170 Journal of Substance Abuse 86–87, 178, 231–32, 269, 273–74, 360–61 374 Publishing Addiction Science 374 native 49, 72, 232 non-English 246 universal 76, 355 Latin America 16, 31, 83 learned societies 4, 55–56 particular 56 legal issues 277, 307 legal review 329 letter 51–52, 103, 107, 129, 136, 142, 197, 199, 224, 239–41, 268, 272, 274, 287, 289, 316 librarians 23, 24, 53, 151 libraries 11, 22–24, 33, 56–57, 63, 66–68, 73, 83, 129 academic 54–55, 147, 149, 151–52 drug 33 electronic 75 professional 147 trade/ industry 23 Library of Congress 147 Library of Congress Subject Headings 152 library subscriptions 56, 69 life’s meaning 362 limitations 58–59, 61, 143. K 144, 162, 169, 174, 184, 234, 236, 336, 345–46 limited access 72 literature 57 95 108 122 140 41 M M M Maimonides 3, 8, 362 manuscripts 54–56, 59, 79, 93–96, 100–101, 103, 136–38 229–35, 237–42, 247–57, 261–62, 268–70, 272–74, 287–92, 307–11 accepted 59 draft 278 improved 256 marginal 238 qualitative 156 rejected 240 reviewing 7, 245, 248 revised 59, 104, 240 student-submitted 100 manuscript style 95–96, 98 manuscript title 164 meaning of scientific life 362–63 mediation 222 MEDLINE, 39–41, 48, 52, 55, 57, 67–68, 73–75, 151–52, 233 MEDLINE database 68 MEDLINE journals 14 Medline/PubMed 176 MEDLINE users 64 meeting abstracts 65 megatrends 12, 30, 62, 85, 363 mental health 33, 68, 73–74, 76, 87–88, 124 mentees 90–91, 101 mentoring opportunity 260 mentor relationship 101 mentors 29, 38, 79, 90, 94, 110, 12 223, 253, 307, 310 MeSH (Medicine controlled vocabulary—Medical Subject Headings) 57, 68, 152 meta-analysis 6, 139, 173, 175, 177 Maimonides 3, 8, 362 manuscripts 54–56, 59, 79, 93–96, 100–101, 103, 136–38, 229–35, 237–42, 247–57, 261–62, 268–70, 272–74, 287–92, 307–11 93–96, 100–101, 103, 136–3 229–35, 237–42, 247–57, 261–62 268–70 272–74 93 96, 100 101, 103, 136 229–35, 237–42, 247–57, 261–62, 268–70, 272–74, p legal issues 277, 307 g legal review 329 accepted 59t accepted 59 draft 278 improved 256 marginal 238 qualitative 156 rejected 240 reviewing 7, 245, 248 revised 59, 104, 240 student-submitted 100 letter 51–52, 103, 107, 129, 136, 142, 197, 199, 224, 239–41, 268, 272, 274, 287, 289, 316 letter 51–52, 103, 107, 129, 136, 142, 197, 199, 224, 239–41, 268, 272, 274, 287, 289, 316 librarians 23, 24, 53, 151 librarians 23, 24, 53, 151 libraries 11, 22–24, 33, 56–57, 63, 66–68, 73, 83, 129 academic 54–55, 147, 149, 151–52 manuscript style 95–96, 98 manuscript title 164i meaning of scientific life 362–63 mediation 222 Library of Congress 147 MEDLINE, 39–41, 48, 52, 55, 57, 67–68, 73–75, 151–52, 233 life’s meaning 362 limitations 58–59, 61, 143. K 144, 162, 169, 174, 184, 234, 236, 336, 345–46 limited access 72 mental health 33, 68, 73–74, 76, literature 57, 95, 108, 122, 140–41, literature 57, 95, 108, 122, 140–41, 164, 166–67, 174, 194–95, 217, 220, 268, 271–73, 307–9, 362 literature 57, 95, 108, 122, 140–41, 164, 166–67, 174, 194–95, 217, 220, 268, 271–73, 307–9, 362 literature 57, 95, 108, 122, 140–41, 164, 166–67, 174, 194–95, 217, 220, 268, 271–73, 307–9, 362 87–88, 124 mentees 90–91, 101 mentoring opportunity 260 mentor relationship 101 background 316 g gray 196 gray 196 grey 348 mentors 29, 38, 79, 90, 94, 110, 125, 223, 253, 307, 310 literature review 96, 105, 140–41, MeSH (Medicine controlled vocabulary—Medical Subject Headings) 57, 68, 152 meta-analysis 6, 139, 173, 175, 177, 180, 184–86, 188–89, 194, 203–5, 269, 271, 273–76, 283, 295–96, 298, 349 MeSH (Medicine controlled vocabulary—Medical Subject Headings) 57, 68, 152 MeSH (Medicine controlled vocabulary—Medical Subject Headings) 57, 68, 152 meta-analysis 6, 139, 173, 175, 177, 180, 184–86, 188–89, 194, 203–5, 269, 271, 273–76, 283, 295–96, 298, 349 143–44, 147, 150, 166, 275, 307, 309 balanced 271 literature review article 140 180, 184–86, 188–89, 194, literature search 69, 121, 125, 129, 151–52, 176, 183, 187, 236, 310 203–5, 269, 271, 273–76, 283, 295–96, 298, 349 295–96, 298, 349 literature search engines 98 methodological weaknesses 231, 236 methodological weaknesses 231, 236 literature search procedure 187 Index 375 Index 375 Methodologic guidelines for systematic reviews 203 methodology 123, 128, 147, 158, 164, 167, 176, 180, 183, 187, 189, 220, 274, 289, 346 methods analytical 162 clear step-by-step 137 data-collection 126 ideal 237 inadequate 167 qualitative 156–58, 167, 169, 234 quantitative 126, 156 scientific 10, 356 standardized 183 uniform 143 validated 237 methods section 143, 168, 275, 277 metrics 55, 61, 69, 104, 201 article-level 201 journal-based 200–201 journal-level 200 misconduct 8, 33, 63, 213–15, 217, 223–24, 238, 267, 269, 279, Methodologic guidelines for systematic reviews 203 Methodologic guidelines for systematic reviews 203 extra 311 scientific director’s 311 team 210 narrative reviews 174 hybrid 175 traditional 174–75 National Center for Responsible Gaming (NCRG) 282 National Institute on Alcohol Abuse and Alcoholism 11, 18, 33 National Institutes 27, 29, 33, 68 National Institutes of Health 68, 222, 225 National Library of Medicine’s methodology 123, 128, 147, 158, 164, 167, 176, 180, 183, 187, 189, 220, 274, 289, 346 methodology 123, 128, 147, 158, 164, 167, 176, 180, 183, 187, 189, 220, 274, 289, 346 methods analytical 162 clear step-by-step 137 data-collection 126 ideal 237 inadequate 167 qualitative 156–58, 167, 169, 234 quantitative 126, 156 scientific 10, 356 standardized 183 uniform 143 validated 237 National Center for Responsible Gaming (NCRG) 282 National Institute on Alcohol Abuse and Alcoholism 11, 18, 33 National Institutes 27, 29, 33, 68 National Institutes of Health 68, 222, 225 National Library of Medicine’s journal citation database 67, 68 methods section 143, 168, 275, 277 networking 19, 21–22, 80, 108, 126 metrics 55, 61, 69, 104, 201 networks 18, 22, 27, 49, 58, 83 collaborative 17 global 23, 80, 339 scholarly citation 200 article-level 201 journal-based 200–201 journal-level 200 j misconduct 8, 33, 63, 213–15, 217, nicotine replacement therapy 271, 283, 295–96, 349 nicotine replacement therapy 271, 283, 295–96, 349 223–24, 238, 267, 269, 279, 291–92, 296, 363 283, 295–96, 349 NIDA (National Institute on Drug Abuse) 11, 18, 22–23, 125 nonmaleficence 301, 305–6, 317 principle of 303, 308 Nordic countries 16, 77, 95 Nordic Studies on Alcohol and Drugs 8, 41, 170, 348 norms 325 accepted ethical 309, 317 institutional 360 participatory 359 notification 102, 117, 268, 275, 287, 292 novice author 135, 137, 174 novice researchers 94–95, 106 O objectivity 285, 330, 339, 347, 356 apparent 202 author’s 272 280 291–92, 296, 363 provable 269 provable 269 provable 269 p misrepresentation 193, 197, 270 misrepresentation 193, 197, 270 mistakes 142–45, 271, 307, 356 researcher’s 166 statistical 272 mixed methods research 156, 171 monographs 169, 177 monograph style 95 moral issues 304, 344 moral jeopardy 343, 348 moral reasoning issues 7, 299, 307 multiauthorship 209, 211, 224 mistakes 142–45, 271, 307, 356 researcher’s 166 statistical 272 esea c e s 66 statistical 272 mixed methods research 156, 171 monographs 169, 177 monograph style 95 moral issues 304, 344 moral jeopardy 343, 348 moral reasoning issues 7, 299, 307 multiauthorship 209, 211, 224 novice author 135, 137, 174 novice researchers 94–95, 106 N N names 91, 95, 101, 104, 211–12, 214, 249, 253, 270, 287, 291, 309, 311, 314, 344–45 names 91, 95, 101, 104, 211–12, 214, 249, 253, 270, 287, 291, 309, 311, 314, 344–45 O objectivity 285, 330, 339, 347, 356 apparent 202 author’s 272, 280 O addiction-related 54 ambiguous 285 Publishing Addiction Science 376 professional judgment 281 scientist’s 330 obligations 280–81, 294, 317, 325, 343 observational studies 139, 180, 189, 205 Office of Research Integrity 288–90, 293, 297 online access 24, 52, 69 online journals 51–52, 104, 108 online publishing 12 online repositories 67 open-access journals 5, 47, 52, 54 63, 69, 108, 263 professional judgment 281 scientist’s 330 plagiarism 7–8, 33, 49, 63, 214, 224, 268–70, 288–89, 291, 295, 301, 304, 307, 316–17, 363 avoiding 289–90, 297 PLoS, 46, 204, 296, 298, 351 PloS Medicine 189, 279, 297, 351 policies 17–19, 25, 27, 29, 66–68, 72–73, 80–82, 92–93, 100, 105–7, 170, 214–15, 217, 284, 294–95 internal 340 open-access 47, 55 organizational 305 populations 26, 31, 74, 105, 152, 157, 234, 271, 302–3, 341 local 127 minority 303 vulnerable 115, 302, 330 postdoctoral fellows 6, 89–90, 92–94, 98, 109, 100, 208, 221, 226, 310 posters 38, 66 power 115–16, 90, 160, 214, 335, 352 explanatory 159 statistical 122, 186, 236, 318 obligations 280–81, 294, 317, 325, 343 304, 307, 316–17, 363 , observational studies 139, 180, 189, 205 Office of Research Integrity 288–90, 293, 297 online access 24, 52, 69 online journals 51–52, 104, 108 online publishing 12 online repositories 67 open-access journals 5, 47, 52, 54, 63, 69, 108, 263 policies 17–19, 25, 27, 29, 66–68, p , , , , , 72–73, 80–82, 92–93, 100, 105–7, 170, 214–15, 217, 284, 294–95 72 73, 80 82, 92 93, 100, 105–7, 170, 214–15, 217, 284, 294–95 105–7, 170, 214–15, 217, 284, 294–95 p j 63, 69, 108, 263 populations 26, 31, 74, 105, 152, 157, 234, 271, 302–3, 341 P local 127 P page charges 13, 39, 52, 104 participants 121, 123–25, 128, 142, 168, 175, 180, 183, 186, 209, 234, 242–44, 286–87, 315, 321 pathological gambling 16, 34, 38, 281, 333 patients 122–24, 126, 131, 139, 203, 209, 287, 313–14, 316 recruited 220 payment 103, 108, 176, 214, 278, 281, 312, 327, 328 peer review 13, 52–53, 58–59, 61–62, 104, 137, 188, 196, 245–48, 250, 252–53, 255, 261–63, 301, 307 exercise 65 guided 253 nonexistent 54 transparent 334 d l P page charges 13, 39, 52, 104 participants 121, 123–25, 128, 142, 168, 175, 180, 183, 186, 209, 234, 242–44, 286–87, 315, 321 pathological gambling 16, 34, 38, 281, 333 patients 122–24, 126, 131, 139, 203, 209, 287, 313–14, 316 recruited 220 payment 103, 108, 176, 214, 278, 281, 312, 327, 328 peer review 13, 52–53, 58–59, 61–62, 104, 137, 188, 196, 245–48, 250, 252–53, 255, 261–63, 301, 307 exercise 65 guided 253 nonexistent 54 transparent 334 i d j l 6 53 65 page charges 13, 39, 52, 104 participants 121, 123–25, 128, page charges 13, 39, 52, 104 participants 121, 123–25, 128, page charges 13, 39, 52, 104 participants 121, 123–25, 128, postdoctoral fellows 6, 89–90, 142, 168, 175, 180, 183, 186, 209, 234, 242–44, 286–87, 92–94, 98, 109, 100, 208, 221, 226, 310 pathological gambling 16, 34, 38, 281, 333 posters 38, 66 pathological gambling 16, 34, 38, 281, 333 PowerPoint presentations 8, 31, 62, 85, 110, 130, 150, 165, 171, 187, 202, 223, 241, 263, 294 predatory publishers 12, 32, 37, 52–55, 58, 62, 104, 108, 110 Preferred Reporting Items for Systematic Reviews and Meta-Analyses 139, 188, 204 PRISMA, 139, 177-180, 185, 188, 204 processing fees 52, 104 professional societies 9–11, 18–19, peer-reviewed journals 6, 53, 65, 21–22, 27, 29–30, 48, 51, 67, 92, 294, 335, 338–39 Project Cork 24, 66 Project Gutenberg 148 promotion 22, 54, 72, 104, 193, 200, 207, 255, 309, 313, 315, 333, 349, 359 21–22, 27, 29–30, 48, 51, 67, 67, 120, 135–37, 186, 203, 230, 245–46, 355, 358 PERIL analysis 342–48 permission 49, 53, 91, 209, 277, 290, 314–15 author’s 248 92, 294, 335, 338–39 promotion 22, 54, 72, 104, 193, 200, 207, 255, 309, 313, 315, piled higher and deeper 91, 111 333, 349, 359 Index 377 Index 377 proofreading 216, 254 proofs 146, 252 ProQuest Dissertations & Theses 98, 99, 177 R randomized controlled trials 32, 111, 124, 203, 234, 242, 271, 275, 283, 295, 349 Redundant publication 273, 276, 309–10 referees 145, 155, 157, 163–64, 166, 167, 170, 246, 316 references 60, 68, 70, 78–79, 114, 135, 144, 151–53, 175, 177, 195–97, 204, 230, 234, 273, 276 Q 196, 271–73, 276, 279, 291, qualitative addiction research 156, 166 qualitative articles 6, 127, 156–57, 163–64, 166–67, 169–70, 186, 234 qualitative research 6, 122–23, 126, 130, 141, 155–61, 166–67, 169–72, 241 qualitative researchers 127, 155, 157–58, 169 317, 325 R randomized controlled trials 32, 111, 124, 203, 234, 242, 271, 275, 283, 295, 349 h PsycINFO, 39–42, 52, 55, 57, 67–69, 73, 75, 151–52, 233 randomized controlled trials 32, 111, 124, 203, 234, 242, 271, 275, 283, 295, 349 275, 283, 295, 349 Publication ethics 225, 268, 288, 290, 295, 300 Publication ethics 225, 268, 288, 290, 295, 300 Publication lag 196 Redundant publication 273, 276, 309–10 Redundant publication 273, 276, 309–10 Publication lag 196 public health 46, 50, 65–66, 72, 171–72, 208, 212, 291, 295, 327, 340, 342, 348, 350, 352 references 60, 68, 70, 78–79, 114, 135, 144, 151–53, 175, publishers 4, 47, 53, 54, 57, 59, 68–69, 73, 96, 104, 146, 200, 270, 275–77, 300, 310, 313 177, 195–97, 204, 230, 234, 273, 276 publishing 3, 5–7, 47, 49–51, 54, 59, 72, 74, 90–91, 94–95, 105–10, 112, 273–74, 316–17, 319–20 academic 198, 205 early-career 94 redundant 276 scientific 5, 7–8, 12, 33, 51, 63, 72–73, 75, 110, 129, 214, 224, 267, 360, 363 publishing career 90, 136 publishing companies 51, 248 open-access 52 Publishing Qualitative Research 157 PubMed 24, 64, 67–68, 102, 106, 151–52, 229, 242, 249, 273 PubMed Central 67–68, 152 publishing 3, 5–7, 47, 49–51, 54, 59 72, 74, 90–91, 94–95, 105–10, 112, 273–74, 316–17, 319–20 academic 198, 205 early-career 94 redundant 276 scientific 5, 7–8, 12, 33, 51, 63, 72–73, 75, 110, 129, 214, 224, 267, 360, 363 publishing career 90, 136 publishing companies 51, 248 open-access 52 Publishing Qualitative Research 157 PubMed 24, 64, 67–68, 102, 106, 151–52, 229, 242, 249, 273 PubMed Central 67–68, 152 reference style 144 reference style 144 reference tool 58 RefWorks 153 rejection 103, 109–10, 231, 233 236–37, 239, 241, 248, 250–51, 258–59, 268, 275, 279, 284, 289, 292 236–37, 239, 241, 248, 250–51, 258–59, 268, 275, 279, 284, 289, 292 258–59, 268, 275, 279, 284, rejection letters 103, 104, 137 rejection letters 103, 104, 137 i 72–73, 75, 110, 129, 214, 224, rejection letters 103, 104, 137 rejection rates 262 reporting guidelines 138–39 reporting observational studies 189 reporting randomized trials 139, 199i rejection rates 262 267, 360, 363 reporting guidelines 138–39 139, 199 139, 199 reporting research findings 49 representativeness 158, 159, 180 reprimand 275–76, 289, 308, 317 re-publication 270, 275 reputations 55, 80, 135, 137, 193, 196, 271–73, 276, 279, 291, 317, 325 colleague’s 194 journal’s 59 scientific 213 research assistants 89, 91, 101, 208, 215, 310–11 research centers 9–10, 16–18, 29–30, 280, 330, 340, 357 research design 72, 93, 124, 128, 131, 158, 185, 215 reporting research findings 49 representativeness 158, 159, 180 reprimand 275–76, 289, 308, 317 re-publication 270, 275 reputations 55, 80, 135, 137, 193, reporting research findings 49 representativeness 158, 159, 180 reprimand 275–76, 289, 308, 317 re-publication 270, 275 reputations 55, 80, 135, 137, 193, PubMed 24, 64, 67–68, 102, 106, 151–52, 229, 242, 249, 273 p re-publication 270, 275 PubMed Central 67–68, 152 reputations 55, 80, 135, 137, 193, Q U right 81 Ulrich’s Periodicals Directory 54 Undeclared COIs 284 Undeserved authorships 208 unethical research 300–301 unfair authorship practices 280, 292 universalism 356, 360 Unpublished manuscript 34, 298 g thesis 67, 90, 92, 94–96, 98–100, 107–9, 113–14, 359 compilation 89 graduate 177 master’s 112, 147 publishing graduate-level 90 thesis advisor 100 thesis authorship 112 thesis defense 100 thesis research 117–18 g thesis 67, 90, 92, 94–96, 98–100, 107–9, 113–14, 359 compilation 89 graduate 177 master’s 112, 147 publishing graduate-level 90 thesis advisor 100 thesis authorship 112 thesis defense 100 thesis research 117–18 qualitative addiction research 156, 166 qualitative addiction research 156, 166 colleague’s 194 journal’s 59i qualitative articles 6, 127, 156–57, 163–64, 166–67, 169–70, scientific 213 215, 310–11 130, 141, 155–61, 166–67, research centers 9–10, 16–18, 29–30, 280, 330, 340, 357 169–72, 241 research design 72, 93, 124, 128, qualitative researchers 127, 155, 157–58, 169 qualitative researchers 127, 155, 157–58, 169 g 131, 158, 185, 215 378 Publishing Addiction Science 378 reviewer feedback 104, 260 reviewer guidelines 129 reviewer instructions 257 reviewers 4, 7, 81, 109, 128–30, 141–46, 158–59, 229, 231–41, 245–62, 269, 271–72, 274, 238, 335–36, 338 potential 214, 249–50, 256 selection of 232, 249 reviewers comments 146, 254 Reviewers’ reports 6, 229, 238, 252 review process 52, 59, 197, 222, 238, 246–48, 250–51, 253, 255–57, 259, 269, 318 peer 245, 356 unblinded 257 revisions 59, 97, 215, 219, 222, 231–32, 238–40, 247, 251, 254, 259, 261–62, 278, 286, 307 final 175, 222 major 98, 237, 248, 258, 308 research fields 60, 77, 191 research findings 25, 97, 273, 288, 292, 325, 336, 338, 346 141–46, 158–59, 229, 231–41, 141–46, 158–59, 229, 231–41, 245–62, 269, 271–72, 274, 238, 245–62, 269, 271–72, 274, 238, research funding 9, 21–22, 28, 114, 135, 142, 257, 281–82, 324–25, 340-342, 347, 351 research funding 9, 21–22, 28, 114, 135, 142, 257, 281–82, 324–25, 340-342, 347, 351 335–36, 338 340-342, 347, 351 research grant applications 252, 288 research grant applications 252, 288 research grants 91, 125, 326, 345 research grants 91, 125, 326, 345 Research in Developing Countries 72 Research in Developing Countries 72 Research in Developing Countries 72 Research in Developing Countries 72 255–57, 259, 269, 318 research infrastructure 16, 31, 27, 105 peer 245, 356 unblinded 257 research integrity 281, 288–90, 293, 295–97, 326, 359 revisions 59, 97, 215, 219, 222, 231–32, 238–40, 247, 251, 254, 259, 261–62, 278, 286, 307 final 175, 222 major 98, 237, 248, 258, 308 research methods 25, 76, 143, 167, 170, 172, 271 research output 63, 69, 191, 195, 197, 200, 202 research participants 168, 269, 286–87, 296, 302–3, 306, 325 S S SAGER Guidelines 181, 188 SALIS, 22–23 SALIS directory 33 sample 78, 125, 142–43, 162, 167, 169, 180, 318 representative 183, 234 small 167 sample size 185–86, 243 inadequate 76 sampling 98, 128, 158, 162 snowball 158 subject-level 186 sampling methods 158 sanctions 238 legal 276 Science Citation Index 59–60, 70 Science Citation Index impact factor 203 science databases 178, 201 Science Editor’s Handbook 85, 263 science writer 52, 211, 278 research practices 267, 269, 275, 279, 292, 294, 332 research productivity 214 research protocols 116, 121, 125, 163, 220, 286, 315 SAGER Guidelines 181, 188 research practices 267, 269, 275, 279, 292, 294, 332 SALIS, 22–23 research protocols 116, 121, 125, 163, 220, 286, 315 research question 121, 128, 142–43, 163–64, 166, 168–69, 175–76, 178, 184–86, 272, 301, 341 research societies 19, 22, 200, 357 joining international 106 Research Society on Alcoholism 19–21, 33, 56 research training 27, 120, 284 resubmission 91, 04, 230, 238–39, 247, 256 retractions 268, 273, 275, 279, 284, 289–90 re-use text 275–76, 295 review articles 48, 89, 173–74, 176, 187, 193, 196, 218, 254, 256, 274, 283, 307, 312–14 research societies 19, 22, 200, 357 joining international 106 Research Society on Alcoholism 19–21, 33, 56 research training 27, 120, 284 resubmission 91, 04, 230, 238–39, 247, 256 retractions 268, 273, 275, 279, 284, 289–90 re-use text 275–76, 295 review articles 48, 89, 173–74, 176, 187, 193, 196, 218, 254, 256, 274, 283, 307, 312–14 sampling methods 158 g resubmission 91, 04, 230, 238–39, sanctions 238 247, 256 legal 276 retractions 268, 273, 275, 279, 284, 289–90 Science Citation Index 59–60, 70 retractions 268, 273, 275, 279, 284, 289–90 re-use text 275–76, 295 review articles 48, 89, 173–74, 176, 187, 193, 196, 218, 254, 256, Science Editor’s Handbook 85, 263 science writer 52, 211, 278 274, 283, 307, 312–14 Index 379 Index 379 self-citation rates 195 self-plagiarism 147, 270, 275–76, 295, 317 services 4, 10, 32, 42, 49, 57–58, 67, 82, 123, 220, 255, 291, 310, 330 list indexing/abstracting 54 online plagiarism detection 297 technical 116, 220 translation 83 self-citation rates 195 self-plagiarism 147, 270, 275–76, 295, 317 scientific articles 3–4, 6, 13, 37, 48, 51, 71, 75, 81, 126, 135–37, 218, 220, 355, 359i services 4, 10, 32, 42, 49, 57–58, 67, 82, 123, 220, 255, 291, 310, 330 scientific communication 4, 6, 38, 48, 56, 71, 87, 148, 357, 359, 361i scientific community 12, 55, 97, 155, 200, 207, 217, 238, 246, 285, 294, 314, 330, 339–40, 355–57i scientific fraud 7, 213, 267–69, 279, 290–92, 304, 318i smoking 111, 140, 165, 203, 271–72, 283, 297, 326–27, 329 scientific fraud 7, 213, 267 69, 279 290–92, 304, 318 scientific integrity 4, 7, 54, 217, 222, 227, 337–38, 355, 357, 359–60i scientific integrity 4, 7, 54, 217, 222, 227, 337–38, 355, 357, 359–60i scientific integrity 4, 7, 54, 217, 222, 227, 337–38, 355, 357, smoking cessation 34, 170, 203, 295, 332, 349 social-aspects/public-relations organizations 331, 333 social media 63, 102, 198, 201, 307 social research 157, 286, 297, 302, 357 social-aspects/public-relations organizations 331, 333 social media 63, 102, 198, 201, 307 scientific literature 50, 129, 194, 196, 200, 215, 338i social media 63, 102, 198, 201, 307 social research 157, 286, 297, 302, 357 scientific misconduct 5, 7, 212, 214, 250, 252, 257–58, 267, 269–71, 274, 276, 292–94, 299, 338, 341 scientific misconduct 5, 7, 212, 214, 250, 252, 257–58, 267, 269–71, 274, 276, 292–94, 299, 338, 341 social sciences 8, 60, 64–65, 69–71, 75, 80, 197, 199, 204, 286, 293, 297 allegations of 213, 290, 292 prevention of 292, 360 Scientific writing 229, 242 Social Sciences Citation Index 70 Social Sciences Citation Index 70 Society for Research on Nicotine 20 Society for Research on Nicotine 20 i scientists 4, 9–10, 12–13, 16, 18, 22, 48–49, 136, 276, 292, 327–28, 340–41, 344–45, 347, 355–58 Society for the Study of Addiction (SSA) 19, 21, 34, 56, 174, 189 174, 189 basic 29 Source-Normalized Impact per Paper (SNIP) 61, 63 specialized addiction-studies programs 26 specialized libraries 10, 22–24, 29 statistical analyses 93, 116, 125, 149, 157, 161, 184, 214, 221, 231, 243, 309, 314, 318 behavioral 330 clinical 27 SCImago Journal Rank 61, 69, 200 search 16, 26, 47, 52, 57, 66–67, 69, 120–21, 129, 151–53, 175, 183, 185, 195, 229, 233, 356–57, 363 keyword 65, 152 search engines 24, 57, 58 search rules 201 search strategies 151–52 search terms 34, 151 self-citation 194–95, 197, 202–3, 271 search 16, 26, 47, 52, 57, 66–67, 69, 120–21, 129, 151–53, 175, 183, 185, 195, 229, 233, 356–57, 363 keyword 65, 152 search engines 24, 57, 58 search rules 201 search strategies 151–52 search terms 34, 151 self-citation 194–95, 197, 202–3, 271 search 16, 26, 47, 52, 57, 66–67, search 16, 26, 47, 52, 57, 66–67, 69, 120–21, 129, 151–53, 175, 183, 185, 195, 229, 233, 356–57, 363 keyword 65, 152 search engines 24, 57, 58 search rules 201 search strategies 151–52 search terms 34, 151 self-citation 194–95, 197, 202–3, 271 149, 157, 161, 184, 214, 221, 231, 243, 309, 314, 318 STROBE (Studies in Epidemiology) 139, 180, 189, 205 student authorship 92–93, 116 students 3, 5, 56, 58, 66, 69, 89–98, 100–101, 105, 109–10, 113– 18, 214, 216, 221, 316–17 postdoctoral 125 STROBE (Studies in Epidemiology) 139, 180, 189, 205 175, 183, 185, 195, 229, 233, 356–57, 363 search rules 201 80 Publishing Addiction Science 380 dissertation or 116 thesis topic 114 Thomson Reuters 52, 60–61, 63, 65, 69–70, 75 titles 24, 38, 57, 67, 69, 142, 144, 147, 149, 152, 164–65, 242, 274, 276 tobacco industry 184, 213, 278, 282, 321, 326–27, 332, 340–41, 351 trainees 90–91, 94, 104, 253, 257 postdoctoral 91 postgraduate 90 training 10, 16, 22, 25, 27, 107, 116, 243, 253, 292, 336, 361 ethical 292, 314 training programs 11, 25, 27, 29, 38 translation 144, 295, 320, 356 treatment research 18, 51, 76 triage 230–31 trials 122, 124, 128, 180, 226, 292, 296, 328, 332 cluster 139 independent 283, 332 industry-supported 283, 332 multicenter 226 multisite 18 nonrandomized controlled 139 pharmaceutical company 215 randomised 189, 242, 244 randomised controlled 123–24, 139, 143, 203 study design 116, 138, 160, 175, 177, 180, 277, 310 p Thomson Reuters 52, 60–61, 63, 65, 69–70, 75 study generalizability 184 style 80, 81, 96–97, 114, 127, 138, 142, 148, 153, 182, 231, 242 style 80, 81, 96–97, 114, 127, 138, 142, 148, 153, 182, 231, 242 titles 24, 38, 57, 67, 69, 142, 144, 147, 149, 152, 164–65, 242, 142, 148, 153, 182, 231, 242 style guide 136–38, 140 submissions 49, 53, 58, 108–9, style guide 136–38, 140 submissions 49, 53, 58, 108–9, 136–37, 169, 208, 213–14, tobacco industry 184, 213, 278, 282, 321, 326–27, 332, 340–41, 351 230–31, 246–52, 255–58, trainees 90–91, 94, 104, 253, 257 postdoctoral 91 postgraduate 90 260–62, 287–88, 310, 316 260–62, 287–88, 310, 316 first 81, 247 training 10, 16, 22, 25, 27, 107, 116, 243, 253, 292, 336, 361 rejected 248 , , , , ethical 292, 314 training programs 11, 25, 27, 29, 38 translation 144, 295, 320, 356 treatment research 18, 51, 76 triage 230–31 trials 122, 124, 128, 180, 226, 292, 296, 328, 332 cluster 139 independent 283, 332 industry-supported 283, 332 multicenter 226 multisite 18 nonrandomized controlled 139 pharmaceutical company 215 randomised 189, 242, 244 randomised controlled 123–24, 139, 143, 203 ethical 292, 314 training programs 11, 25, 27, 29, 38 translation 144, 295, 320, 356 treatment research 18, 51, 76 triage 230–31 trials 122, 124, 128, 180, 226, 292, 296, 328, 332 cluster 139 independent 283, 332 industry-supported 283, 332 multicenter 226 multisite 18 nonrandomized controlled 139 pharmaceutical company 215 randomised 189, 242, 244 randomised controlled 123–24, 139, 143, 203 274, 276, 290 Substance Abuse Librarians & Information Specialists 22, 70 survey findings 309 survey results 38 survey studies 269, 287 systematic review and meta- analysis 6, 173, 175, 188, 205 systematic reviews 62, 64, 107, 139–40, 174–78, 181, 184–89, 193, 202–4, 296, 351 Substance Abuse Librarians & Information Specialists 22, 70i trials 122, 124, 128, 180, 226, 292, survey findings 309 survey studies 269, 287 industry-supported 283, 332 y pp multicenter 226 systematic reviews 62, 64, 107, 139–40, 174–78, 181, 184–89, 193, 202–4, 296, 351 systematic reviews 62, 64, 107, 139–40, 174–78, 181, 184–89, 193, 202–4, 296, 351 T tenure 54, 91, 255, 325 terminology 10, 49, 110, 174, 177, 183, 196 right 81 T tenure 54, 91, 255, 325 terminology 10, 49, 110, 174, 177, 183, 196 V verbatim material 310 verbs, strong 148 Index 381 Index 381 writing assistance 216 writing book chapters 347 writing comments 257 writing habits 290 writing handicaps 148 writing letters 347 writing principles 148 writing reviews 174 writing style 137, 142, 146–48 W W weaknesses 144, 232, 236, 248, 253–54, 259, 261–62 weaknesses 144, 232, 236, 248, 253–54, 259, 261–62 weaknesses 144, 232, 236, 248, 253–54, 259, 261–62 Web of Science 39–41, 47–48, 52, 54–55, 57, 69–70, 176, 183, 199, 204 women 31, 165, 349 pregnant 165 words 57, 137, 149, 152, 161, 174, 239, 246, 288–89, 363 nonstigmatizing 49 World Health Organization 50, 72, 87–88, 108, 120, 331, 348, 360–61 Web of Science 39–41, 47–48, 52, 54–55, 57, 69–70, 176, 183, 199, 204 women 31, 165, 349 pregnant 165 women 31, 165, 349 pregnant 165 women 31, 165, 349 pregnant 165 words 57, 137, 149, 152, 161, 174, 239 246 288–89 363 words 57, 137, 149, 152, 161, 174, 239, 246, 288–89, 363 nonstigmatizing 49 younger researchers 359 World Health Organization 50, 72, 87–88, 108, 120, 331, 348, 360–61 Publishing Addiction Science is a comprehensive guide for addiction scientists facing the complex process of contributing to scholarly journals. Written by an international group of addiction journal editors and their colleagues, it discusses how to write research articles and systematic reviews, choose a journal, respond to reviewers’ reports, become a reviewer, and resolve the often difficult author ship, ethical and citation issues that arise in addiction science publishing. As a “Guide for the Perplexed,” Publishing Addiction Science helps novice as well as experienced researchers to deal with these challenges. It is suitable for univer sity courses and forms the basis of the training workshops offered by the Interna tional Society of Addiction Journal Editors (ISAJE). Co-sponsored by ISAJE and the scientific journal Addiction, the third edition of Publishing Addiction Science gives special attention to the challenges faced by researchers from developing and non-English-speaking countries and features new chapters on guidance for clinician-scientists and the growth of infrastructure and career opportunities in addiction science. P R A I S E F O R T H E S E C O N D E D I T I O N : …this is an easy to read and valuable book that serves to demystify the publication pro cess. …this is an easy to read and valuable book that serves to demystify the publication pro cess. Heather Black, Research Assistant, Queen Margaret University, UK; Alcohol & Alcoholism …a remarkable compendium of practical tips combined with experience and wisdom. Nidal Moukaddam, MD, PhD, University of Texas Health Science Center, USA; Addictive Disorders and their Treatment PAS [Publishing Addiction Science] shares editors’ insights into publishing while raising re searchers’ consciousness regarding the global issues of addiction and the political threats to scientific integrity. PAS is an invaluable tool for novice researchers and a must read for addiction researchers from LAMI countries…. Brett Engle, PhD, LCSW, Assistant Professor, Barry University School of Social Work, USA; Journal of Groups in Addiction & Recovery www. u biqu it ypr e ss. c o m www. u biqu it ypr e ss. c o m
https://openalex.org/W2778166711	http://thescipub.com/pdf/10.3844/ajeassp.2017.900.907	English	null	Adaptive Video Quality Model for UHD Video Broadcasting Using the Principle of Inclusion	American journal of engineering and applied sciences	2,017	cc-by	5,075	Higher Dynamic Range (HDR), WCG & Rec.2020 UHD technology allows for a greater array of colours to be perceived by viewers. Rec.709 captures 35% of the natural view, while Rec.2020 captures 75%. The wide range of colours is going to radically enhance the picture quality of a UHD video (Kenichiro et al., 2014). Keywords: Broadcast, DVB, UHD, BER Keywords: Broadcast, DVB, UHD, BER Urvashi Pal and Horace King College of Engineering and Science, Victoria University, Melbourne, Australia Article history Received: 24-08-2017 Revised: 25-10-2017 Accepted: 18-11-2017 Corresponding Author: Urvashi Pal College of Engineering and Science, Victoria University, Melbourne, Australia Email: Urvashi.pal@live.vu.edu.au Article history Received: 24-08-2017 Revised: 25-10-2017 Accepted: 18-11-2017 Abstract: In this study, an adaptive video quality algorithm is developed for Ultra High Definition (UHD) video broadcasting through Digital Video Broadcasting by Satellite 2nd Generation (DVB-S2), where three conditions are responsible for enhancing or reducing the quality of a video signal received by the DVB-S2 Set-Top-Box (STB). The conditions are: Coverage area, Distance between transmitter and receiver and Separation distance. These conditions are responsible for the required Signal to Noise Ratio (SNR), resultant Bit Error Rate (BER) and the overall capacity of the system. Based on these conditions, received parameters of an HD or UHD video vary; and the quality viewed by the user changes. Therefore, in this study, we have proposed an algorithm based on the future broadcast scenario where the broadcasters will be dealing with simulcasting of multiple video standards of HD and UHD, varying in resolution, frame rate, codec and more. This algorithm is developed using the Principle of Inclusion. Corresponding Author: Urvashi Pal College of Engineering and Science, Victoria University, Melbourne, Australia Email: Urvashi.pal@live.vu.edu.au Video Compression At present, MPEG-4 video compression format is being used to watch HD channels on our HDTVs. HEVC is the new video compression method, developed especially to compress the huge data of UHD and has been adopted for its transmission by DVB (Advanced Television, 2014). HEVC offers 50% higher video compression and quality as compared to MPEG-4 and therefore, will make the transmission of UHD content more efficient by saving the bandwidth significantly. © 2017 Urvashi Pal and Horace King. This open access article is distributed under a Creative Commons Attribution (CC- BY) 3.0 license. High Frame Rates (HFR) Frame rate used till now for HD is 25fps (frames per second), but for UHD we will be dealing with 50fps, 100fps or even higher. Increasing the frame rate increases the smoothness of a video, especially for high motion contents (Limmer and Chabrol, 2014). In other words, increased information per second of the video with more frames enhances the smoothness and colour rendition (Pal and King, 2015a). The future of video broadcasting not just lies in resolution, but also in new technologies like High Frame Rate (HFR), Wide Colour Gamut (WCG), High Efficiency Video Coding (HEVC) and more. These video parameters contribute towards the ultimate viewing experience (Vignelles and Marshall, 2015). However, the availability of required resources for the bandwidth hungry Ultra High Definition (UHD) video will be a challenge that the broadcasters need to overcome. In such a scenario, allocating dedicated resources to maintain a fixed quality of video will result in the wastage of resources or video outage, in case the resources are not enough to deliver the promised quality. An adaptive video quality model using the principle of inclusion as given in this study, resolves this problem by efficiently allocating the available resources (channel capacity, coverage area, distance between transmitter and receiver and separation distance), by trading off the video quality. American Journal of Engineering and Applied Sciences American Journal of Engineering and Applied Sciences BER vs. SNR Results The results show that for a Rician Fading Channel (K = 5), BER decreases to 10−6 level for most of the MODCOD schemes, except 32APSK, which is a complex modulation scheme to be decoded successfully in the presence of heavy noise. Coverage Area: Distance between Transmitter and Receiver Coverage Area: Distance between Transmitter and Receiver The link budget model according to Friss-Free-Space Path Loss formula is: r t t r L P P G G P = + + − (2) (2) Problem Formulation Correlation of Channel Capacity and BER vs. SNR Results Correlation of Channel Capacity and BER vs. SNR Results For a successful transmission and reception of a UHD video, it is important that every block in the broadcast chain must be upgraded. This will lead to an overall increase in the cost of production and broadcasting but the enhanced video quality with richer colours and dynamic motion range makes the effort totally worth it (Pal and King, 2017). Still, at the moment, broadcasters will opt for a trade off in video quality by artificially upscaling a lower resolution content rather than using the original high resolution content in the initial phase of broadcasting (Intelsat, 2015). Using Shannon Capacity Theorem and SNR results from the above experiment, capacity of the channel is calculated using Equation 1 and plotted against its BER values, in Fig. 4: 2 1 C S log B N   = +     (1) (1) Where: Where: Where: C = Capacity of the channel in bits/second B = Bandwidth of the channel in Hertz S = Signal power in Watts N = Noise power in Watts C/B = Bits/seconds/hertz C = Capacity of the channel in bits/second The availability of numerous options to select from for a UHD and HD video will itself create confusion in the future broadcast scenario for the Direct to Home (DTH) operators. It is also important that advanced hardwares support interoperability at every stage, which will take time. Many video standards with varying resolutions, frame rates and compression, as depicted in Fig. 1, will have to support future transmissions (SES, 2013). Therefore, an adaptive stream methodology is proposed in this study, which uses a statistical approach to assign the best signal stream as per the reception criteria. p y B = Bandwidth of the channel in Hertz S = Signal power in Watts N = Noise power in Watts C/B = Bits/seconds/hertz B = Bandwidth of the channel in Hertz Figure 4 shows that the maximum capacity of a channel is reached at 10−6 for a Rician Fading Channel. Also, the maximum capacity is reached earlier by 32APSK and 16APSK, as compared to 8PSK and QPSK. This shows that, even though M-PSK has a lower symbol rate than M-APSK, its probability of error is also low. Therefore, more reliable information can be transmitted though M-PSK than M-APSK. Resolution UHDTV has a resolution of 3840×2160 pixels, which is four times the resolution of HDTV. This means that there is four times more information displayed on screen, which is one of the factors to enhance the video quality (Eutelsat, 2015). Urvashi Pal and Horace King / American Journal of Engineering and Applied Sciences 2017, 10 (4): 900.907 DOI: 10.3844/ajeassp.2017.900.907 (DVB, 2005). The results achieved are for a Rician Fading Channel, at K = 5 and SNR = 20dB. The noise channel consists of a Rician Fading Channel, Correlated Phase Noise and AWGN. BER vs. SNR graph is generated for a range of modulation and coding schemes, as given in Fig. 3. However, to use HEVC, broadcasters will have to invest in upgraded infrastructure, which will take time and cost a lot of money. Television Size The ideal size of a UHDTV is supposed to be around 55” to 80”. Based on the size of television, viewing distance is calculated to maintain the maximum perceived angular resolution because there are limits to what an eye can perceive (TT, 2013). If you sit too close to the TV, you will be seeing the unwanted individual pixels and if you sit too far, you won't be able to observe all the details in the video. That means, if you sit too far away from a UHDTV, the UHD content will look like HD. As a result, the viewing distance for a UHDTV is half of what is required for HDTV. BER vs. SNR Results Experimental Model and Results The following experiment is performed to understand the signal behaviour in a broadcast communication channel. Using the BER vs. SNR results, its correlation with the channel capacity, separation distance and coverage area is analysed. Based on this analysis, a statistical adaptive video quality algorithm is developed for UHD video broadcasting through DVB-S2, using the principle of inclusion. Analysis of Service Area Separation Distance Suppose, frequency range from 57 to 64 GHz is being used, the constraint on transmit power is Pt ≤ 40dBm. If thermal noise is the primary source of interference, the required sensitivity (Sr) at the receiver can be calculated as: Spectrum efficiency is a function of the size of the broadcaster’s coverage area and the separation distance between these coverage areas. To obtain the maximum achievable efficiency of spectrum use, which is a function of both the size of the broadcaster’s coverage area and the distance separating them, broadcasters are packed in a regular hexagonal constellation, as shown in Fig. 6, to achieve the highest average density of broadcasters on a per area basis (Bettancourt and Peha, 2015). Consider a statistical path loss model where the median path loss depends only on the distance from each transmitter. For a traditional broadcaster, a circle in the hexagon represents the interference-limited coverage area, centred at the transmitter, with radius Rtrad equal to the distance between the transmitter and the nearest point on the edge of the coverage area. Where, Ctrad is the minimum distance between coverage areas of two traditional broadcasters. rS NF F SNR = + + (4) (4) Where: NF = The noise floor calculated by thermal noise: N = kTWF NF = The noise floor calculated by thermal noise: N = kTWF NF = The noise floor calculated by thermal noise: N = kTWF F = The noise figure (optimistically) assumed to be 0 dB F = The noise figure (optimistically) assumed to be 0 dB F = The noise figure (optimistically) assumed to be 0 dB SNR = The Signal to Noise Ratio at the receiver SNR = The Signal to Noise Ratio at the receiver k = Boltzmann’s constant k = Boltzmann’s constant T = The room temperature (typically 290K) For the 60 GHz systems, the noise floor is calculated as -76 dBm. To ensure adequate performance at the receiver, the minimum received power should be greater than or equal to the required sensitivity as expressed in Equation 5: The maximum fraction of area that can be covered by traditional broadcasters divided by the area of their respective hexagonal tile in the lattice, is given by: 2 10 4 116 d SNR log π   ≤ −   λ   (5) (5) ( ) 2 2 . DVB-S2 Model = Transmit power Pt = Transmit power Pr = Received power at distance ‘d’ Using the generic DVB-S2 model, as explained in detail in (Pal and King, 2015b), information bits are extracted from a UHD video and transmitted through the MATLAB built DVB-S2 model, as given in Fig. 2 Gt and Gr = Antenna gain for transmit and receive antennas respectively (both assumed to be 0 dB for simplicity) 901 Urvashi Pal and Horace King / American Journal of Engineering and Applied Sciences 2017, 10 (4): 900.907 DOI: 10.3844/ajeassp.2017.900.907 Urvashi Pal and Horace King / American Journal of Engineering and Applied Sciences 2017, 10 (4): 900.907 DOI: 10.3844/ajeassp.2017.900.907 Fig. 1: Co-existence of multiple video standards Fig. 2: DVB-S2 block schematic 902 Fig. 1: Co-existence of multiple video standards Fig. 2: DVB-S2 block schematic Fig. 3: BER vs. SNR graph Fig. 1: Co-existence of multiple video standards Fig 2: DVB S2 block schematic Fig. 2: DVB-S2 block schematic g 902 Urvashi Pal and Horace King / American Journal of Engineering and Applied Sciences 2017, 10 (4): 900.907 DOI: 10.3844/ajeassp.2017.900.907 Fig. 4: Capacity vs. BER graph for rayleigh and rician fading channel (legend same as Fig. 3.) Fig. 4: Capacity vs. BER graph for rayleigh and rician fading channel (legend same as Fig. 3.) The received signal strength is dominated by the distance from the transmitter and the receiver and the general path loss model can be expressed as in Equation 3 where ‘λ’ is the wavelength corresponding to the center frequency fc and ‘n’ is the path loss exponent which can be approximated as 2 (Wang and Zhang, 2012): taking into account the contribution by SNR in Equation 5. Substituting the values of Shannon Capacity ‘C’ from Equation 1 into Equation 6, ‘d’ is calculated. Using SNR values from the above experiment, we plot Distance ‘d’ between the Transmitter and Receiver vs. BER graph for Rician Fading Channel. The results in Fig. 5 show that as ‘d’ decreases, Signal strength increases and errors decrease. (Values assumed: n = 2, λ = 10, π = 3.14). ( ) 10 4 10 n L d P dB log π   =   λ   (3) (3) Analysis of Service Area Separation Distance When the separation distance is high, error probability from the adjacent coverage area is low. But when the separation distance is small, noise is high and coverage area is small. Large coverage areas require larger separation distance to maintain low interference from adjacent cells. Therefore, there is a trade-off between transmit power and noise as spectrum efficiency increases with coverage area and decreases with separation distance. Hence, the larger the coverage area, the lower the spectrum efficiency. As a result, it is efficient in terms of spectrum Analysis of Service Area Separation Distance 2 3 0.5 trad trad trad R R C π η = + (7) (7) Channel capacity can be calculated according to the Shannon capacity and the relationship between the capacity and communication distance is then given by: Where: Where: η = Spectral Efficiency (Modulation Efficiency of the MODCOD scheme) 10 4 116 10 10 2 1 10 n d log C Blog π   −   λ       ≤ +       (6) (6) Rtrad = Distance between transmitter and receiver Ctrad = Separation distance between two coverage areas 903 Urvashi Pal and Horace King / American Journal of Engineering and Applied Sciences 2017, 10 (4): 900.907 DOI: 10.3844/ajeassp.2017.900.907 Fig. 5: Distance between transmitter and receiver vs. BER for Rician (legend same as Fig. 3.) Fig. 6: Hexagonal packing of co-channel traditional broadcasters Fig. 5: Distance between transmitter and receiver vs. BER for Rician (legend same as Fig. 3.) Fig. 5: Distance between transmitter and receiver vs. BER for Rician (legend same as Fig. 3.) Fig. 5: Distance between transmitter and receiver vs. BER for Rician (legend same as Fig. 3.) Fig. 6: Hexagonal packing of co-channel traditional broadcasters Fig. 6: Hexagonal packing of co-channel traditional broadcasters Substituting the values of spectral efficiency (DVB, 2005) and distance between transmitter and receiver from the previous section, in Equation 7, Ctrad is calculated and plotted again the BER. efficiency to provide TV service to a given area by using many small individual coverage areas rather than few large coverage areas. The graph for separation distance vs. BER is plotted in Fig. 7, which shows that as the separation area decreases, BER or noise increases. As the distance between the transmitter and receiver increases, required transmit power to maintain a low BER increases. As the transmit power increases, the coverage area increases and the separation distance between two coverage areas decreases. When the separation distance is high, error probability from the adjacent coverage area is low. But when the separation distance is small, noise is high and coverage area is small. As the distance between the transmitter and receiver increases, required transmit power to maintain a low BER increases. As the transmit power increases, the coverage area increases and the separation distance between two coverage areas decreases. further simplifies Equation 9 such that: when they satisfy other conditions bj for ≠ . Therefore for any , ∈ 1,2,3,…, where ≠ Z (bi bj) denotes the number of elements in K that satisfy both of the conditions bi and bj. If 1 ≤ i, , ≤ are three distinct values, then (i j k) denotes the number of elements in ‘K’ satisfying each of the conditions bi, bj and bk. Therefore, for each 1 1 ' i i t,Z(b ) Z Z(b ) ≤≤ = − will denote the number of elements in ‘K’ that do not satisfy condition bi. However, if 1≤i, j≤t with ' ' i j i j,(b b ) ≠ equates to the number of elements in ‘K’ that do not satisfy either of the conditions bi or bj. Hence: when they satisfy other conditions bj for ≠ . Therefore for any , ∈ 1,2,3,…, where ≠ Z (bi bj) denotes the number of elements in K that satisfy both of the conditions bi and bj. If 1 ≤ i, , ≤ are three distinct values, then (i j k) denotes the number of elements in ‘K’ satisfying each of the conditions bi, bj and bk. Therefore, for each 1 1 ' i i t,Z(b ) Z Z(b ) ≤≤ = − will denote the number of elements in ‘K’ that do not satisfy condition bi. However, if 1≤i, j≤t with ' ' i j i j,(b b ) ≠ equates to the number of elements in ‘K’ that do not satisfy either of the conditions bi or bj. Hence: 1 2 1 1 2 ( ) ( ) ... ( )] [ ( )],1 ( ) k t k i i i K Z b Z b Z b K Z b b b k t t = + + + = ∑ ≤ ≤ (10) (10) The summation in Equation 10 is taken overall selections of size ‘k’ from the collection of ‘t’ conditions and Kk has J J t k       summands in it. Equation 9 and 10 can be used to establish whether all the conditions that enhance the video quality are met. If one of the conditions is not met then the user cannot view a video having the best quality parameters. further simplifies Equation 9 such that: This may mean a change in video parameters to the active set or may necessitate requiring more resources to be allocated. ' ' i j i j i j Z(b b ) Z [ Z(b ) Z(b ) Z(b b ) = − + + (8) (8) cannot view a video having the best quality parameters. This may mean a change in video parameters to the active set or may necessitate requiring more resources to be allocated. The 3rd term in Equation 8 is added because it is eliminated twice in the second term [Z (bi) + Z (bj)]. From Equation 8, it is possible to determine the number of elements of ‘K’ that satisfy none of the conditions bi, for 1≤i≤t. This is denoted by 1 2 3 ' ' ' ' t Z' Z(b b b ...b ) = and by expansion: Proposed Adaptive Video Quality Model for UHD Video Broadcasting Applying the Principle of Inclusion Suppose, number of cells in active set ≤ 4; respectively represented by b1, b2 and b3. Let ‘K’ be a set with \|K\| = Z in service area J and let b1, b2…bt be a collection of conditions, such as Coverage area, Distance between transmitter and receiver and Separation distance, satisfied by some or all of the elements of ‘K’. Some elements of ‘K’, such as SNR, BER and Capacity, may satisfy more than one of the conditions, whereas others may not satisfy any of them (King and Pal, 2015). Large coverage areas require larger separation distance to maintain low interference from adjacent cells. Therefore, there is a trade-off between transmit power and noise as spectrum efficiency increases with coverage area and decreases with separation distance. Hence, the larger the coverage area, the lower the spectrum efficiency. As a result, it is efficient in terms of spectrum Denote the number of elements in ‘K’ that satisfy condition bi for 1≤ i ≤ t by Z (bi). Elements of ‘K’ are only valid when they satisfy only condition bi as well as 904 Urvashi Pal and Horace King / American Journal of Engineering and Applied Sciences 2017, 10 (4): 900.907 DOI: 10.3844/ajeassp.2017.900.907 further simplifies Equation 9 such that: further simplifies Equation 9 such that: Proposed Adaptive Video Quality Model for UHD Video Broadcasting In Table 1, the best-case scenario is represented by case 1, where the coverage area is small, separation distance is big and the distance between transmitter and receiver is also small. Due to these factors, it is possible to achieve the BER of 10-6 at a SNR ≥ 6dB. Therefore, the capacity consumed is ≤ 75%. As a result of these conditions, the video quality viewed on TV has a resolution and frame rate of 2160p/50, colour profile of Rec.2020, with HEVC codec. Such a video must be viewed on TV screen of size ≥ 55”. However, as the conditions vary, the resultant video quality also varies, as given in the table below. 1 1 1 2 3 1 ' ( ) ( ) ( ) ..... ( 1) ( ..... ) i i j i t j t t i j k t i j k t Z Z Z b Z b b Z b b b Z b b b b ≤≤ ≤≤ ≤< < ≤ = − + − + + − ∑ ∑ ∑ (9) (9) Using Equation 9 for ‘’ ∈ and that ‘s’ satisfies none of the conditions in Equation 9; it is clear that ‘s’ is counted once in Z’ and once in Z but will not be counted in any of the other three terms in Equation 9. It is evident that the number of elements in ‘K’ that satisfy at least one of the conditions i where 1 ≤ ≤ is given by Z (b1 or b2 or … or bt) = Z – Z’. The following notation 905 Fig. 7: Separation distance vs. BER graph for Rician (legend same as Fig. 3) Fig. 7: Separation distance vs. BER graph for Rician (legend same as Fig. Proposed Adaptive Video Quality Model for UHD Video Broadcasting 3) 905 Urvashi Pal and Horace King / American Journal of Engineering and Applied Sciences 2017, 10 (4): 900.907 DOI: 10.3844/ajeassp.2017.900.907 Urvashi Pal and Horace King / American Journal of Engineering and Applied Sciences 2017, 10 (4): 900.907 DOI: 10.3844/ajeassp.2017.900.907 Table 1: Video quality result in different scenarios applying the principle of inclusion Scenario Video result 1 Coverage area = Low Res/fps = 2160p/50 Separation distance = High Colour = Rec2020 d = Low Codec = HEVC BER = 10−6 Ideal TV Size ≥ 55” SNR≥6dB Best video quality using Capacity ≤ 75% future resources 2 Coverage area = Low Separation distance = Low Res/fps = 1080/50p d = Low Colour = Rec2020 BER = 10−6 Codec = HEVC SNR≥6dB Ideal TV Size = 45-55” Capacity ≤75% Using many resources 3 Coverage area = Low Separation distance = Low Res/fps = 1080/25p d = High Colour = Rec709 BER = 10−5 Codec = MPEG-4 SNR ≥ 6dB Ideal TV Size = 40-50” Capacity ≤75% Using available resources 4 Coverage area = High Res/fps = 1080/25i Separation distance = Low Colour = Rec709 d = High Codec = MPEG-4 BER = 10−4 Ideal TV Size = 30-40” SNR ≥ 5dB Resources used more than necessary Capacity ≤75% 5 Coverage area = Very high Res/fps = 720/25i Separation distance = Very low Colour = Rec709 d = Low Codec = MPEG-4 BER = 10−4 Ideal TV Size = 20-30” SNR ≥ 4dB Unacceptable resource usage Capacity > 75% 6 Coverage area = Very high Separation distance = Very Low No video received d = Very high Video outage BER = 10−2 Should not be allowed to happen SNR ≥ 20dB Capacity > 75% * d = Distance between transmitter and receiver, Res/fps = Resolution/frame rate Table 1: Video quality result in different scenarios applying the principle of inclusion Scenario Video result 1 Coverage area = Low Res/fps = 2160p/50 Separation distance = High Colour = Rec2020 d = Low Codec = HEVC BER = 10−6 Ideal TV Size ≥ 55” SNR≥6dB Best video quality using Capacity ≤ 75% future resources 2 Coverage area = Low Separation distance = Low Res/fps = 1080/50p d = Low Colour = Rec2020 BER = 10−6 Codec = HEVC SNR≥6dB Ideal TV Size = 45-55” Capacity ≤75% Using many resources 3 Coverage area = Low Separation distance = Low Res/fps = 1080/25p d = High Colour = Rec709 BER = 10−5 Codec = MPEG-4 SNR ≥ 6dB Ideal TV Size = 40-50” Capacity ≤75% Using available resources 4 Coverage area = High Res/fps = 1080/25i Separation distance = Low Colour = Rec709 d = High Codec = MPEG-4 BER = 10−4 Ideal TV Size = 30-40” SNR ≥ 5dB Resources used more than necessary Capacity ≤75% 5 Coverage area = Very high Res/fps = 720/25i Separation distance = Very low Colour = Rec709 d = Low Codec = MPEG-4 BER = 10−4 Ideal TV Size = 20-30” SNR ≥ 4dB Unacceptable resource usage Capacity > 75% 6 Coverage area = Very high Separation distance = Very Low No video received d = Very high Video outage BER = 10−2 Should not be allowed to happen SNR ≥ 20dB Capacity > 75% * d = Distance between transmitter and receiver, Res/fps = Resolution/frame rate Table 1: Video quality result in different scenarios applying the principle of inclusion Scenario Video result 1 Coverage area = Low Res/fps = 2160p/50 Separation distance = High Colour = Rec2020 d = Low Codec = HEVC BER = 10−6 Ideal TV Size ≥ 55” SNR≥6dB Best video quality using Capacity ≤ 75% future resources 2 Coverage area = Low Separation distance = Low Res/fps = 1080/50p d = Low Colour = Rec2020 BER = 10−6 Codec = HEVC SNR≥6dB Ideal TV Size = 45-55” Capacity ≤75% Using many resources 3 Coverage area = Low Separation distance = Low Res/fps = 1080/25p d = High Colour = Rec709 BER = 10−5 Codec = MPEG-4 SNR ≥ 6dB Ideal TV Size = 40-50” Capacity ≤75% Using available resources 4 Coverage area = High Res/fps = 1080/25i Separation distance = Low Colour = Rec709 d = High Codec = MPEG-4 BER = 10−4 Ideal TV Size = 30-40” SNR ≥ 5dB Resources used more than necessary Capacity ≤75% 5 Coverage area = Very high Res/fps = 720/25i Separation distance = Very low Colour = Rec709 d = Low Codec = MPEG-4 BER = 10−4 Ideal TV Size = 20-30” SNR ≥ 4dB Unacceptable resource usage Capacity > 75% 6 Coverage area = Very high Separation distance = Very Low No video received d = Very high Video outage BER = 10−2 Should not be allowed to happen SNR ≥ 20dB Capacity > 75% * d = Distance between transmitter and receiver, Res/fps = Resolution/frame rate Fig. Acknowledgement Kenichiro, M., T. Yamashita, Y. Nishida and M. Sugawara, 2014. Color management for wide-color- gamut UHDTV production. SMPTE Motion Imaging J., 124: 19-27. DOI: 10.5594/j18528 Victoria University colleagues that helped in the detailed discussion during the research period are greatly acknowledged. Limmer, C. and M. Chabrol, 2014. Ultra high definition market outlook and next steps. Proposed Adaptive Video Quality Model for UHD Video Broadcasting 8: Required distance between transmitter and receiver to maintain 3×10-5 BER based on Fig. 7 results (approximate depiction) 2 Fig. 8: Required distance between transmitter and receiver to maintain 3×10-5 BER based on Fig. 7 results (approximate depiction) Fig. 8: Required distance between transmitter and receiver to maintain 3×10-5 BER based on Fig. 7 results (approximate depiction) 906 Urvashi Pal and Horace King / American Journal of Engineering and Applied Sciences 2017, 10 (4): 900.907 DOI: 10.3844/ajeassp.2017.900.907 Urvashi Pal and Horace King / American Journal of Engineering and Applied Sciences 2017, 10 (4): 900.907 DOI: 10.3844/ajeassp.2017.900.907 DVB, 2005. User guidelines for the second generation system for broadcasting, interactive services, News Gathering and other broadband satellite applications (DVB-S2). Digital Video Broadcasting. Ethics SES, 2013. Next generation of TV viewing: Developing the path for Ultra HD. Publication of this paper will not lead to any ethical issue. TT, 2013. The trade-off between image resolution and field of view: The influence of lens selection. Vignelles, A. and D. Marshall, 2015. Broadcast in the age of disruption. Conclusion In this study, experimental results obtained for BER vs. SNR for a UHD video signal, have been used to calculate the system capacity, spectral efficiency and the distance between transmitter and receiver. Using the results of these parameters, an adaptive video quality scenario is proposed for load balancing, using the principle of inclusion. Such a model will help in varying the video parameters as per the available resources, to prevent any kind of signal outage in the future broadcast scenario. Eutelsat, 2015. Ultra HD Guidebook. Eutelsat, 2015. Ultra HD Guidebook. Intelsat, 2015. At the forefront of 4K: Live, True 4K ultra high definition television, end-to-end video transmission over satellite. King, H. and U. Pal, 2015. A statistical approach to determine handover success using the principle of inclusion and load variation on links in wireless networks. IJICTA. Author’s Contributions Urvashi Pal: Has contributed towards the research plan development, problem formulation, experimental simulation, data analysis and writing of the manuscript. Pal, U. and H. King, 2017. Cost increase due to UHD video broadcasting as compared to HD. SMPTE Conf. Pal, U. and H. King, 2015a. DVB-S2 channel estimation and decoding in the presence of phase noise for non- linear channels. IJICTA. y g p Horace King: Has contributed towards the research plan development, problem formulation and data analysis and manuscript editing. Pal, U. and H. King, 2015b. Effect of UHD HFR on video transmission. SMPTE Conference. References Advanced Television, 2014. DVB approves UHDTV HEVC delivery profile. Wang, J. and H. Zhang, 2012. Capacity on 60 GHz wireless communication system over fading channels. J. Networks. Bettancourt, R.E. and H.M. Peha, 2015. On the trade-off between spectrum efficiency and transmission cost in traditional and SFN-based broadcast television. IEEE DySPAN. 907
https://openalex.org/W2766128433	https://europepmc.org/articles/pmc5653814?pdf=render	English	null	Regiospecific Synthesis of Ring A Fused Withaferin A Isoxazoline Analogues: Induction of Premature Senescence by W-2b in Proliferating Cancer Cells	Scientific reports	2,017	cc-by	18,877	Regiospecific Synthesis of Ring A Fused Withaferin A Isoxazoline Analogues: Induction of Premature Senescence by W-2b in Proliferating Cancer Cells Received: 19 May 2017 Accepted: 19 September 2017 Published: xx xx xxxx Faheem Rasool1,2, Debasis Nayak1,3, Archana Katoch1,3, Mir Mohd Faheem1,3, Syed Khalid Yousuf1,4, Nazar Hussain1,2, Chetan Belawal5, N. K. Satti5, Anindya Goswami1,3 & Debaraj Mukherjee1,2 Induction of premature senescence represents a novel functional strategy to curb the uncontrolled proliferation of malignant cancer cells. This study unveils the regiospecific synthesis of novel isoxazoline derivatives condensed to ring A of medicinal plant product Withaferin-A. Intriguingly, the cis fused products with β-oriented hydrogen exhibited excellent cytotoxic activities against proliferating human breast cancer MCF7 and colorectal cancer HCT-116 cells. The most potent derivative W-2b triggered premature senescence along with increase in senescence-associated β-galactosidase activity, G2/M cell cycle arrest, and induction of senescence-specific marker p21Waf1/Cip1 at its sub-toxic concentration. W-2b conferred a robust increase in phosphorylation of mammalian checkpoint kinase-2 (Chk2) in cancer cells in a dose-dependent manner. Silencing of endogenous Chk2 by siRNA divulged that the amplification of p21 expression and senescence by W-2b was Chk2-dependent. Chk2 activation (either by ectopic overexpression or through treatment with W-2b) suppressed NM23-H1 signaling axis involved in cancer cell proliferation. Finally, W-2b showed excellent in vivo efficacy with 83.8% inhibition of tumor growth at a dose of 25 mg/kg, b.w. in mouse mammary carcinoma model. Our study claims that W-2b could be a potential candidate to limit aberrant cellular proliferation rendering promising improvement in the treatment regime in cancer patients. Natural products, particularly steroids, have been employed as a powerful tool for deciphering new biological tar- gets1,2. In the last two decades, the search for biologically active steroids has led to the successful development of emerging heterocyclic steroid derivatives3,4. The main driving force towards the preparation of such compounds primarily confers upon the modification of the receptor-binding ability by chemical transformation of the extant functional groups for the reduction or elimination of the undesirable side effects and also modulation of phar- macodynamic and pharmacokinetic properties5. Indeed, transforming parent bioactive natural steroids to more/ new bioactive ones via semisynthetic approach has enlightened researchers for paving way of drug development. Withaferin A (WA) is a naturally occurring steroidal lactone, the first member of the withanolide class of compounds derived from the medicinal plant Withania somnifera, commonly known as Ashwagandha or Indian winter cherry6. The presence of the steroidal framework has endowed WA with antiangiogenic prop- erties. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 19 May 2017 Accepted: 19 September 2017 Published: xx xx xxxx Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x Regiospecific Synthesis of Ring A Fused Withaferin A Isoxazoline Analogues: Induction of Premature Senescence by W-2b in Proliferating Cancer Cells Its tremendous potential to modulate various oncogenes and tumor-suppressor genes with appreciable in vivo activities, bioavailability and less toxicity have conferred the molecule a suitable anticancer candidate7,8. 1Academy of Scientific and Innovative Research (AcSIR), New Delhi, India. 2Natural Product Chemistry-Microbes, CSIR-Indian Institute of Integrative Medicine, Jammu, 180001, India. 3Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, 180001, India. 4Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Srinagar, 190005, India. 5Natural Product Chemistry-Plants, CSIR-Indian Institute of Integrative Medicine, Jammu, 180001, India. Faheem Rasool and Debasis Nayak contributed equally to this work. Correspondence and requests for materials should be addressed to A.G. (email: agoswami@iiim.ac.in) or D.M. (email: dmukherjee@iiim.ac.in) Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 1 www.nature.com/scientificreports/ Figure 1. Standardization of the reaction conditions for the cis-fused isoxazoline derivatives of WA.a aIn all cases WA (1 equiv), N-hydroxy-4-chlorobenzenecarboximidoyl chloride (1.2 equiv) in DMF was tak bIsolated yield after column chromatography. Figure 1. Standardization of the reaction conditions for the cis-fused isoxazoline derivatives of WA.a aIn all cases WA (1 equiv), N-hydroxy-4-chlorobenzenecarboximidoyl chloride (1.2 equiv) in DMF was bIsolated yield after column chromatography. Figure 1. Standardization of the reaction conditions for the cis-fused isoxazoline derivatives of WA.a aIn all cases WA (1 equiv) N hydroxy 4 chlorobenzenecarboximidoyl chloride (1 2 equiv) in DMF was Figure 1. Standardization of the reaction conditions for the cis-fused isoxazoline derivatives of WA.a aIn all cases WA (1 equiv), N-hydroxy-4-chlorobenzenecarboximidoyl chloride (1.2 equiv) in DMF was taken. bIsolated yield after column chromatography. g aIn all cases WA (1 equiv), N-hydroxy-4-chlorobenzenecarboximidoyl chloride (1.2 equiv) in DMF was taken. bIsolated yield after column chromatography. Among the five-membered heterocyclic compounds, 2-isoxazolines have gained tremendous attention from the medicinal chemists as structural building blocks of biologically active molecules and versatile intermedi- ates in organic synthesis9. The importance of isoxazolines also stem from their utility as precursors in the syn- thesis of 1,3-aminoalcohols, which are excellent starting materials for a wide variety of natural products and related compounds such as alkaloids and nucleoside antibiotics10. Thus, the isoxazoline ring system could be semi-synthetically manipulated in presence of bioactive natural product WA for the discovery of novel leads with anticancer therapeutic potential. Cellular senescence is regarded as a safeguard mechanism to protect the organism by preventing uncon- trolled proliferation of malignant cancer cells11. Regiospecific Synthesis of Ring A Fused Withaferin A Isoxazoline Analogues: Induction of Premature Senescence by W-2b in Proliferating Cancer Cells Senescent cells possess characteristic features including growth arrest, flattened cellular morphology, SA-β-gal activity, and augmentation of cell-cycle specific marker such as cyclin-dependent kinase inhibitor p2112. Premature senescence occurs in response to various exogenous and endogenous insults including DNA damage and reactive oxygen species (ROS) generation etc., which is inde- pendent of telomere length and number of replications13. Checkpoint kinase 2 (Chk2) is a universal tumor suppressor gene that is activated in response to various genotoxic threats including ionizing radiation (IR) or chemotherapies14. DNA double-strand breaks (DSBs) activate ataxia telangiectasia mutated (ATM) protein kinase that phosphorylates Chk2 at Thr68 and activates it15. The ATM and Chk2 act in a linear fashion to stabilize tumor suppressor p53 leading to either cell-cycle arrest or apoptosis15. Chk2 is an essential component to induce both replicative and premature senescence through cell-cycle arrest by activating p21 in a p53 dependent manner16. However, studies also found that Chk2 can activate senescence in cancer cells by inducing p21, independent of the p53 status of the cell17,18. Hence, Chk2 is a lucratic target that can be manipulated to promote senescence in proliferating cancer cells.h p g Though therapeutic agents and small molecule natural products such as doxorubicin, camptothecin, resvera- trol, triptolide etc., are reported to induce senescence by augmenting p21 through various mechanisms in human cancer cells19,20, the effect of WA and its derivatives on induction of premature senescence is yet to be examined. In this endeavour, we sought to examine the potential of fused 2-isoxazoline derivatives of WA to induce cyto- toxicity in human cancer cells by abrogating cell proliferation through the induction of premature senescence. Results Ch i Temperature played a vital role in obtaining one stereoisomer over other as major product, such as by decreasing the temperature from rt to 0 °C, stereoisomer W-1b, in which both H2 and H3 are β was obtained in major quantity (entries 5–7). Thus, from the optimization study we concluded that WA (1 equiv), triethylamine (0.1 equiv), aromatic hydroximidoyl chloride (1.2 equiv) in DMF at 0 °C for 3 h was the optimal reaction condi- tion for this cycloaddition reaction.h entry 8). Temperature played a vital role in obtaining one stereoisomer over other as major product, such as by decreasing the temperature from rt to 0 °C, stereoisomer W-1b, in which both H2 and H3 are β was obtained in major quantity (entries 5–7). Thus, from the optimization study we concluded that WA (1 equiv), triethylamine (0.1 equiv), aromatic hydroximidoyl chloride (1.2 equiv) in DMF at 0 °C for 3 h was the optimal reaction condi- tion for this cycloaddition reaction.h y The structure of the products W-1a and W-1b were elucidated by 1D and 2D NMR analysis (Tables 1 and 2). 1H and 13C NMR spectra of major strereoisomer W-1b revealed that the signals relating to B/C/D ring sys- tems remained largely unaltered. But the profound shifting of resonance positions of H2 and H3 from δ 6.0 and δ 7.0 to δ 4.82 and δ 5.10 respectively in the 1H NMR combined with the shifting of C2 and C3 signals from δ 132.8 and 146.7 to 59.1 and 84.2 respectively in 13C NMR provided the clear indication of formation of 2-isoxazoline ring in W-1b (Table 1). The coupling constant of 11.9 Hz between H2 and H3 was consistent with the 2,3-(cis)-annulations of the heteroring. Similar types of signal shifts were observed for the other isomer W-1a (Table 2). However in the NOESY of W-1a, peaks for H2 and H4 showed a strong correlation whereas no such correlation was observed in the spectrum of W-1b indicating β,β ring juncture (Fig. 2A). From the mechanistic point of view, depending on the dipole orientation of nitrile oxide relative to the double bond, four diastere- omers (two cis and two trans) are possible from the cycloaddition. Further, the regioisomer in which the oxygen of the nitrile oxide is attached to the β-carbon of the α,β-unsaturated system is preferred due to the favourable Large-Large HOMO-LUMO favourable orbital interaction (Fig. 2B). Results Ch i Chemistry of Withaferin A isoxazolines. Although there are plenty of reports available in the literature for 1,3-dipolar cycloaddition of nitrile oxide with alkenes21, but there are limited reports of it when the alk- ene is a part of internal α,β-unsaturated cyclic system22. Fused 2-isoxazoline derivatives of WA were prepared by reacting WA with aromatic hydroximidoyl chlorides (precursors of nitrile oxides), obtained from the corre- sponding aromatic aldehydes via two steps. We initiated our optimization study by taking WA and N-hydroxy-4- chlorobenzenecarboximidoyl chloride in DMF as summarized in Fig. 1. Initially, two stereoisomeric isoxazoline products namely W-1a, W-1b were obtained in lower yield (20%) using diisopropylethylamine (DIPEA) as base (Fig. 1, entry 1) at elevated temperature. In order to increase the yield of the reaction, we explored various bases in different proportion to find that triethylamine was the most appropriate for this cycloaddition (Fig. 1, entries 1–7). Again triethylamine in catalytic amount (10 mol %) was more effective than stoichiomeric amount (Fig. 1, Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 2 www.nature.com/scientificreports/ Position WA 13C NMR W-1b 13C NMR 1H NMR (DMSO, 400 MHz) 1H NMR (DMSO, 400 MHz) 1 — 203.3 — 203.6 2 6.0 (d, J = 9.8 Hz) 132.8 4.82 (d, J = 12 Hz) 59.1 3 7.0 (dd, J = 9.8, 6.3 Hz) 146.7 5.10 (dd, J = 12.00, 3.6 Hz) 84.2 4 3.5 (dd, J = 6.3, 4.2 Hz) 70.1 3.46 (d, J = 3.4 Hz) 72.3 4-OH 5.6 (d, J = 4.2 Hz) 6.13 (d, J = 3.3 Hz) 5 — 64.7 — 62.4 6 3.1 (brs) 60.2 3.3 (bs) 57.2 18 0.5 (s) 12.8 0.48 (s) 11.6 19 1.2 (s) 17.7 1.12 (s) 15.2 21 0.7 (d, J = 6.3 Hz) 16.4 0.78 (d, J = 6.6 Hz) 13.5 22 4.2 (m) 79.0 4.2 (d, J = 12.3 Hz) 77.9 23 2.5 (m) 28.8 2.5 (m) 29.7 24 — 156.3 — 155.0 25 — 126.9 — 125.9 26 — 166.9 — 165.7 27a,b 4.02 (dq, J = 16.6, 5.3) 56.0 4.10 (dq, J = 16.6; 5.3) 56.3 27-OH 4.52 (t, J = 5.3) 4.5 (t, J = 5.3) — 28 1.88 (s) 21.4 2.04 (s) 20.3 Table 1. 1H NMR and 13C NMR assignments of WA and W-1b. Table 1. 1H NMR and 13C NMR assignments of WA and W-1b. entry 8). Results Ch i The attack of the dipole from above the general plane of the sterane framework (the β side) is less feasible in WA because of steric interactions with 4,5,10 substituents, forming the stereoisomer having β,β-ring juncture in major quantity. Finally, we established the structures of both the stereoisomers by HMBC and HSQC and all signals are listed in Tables 1 and 2. The reaction proceeded well with other substituted benzonitrile oxides, most of which formed the fused isoxazoline having the β,β-ring juncture as the major stereoisomer. The para substituted benzonitrile oxides reacted much more effec- tively than the meta or ortho substitution ones (Fig. 3). Among para substituted aromatic aldehydes, those with a electron withdrawing group (EWG) reacted much faster and with better yield. With aliphatic hydroximidoyl chloride, the reaction yielded a complex mixture of different products which could not be characterized. Withaferin A isoxazolines are cytotoxic and the most potent derivative (W-2b) induces prema- ture senescence in proliferating cancer cells. Recent reports reveal that WA possesses anti-tumor activ- ities against breast and colorectal cancer models23–26. In order to check the cytotoxic activities of WA isoxazoline derivatives against human breast cancer MCF7 and colorectal cancer HCT-116 cells, we performed cell viability assay through MTT dye reduction method and the cytotoxicity results are presented in terms of IC50 values in Table 3. The parent WA and isoxazoline derivatives of WA (W-1a to W-12b) displayed significant cytotoxic activ- ities against both the MCF7 and HCT-116 cells. In general, the fused isoxazolines having β,β-ring juncture were found to be more potent than the other corresponding stereoisomer having α,α-ring juncture. The nitro deriv- ative W-2b showed the most promising cytotoxic effects among the synthesized molecules in both MCF7 and HCT-116 cell lines with comparatively less toxicity towards human normal breast epithelial (fR2) cells (Fig. 4A,B). We examined the cytotoxicity of W-2b in both dose-dependent as well as time-dependent manner in these three cell lines (Table 4). Results Ch i The 24 h IC50 values of W-2b were 0.881 ± 0.052 µM and 1.48 ± 0.129 µM, the 48 h IC50 values: 0.705 ± 0.059 µM and 1.25 ± 0.156 µM, and the 72 h IC50 values: 0.682 ± 0.075 µM and 1.03 ± 0.33 µM in MCF7 Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 3 www.nature.com/scientificreports/ position WA 13C NMR W-1a 13C NMR 1H NMR (DMSO, 400 MHz) 1H NMR (DMSO, 400 MHz) 1 — 203.3 — 204.2 2 6.0 (d, J = 9.8 Hz) 132.8 4.5 (m) 55.8 3 7.0 (dd, J = 9.8, 6.3 Hz) 146.7 4.9 (d, J = 10.9) 85.2 4 3.5 (dd, J = 6.3, 4.2 Hz) 70.1 3.3 (s) 71.8 4-OH 5.6 (d, J = 4.2 Hz) — 6.0 (s) — 5 — 64.7 — 63.1 6 3.1 (brs) 60.2 3.3 (s) 56.6 18 0.5 (s) 12.8 3.3 (s) 11.6 19 1.2 (s) 17.7 1.2 (s) 15.6 21 0.7 (d, J = 6.3 Hz) 16.4 0.9 (d, J = 6.3 Hz) 13.5 22 4.2 (m) 79.0 4.2 (d, J = 12.3 Hz) 78.0 24 — 156.3 — 155.1 25 — 126.9 — 125.9 26 — 166.9 — 165.8 27a,b 4.02 (dq, J = 16.6; 5.3) 56.0 4.1 (m) 54.9 27-OH 4.52 (t, J = 5.3) — 4.5 (m) — 28 1.88 (s) 21.4 2.0 (s) 20.3 Figure 2. (A) NOESY of W-1a and W-1b. (B) Regioselectivity in 1,3-dipolar cycloaddition reaction of aromatic nitrile oxides with α,β-unsaturated ketone of ring A of WA. Figure 2. (A) NOESY of W-1a and W-1b. (B) Regioselectivity in 1,3-dipolar cycloaddition reaction of aromatic nitrile oxides with α,β-unsaturated ketone of ring A of WA. Figure 2. (A) NOESY of W-1a and W-1b. (B) Regioselectivity in 1,3-dipolar cycloaddition reaction of arom nitrile oxides with α,β-unsaturated ketone of ring A of WA. and HCT-116 cells respectively (Table 4). However, the IC50 values of W-2b in fR2 cells were 39.66 ± 7.3 µM, 35.5 ± 6.12 µM, and 33.9 ± 8.23 µM in 24 h, 48 h, and 72 h respectively (Table 4). Senescence is an important tumor suppressive mechanism that works as a barrier to uncontrolled cell proliferation11. SA-β-gal activity is regarded as a specific marker for cells undergoing senescence12. Table 2. 1H NMR and 13CNMR of WA and W-1a. Results Ch i In order to check the ability of our test com- pound to induce premature senescence, we carried out SA-β-gal activity assays in MCF7 and HCT-116 cells following treatment with indicated concentrations of W-2b along with positive control doxorubicin for five days. Indeed, we noticed a remarkable increase in SA-β-gal positive cells (58% in MCF7 and 53% in HCT-116) follow- ing treatment with the sub-toxic concentrations of W-2b and characteristic senescent features - flattened cellular Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 4 www.nature.com/scientificreports/ Figure 3. Reaction of WA with various N-hydroxy benzenecarboximidoyl chlorides.a,b aReaction condition: WA (1 equiv), aromatic hydroximidoyl chloride (1.2 equiv), Triethylamine (0.1 equiv), in DMF at 0 °C for 3 h. bYields refer to the isolated yields after column chromatography. Figure 3. Reaction of WA with various N-hydroxy benzenecarboximidoyl chlorides.a,b aReaction condition: WA (1 equiv), aromatic hydroximidoyl chloride (1.2 equiv), Triethylamine (0.1 equiv), in DMF at 0 °C for 3 h. bYields refer to the isolated yields after column chromatography. Compounds MCF7 HCT-116 fR2 W-1a 3.8 ± 0.378 5.62 ± 0.412 45.8 ± 5.19 W-1b 1.64 ± 0.267 16.2 ± 0.676 39.2 ± 3.81 W-2a 5.82 ± 0.418 8.2 ± 0.211 52.1 ± 6.6 W-2b 0.705 ± 0.059 1.25 ± 0.156 35.5 ± 6.12 W-3a 95.5 ± 5.412 45.8 ± 2.343 >100 W-3b 1.82 ± 0.33 15.9 ± 1.67 85.3 ± 9.5 W-4a >100 19.6 ± 1.81 >100 W-4b >100 >100 >100 W-5a 3.52 ± 0.185 63.8 ± 5.433 >100 W-5b 9.7 ± 0.55 42.4 ± 3.189 91.25 ± 7.33 W-6a 2.28 ± 0.265 >100 >100 W-6b >100 11.51 ± 2.21 63.3 ± 2.5 W-7a 70.64 ± 5.18 8.1 ± 1.655 >100 W-8a 17.6 ± 1.676 7.5 ± 1.129 28.2 ± 4.21 W-9b >100 15.8 ± 3.311 66.23 ± 7.63 W-10a 58.3 ± 3.24 5.28 ± 0.561 98.1 ± 7.18 W-11a >100 24.5 ± 2.55 >100 W-11b 83.3 ± 6.512 4.16 ± 0.344 >100 W-12b 26.9 ± 2.224 5.29 ± 0.151 75.6 ± 4.82 WA 1.75 ± 0.376 2.58 ± 0.415 43.32 ± 3.77 Table 3. Cytotoxicity of WA and derivatives (48 h) in MCF7, HCT-116, and fR2 cells; IC50: µM. IC50 values are indicated as mean ± standard deviation of three independent experiments performed. Results Ch i Compounds MCF7 HCT-116 fR2 W-1a 3.8 ± 0.378 5.62 ± 0.412 45.8 ± 5.19 W-1b 1.64 ± 0.267 16.2 ± 0.676 39.2 ± 3.81 W-2a 5.82 ± 0.418 8.2 ± 0.211 52.1 ± 6.6 W-2b 0.705 ± 0.059 1.25 ± 0.156 35.5 ± 6.12 W-3a 95.5 ± 5.412 45.8 ± 2.343 >100 W-3b 1.82 ± 0.33 15.9 ± 1.67 85.3 ± 9.5 W-4a >100 19.6 ± 1.81 >100 W-4b >100 >100 >100 W-5a 3.52 ± 0.185 63.8 ± 5.433 >100 W-5b 9.7 ± 0.55 42.4 ± 3.189 91.25 ± 7.33 W-6a 2.28 ± 0.265 >100 >100 W-6b >100 11.51 ± 2.21 63.3 ± 2.5 W-7a 70.64 ± 5.18 8.1 ± 1.655 >100 W-8a 17.6 ± 1.676 7.5 ± 1.129 28.2 ± 4.21 W-9b >100 15.8 ± 3.311 66.23 ± 7.63 W-10a 58.3 ± 3.24 5.28 ± 0.561 98.1 ± 7.18 W-11a >100 24.5 ± 2.55 >100 W-11b 83.3 ± 6.512 4.16 ± 0.344 >100 W-12b 26.9 ± 2.224 5.29 ± 0.151 75.6 ± 4.82 WA 1.75 ± 0.376 2.58 ± 0.415 43.32 ± 3.77 Table 3. Cytotoxicity of WA and derivatives (48 h) in MCF7, HCT-116, and fR2 cells; IC50: µM. IC50 values are indicated as mean ± standard deviation of three independent experiments performed. Compounds MCF7 HCT-116 fR2 W-1a 3.8 ± 0.378 5.62 ± 0.412 45.8 ± 5.19 W-1b 1.64 ± 0.267 16.2 ± 0.676 39.2 ± 3.81 W-2a 5.82 ± 0.418 8.2 ± 0.211 52.1 ± 6.6 W-2b 0.705 ± 0.059 1.25 ± 0.156 35.5 ± 6.12 W-3a 95.5 ± 5.412 45.8 ± 2.343 >100 W-3b 1.82 ± 0.33 15.9 ± 1.67 85.3 ± 9.5 W-4a >100 19.6 ± 1.81 >100 W-4b >100 >100 >100 W-5a 3.52 ± 0.185 63.8 ± 5.433 >100 W-5b 9.7 ± 0.55 42.4 ± 3.189 91.25 ± 7.33 W-6a 2.28 ± 0.265 >100 >100 W-6b >100 11.51 ± 2.21 63.3 ± 2.5 W-7a 70.64 ± 5.18 8.1 ± 1.655 >100 W-8a 17.6 ± 1.676 7.5 ± 1.129 28.2 ± 4.21 W-9b >100 15.8 ± 3.311 66.23 ± 7.63 W-10a 58.3 ± 3.24 5.28 ± 0.561 98.1 ± 7.18 W-11a >100 24.5 ± 2.55 >100 W-11b 83.3 ± 6.512 4.16 ± 0.344 >100 W-12b 26.9 ± 2.224 5.29 ± 0.151 75.6 ± 4.82 WA 1.75 ± 0.376 2.58 ± 0.415 43.32 ± 3.77 Table 3. Cytotoxicity of WA and derivatives (48 h) in MCF7, HCT-116, and fR2 cells; IC50: µM. Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x Results Ch i IC50 values are indicated as mean ± standard deviation of three independent experiments performed. Table 3. Cytotoxicity of WA and derivatives (48 h) in MCF7, HCT-116, and fR2 cells; IC50: µM. IC50 values are indicated as mean ± standard deviation of three independent experiments performed. morphology and bluish nuclear stains were distinctly visible in both the cell lines compared to the vehicle treated cells (Fig. 4C,D). Albeit, the results of SA-β-gal staining demonstrated a substantial increase in the number of cells with distinct senescence phenotype in doxorubicin (100 nM) treated cells whereas very less number of senescent Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 5 www.nature.com/scientificreports/ Figure 4. W-2b is cytotoxic and induces premature senescence in cancer cells. (A) Structure of novel isoxazoline derivative of withaferin A (W-2b). (B) Graphs showing the percent cell viability of MCF7, HCT-116 and fR2 cells in response to logarithmic concentrations of W-2b for 24 h, 48 h, and 72 h. (C) Effect of vehicle, doxorubicin (100 nM), WA, and various concentrations of W-2b on SA-β-gal activity in MCF7, HCT-116, and fR2 cells. Original magnification 20x. (D) Bar graph showing quantification of SA-β-gal positive cells. Error bars: mean ± s.d. P < 0.05. (E) After treatment, MCF7 and HCT-116 cells were stained with DAPI containing mounting media for 15–20 min and observed for the formation of SAHF under Floid Cell Imaging Station. Insets (red boxes) showing magnified images for proper visualization of SAHF. Data are representative of three independent experiments. Original magnification 20x. Figure 4. W-2b is cytotoxic and induces premature senescence in cancer cells. (A) Structure of novel isoxazoline derivative of withaferin A (W-2b). (B) Graphs showing the percent cell viability of MCF7, HCT-116 and fR2 cells in response to logarithmic concentrations of W-2b for 24 h, 48 h, and 72 h. (C) Effect of vehicle, doxorubicin (100 nM), WA, and various concentrations of W-2b on SA-β-gal activity in MCF7, HCT-116, and fR2 cells. Original magnification 20x. (D) Bar graph showing quantification of SA-β-gal positive cells. Error bars: mean ± s.d. P < 0.05. (E) After treatment, MCF7 and HCT-116 cells were stained with DAPI containing mounting media for 15–20 min and observed for the formation of SAHF under Floid Cell Imaging Station. Insets (red boxes) showing magnified images for proper visualization of SAHF Data are representative of three Figure 4. W-2b is cytotoxic and induces premature senescence in cancer cells. Results Ch i (A) Structure of novel isoxazoline derivative of withaferin A (W-2b). (B) Graphs showing the percent cell viability of MCF7, HCT-116 and fR2 cells in response to logarithmic concentrations of W-2b for 24 h, 48 h, and 72 h. (C) Effect of vehicle, doxorubicin (100 nM), WA, and various concentrations of W-2b on SA-β-gal activity in MCF7, HCT-116, and fR2 cells. Original magnification 20x. (D) Bar graph showing quantification of SA-β-gal positive cells. Error bars: mean ± s.d. P < 0.05. (E) After treatment, MCF7 and HCT-116 cells were stained with DAPI containing mounting media for 15–20 min and observed for the formation of SAHF under Floid Cell Imaging Station. Insets (red boxes) showing magnified images for proper visualization of SAHF. Data are representative of three independent experiments. Original magnification 20x. Compounds MCF7 HCT-116 fR2 24 h, IC50: µM WA 9.18 ± 1.358 4.38 ± 1.673 51.12 ± 5.18 W-2b 0.881 ± 0.052 1.48 ± 0.129 39.66 ± 7.3 48 h, IC50: µM WA 1.75 ± 0.376 2.58 ± 0.415 43.32 ± 3.77 W-2b 0.705 ± 0.059 1.25 ± 0.156 35.5 ± 6.12 72 h, IC50: µM WA 1.46 ± 0.291 2.13 ± 0.55 38.34 ± 5.22 W-2b 0.682 ± 0.075 1.03 ± 0.33 33.9 ± 8.23 Table 4. Time-dependent cytotoxicity of WA and W-2b in MCF7, HCT-116, and fR2 cells. IC50 values are indicated as mean ± standard deviation of three independent experiments performed. Compounds MCF7 HCT-116 fR2 24 h, IC50: µM WA 9.18 ± 1.358 4.38 ± 1.673 51.12 ± 5.18 W-2b 0.881 ± 0.052 1.48 ± 0.129 39.66 ± 7.3 48 h, IC50: µM WA 1.75 ± 0.376 2.58 ± 0.415 43.32 ± 3.77 W-2b 0.705 ± 0.059 1.25 ± 0.156 35.5 ± 6.12 72 h, IC50: µM WA 1.46 ± 0.291 2.13 ± 0.55 38.34 ± 5.22 W-2b 0.682 ± 0.075 1.03 ± 0.33 33.9 ± 8.23 Table 4. Time-dependent cytotoxicity of WA and W-2b in MCF7, HCT-116, and fR2 cells. IC50 values are indicated as mean ± standard deviation of three independent experiments performed. Table 4. Time-dependent cytotoxicity of WA and W-2b in MCF7, HCT-116, and fR2 cells. IC50 values are indicated as mean ± standard deviation of three independent experiments performed. Table 4. Time-dependent cytotoxicity of WA and W-2b in MCF7, HCT-116, and fR2 cells. IC50 values are indicated as mean ± standard deviation of three independent experiments performed. Results Ch i cells were observed in presence of the parent molecule WA even at the higher concentration (2–3 µM). However, some early apoptotic population of cells were visible in WA treatment conditions (Fig. 4C,D). We also confirmed the effect of W-2b on fR2 cells through SA-β-gal staining and the results indicated significantly lower number of SA-β-gal positive cells (15–19%) at the sub-toxic doses of W-2b (1–2 µM) unveiling the selectivity of the molecule Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 6 www.nature.com/scientificreports/ towards proliferating cancer cells (Fig. 4C,D). For further confirmation, we performed senescence-associated heterochromatin foci (SAHF) formation assay through nuclear staining of the cells by DAPI containing mounting media12,27. We found a notable increase in SAHF formation with appearance of typical beaded nucleus in cells treated with W-2b/doxorubicin, whereas the nucleus of the cells treated with vehicle (DMSO) were totally devoid of such beaded appearances (Fig. 4E). Although there observed some SAHF formation in WA treated cells but the features of early apoptosis were predominating in these cells (Fig. 4E). The unlimited proliferative capability of cancer cells supports the growth of primary tumor that eventually leads to cancer progression and morbidity of the patient28. However, the onset of senescence further hinders the division and colony forming ability of prolif- erating cancer cells29. To check the effect of W-2b on proliferation of MCF7 and HCT-116 cells, we then employed colony formation assay. Our results obtained a significant number of colonies in vehicle treated wells, whereas, the colony formation was inhibited gradually at the lower doses (0.5 µM for MCF7 and 1.0 µM for HCT-116) and significantly (>70%) at the higher doses (1.0 µM for MCF7 and 1.5 µM for HCT-116) similar to the positive con- trol doxorubicin (100 nM) (Supplementary Fig. S1). As in most cases, the generation of reactive oxygen species (ROS) is linked with loss of cell proliferation and senescence30, we sought to determine the effect of W-2b on ROS generation in MCF7 and HCT-116 cells. Our results revealed that W-2b triggered sufficient quantity of mitochon- drial ROS at sub-toxic doses of the molecule within 48 h of treatment similar to the positive control H2O2 (10 µM), which further supports its anti-proliferative effects in these cells (Supplementary Fig. S2). Together, these findings demonstrate that W-2b is potentially cytotoxic and induces premature senescence in proliferating cancer cells. W-2b triggers cell-cycle arrest and p21Waf1/Cip1 upregulation. Results Ch i Though, therapeutics induced prema- ture senescence is directly correlated with cell-cycle arrest (preferably in the G0/G1 phase), studies also uncovered that senescence can be induced through G2/M arrest31. Our cell-cycle analysis experiments through propidium iodide staining demonstrated that W-2b (1.0 µM) arrested the MCF7 cells in G2/M phase (45.5%) compared to 13.03% in the vehicle treated cells (Fig. 5A). Alterations in the expression of vital genes occur in the cells undergoing senescence. Cyclin-dependent kinase inhibitor p21 is regarded as a senescent-specific marker, as its upregulation has been documented in almost all cells undergoing senescence12,18,30. The expression of p16INK4a (another cyclin-dependent kinase inhibitor) is also an indicative marker of senescence12. The p16-mediaed senes- cence takes place through the retinoblastoma (Rb) pathway suppressing the cyclin-dependent kinases leading to cell-cycle arrest32,33. The tumor suppressor p53 (also called as the guardian of the genome) is too considered as a molecular marker of cellular senescence. In response to DNA-damage, the p53 gets activated in cells trans- mitting directly signals to p21 for the execution of cell-cycle arrest, apoptosis, and/or senescence33,34. Induction of p21 suppresses the cyclin-dependent kinases (CDKs), thereby leading to cell cycle arrest and loss of cell pro- liferation12. Correspondingly, our western blotting experiments showed a robust increase in expression of p21 in both the MCF7 and HCT-116 cells along with upregulation of p16, p53, and concomitant downregulation of CDK-2 and CDK-4 expression with increasing concentrations of W-2b (Fig. 5B,C). The immunocytochemistry results further confirmed that W-2b could induce the expression and nuclear localization of p21 in these cells (Fig. 5D). These collective results strongly support that W-2b is a potential inducer of premature senescence through cell-cycle arrest and activation of p21 in proliferating cancer cells. W-2b confers induction of Chk2 in proliferating cancer cells. Chk2 kinase is an important tumor suppressor protein that preserves genomic stability of the organisms during critical situations such as DNA dam- age response by inducing cell-cycle arrest to facilitate either DNA repair or apoptosis or senescence15,16,18. It serves as a key target for small molecules from natural product/synthetic sources that can be modulated to circumvent cancer cell proliferation, invasion, and metastasis35,36. Though our results underscored a substantial provocation of premature senescence in two rapidly proliferating epithelial cancer cells (MCF7 and HCT-116), we hypothe- sized that W-2b might be targeting Chk2 at the molecular level. Results Ch i We carried out western blotting experiments with whole cell lysates prepared from MCF7 and HCT-116 cells after treatment with W-2b for 48 h. Interestingly, we found a significant increase in phosphorylation of Chk2 (Thr68) with increasing concentrations of W-2b, whereas no/negligible expression of pChk2 were observed in vehicle treated conditions (Fig. 6A,B). We also found a pro- portionate induction in expression of total Chk2 in a dose-dependent manner in both these cell lines (Fig. 6A,B). These results unveiled that W-2b causes phosphorylation-mediated induction of Chk2 in proliferating cancer cells. SiRNA-mediated knockdown of Chk2 abrogates the induction of senescence by W-2b. To confirm whether the molecule (W-2b) targets and induces the expression of Chk2 to provoke senescence, we performed SiRNA mediated knockdown of Chk2 in MCF7 cells followed by treatment with W-2b. The western blotting results disclosed three fold decrease in expression of Chk2 in Si-Chk2 treated wells with concomitant decrease in p21 expression. However, W-2b became unable to induce sufficient Chk2 and, thereby, p21 expres- sion in Si-Chk2 plus W-2b (1.0 µM) treated samples (Fig. 7A,B). The CDK-2 expression also remained unaffected in the Si-Chk2 plus W-2b treated conditions (Fig. 7A,B). The SA-β-gal assay further verified no such increase in SA-β-gal positive cells in Si-Chk2 plus W-2b (1.0 µM) treated cells compared to the W-2b treatment alone (Fig. 7C,D). These results strongly demonstrate that W-2b promotes premature senescence in a Chk2-dependent manner to limit aberrant cellular proliferation. Chk2 activation negatively regulates NM23-H1 signaling axis to control cell proliferation. To explore the molecular mechanism behind this regulation of cell proliferation and induction of senescence by W-2b, and how it induces p21 at the molecular level, we were curious to look at some regulators of cancer cell pro- liferation and malignancy. Emerging evidences demonstrate that NM23-H1 is an important regulator expressed Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 7 www.nature.com/scientificreports/ Figure 5. W-2b confers cell-cycle arrest and p21 induction in proliferating cancer cells. (A) Flow cytometric cell cycle analysis in MCF7 cells treated with vehicle and W-2b (1.0 µM) for 48 h. (B) MCF7 and HCT- 116 cells were treated with vehicle and increasing concentrations of W-2b for 48 h; whole cell lysates were prepared and subjected to western blot analysis for the expression of p21, p16, p53, CDK-2, CDK-4 and β-actin. (C) Densitometry analysis showing relative protein expression for the above western blots. Results Ch i (D) Immunofluorescence analysis results depicting the effect of vehicle, doxorubicin and W-2b on induction of p21 in MCF7 cells. Original magnification 20x. Data are representatives of two independent experiments. Figure 5. W-2b confers cell-cycle arrest and p21 induction in proliferating cancer cells. (A) Flow cytometric cell cycle analysis in MCF7 cells treated with vehicle and W-2b (1.0 µM) for 48 h. (B) MCF7 and HCT- 116 cells were treated with vehicle and increasing concentrations of W-2b for 48 h; whole cell lysates were prepared and subjected to western blot analysis for the expression of p21, p16, p53, CDK-2, CDK-4 and β-actin. (C) Densitometry analysis showing relative protein expression for the above western blots. (D) Immunofluorescence analysis results depicting the effect of vehicle, doxorubicin and W-2b on induction of p21 in MCF7 cells. Original magnification 20x. Data are representatives of two independent experiments. at the S-phase of the cell cycle leading to cell proliferation in human epithelial breast cancer cell line MCF- 10A and human peripheral blood lymphocytes37. To assess whether Chk2 activation could affect the intracellu- lar NM23-H1, we transiently overexpressed Chk2 with the help of GFP-Chk2 plasmid construct in MCF7 and HCT-116 cells. Western blotting of the whole cell lysates prepared from the above transfected cells revealed that ectopically overexpressed Chk2 strongly suppressed NM23-H1 expression in both these cell lines compared to the vector/GFP transfected cells (Fig. 8A,B). We also checked the effect of W-2b on NM23-H1expression; the results found a steady downregulation in the expression of NM23-H1 in a dose-dependent manner in MCF7 and HCT-116 cells after 48 h of treatment (Fig. 8C,D). We, then, investigated the expression of few downstream target genes of NM23-H1 involved in cell proliferation and tumor growth such as NF-kB, c-Myc and Cyclin D1; the synchronization of these genes regulate CDKs and p21. Our immunoblot results further validated a consistent downregulation in the expression of NF-kB (p65), c-Myc and Cyclin D1 expression in a dose-dependent treat- ment of W-2b in both these cell lines (Fig. 8C,D). Collectively, these data envisaged that Chk2 activation (either by ectopic overexpression or through treatment with W-2b) hinders NM23-H1 function and its target genes to regulate CDKs and p21 expression. W-2b is an effective inhibitor of tumor growth in vivo. Discussion We earlier demonstrated that blocking the irreversible covalent binding in active sites of WA by biological nucleo- philes via Michael addition at the β-position retains or even enhance its anticancer potential with minimal side effect making it more target specific38. Our recent approach towards the development of ring A modified deriva- tives of withaferin A successfully generated a 3-azido analogue with strong anticancer activities. Studies from our laboratory demonstrate that 3-azido withaferin A (3-AWA) inhibits invasion of cervical and prostate cancer cells and angiogenesis by modulating extracellular prostate-apoptosis response-4 (Par-4)39. 3-AWA mediated induc- tion of Par-4 regulates cellular β-catenin to control EMT and invasion in prostate cancer PC-3 and breast cancer MCF7 cells40. Another study by Rah et al. demonstrated that Par-4 mediated suppression of Bcl-2 by 3-AWA promotes switching of autophagy to apoptosis in prostate cancer cells41. Rasool et al. described that 3-AWA con- fers translational attenuation through dephosphorylation of eukaryotic translation initiation factor 4E (eIF4E), that results in inhibition of tumor growth and metastasis42. Keeping in mind the potential anticancer activities of 3-AWA, we contemplated that addition at both the α and β-position of the α,β-unsaturated carbonyl system may modify the biological activity further. Considering the promising biological importance of isoxazoline ring system, we intriguingly explored the synthesis of a combined motif involving ring A of WA using 1,3-dipolar cycloaddition reaction on arylnitrile oxides. Another goal was to investigate the regio- and stereoselectivity of the processes vis-à-vis the influence of steric and electronic factors on the ring closures and compare the reactivity of WA ring A against various nitrile oxides. We envisioned that the addition of steric bulk adjacent to the extant functional groups on C-3, essential for hormone-receptor binding, may contribute to a change in biological activ- ity and these derivatives may therefore deserve attention from a pharmacological aspect. Moreover, the vicinity of the angular methyl groups (C-19) to the reaction centre and also the rigidity of the sterane skeleton overall was thought to have a significant influence on the stereo and regiocontrol of the process. In this regard, our medicinal chemistry approach with the ring A modified WA isoxazolines found out a potential lead molecule (W-2b) with strong antiproliferative and antitumor activities. g Senescence is an important biological phenomenon in normal as well as cancer cells that facilitates as a barrier to control aberrant cell proliferation11. Results Ch i Though onset of premature senescence impedes the growth of primary tumor and further cancer progression, we were interested to evaluate the in vivo efficacy of W-2b on tumor growth in 4T1 mouse mammary carcinoma model. Upon intraperitoneal administra- tion of 25 mg/kg, b.w. of W-2b in each alternate day for two weeks, we found 83.8% inhibition in tumor volume compared to the 78.4% inhibition in 5-FU (25 mg/kg, b.w.) treated group (Fig. 9A,B). The tumor weight also reduced significantly and the results showed 91.2% inhibition in tumor weight in W-2b treated group whereas 86.7% in 5-FU treated group of animals compared to the normal saline treated group (Fig. 9C). Moreover, the ani- mals remained healthy without any serious side effects or mortality throughout the experimental period. These data strongly imply that W-2b is a potential and tolerable inhibitor of tumor growth similar to or more efficacious than the standard anticancer drug 5-fluorouracil. Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 8 www.nature.com/scientificreports/ Figure 6. W-2b triggers Chk2 activation in cancer cells. (A) MCF7 and HCT-116 cells were treated with vehicle and increasing concentrations of W-2b for 48 h; whole cell lysates were prepared and subjected to western blot analysis for the expression of pChk2 (T68), Chk2 and β-actin. (B) Densitometry analysis of the bands obtained from above experiments. Data are representatives of three independent experiments. Error bars: mean ± s.d. P < 0.05, *P < 0.01. Figure 6. W-2b triggers Chk2 activation in cancer cells. (A) MCF7 and HCT-116 cells were treated with vehicle and increasing concentrations of W-2b for 48 h; whole cell lysates were prepared and subjected to western blot analysis for the expression of pChk2 (T68), Chk2 and β-actin. (B) Densitometry analysis of the bands obtained from above experiments. Data are representatives of three independent experiments. Error bars: mean ± s.d. P < 0.05, *P < 0.01. Figure 6. W-2b triggers Chk2 activation in cancer cells. (A) MCF7 and HCT-116 cells were treated with vehicle and increasing concentrations of W-2b for 48 h; whole cell lysates were prepared and subjected to western blot analysis for the expression of pChk2 (T68), Chk2 and β-actin. (B) Densitometry analysis of the bands obtained from above experiments. Data are representatives of three independent experiments. Error bars: mean ± s.d. P < 0.05, *P < 0.01. Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x Discussion Our recent approach in this direction uncovered 4′-Demethyl deoxypodophyllotoxin glucoside (4DPG), a natural podophyllotoxin congener from the medicinal plant Podophyllum hexandrum as a strong anticancer candidate that modulates Chk2 activity to suppress proliferation, invasion and metastasis in aggressive cancer cells. The molecule (4DPG) also induces premature senescence in p53-defective invasive cancer cells35,36. In this study, our hunch for small molecule inducers of Chk2 found out a potential lead from Withaferin A isoxazoline derivatives (W-2b) that phosphorylates Chk2 (T68) and induces its expression in two rapidly proliferating cancer cells from diverse tissue origin (MCF7 and HCT-116). Evidence suggests that sub-lethal level of intracellular ROS generation could initiate premature senescence by inducing p21 expression through G1 arrest30. Being a key regulator of the cell cycle machinery, p21 controls cell proliferation and DNA replication through regulation of cyclin-dependent kinases (CDKs)30. Although p53 is a major transcription factor that regulates p21, studies also found that Chk2 can induce senescence in cancer cells via p21, irrespective of the p53 status of the cell18. Indeed, W-2b causes a significant increase in senescence phenotypes with remarkable SA-β-gal activity coupled with G2/M cell cycle arrest and induction of p21 in a dose-dependent manner (Fig. 10). Chk2, in turn, activates and stabilizes major tumor suppressor proteins viz. p53 to carry out antitumor activities by inducing cell cycle arrest and apoptosis15. Ample evidences demonstrate that Chk2 is a suitable target that can be modulated to promote senescence in proliferating cancer cells17,18. Though many small molecules from natural, semi-synthetic as well as synthetic sources are reported to induce premature senescence19, the finding of a potential compound that can activate Chk2 to limit uncontrolled proliferation in cancer cells is extremely limited. Our recent approach in this direction uncovered 4′-Demethyl deoxypodophyllotoxin glucoside (4DPG), a natural podophyllotoxin congener from the medicinal plant Podophyllum hexandrum as a strong anticancer candidate that modulates Chk2 activity to suppress proliferation, invasion and metastasis in aggressive cancer cells. The molecule (4DPG) also induces premature senescence in p53-defective invasive cancer cells35,36. In this study, our hunch for small molecule inducers of Chk2 found out a potential lead from Withaferin A isoxazoline derivatives (W-2b) that phosphorylates Chk2 (T68) and induces its expression in two rapidly proliferating cancer cells from diverse tissue origin (MCF7 and HCT-116). Evidence suggests that sub-lethal level of intracellular ROS generation could initiate premature senescence by inducing p21 expression through G1 arrest30. Discussion In response to various cellular stresses, including genotoxic stress by DNA damaging agents, proliferating cells cease to divide permanently and attain an enlarged morphology. Growth arrest occurs usually in the G1 or G2/M phases of the cell cycle. Senescence prevents the growth of damaged or stressed cells that are harmful for the organism12. Chk2 kinase is an important component of the DNA dam- age checkpoint signaling pathway, which is activated directly by ATM in response to the ionizing radiation14. Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 9 www.nature.com/scientificreports/ hk d b l Figure 7. Effect of silencing of Chk2 on induction of senescence by W-2b. (A) MCF7 cells were either transfected with scramble, Si-Chk2 or treated with vehicle, W-2b (1.0 µM) and Si-Chk2 plus W-2b (1.0 µM) for 48 h; the whole cell lysates were analysed for the expression of Chk2, p21, and CDK-2 through western blotting. (B) Graph shows densitometry analysis of the bands obtained from above experiments. (C,D) MCF7 cells were treated under above mentioned conditions and subjected to SA-β-gal staining. Original magnification 20x. Bar graphs: mean ± s.d. of three independent experiments. P < 0.05. Figure 7. Effect of silencing of Chk2 on induction of senescence by W-2b. (A) MCF7 cells were either transfected with scramble, Si-Chk2 or treated with vehicle, W-2b (1.0 µM) and Si-Chk2 plus W-2b (1.0 µM) for 48 h; the whole cell lysates were analysed for the expression of Chk2, p21, and CDK-2 through western blotting. (B) Graph shows densitometry analysis of the bands obtained from above experiments. (C,D) MCF7 cells were treated under above mentioned conditions and subjected to SA-β-gal staining. Original magnification 20x. Bar graphs: mean ± s.d. of three independent experiments. P < 0.05. Chk2, in turn, activates and stabilizes major tumor suppressor proteins viz. p53 to carry out antitumor activities by inducing cell cycle arrest and apoptosis15. Ample evidences demonstrate that Chk2 is a suitable target that can be modulated to promote senescence in proliferating cancer cells17,18. Though many small molecules from natural, semi-synthetic as well as synthetic sources are reported to induce premature senescence19, the finding of a potential compound that can activate Chk2 to limit uncontrolled proliferation in cancer cells is extremely limited. Discussion specific manner, its role in regulating metastasis and its loss of control in advance stages of the tumor progres- sion has been understood substantially47, how this protein can be modulated to induce premature senescence in proliferating cancer cells has not been explored till date. In this context, we hypothesized that Chk2 activation could suppress the oncogenic signaling of NM23-H1 in proliferating cancer cells. Surprisingly, our study found that ectopically induced Chk2 downregulates NM23-H1 expression substantially in both the MCF7 and HCT- 116 cells after 48 h of post transfection. We also found a steady downregulation in the expression of NM23-H1 in MCF7 and HCT-116 cells after 48 h of treatment with increasing concentrations of W-2b compared to the vehicle treated cells (Fig. 8C). The nuclear factor kB (NF-kB), one of the major transcription factors, is known to induce inflammatory responses, cancer cell survival, proliferation and tumor progression48. Though a splicing variant of NM23-H1 is reported to negatively regulate NF-kB signalling49, the effect of NM23-H1 itself on the regulation of NF-kB is poorly understood. The expression of c-Myc oncogene is associated with growth, differentiation and advancement of many tumors50. Studies also reported that c-Myc oncogene contains two responsive elements on its promoter for the NF-kB family of transcription factors and classical NF-kB (p65/p50) is a potential activator of the c-Myc promoter51. Accumulating evidence revealed that NF-kB activates Cyclin D1 expression at the tran- scriptional level through direct binding of NF-kB to multiple sites in the Cyclin D1 promoter and promote G1 to S phase transition52. Cell-cycle progression through G1 phase of the cell cycle requires the association of specific cyclin: cyclin-dependent kinase (CDK). Cyclin D1, CDK-2 and CDK-4 are the key players in this regard, forming stable complexes leading to G1/S transition53. In mammalian cells, p21 binds to and inhibits the kinase activity of several cyclin-dependent kinases including CDK-2 and CDK-4 leading to growth arrest at specific phases of the cell cycle54. Rationally, W-2b treatment suppressed the expression of NF-KB (p65), c-Myc, Cyclin D1 along with CDK-2 and CDK-4 at its sub-toxic doses in both MCF7 and HCT-116 cells (Fig. 10). These results also support the induction of p21 in these cells by W-2b in a dose-dependent manner. Discussion Being a key regulator of the cell cycle machinery, p21 controls cell proliferation and DNA replication through regulation of cyclin-dependent kinases (CDKs)30. Although p53 is a major transcription factor that regulates p21, studies also found that Chk2 can induce senescence in cancer cells via p21, irrespective of the p53 status of the cell18. Indeed, W-2b causes a significant increase in senescence phenotypes with remarkable SA-β-gal activity coupled with G2/M cell cycle arrest and induction of p21 in a dose-dependent manner (Fig. 10). y g NM23 gene belongs to the family of nucleoside diphosphate kinases (NDPKs) that catalyze the phosphoryla- tion of nucleoside diphosphates to their corresponding nucleoside triphosphates through oxidative phosphoryl- ation43. There are ten NM23 isoforms characterized so far; NM23-H1, encoding for a 17 kDa protein in human, is the most studied44. It was initially identified as a metastasis suppressor gene because of its reduced expression in highly metastatic mouse melanoma cells45. However, handful of evidence clearly demonstrates that overex- pression of NM23-H1 is associated with disease progression and poor patient survival in thyroid carcinomas, cervical cancer, neuroblastomas and osteosarcoma patients46. Though the expression of NM23-H1 in a cell cycle Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 10 www.nature.com/scientificreports/ Figure 8. Effect of Chk2 activation on NM23-H1 signaling. (A) MCF7 and HCT-116 cells were transfected transiently with GFP and GFP-Chk2 plasmid construct for 48 h. Whole cell lysates were prepared and checked for the expression of Chk2, NM23-H1 and β-actin. (B) Bar graph showing relative protein expression as determined by densitometry analysis of the bands. (C) Cells were treated with indicated concentrations of W-2b for 48 h; whole cell lysates were subjected to immunoblotting experiments for checking the expression of NM23-H1, NF-kB, c-Myc, Cyclin D1 and β-actin. (D) Bar graph showing relative protein expression as determined by densitometry analysis of the bands. Blots are representatives of three independent experiments. Figure 8. Effect of Chk2 activation on NM23-H1 signaling. (A) MCF7 and HCT-116 cells were transfected transiently with GFP and GFP-Chk2 plasmid construct for 48 h. Whole cell lysates were prepared and checked for the expression of Chk2, NM23-H1 and β-actin. (B) Bar graph showing relative protein expression as determined by densitometry analysis of the bands. (C) Cells were treated with indicated concentrations of W-2b for 48 h; whole cell lysates were subjected to immunoblotting experiments for checking the expression of NM23-H1, NF-kB, c-Myc, Cyclin D1 and β-actin. Discussion p y p In conclusion, our study reports a potential lead from Withaferin A isoxazoline derivatives (W-2b) that induces premature senescence as an antitumor safeguard mechanism against proliferating cancer cells through activation of tumor suppressor Chk2. It’s (W-2b) strong in vivo efficacy and tolerability claim for its further devel- opment as a therapeutically relevant anticancer candidate. Discussion (D) Bar graph showing relative protein expression as determined by densitometry analysis of the bands. Blots are representatives of three independent experiments. specific manner, its role in regulating metastasis and its loss of control in advance stages of the tumor progres- sion has been understood substantially47, how this protein can be modulated to induce premature senescence in proliferating cancer cells has not been explored till date. In this context, we hypothesized that Chk2 activation could suppress the oncogenic signaling of NM23-H1 in proliferating cancer cells. Surprisingly, our study found that ectopically induced Chk2 downregulates NM23-H1 expression substantially in both the MCF7 and HCT- 116 cells after 48 h of post transfection. We also found a steady downregulation in the expression of NM23-H1 in MCF7 and HCT-116 cells after 48 h of treatment with increasing concentrations of W-2b compared to the vehicle treated cells (Fig. 8C). The nuclear factor kB (NF-kB), one of the major transcription factors, is known to induce inflammatory responses, cancer cell survival, proliferation and tumor progression48. Though a splicing variant of NM23-H1 is reported to negatively regulate NF-kB signalling49, the effect of NM23-H1 itself on the regulation of NF-kB is poorly understood. The expression of c-Myc oncogene is associated with growth, differentiation and advancement of many tumors50. Studies also reported that c-Myc oncogene contains two responsive elements on its promoter for the NF-kB family of transcription factors and classical NF-kB (p65/p50) is a potential activator of the c-Myc promoter51. Accumulating evidence revealed that NF-kB activates Cyclin D1 expression at the tran- scriptional level through direct binding of NF-kB to multiple sites in the Cyclin D1 promoter and promote G1 to S phase transition52. Cell-cycle progression through G1 phase of the cell cycle requires the association of specific cyclin: cyclin-dependent kinase (CDK). Cyclin D1, CDK-2 and CDK-4 are the key players in this regard, forming stable complexes leading to G1/S transition53. In mammalian cells, p21 binds to and inhibits the kinase activity of several cyclin-dependent kinases including CDK-2 and CDK-4 leading to growth arrest at specific phases of the cell cycle54. Rationally, W-2b treatment suppressed the expression of NF-KB (p65), c-Myc, Cyclin D1 along with CDK-2 and CDK-4 at its sub-toxic doses in both MCF7 and HCT-116 cells (Fig. 10). These results also support the induction of p21 in these cells by W-2b in a dose-dependent manner. Materials and Methods Biology. Cell culture and reagents. The cell lines used in this study were procured from American Type Culture Collection (ATCC), Manassas, USA and European Collection of Authenticated Cell Cultures (ECACC), Porton Down, Salisbury, UK. The MCF7, HCT-116, and fR2 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Sigma) in a humid- ified CO2 incubator (New Brunswick Galaxy 170 R) with 5% CO2. For the treatment, withaferin A and its Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 11 www.nature.com/scientificreports/ Figure 9. In vivo anti-tumor activity of W-2b in 4T1 mouse mammary carcinoma model. (A) After tumors were grown over the mammary fat pad, animals were injected with either vehicle (normal saline) or 5-FU (25 mg/kg, b.w.) or W-2b (25 mg/kg, b.w.) in each alternate days for two weeks. The tumors were dissected out carefully and photographed. (B) Bar graph showing tumor volume recorded at the end of the experiment. (C) Tumor weight was measured after sacrificing the animals at the end of the experiment. Error bars: mean ± s.d. P < 0.05. Figure 9. In vivo anti-tumor activity of W-2b in 4T1 mouse mammary carcinoma model. (A) After tumors were grown over the mammary fat pad, animals were injected with either vehicle (normal saline) or 5-FU (25 mg/kg, b.w.) or W-2b (25 mg/kg, b.w.) in each alternate days for two weeks. The tumors were dissected out carefully and photographed. (B) Bar graph showing tumor volume recorded at the end of the experiment. (C) Tumor weight was measured after sacrificing the animals at the end of the experiment. Error bars: mean ± s.d. P < 0.05. Figure 9. In vivo anti-tumor activity of W-2b in 4T1 mouse mammary carcinoma model. (A) After tumors were grown over the mammary fat pad, animals were injected with either vehicle (normal saline) or 5-FU (25 mg/kg, b.w.) or W-2b (25 mg/kg, b.w.) in each alternate days for two weeks. The tumors were dissected out carefully and photographed. (B) Bar graph showing tumor volume recorded at the end of the experiment. (C) Tumor weight was measured after sacrificing the animals at the end of the experiment. Error bars: mean ± s.d. P < 0.05. Figure 10. Schematic diagram represents the proposed mechanism of action of W-2b. W-2b triggers Chk2 activation in cancer cells, thereby, inhibiting cell proliferation by NM23-H1/NF-kB/c-Myc/Cyclin D1/CDK-2/ CDK-4 signaling axis. Materials and Methods Optical density was measured with the help of a UV-visible spectrophotom- eter coupled with microplate reader (TECAN, Infinite M200 Pro), the percent inhibition was calculated and IC50 values were determined with the help of GraphPad Prism software (GraphPad software Inc. CA, USA). SA-β-gal assay. The procedure was followed as described previously with some modifications27. Briefly, MCF7 and HCT-116 cells (0.4 × 106 per well) were seeded in six-well plates and treated with indicated concentrations of W-2b along with WA, vehicle and positive control doxorubicin for five days. Cells were accordingly washed twice with PBS, fixed with 4% paraformaldehyde for 5 min, rinsed twice with PBS and incubated with freshly prepared staining solution (40 mM citric acid/Na phosphate buffer, 5 mM K4[Fe(CN)6] 3H2O, 5 mM K3[Fe(CN)6], 150 mM sodium chloride, 2 mM magnesium chloride and 1 mg/ml X-gal in distilled water, pH 6.0) for 48 h at 37 °C. Stained cells were thoroughly washed and air dried in dark. Cells were then observed under bright field microscope (NIKON) for the SA-β-gal positive cells and images were captured at 20x magnification. SAHF detection. The senescence-associated heterochromatin foci (SAHF) detection method was carried out as previously described by our group27. About 20 × 103 cells/well were seeded in 8 well chamber slides and treated with vehicle, doxorubicin, WA, and W-2b for five days. Subsequently, these cells were washed with PBS and fixed with 4% paraformaldehyde (w/v) at room temperature for 10 min. Cells were then washed with PBS, stained and mounted with DAPI containing mounting media (Invitrogen). Fluorescence images were captured with Floid cell imaging station (Thermo Scientific) using 20x objective. Flow cytometric cell cycle analysis. MCF7 cells were seeded at a density of 0.4 × 106 cells/well in 35 mm dishes and incubated overnight at 37 °C and 5% CO2. Next day, cells were treated with vehicle and W-2b at indicated concentrations for 48 h. They were then trypsinized, centrifuged at 2000 rpm for 3 min, washed twice with ice-cold PBS and fixed with ice-cold 70% ethanol (v/v) for 1 hour at 4 °C. Cells were then pelleted down at 2000 rpm for 3 min, washed with PBS and incubated first with 200 μg/ml of RNase A in PBS at 37 °C water bath for 90 min and then with 50 μg/mL of propidium iodide (PI) at room temperature for 30 min in dark. Materials and Methods These effects of W-2b further induce senescence-specific marker p21 expression and its nuclear localization to promote premature senescence in proliferating cancer cells. Figure 10. Schematic diagram represents the proposed mechanism of action of W-2b. W-2b triggers Chk2 activation in cancer cells, thereby, inhibiting cell proliferation by NM23-H1/NF-kB/c-Myc/Cyclin D1/CDK-2/ CDK-4 signaling axis. These effects of W-2b further induce senescence-specific marker p21 expression and its nuclear localization to promote premature senescence in proliferating cancer cells. derivatives were solubilized in dimethylsulfoxide (DMSO) and delivered to the cells in culture through com- plete medium. The DMSO was treated as vehicle in each experiments performed. Reagents such as paraform- aldehyde, Triton X-100, phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), DMSO, crystal violet, 2′,7′-dichlorofluorescin diacetate (DCFDA) and Bradford’s reagent were purchased from Sigma-Aldrich. X-gal Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 12 www.nature.com/scientificreports/ (5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside) substrate was obtained from Thermo Fisher Scientific. Protease inhibitor cocktail was procured from Roche. The UltraCruz DAPI mounting media and antibodies for human p21Waf1/Cip1, NM23-H1, NF-kB, c-Myc, Cyclin D1, CDK-2, CDK-4 and BCL-2 were procured from Santa-Cruz Biotechnology. The anti-β-actin antibody and the secondary antibodies such as anti-rabbit IgG and anti-mouse IgG were procured from Sigma-Aldrich. Fluorescence-conjugated anti-mouse secondary antibody Alexa-Fluor 488 (green) was purchased from Thermo Fisher Scientific. (5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside) substrate was obtained from Thermo Fisher Scientific. Protease inhibitor cocktail was procured from Roche. The UltraCruz DAPI mounting media and antibodies for human p21Waf1/Cip1, NM23-H1, NF-kB, c-Myc, Cyclin D1, CDK-2, CDK-4 and BCL-2 were procured from Santa-Cruz Biotechnology. The anti-β-actin antibody and the secondary antibodies such as anti-rabbit IgG and anti-mouse IgG were procured from Sigma-Aldrich. Fluorescence-conjugated anti-mouse secondary antibody Alexa-Fluor 488 (green) was purchased from Thermo Fisher Scientific. Cell viability assay. The cell viability assay was performed according to the procedure previously described with minor modifications39. Briefly, MCF7, HCT-116 and fR2 cells were plated in 96 well plates at a density of 5 × 103 cells per well and incubated overnight. On the next day, varying concentrations (100, 10, 1 and 0.1 µM) of WA and the isoxazoline derivatives were added along with DMSO as vehicle for 48 h. MTT dye solution (2.5 mg/mL) was introduced to the cells in medium 4 h before the completion of the treatment period and the formed formazan crystals were solubilized with DMSO. Materials and Methods Cells were then washed, fixed with 4% paraformaldehyde for 10 min, rewashed twice and stained with 0.25% crystal violet solution for 1 h. The wells containing the cells were then washed thoroughly with distilled water to remove any extra stains and then air dried overnight. The plate was then observed under an inverted microscope and colonies from three random fields were counted, averaged and photographed with NIKON camera (D3100) at 4x magnification. concentrations of W-2b for five days. Cells were then washed, fixed with 4% paraformaldehyde for 10 min, rewashed twice and stained with 0.25% crystal violet solution for 1 h. The wells containing the cells were then washed thoroughly with distilled water to remove any extra stains and then air dried overnight. The plate was then observed under an inverted microscope and colonies from three random fields were counted, averaged and photographed with NIKON camera (D3100) at 4x magnification. ROS determination assay. The procedure followed was according to the protocol previously described by our group41. Accordingly, cells were plated in 12-well plates at a density of 50 × 103 cells/well, incubated overnight and then treated with vehicle and increasing concentrations of W-2b for 48 h. Two hours before the completion, H2O2 (+ve control) was added to the cells in indicated wells and then ROS dye (DCFDA) was added to the cells and further incubation was done in dark for 30 min. Cells were successively washed with PBS thoroughly and images were captured with Floid Cell Imaging Station (Thermo Scientific) at 20x magnification. Fluorescent intensity was measured with the help of a fluorescence spectrometer coupled with microplate reader (TECAN, Infinite M200 Pro). Experimental animals. All animals used in this study were bred and maintained at the central animal facility of Indian Institute of Integrative Medicine, Jammu, India. Animals were maintained at 20–25 °C in a 12 h light dark cycle, routinely monitored for their diet and water consumption and proper sanitations were maintained to avoid any risk of possible pathogenic contamination. Animal studies were performed in accordance with the experimental guidelines that were approved by the Animal Ethics Committee of the institute “CPCSEA” (IAEC No. 51/02/15). During the animal experiments, special handling and care were taken in a humane way, so that no extra pains/injuries were imparted to the animals. Materials and Methods To minimize the mortality of animals during experimentation, only a limited number of animals were employed to yield the statistically significant results. In vivo studies for tumor growth. The experiment was performed according to the pre-standardized protocol with minor modifications42. To evaluate the in vivo anti-tumor efficacy of W-2b, healthy female Balb/c mice (25–30 g) were taken. Animals were randomized into three groups, and six animals were taken per group. For the tumor cells implantation, mouse mammary carcinoma 4T1 cells (1 × 106 per 200 μL) diluted in serum-free RPMI medium were injected subcutaneously into the mammary pad of each mouse around the second right mammary gland. A week after tumor cell implantation, when the palpable mammary tumors develop, mice were injected intraperitoneally with either vehicle (normal saline) or 5-FU (25 mg/kg/b.w.) or W-2b (25 mg/kg/b.w.) in each alternative day for two weeks. Tumor sizes were measured in each alternate day after tumor cell injection, and the body weight was recorded once in a week. Mice were sacrificed on the 15th day after treatment initiation, and tumors were dissected out carefully from the mammary pad area. Statistical analysis. Data were expressed as the mean ± standard deviation of three independent experiments performed and analyzed by Student’s t-test. IC50 values were determined with the help of GraphPad Prism soft- ware Version 5.0 (GraphPad Software, Inc., USA) by taking the log of inhibitor vs. response. A 2-sided value of *P < 0.05 was considered significant in all cases. Chemistry. General information. 1H and 13C NMR spectra were recorded on 400 and 500 MHz spectrome- ters with TMS as internal standard. Chemical shifts are expressed in parts per million (δ ppm). J values are given in Hz and s, d, dd, t, q, m abbreviations correspond to singlet, doublet, doublet of doublet, triplet, quartet, mul- tiplet respectively. Silica gel coated aluminium plates were used for TLC. The products were purified by column chromatography on silica gel (100–200 mesh) using petroleum ether–ethyl acetate as the eluent to obtain the pure products. Exact mass of all products were analysed by using HRMS having QTOF analyser. Reagents used were mostly purchased from Sigma Aldrich. General Procedure for the synthesis of the cis-fused isoxazoline derivatives of Withaferin A. To a solution of aro- matic hydroximidoyl chloride (1.2 equiv) in DMF at 0 °C was added Et3N (0.1 equiv) first and then WA (1 equiv) after ten minutes. Materials and Methods The samples were then analyzed by BD Accuri C6 flow cytometer (BD Biosciences). Western blotting. Western blot analysis was carried out with MCF7 and HCT-116 cells as per the indicated conditions (figure legends) as previously described39. Briefly, cells (0.5 × 106/well in 6 well plates) after treat- ment were harvested, washed with chilled PBS and lysed with lysis buffer containing; HEPES 1 mM, KCl 60 mM, NP-40 0.3%, EDTA 1 mM, DTT 1 mM, sodium orthovanadate 1 mM, PMSF 0.1 mM, protease inhibitor cock- tail. Cell extracts were centrifuged at 12,000 rpm for 10 min at 4 °C, the supernatants were collected and pro- tein estimation was performed with Bradford’s reagent. Equal quantity of protein (20 µg) from each sample was employed for gel electrophoresis, transferred to PVDF membranes, blocked with 5% non-fat milk and incubated with primary antibody (1: 1000 dilution) overnight at 4 °C. Membranes were subsequently washed and probed with species-specific secondary antibodies coupled to horse-radish peroxidase. Immunoreactive proteins were detected with the help of Western Bright ECL chemiluminescent HRP substrate (Advansta Inc. CA, USA) and exposed over the CL-XPosure film (Thermo Scientific). Immunofluorescence staining. MCF7 cells were seeded in 8 well chamber slides at a density of 0.5 × 105 cells/ well. Cells were further treated with vehicle, doxorubicin and W-2b as per the indicated conditions for 48 h. Subsequently, immunocytochemical analysis was carried out following the published protocol40. Images were captured under Floid Cell Imaging Station (Thermo Scientific) at 20x magnification. siRNA knockdown experiments. MISSION® esiRNAs for human CHEK2 (EHU158481), were procured from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA) and transfection experiments were performed using oligo- fectamine transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Transient transfection. MCF7 and HCT-116 cells were harvested and transfected with GFP and GFP-Chk2 plas- mid construct (generously gifted by Dr. Domenico Delia, Fondazione IRCCS Istituto Nazionale Tumori, Italy) using Neon Transfection System (Invitrogen) according to the manufacturer’s instruction. Clonogenic assay. The experiment was carried out according to the standardized protocol with some modi- fications40. MCF7 and HCT-116 cells were trypsinized properly, seeded in 6 well plates at a density of 1 × 103 cells/well and incubated overnight. Then treatment was given to the cells with vehicle, doxorubicin and various Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 13 www.nature.com/scientificreports/ concentrations of W-2b for five days. Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x Materials and Methods 13C NMR (126 MHz, DMSO) δ 204.29 (s), 165.80 (s), 156.58 (s), 155.13 (s), 135.88 (s), 129.98 (s), 129.07 (s), 126.90 (s), 125.93 (s), 85.21 (s), 78.01 (s), 71.89 (s), 63.16 (s), 56.69 (s), 56.18 (s), 55.86 (s), 54.99 (s), 51.46 (s), 49.06 (s), 43.22 (s), 42.52 (s), 38.90 (s), 38.87 (s), 30.95 (s), 29.74 (s), 29.52 (s), 27.00 (s), 24.36 (s), 20.47 (s), 20.39 (s), 15.62 (s), 13.53 (s), 11.69 (s). HRMS ESI: m/z calcd. for C35H43ClNO7 (M + H)+ 624.2728, found 624.2708. 4-chlorophenyl-2-isoxazoline withaferin A (W-1b). White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 8.5 Hz, 2H), 7.39 (d, J = 8.5 Hz, 2H), 5.17 (dd, J = 11.9, 3.4 Hz, 1H), 4.60 (d, J = 11.9 Hz, 1H), 4.4–4.3 (m, 3H), 3.70 (d, J = 3.4 Hz, 1H), 3.36 (s, 1H), 2.55–2.39 (m, 1H), 2.18 (d, J = 11.9 Hz, 1H), 2.06 (s, 3H), 1.97 (d, J = 2.8 Hz, 1H), 1.91 (d, J = 9.2 Hz, 2H), 1.62–1.54 (m, 3H), 1.33 (s, 3H), 1.26 (s, 3H), 1.17–1.03 (m, 3H), 0.88 (d, J = 6.5 Hz, 3H), 0.85–0.78 (m, 1H), 0.62 (d, J = 8.2 Hz, 2H), 0.53 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 203.04 4-chlorophenyl-2-isoxazoline withaferin A (W-1b). White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 8.5 Hz, 2H), 7.39 (d, J = 8.5 Hz, 2H), 5.17 (dd, J = 11.9, 3.4 Hz, 1H), 4.60 (d, J = 11.9 Hz, 1H), 4.4–4.3 (m, 3H), 3.70 (d, J = 3.4 Hz, 1H), 3.36 (s, 1H), 2.55–2.39 (m, 1H), 2.18 (d, J = 11.9 Hz, 1H), 2.06 (s, 3H), 1.97 (d, J = 2.8 Hz, 1H), 1.91 (d, J = 9.2 Hz, 2H), 1.62–1.54 (m, 3H), 1.33 (s, 3H), 1.26 (s, 3H), 1.17–1.03 (m, 3H), 0.88 (d, J = 6.5 Hz, 3H), 0.85–0.78 (m, 1H), 0.62 (d, J = 8.2 Hz, 2H), 0.53 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 203.04 4-chlorophenyl-2-isoxazoline withaferin A (W-1b). Materials and Methods The reaction was allowed to stir for 3 hours at 0 °C and after completion of the reaction; the reaction mixture was diluted with ethyl acetate and extracted with water (5 mL) and brine (5 mL). The organic layer was evaporated and the residue was purified by flash column chromatography (petroleum ether/EtOAc) (7:3) to afford the product as white solid powder. ( )f p p 4-chlorophenyl-2-isoxazoline withaferin A (W-1a). White solid powder; 1H NMR (500 MHz, DMSO) δ 7.73 (d, J = 7.7 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 6.06 (s, 1H), 4.93 (d, J = 10.9 Hz, 1H), 4.63–4.54 (m, 2H), 4.29 (d, J = 12.4 Hz, 1H), 4.16–4.12 (m, 2H), 3.34 (bs, 2H), 2.42 (d, J = 16.9 Hz, 2H), 2.11 (d, J = 17.5 Hz, 1H), 2.02 (s, 3H), 1.89 (d, J = 11.0 Hz, 1H), 1.76 (d, J = 31.6 Hz, 2H), 1.55 (d, J = 9.8 Hz, 2H), 1.31–1.29 (m, 3H), 1.23 (s, 3H), 1.20 (s, 3H), 1.15 (d, J = 13.1 Hz, 4H), 1.04 (d, J = 9.4 Hz, 3H), 0.91 (d, J = 5.3 Hz, 3H), 0.62 (s, 3H). 13C NMR (126 MHz, DMSO) δ 204.29 (s), 165.80 (s), 156.58 (s), 155.13 (s), 135.88 (s), 129.98 (s), 129.07 (s), 126.90 (s), 125.93 (s), 85.21 (s), 78.01 (s), 71.89 (s), 63.16 (s), 56.69 (s), 56.18 (s), 55.86 (s), 54.99 (s), 51.46 (s), 49.06 (s), 43.22 (s), 42.52 (s), 38.90 (s), 38.87 (s), 30.95 (s), 29.74 (s), 29.52 (s), 27.00 (s), 24.36 (s), 20.47 (s), 20.39 (s), 15.62 (s), 13.53 (s), 11.69 (s). HRMS ESI: m/z calcd. for C35H43ClNO7 (M + H)+ 624.2728, found 624.2708. f p p 4-chlorophenyl-2-isoxazoline withaferin A (W-1a). White solid powder; 1H NMR (500 MHz, DMSO) δ 7.73 (d, J = 7.7 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 6.06 (s, 1H), 4.93 (d, J = 10.9 Hz, 1H), 4.63–4.54 (m, 2H), 4.29 (d, J = 12.4 Hz, 1H), 4.16–4.12 (m, 2H), 3.34 (bs, 2H), 2.42 (d, J = 16.9 Hz, 2H), 2.11 (d, J = 17.5 Hz, 1H), 2.02 (s, 3H), 1.89 (d, J = 11.0 Hz, 1H), 1.76 (d, J = 31.6 Hz, 2H), 1.55 (d, J = 9.8 Hz, 2H), 1.31–1.29 (m, 3H), 1.23 (s, 3H), 1.20 (s, 3H), 1.15 (d, J = 13.1 Hz, 4H), 1.04 (d, J = 9.4 Hz, 3H), 0.91 (d, J = 5.3 Hz, 3H), 0.62 (s, 3H). www.nature.com/scientificreports/ White solid powder; 1H NMR (400 MHz, Pyr) δ 7.89 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 5.47 (d, J = 11.2 Hz, 1H), 4.92 (dd, J = 16.1, 7.0 Hz, 2H), 4.79 (d, J = 11.7 Hz, 1H), 4.41 (d, J = 13.2 Hz, 1H), 4.07 (d, J = 2.0 Hz, 1H), 2.86 (s, 1H), 2.42–2.40 (m, 1H), 2.21 (s, 3H), 2.18–2.03 (m, 2H), 1.92 (bd, J = 36.4 Hz, 2H), 1.78 (s, 3H), 1.53 (d, J = 9.8 Hz, 2H), 1.50–1.36 (m, 4H), 1.27 (bs, 1H), 1.12 (d, J = 11.1 Hz, 1H), 0.96 (d, J = 6.6 Hz, 3H), 0.89–0.87 (m, 3H), 0.77–0.65 (m, 1H), 0.52 (s, 3H). 13C NMR (126 MHz, Pyr) δ 206.08 (s), 168.09 (s), 158.50 (s), 155.82 (s), 134.53 (s), 130.73 (s), 129.38 (s), 128.93 (s), 126.75 (s), 87.83 (s), 79.92 (s), 74.37 (s), 65.92 (s), 58.97 (s), 58.27 (s), 57.73 (s), 57.22 (s), 53.23 (s), 51.46 (s), 45.32 (s), 44.15 (s), 40.66 (s), 40.60 (s), 32.80 (s), 31.70 (s), 31.50 (s), 28.75 (s), 25.87 (s), 22.48 (s), 21.82 (s), 17.85 (s), 15.03 (s), 12.99 (s). HRMS ESI: m/z calcd. for C35H43BrNO7 (M + H)+ 668.2223, found 668.2270. ( ), ( ) ( ) , 4-bromophenyl-2-isoxazoline withaferin A (W-3b). White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 5.17 (dd, J = 11.9, 3.4 Hz, 1H), 4.60 (d, J = 11.9 Hz, 1H), 4.45–4.30 (m, 3H), 3.70 (bs, 1H), 3.35 (s, 1H), 2.58 (bs, 1H), 2.54–2.43 (m, 1H), 2.18 (d, J = 12.4 Hz, 1H), 2.05 (s, 3H), 1.98–1.90 (m, 2H), 1.59 (s, 3H), 1.56 (bs, 1H), 1.30 (d, J = 11.5 Hz, 1H), 1.27–1.19 (m, 4H), 1.20–1.05 (m, 2H), 0.89 (d, J = 6.6 Hz, 3H), 0.80 (d, J = 9.9 Hz, 1H), 0.65–0.58 (m, 2H), 0.53 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 202.87 (s), 166.82 (s), 153.44 (s), 152.69 (s), 131.97 (s), 128.28 (s), 127.37 (s), 125.43 (s),124.82 (s), 82.97 (s), 78.42 (s), 73.49 (s), 62.43 (s), 58.99 (s), 58.05 (s), 57.26 (s), 56.21 (s), 51.60 (s), 50.73 (s), 42.09 (s), 41.89 (s), 38.37 (s), 37.39 (s), 30.86 (s), 29.62 (s), 29.50 (s), 26.97 (s), 23.88 (s), 19.88 (s), 19.83 (s), 15.05 (s), 13.08 (s), 11.14 (s). HRMS ESI: m/z calcd. www.nature.com/scientificreports/ for C35H43BrNO7 (M + H)+ 668.2223, found 668.2256.l ( + ) , 4-fluorophenyl-2-isoxazoline withaferin A (W-4a). White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.81 (dd, J = 8.5, 5.4 Hz, 2H), 7.11 (t, J = 8.5 Hz, 2H),5.16 (dd, J = 11.9, 3.3 Hz, 1H), 4.61 (d, J = 11.9 Hz, 1H), 4.38–4.35 (m, 3H),), 3.70 (bs, 1H), 3.35 (s, 1H), 2.58 (bs, 1H), 2.54–2.43 (m, 1H), 2.18 (d, J = 12.4 Hz, 1H), 2.05 (s, 3H), 1.98–1.90 (m, 2H), 1.59 (s, 3H), 1.56 (bs, 1H), 1.30 (d, J = 11.5 Hz, 1H), 1.27–1.19 (m, 4H), 1.20–1.05 (m, 2H), 0.89 (d, J = 6.6 Hz, 3H), 0.80 (d, J = 9.9 Hz, 1H), 0.65–0.58 (m, 2H), 0.53 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 203.48 (s), 179.85, 168.05 (d), 155.50 (s), 152.83 (s), 128.96(d), 127.06 (s), 125.72 (s), 116.69(d), 84.87 (s), 78.73 (s), 73.22 (s), 63.47 (s), 57.97 (s), 57.50 (s), 55.73 (s), 55.17 (s), 51.82 (s), 49.34 (s), 42.90 (s), 42.72 (s), 38.82 (s), 31.93 (s), 30.82 (s), 29.36 (s), 27.37 (s), 24.23 (s), 22.70 (s), 20.29 (s), 20.02 (s), 15.60 (s), 13.37 (s), 11.47 (s). HRMS ESI: m/z calcd. for C35H43FNO7 (M + H)+ 608.3024, found 608.3017.l ( ) , 4-fluorophenyl-2-isoxazoline withaferin A (W-4b). White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.81 (dd, J = 8.5, 5.4 Hz, 2H), 7.11 (t, J = 8.5 Hz, 2H), 5.16 (dd, J = 11.9, 3.6 Hz, 1H), 4.61 (d, J = 11.9 Hz, 1H), 4.38–4.32 (m, 3H), 3.72 (d, J = 3.3 Hz, 1H), 3.36 (s, 1H), 2.89 (bs, 1H), 2.66 (bs, 1H), 2.52–2.41 (m, 1H), 2.19 (d, J = 11.0 Hz, 1H), 2.05 (s, 3H), 1.94 (d, J = 18.3 Hz, 2H), 1.29 (s, 3H), 1.27 (s, 3H), 1.25 (s, 3H), 0.87 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 10.1 Hz, 2H), 0.73–0.58 (m, 2H), 0.53 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 203.01 (s), 179.05, 167.40 (d), 155.09 (s), 152.08 (s), 128.50(d), 126.68(s), 125.25 (s), 116.22(d), 84.40 (s), 78.26 (s), 72.75 (s), 63.00 (s), 57.50 (s), 57.03 (s), 55.26 (s), 54.70 (s), 51.35 (s), 48.87 (s), 42.43 (s), 42.25(s), 38.35 (s), 31.47 (s), 30.35 (s), 28.90 (s), 26.90 (s), 22.23 (s), 20.02 (s), 19.82 (s), 19.55 (s), 15.13 (s), 12.90 (s), 11.00 (s). HRMS ESI: m/z calcd. for C35H43FNO7 (M + H)+ 608.3024, found 608.3058. www.nature.com/scientificreports/ www.nature.com/scientificreports/ (s), 166.98 (s), 153.59 (s), 152.82 (s), 136.71 (s), 129.21 (s), 128.32 (s), 127.12 (s), 125.71 (s), 83.17 (s), 78.66 (s), 73.69 (s), 62.74 (s), 59.27 (s), 58.34 (s), 57.47 (s), 56.39 (s), 51.85 (s), 51.00 (s), 42.34 (s), 42.21 (s), 38.60 (s), 38.24 (s), 31.15 (s), 29.88 (s), 29.77 (s), 29.71 (s), 27.21 (s), 24.13 (s), 20.09 (s), 20.04 (s), 15.29 (s), 13.31 (s), 11.37 (s). HRMS-ESI: m/z calcd. for C35H43ClNO7 (M + H)+ 624.2728, found 624.2728. (s), 166.98 (s), 153.59 (s), 152.82 (s), 136.71 (s), 129.21 (s), 128.32 (s), 127.12 (s), 125.71 (s), 83.17 (s), 78.66 (s), 73.69 (s), 62.74 (s), 59.27 (s), 58.34 (s), 57.47 (s), 56.39 (s), 51.85 (s), 51.00 (s), 42.34 (s), 42.21 (s), 38.60 (s), 38.24 (s), 31.15 (s), 29.88 (s), 29.77 (s), 29.71 (s), 27.21 (s), 24.13 (s), 20.09 (s), 20.04 (s), 15.29 (s), 13.31 (s), 11.37 (s). HRMS-ESI: m/z calcd. for C35H43ClNO7 (M + H)+ 624.2728, found 624.2728. ( + ) , 4-nitrophenyl-2-isoxazoline withaferin A (W-2a). White solid powder; 1H NMR (400 MHz, Pyr) δ 8.33 (d, J = 8.6 Hz, 2H), 8.18 (d, J = 8.7 Hz, 2H), 5.56 (d, J = 11.2 Hz, 1H), 5.04 (dd, J = 11.3, 2.1 Hz, 2H), 4.91 (d, J = 11.7 Hz, 1H), 4.80 (d, J = 11.7 Hz, 1H), 4.42 (d, J = 13.1 Hz, 1H), 4.12 (s, 1H), 2.88 (s, 1H), 2.51–2.38 (m, 1H), 2.21 (s, 3H), 2.14 (dd, J = 18.2, 3.3 Hz, 2H), 1.99–1.86 (m, 2H), 1.80 (s, 3H), 1.56 (s, 1H), 1.52 (s, 2H), 1.44 (dd, J = 17.6, 8.6 Hz, 3H), 1.33 (dd, J = 16.2, 9.1 Hz, 2H), 1.27 (s, 3H), 1.12 (dd, J = 12.9, 5.7 Hz, 1H), 0.97 (d, J = 6.6 Hz, 3H), 0.94–0.86 (m, 3H), 0.53 (s, 3H). 13C NMR (126 MHz, Pyr) δ 205.68 (s), 168.02 (s), 158.34 (s), 155.67 (s), 136.20 (s), 129.99 (s), 128.93 (s), 126.45 (s), 124.74 (s), 88.37 (s), 79.94 (s), 74.03 (s), 65.86 (s), 58.99 (s), 57.97 (s), 57.73 (s), 57.23 (s), 53.28 (s), 51.54 (s), 45.37 (s), 44.17 (s), 40.61 (s), 32.98 (s), 32.80 (s), 31.74 (s), 31.57 (s), 28.76 (s), 25.88 (s), 22.48 (s), 21.79 (s), 17.80 (s), 15.04 (s), 12.91 (s). HRMS ESI: m/z calcd. for C35H43N2O9 (M + H)+ 635.2969, found 635.2959. 4-nitrophenyl-2-isoxazoline withaferin A (W-2b). www.nature.com/scientificreports/ White solid powder; 1H NMR (400 MHz, Pyr) δ 8.37 (d, J = 8.7 Hz, 2H), 8.28 (d, J = 8.7 Hz, 2H), 5.66 (dd, J = 11.9, 3.6 Hz, 1H), 5.16 (d, J = 11.9 Hz, 1H), 4.86 (d, J = 11.7 Hz, 1H), 4.75 (d, J = 11.7 Hz, 1H), 4.29 (d, J = 12.9 Hz, 1H), 4.14 (d, J = 3.5 Hz, 1H), 3.62 (s, 1H), 3.58 (s, 1H), 2.18 (d, J = 13.1 Hz, 2H), 2.07 (s, 3H), 1.87 (s, 2H), 1.71 (s, 3H), 1.46–1.43(m, 4H), 1.40–1.19 (m, 4H), 1.07 (d, J = 9.5 Hz, 1H), 0.91 (d, J = 11.1 Hz, 3H), 0.79 (d, J = 8.9 Hz, 1H), 0.73 (d, J = 6.6 Hz, 3H), 0.60 (bs, 1H), 0.40 (s, 3H). 13C NMR (101 MHz, Pyr) δ 204.28 (s), 166.76 (s), 154.54 (s), 154.31 (s), 149.45 (s), 128.90 (s), 127.76 (s), 125.01 (s), 124.49 (s), 86.35 (s), 78.59 (s), 73.53 (s), 63.82 (s), 59.96 (s), 58.39 (s), 56.98 (s), 56.61 (s), 52.25 (s), 52.01 (s), 43.35 (s), 42.87 (s), 39.38 (s), 39.18 (s), 32.19 (s), 30.93 (s), 30.15 (s), 27.60 (s), 24.77 (s), 21.11 (s), 20.54 (s), 16.33 (s), 13.72 (s), 11.79 (s). HRMS ESI: m/z calcd. for C35H43N2O9 (M + H)+ 635.2969, found 635.2961. 4-bromophenyl-2-isoxazoline withaferin A (W-3a). White solid powder; 1H NMR (400 MHz, Pyr) δ 7.89 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 5.47 (d, J = 11.2 Hz, 1H), 4.92 (dd, J = 16.1, 7.0 Hz, 2H), 4.79 (d, J = 11.7 Hz, 1H), 4.41 (d, J = 13.2 Hz, 1H), 4.07 (d, J = 2.0 Hz, 1H), 2.86 (s, 1H), 2.42–2.40 (m, 1H), 2.21 (s, 3H), 2.18–2.03 (m, 2H), 1.92 (bd, J = 36.4 Hz, 2H), 1.78 (s, 3H), 1.53 (d, J = 9.8 Hz, 2H), 1.50–1.36 (m, 4H), 1.27 (bs, 1H), 1.12 (d, J = 11.1 Hz, 1H), 0.96 (d, J = 6.6 Hz, 3H), 0.89–0.87 (m, 3H), 0.77–0.65 (m, 1H), 0.52 (s, 3H). www.nature.com/scientificreports/ 13C NMR (126 MHz, Pyr) δ 206.08 (s), 168.09 (s), 158.50 (s), 155.82 (s), 134.53 (s), 130.73 (s), 129.38 (s), 128.93 (s), 126.75 (s), 87.83 (s), 79.92 (s), 74.37 (s), 65.92 (s), 58.97 (s), 58.27 (s), 57.73 (s), 57.22 (s), 53.23 (s), 51.46 (s), 45.32 (s), 44.15 (s), 40.66 (s), 40.60 (s), 32.80 (s), 31.70 (s), 31.50 (s), 28.75 (s), 25.87 (s), 22.48 (s), 21.82 (s), 17.85 (s), 15.03 (s), 12.99 (s). HRMS ESI: m/z calcd. for C35H43BrNO7 (M + H)+ 668.2223, found 668.2270. ( ) h l d d 1 ( l ) 4-nitrophenyl-2-isoxazoline withaferin A (W-2b). White solid powder; 1H NMR (400 MHz, Pyr) δ 8.37 (d, J = 8.7 Hz, 2H), 8.28 (d, J = 8.7 Hz, 2H), 5.66 (dd, J = 11.9, 3.6 Hz, 1H), 5.16 (d, J = 11.9 Hz, 1H), 4.86 (d, J = 11.7 Hz, 1H), 4.75 (d, J = 11.7 Hz, 1H), 4.29 (d, J = 12.9 Hz, 1H), 4.14 (d, J = 3.5 Hz, 1H), 3.62 (s, 1H), 3.58 (s, 1H), 2.18 (d, J = 13.1 Hz, 2H), 2.07 (s, 3H), 1.87 (s, 2H), 1.71 (s, 3H), 1.46–1.43(m, 4H), 1.40–1.19 (m, 4H), 1.07 (d, J = 9.5 Hz, 1H), 0.91 (d, J = 11.1 Hz, 3H), 0.79 (d, J = 8.9 Hz, 1H), 0.73 (d, J = 6.6 Hz, 3H), 0.60 (bs, 1H), 0.40 (s, 3H). 13C NMR (101 MHz, Pyr) δ 204.28 (s), 166.76 (s), 154.54 (s), 154.31 (s), 149.45 (s), 128.90 (s), 127.76 (s), 125.01 (s), 124.49 (s), 86.35 (s), 78.59 (s), 73.53 (s), 63.82 (s), 59.96 (s), 58.39 (s), 56.98 (s), 56.61 (s), 52.25 (s), 52.01 (s), 43.35 (s), 42.87 (s), 39.38 (s), 39.18 (s), 32.19 (s), 30.93 (s), 30.15 (s), 27.60 (s), 24.77 (s), 21.11 (s), 20.54 (s), 16.33 (s), 13.72 (s), 11.79 (s). HRMS ESI: m/z calcd. for C35H43N2O9 (M + H)+ 635.2969, found 635.2961. 4-bromophenyl-2-isoxazoline withaferin A (W-3a). White solid powder; 1H NMR (400 MHz, Pyr) δ 7.89 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 5.47 (d, J = 11.2 Hz, 1H), 4.92 (dd, J = 16.1, 7.0 Hz, 2H), 4.79 (d, J=11.7Hz, 1H), 4.41 (d, J=13.2Hz, 1H), 4.07 (d, J=2.0Hz, 1H), 2.86 (s, 1H), 2.42–2.40 (m, 1H), 2.21 (s, 3H), ( ), ( ), ( ), ( ) / ( + ) , 4-bromophenyl-2-isoxazoline withaferin A (W-3a). Materials and Methods White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 8.5 Hz, 2H), 7.39 (d, J = 8.5 Hz, 2H), 5.17 (dd, J = 11.9, 3.4 Hz, 1H), 4.60 (d, J = 11.9 Hz, 1H), 4.4–4.3 (m, 3H), 3.70 (d, J = 3.4 Hz, 1H), 3.36 (s, 1H), 2.55–2.39 (m, 1H), 2.18 (d, J = 11.9 Hz, 1H), 2.06 (s, 3H), 1.97 (d, J = 2.8 Hz, 1H), 1.91 (d, J = 9.2 Hz, 2H), 1.62–1.54 (m, 3H), 1.33 (s, 3H), 1.26 (s, 3H), 1.17–1.03 (m, 3H), 0.88 (d, J = 6.5 Hz, 3H), 0.85–0.78 (m, 1H), 0.62 (d, J = 8.2 Hz, 2H), 0.53 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 203.04 Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 14 www.nature.com/scientificreports/ White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.83–7.79 (m, 1H), 7.62 (dd, J = 8.6, 6.5 Hz, 2H), 7.56 (d, J = 8.3 Hz, 1H), 5.00 (d, J = 10.6 Hz, 1H), 4.41 (dt, J = 13.2, 3.4 Hz, 1H), 4.37–4.30 (m, 2H), 4.10 (dd, J = 14.3, 7.1 Hz, 1H), 3.23 (bs, 1H), 2.95 (s, 1H), 2.55–2.43 (m, 1H), 2.23 (s, 1H), 2.17 (d, J = 8.9 Hz, 1H), 2.03 (s, 3H), 1.99 (d, J = 3.3 Hz, 1H), 1.95 (d, J = 3.2 Hz, 1H), 1.73– 1.63 (m, 3H), 1.55 (bs, 3H), 1.43–1.37 (m, 2H), 1.32 (s, 3H), 1.23 (s, 3H), 1.15–1.07 (m, 2H), 0.99 (d, J = 6.6 Hz, 3H), 0.88–0.83 (m, 1H), 0.66 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 203.63 (s), 167.22 (s), 156.39 (s), 153.03 (s), 132.62 (s), 131.68 (s), 130.97 (s), 129.06 (s), 127.80 (d, J = 5.8 Hz), 126.34 (s), 125.93 (s), 125.23 (s), 85.10 (s), 78.95 (s), 72.60 (s), 63.82 (s), 58.62 (s), 57.73 (s), 57.29 (s), 55.98 (s), 52.03 (s), 49.60 (s), 42.95 (s), 42.83 (s), 39.04 (s), 30.98 (s), 30.04 (s), 29.91 (s), 29.80 (s), 27.60 (s), 24.46 (s), 20.56 (s), 20.22 (s), 15.79 (s), 13.57 (s), 11.70 (s). HRMS ESI: m/z calcd. for C36H43F3NO7 (M + H)+ 658.2992, found 658.2973.l ( + ) , 2-methoxy-4-trifluorophenyl-2-isoxazoline withaferin A (W-9b). White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.81 (d, J = 8.1 Hz, 1H), 7.23 (d, J = 6.1 Hz, 2H), 5.13 (dd, J = 12.2, 3.2 Hz, 1H), 4.96 (d, J = 12.2 Hz, 1H), 4.35 (q, J = 12.3 Hz, 3H), 3.92 (s, 3H), 3.60 (d, J = 3.1 Hz, 1H), 3.32 (s, 1H), 2.44 (dd, J = 17.3, 13.8 Hz, 1H), 2.28 (bs, 1H), 2.04 (s, 3H), 1.92 (dd, J = 18.2, 2.7 Hz, 2H), 1.39 (s, 3H), 1.26 (bs, 6H), 1.23 (s, 3H), 1.19 (s, 3H), 0.87 (s, 1H), 0.85 (s, 2H), 0.83 (d, J = 6.4 Hz, 3H), 0.66–0.60 (m, 1H), 0.49 (s, 3H). www.nature.com/scientificreports/ ( + ) , pyridenyl-2-isoxazoline withaferin A (W-5a). White solid powder; 1H NMR (400 MHz, CDCl3) δ 8.70 (s, 2H) H), 7.67 (d, J = 5.1 Hz, 2H), 5.24 (dd, J = 12.0, 3.5 Hz, 1H), 4.60 (d, J = 12.0 Hz, 1H), 4.41–4.32 (m, 3H), 3.73 (d, J = 3.5 Hz, 1H), 3.35 (s, 1H), 2.52–2.39 (m, 1H), 2.18 (d, J = 13.8 Hz, 1H), 2.04 (s, 3H), 1.91 (dd, J = 18.2, 2.9 Hz, 2H), 1.73 (bs, 1H), 1.63–1.53 (m, 3H), 1.29 (s, 3H), 1.25 (s, 3H), 1.20 (dd, J = 13.0, 8.0 Hz, 2H), 1.13–0.99 (m, 2H), 0.86 (d, J = 6.6 Hz, 3H), 0.82–0.76 (m, 1H), 0.67 (m, 1H), 0.53 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 202.57 (s), 166.95 (s), 152.97 (s), 152.90 (s), 150.53 (s), 136.07 (s), 125.66 (s), 120.59 (s), 83.91 (s), 78.61 (s), 73.41 (s), 62.53 (s), 58.54 (s), 58.18 (s), 57.42 (s), 56.29 (s), 51.55 (s), 51.00 (s), 42.39 (s), 42.32 (s), 38.79 (s), 38.28 (s), 31.16 (s), 29.91 (s), 29.69 (s), 27.28 (s), 24.19 (s), 20.04 (s), 20.01 (s), 15.37 (s), 13.19 (s), 11.31 (s). HRMS ESI: m/z calcd. for C34H43N2O7 (M +H)H)+ 591.3070, found 591.3053. Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 15 www.nature.com/scientificreports/ pyridenyl-2-isoxazoline withaferin A (W-5b). White solid powder; 1H NMR (400 MHz, CDCl3) δ 8.75 (bs, 2H), 7.58 (bs, 2H), 5.04 (d, J = 10.8 Hz, 1H), 4.47–4.26 (m, 4H), 3.53 (s, 1H), 2.66 (s, 1H), 2.56–2.45 (m, 1H), 2.10 (d, J = 14.6 Hz, 1H), 2.05 (s, 3H), 2.02–1.95 (m, 3H), 1.65 (d, J = 6.3 Hz, 3H), 1.42 (s, 2H), 1.34 (s, 3H), 1.29 (s, 2H), 1.25 (s, 4H), 1.13 (d, J = 11.8 Hz, 2H), 1.00 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 7.0 Hz, 1H), 0.67 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 202.83(s), 167.12 (s), 155.05 (s), 153.09 (s), 150.91 (s), 135.15 (s), 125.70 (s), 123.48 (s), 85.47 (s), 78.73 (s), 72.84 (s), 63.43 (s), 57.86 (s), 57.46 (s), 55.69 (s), 54.19 (s), 51.75 (s), 49.39 (s), 42.91 (s), 42.69 (s), 38.77 (s), 31.64 (s), 30.77 (s), 29.79 (s), 27.35 (s), 24.21 (s), 22.73 (s), 20.26 (s), 20.10 (s), 15.65 (s), 13.39 (s), 11.49 (s). HRMS ESI: m/z calcd. for C34H43N2O7 (M + H)+ 591.3070, found 591.3079. ( ) ( ) 2,6-dichlorophenyl-2-isoxazoline withaferin A (W-6a). www.nature.com/scientificreports/ 13C NMR (126 MHz, CDCl3) δ 201.54 (s), 166.28 (s), 152.17 (s), 151.34 (s), 135.86 (s), 132.89 (s), 130.96 (s), 130.55 (s), 126.66 (s), 124.88 (s), 124.16 (s), 81.38 (s), 77.93 (s), 72.54 (s), 62.02 (s), 59.04 (s), 57.66 (s), 56.72 (s), 54.77 (s), 51.17 (s), 49.94 (s), 41.59 (s), 40.89 (s), 37.81 (s), 37.53 (s), 30.28 (s), 29.04 (s), 28.89 (s), 26.45 (s), 23.50 (s), 19.34 (s), 19.26 (s), 14.61 (s), 12.63 (s), 10.46 (s). HRMS ESI: m/z calcd. for C35H42Cl2NO7 (M + H)+ 658.2338, found 658.2396. b h h l i li i h f i A (W ) Wh l d d 1H NMR ( MH 3-bromo-6-methoxyphenyl-2-isoxazoline withaferin A (W-7a). White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 2.4 Hz, 1H), 7.43 (dd, J = 8.9, 2.4 Hz, 1H), 6.80 (d, J = 8.9 Hz, 1H), 5.25 (d, J = 14.8 Hz, 1H), 5.07 (dd, J = 12.2, 3.2 Hz, 1H), 4.95 (d, J = 12.2 Hz, 1 H), 4.82 (q, J = 11.9 Hz, 2H), 4.71 (d, J = 3.2 Hz, 1H), 4.33 (d, J = 13.1 Hz, 1H), 3.84 (s, 3H), 3.35 (s, 1H), 2.86 (s, 1H), 2.58 (s, 1H), 2.53–2.42 (m, 1H), 2.18 (d, J = 12.4 Hz, 1H), 2.05 (d, J = 4.8 Hz, 3H), 1.94 (dd, J = 25.4, 7.2 Hz, 2H), 1.59 (s, 5H), 1.25 (s, 3H), 1.17–0.99 (m, 2H), 0.89 (d, J = 6.6 Hz, 3H), 0.80 (d, J = 9.9 Hz, 1H), 0.61 (s, 2 H), 0.53 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 201.80 (s), 165.26 (s), 156.99 (s), 156.88 (s), 153.23 (s), 134.52 (s), 133.29 (s), 121.94 (s), 118.89 (s), 113.92 (s), 112.74 (s), 80.53 (s), 78.09 (s), 74.40 (s), 59.79 (s), 59.71 (s), 58.20 (s), 58.02 (s), 56.54 (s), 56.03 (s), 51.76 (s), 51.21 (s), 42.27 (s), 41.57 (s), 38.57 (s), 38.07 (s), 31.04 (s), 30.00 (s), 29.69 (s), 27.22 (s), 24.07 (s), 20.02 (s), 14.34 (s), 13.26 (s), 11.34 (s). HRMS ESI: m/z calcd. C36H45BrNO8 (M + H)+ 698.2329, found 698.2317. 2-trifluoromethylphenyl-2-isoxazoline withaferin A (W-8a). www.nature.com/scientificreports/ White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.51 (d, J = 8.4 Hz, 1H), 7.46 (d, J = 1.9 Hz, 1H), 7.28 (d, J = 11.2 Hz, 1H), 4.96 (d, J = 11.2 Hz, 1H), 4.64 (dd, J = 11.2, 2.0 Hz, 1H), 4.37–4.30 (m, 2H), 4.05 (q, J = 7.1 Hz, 2H), 3.25 (bs, 1H), 2.81 (bs, 1H), 2.43–2.39 (m, 1H), 2.08 (d, J = 9.3 Hz, 1H), 1.98 (s, 3H), 1.53 (s, 3H), 1.37–1.30 (m, 3H), 1.25 (s, 3H), 1.19 (d, J = 2.3 Hz, 4H), 1.11–1.04 (m, 1H), 0.93 (d, J = 6.6 Hz, 2H), 0.83–0.79 (m, 2H), 0.60 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 201.16 (s), 165.14, 152.94, 151.01, 135.40, 131.66, 129.57, 129.44, 126.05, 123.69, 123.07, 82.69, 76.78, 61.61, 58.51, 56.14, 55.55, 53.89, 53.79, 59.82, 47.46, 40.95, 40.95, 40.75, 36.84, 28.92, 27.63, 25.41, 22.28, 18.36, 18.13, 18.09, 13.68, 12.27, 11.43, 9.52. HRMS ESI: m/z calcd. for C35H42Cl2NO7 (M + H)+ 658.2338, found 658.2338. . 7, . 3, 9.5 . S S : / ca cd. o C35 C O7 ( + ) 658. 338, ou d 658. 338. 2,6-dichlorophenyl-2-isoxazoline withaferin A (W-6b). White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.55 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.3 Hz, 1H), 5.17 (dd, J = 12.0, 3.4 Hz, 1H), 4.82 (d, J = 12.0 Hz, 1H), 4.82 (d, J = 12.0 Hz, 1H), 4.41–4.38 (m, 3H), 3.72 (bs, 2H), 3.37 (s, 1 H), 2.90 (t, J = 6.5 Hz, 1H), 2.69 (bs, 1 H), 2.57–2.45 (m, 1H), 2.20 (d, J = 13.9 Hz, 1H), 2.09 (s, 3H), 1.96 (d, J = 21.5 Hz, 1H), 1.61 (s, 7H), 1.48–1.38 (m, 1H), 1.36– 1.31 (m, 1H), 1.25 (s, 3H), 1.12 (s, 2H), 0.91 (d, J = 6.3 Hz, 3H), 0.75 (bs, 1H), 0.55 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 201.54 (s), 166.28 (s), 152.17 (s), 151.34 (s), 135.86 (s), 132.89 (s), 130.96 (s), 130.55 (s), 126.66 (s), 124.88 (s), 124.16 (s), 81.38 (s), 77.93 (s), 72.54 (s), 62.02 (s), 59.04 (s), 57.66 (s), 56.72 (s), 54.77 (s), 51.17 (s), 49.94 (s), 41.59 (s), 40.89 (s), 37.81 (s), 37.53 (s), 30.28 (s), 29.04 (s), 28.89 (s), 26.45 (s), 23.50 (s), 19.34 (s), 19.26 (s), 14.61 (s), 12.63 (s), 10.46 (s). HRMS ESI: m/z calcd. for C35H42Cl2NO7 (M + H)+ 658.2338, found 658.2396. 3-bromo-6-methoxyphenyl-2-isoxazoline withaferin A (W-7a). www.nature.com/scientificreports/ 13C NMR (101 MHz, CDCl3) δ 203.20 (s), 167.36 (s), 153.79 (s), 153.22 (s), 132.74 (s), 132.42 (s), 127.74 (s), 126.32 (dd, J = 7.4, 3.6 Hz), 126.10 (s), 122.75 (s), 84.01 (s), 78.95 (s), 74.16 (s), 62.87 (s), 59.51 (s), 58.56 (s), 57.87 (s), , 2,6-dichlorophenyl-2-isoxazoline withaferin A (W-10b). White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.42 (t, J = 7.0 Hz, 2H), 7.36–7.32 (m, 1H), 5.14 (d, J = 10.0 Hz, 1H), 4.46–4.44 (m, 1H), 4.40–4.33 (m, 3H), 3.48 (s, 1H), 3.46 (s, 1H), 2.57–2.46 (m, 1H), 2.18 (d, J = 11.2 Hz, 1H), 2.05 (s, 3H), 2.03–1.94 (m, 3H), 1.67 (s, 5H), 1.36 (bs, 5H), 1.25 (s, 3H), 1.15 (d, J = 9.7 Hz, 2H), 1.01 (d, J = 6.6 Hz, 3H)H), 0.93–0.85 (m, 2H), 0.69 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 202.68 (s), 165.42 (s), 152.86 (s), 151.41 (s), 133.34 (s), 129.84 (s), 127.25 (s), 124.86 (s), 123.88 (s), 82.63 (s), 77.04 (s), 68.99 (s), 62.58 (s), 57.26 (s), 55.75 (s), 55.18 (s), 54.04 (s), 51.78 (s), 50.09 (s), 47.61 (s), 41.01 (s), 39.84 (s), 37.12 (s), 37.08 (s), 28.75 (s), 28.06 (s), 27.39 (s), 25.61 (s), 22.51 (s), 18.88 (s), 18.37 (s), 13.89 (s), 11.67 (s). HRMS ESI: m/z calcd. for C35H42Cl2NO7 (M +H)+ 658.2338, found 658.2339.l 4-trifluorophenyl-2-isoxazoline withaferin A (W-11a). White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 8.3 Hz, 2H), 5.00 (d, J = 10.7 Hz, 1H), 4.43–4.28 (m, 4H), 3.50 (s, 1H), 2.65 (s, 1H), 2.54–2.45 (m, 1H), 2.12–2.07 (m, 1H), 2.03 (s, 3H), 2.00–1.92 (m, 3H), 1.66 (dd, J = 16.8, 5.9 Hz, 4H), 1.31 (s, 3H), 1.24 (s, 5H), 1.16–1.06 (m, 3H), 0.99 (d, J = 6.6 Hz, 3H), 0.85 (dd, J = 16.2, 9.3 Hz, 2H), 0.65 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 203.20 (s), 167.36 (s), 153.79 (s), 153.22 (s), 132.74 (s), 132.42 (s), 127.74 (s), 126.32 (dd, J = 7.4, 3.6 Hz), 126.10 (s), 122.75 (s), 84.01 (s), 78.95 (s), 74.16 (s), 62.87 (s), 59.51 (s), 58.56 (s), 57.87 (s), 4-trifluorophenyl-2-isoxazoline withaferin A (W-11a). www.nature.com/scientificreports/ 13C NMR (101 MHz, CDCl3) δ 202.46 (s), 167.17 (s), 157.82 (s), 153.12 (s), 152.99 (s), 133.63 (s), 131.06 (s), 125.86 (s), 121.35 (s), 117.60 (d, J = 3.8 Hz), 114.24 (s), 108.97 (d, J = 3.8 Hz), 83.38 (s), 78.74 (s), 73.93 (s), 63.00 (s), 59.95 (s), 58.40 (s), 57.60 (s), 56.54 (s), 56.16 (s), 52.13 (s), 50.84 (s), 42.47 (s), 41.49 (s), 38.83 (s), 38.30 (s), 32.10 (s), 31.33 (s), 27.40 (s), 24.27 (s), 22.87 (s), 20.15 (s), 15.32 (s), 14.29 (s), 13.22 (s), 11.39 (s). HRMS ESI: m/z calcd. for C37H45F3NO8 (M + H)+ 688.3097, found 688.3099. , 2,6-dichlorophenyl-2-isoxazoline withaferin A (W-10b). White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.42 (t, J = 7.0 Hz, 2H), 7.36–7.32 (m, 1H), 5.14 (d, J = 10.0 Hz, 1H), 4.46–4.44 (m, 1H), 4.40–4.33 (m, 3H), 3.48 (s, 1H), 3.46 (s, 1H), 2.57–2.46 (m, 1H), 2.18 (d, J = 11.2 Hz, 1H), 2.05 (s, 3H), 2.03–1.94 (m, 3H), 1.67 (s, 5H), 1.36 (bs, 5H), 1.25 (s, 3H), 1.15 (d, J = 9.7 Hz, 2H), 1.01 (d, J = 6.6 Hz, 3H)H), 0.93–0.85 (m, 2H), 0.69 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 202.68 (s), 165.42 (s), 152.86 (s), 151.41 (s), 133.34 (s), 129.84 (s), 127.25 (s), 124.86 (s), 123.88 (s), 82.63 (s), 77.04 (s), 68.99 (s), 62.58 (s), 57.26 (s), 55.75 (s), 55.18 (s), 54.04 (s), 51.78 (s), 50.09 (s), 47.61 (s), 41.01 (s), 39.84 (s), 37.12 (s), 37.08 (s), 28.75 (s), 28.06 (s), 27.39 (s), 25.61 (s), 22.51 (s), 18.88 (s), 18.37 (s), 13.89 (s), 11.67 (s). HRMS ESI: m/z calcd. for C35H42Cl2NO7 (M +H)+ 658.2338, found 658.2339. 4-trifluorophenyl-2-isoxazoline withaferin A (W-11a). White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 8.3 Hz, 2H), 5.00 (d, J = 10.7 Hz, 1H), 4.43–4.28 (m, 4H), 3.50 (s, 1H), 2.65 (s, 1H), 2.54–2.45 (m, 1H), 2.12–2.07 (m, 1H), 2.03 (s, 3H), 2.00–1.92 (m, 3H), 1.66 (dd, J = 16.8, 5.9 Hz, 4H), 1.31 (s, 3H), 1.24 (s, 5H), 1.16–1.06 (m, 3H), 0.99 (d, J = 6.6 Hz, 3H), 0.85 (dd, J = 16.2, 9.3 Hz, 2H), 0.65 (s, 3H). Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x www.nature.com/scientificreports/ White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 2.4 Hz, 1H), 7.43 (dd, J = 8.9, 2.4 Hz, 1H), 6.80 (d, J = 8.9 Hz, 1H), 5.25 (d, J = 14.8 Hz, 1H), 5.07 (dd, J = 12.2, 3.2 Hz, 1H), 4.95 (d, J = 12.2 Hz, 1 H), 4.82 (q, J = 11.9 Hz, 2H), 4.71 (d, J = 3.2 Hz, 1H), 4.33 (d, J = 13.1 Hz, 1H), 3.84 (s, 3H), 3.35 (s, 1H), 2.86 (s, 1H), 2.58 (s, 1H), 2.53–2.42 (m, 1H), 2.18 (d, J = 12.4 Hz, 1H), 2.05 (d, J = 4.8 Hz, 3H), 1.94 (dd, J = 25.4, 7.2 Hz, 2H), 1.59 (s, 5H), 1.25 (s, 3H), 1.17–0.99 (m, 2H), 0.89 (d, J = 6.6 Hz, 3H), 0.80 (d, J = 9.9 Hz, 1H), 0.61 (s, 2 H), 0.53 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 201.80 (s), 165.26 (s), 156.99 (s), 156.88 (s), 153.23 (s), 134.52 (s), 133.29 (s), 121.94 (s), 118.89 (s), 113.92 (s), 112.74 (s), 80.53 (s), 78.09 (s), 74.40 (s), 59.79 (s), 59.71 (s), 58.20 (s), 58.02 (s), 56.54 (s), 56.03 (s), 51.76 (s), 51.21 (s), 42.27 (s), 41.57 (s), 38.57 (s), 38.07 (s), 31.04 (s), 30.00 (s), 29.69 (s), 27.22 (s), 24.07 (s), 20.02 (s), 14.34 (s), 13.26 (s), 11.34 (s). HRMS ESI: m/z calcd. C36H45BrNO8 (M + H)+ 698.2329, found 698.2317. ( ) 2,6-dichlorophenyl-2-isoxazoline withaferin A (W-6b). White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.55 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.3 Hz, 1H), 5.17 (dd, J = 12.0, 3.4 Hz, 1H), 4.82 (d, J = 12.0 Hz, 1H), 4.82 (d, J = 12.0 Hz, 1H), 4.41–4.38 (m, 3H), 3.72 (bs, 2H), 3.37 (s, 1 H), 2.90 (t, J = 6.5 Hz, 1H), 2.69 (bs, 1 H), 2.57–2.45 (m, 1H), 2.20 (d, J = 13.9 Hz, 1H), 2.09 (s, 3H), 1.96 (d, J = 21.5 Hz, 1H), 1.61 (s, 7H), 1.48–1.38 (m, 1H), 1.36– 1.31 (m, 1H), 1.25 (s, 3H), 1.12 (s, 2H), 0.91 (d, J = 6.3 Hz, 3H), 0.75 (bs, 1H), 0.55 (s, 3H). www.nature.com/scientificreports/ www.nature.com/scientificreports/ 57.07 (s), 52.23 (s), 51.25 (s), 42.67 (s), 42.39 (s), 39.03 (s), 38.53 (s), 31.47 (s), 30.25 (s), 29.94 (s), 27.61 (s), 24.44 (s), 20.36 (s), 20.34 (s), 15.64 (s), 13.42 (s), 11.72 (s). HRMS ESI: m/z calcd. for C36H43F3NO7 (M + H)+ 658.2992, found 658.2984.l 57.07 (s), 52.23 (s), 51.25 (s), 42.67 (s), 42.39 (s), 39.03 (s), 38.53 (s), 31.47 (s), 30.25 (s), 29.94 (s), 27.61 (s), 24.44 (s), 20.36 (s), 20.34 (s), 15.64 (s), 13.42 (s), 11.72 (s). HRMS ESI: m/z calcd. for C36H43F3NO7 (M + H)+ 658.2992, found 658.2984.l 4-trifluorophenyl-2-isoxazoline withaferin A (W-11b). White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 8.3 Hz, 2H), 5.20 (dd, J = 12.0, 3.3 Hz, 1H), 4.63 (d, J = 12.0 Hz, 1H), 4.42–4.25 (m, 3H), 3.68 (d, J = 3.3 Hz, 1H), 3.33 (s, 1H), 2.47–2.34 (m, 1H), 2.16 (d, J = 12.9 Hz, 1H), 2.03 (s, 3H), 1.88 (dd, J = 17.9, 3.2 Hz, 2H), 1.57–1.48 (m, 4H), 1.22 (s, 3H), 1.23 (s, 3H), 1.19 (d, J = 12.9 Hz, 2H), 1.15–0.97 (m, 3H), 0.86 (s, 1H), 0.80 (d, J = 6.6 Hz, 3H), 0.68 (dd, J = 19.6, 9.6 Hz, 1H), 0.59 (dd, J = 14.3, 3.4 Hz, 1H), 0.49 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 203.00 (s), 167.16 (s), 153.59 (s), 153.02 (s), 132.55 (s), 132.22 (s), 127.54 (s), 126.12 (dd, J = 7.4, 3.6 Hz), 125.90 (s), 122.55 (s), 83.81 (s), 78.75 (s), 73.96 (s), 62.67 (s), 59.31 (s), 58.37 (s), 57.67 (s), 56.87 (s), 52.03 (s), 51.05 (s), 42.47 (s), 42.19 (s), 38.83 (s), 38.34 (s), 31.27 (s), 30.06 (s), 29.91 (s), 29.74 (s), 27.41 (s), 24.24 (s), 20.17 (s), 15.44 (s), 13.22 (s), 11.52 (s). HRMS ESI: m/z calcd. for C36H43F3NO7 (M + H)+ 658.2992, found 658.2981.l , 4-fluorophenyl-2-isoxazoline 4,27-acetylwithaferin A (W-12b). www.nature.com/scientificreports/ White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.78 (dd, J = 8.6, 5.3 Hz, 2H), 7.13 (t, J = 8.5 Hz, 2H), 5.17 (dd, J = 11.9, 3.2 Hz, 1H), 4.93–4.83 (m, 2H), 4.79 (d, J = 3.2 Hz, 1H), 4.58 (d, J = 11.9 Hz, 1H), 4.35 (bd, J = 13.1 Hz, 1H), 3.43 (s, 1H), 2.77 (s, 1H), 2.57–2.42 (m, 1H), 2.19 (d, J = 12.0 Hz, 1H), 2.12 (s, 3H), 2.09 (s, 3H), 2.06 (s, 3H), 2.00–1.91 (m, 2H), 1.58 (dd, J = 17.7, 5.3 Hz, 4H), 1.29 (s, 2H), 1.25 (s, 4H), 1.25 (s, 3H), 1.10–1.05 (m, 1H), 0.87 (d, J = 6.7 Hz, 3H), 0.70–0.62 (m, 1H), 0.53 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 202.78 (s), 170.96 (s), 169.12 (s), 165.28 (s), 163.04 (s), 157.03 (s), 153.25 (s), 129.06 (d, J = 8.5 Hz), 124.59 (d, J = 3.4 Hz), 121.85 (s), 116.21 (d, J = 22.0 Hz), 80.59 (s), 78.10 (s), 74.37 (s), 59.75 (s), 59.65 (s), 58.22 (s), 58.01 (s), 56.14 (s), 51.78 (s), 51.60 (s), 42.33 (s), 42.15 (s), 38.57 (s), 38.23 (s), 31.10 (s), 27.21 (s), 24.13 (s), 20.96 (s), 20.93 (s), 20.64 (s), 20.02 (s), 14.52 (s), 13.29 (s), 11.37 (s).). HRMS ESI: m/z calcd. for C41H49F3NO10 (M + H)+ 772.3309, found 772.3343. References References 1. Hanson, J. R. Steroids: partial synthesis in medicinal chemistry. Nat. Prod. Rep. 27, 887–899 (2010). 1. Hanson, J. R. Steroids: partial synthesis in medicinal chemistry. Nat. Prod. Rep. 27, 887–899 (2010). J p y y . Djerassi, C. A Steroid autobiography. Steroids 43, 351–361 (1984) j g p y ( ) 3. Dua, R., Shrivastava, S., Sonwane, S. K. & Srivastava, S. K. Pharmacological significance of synthetic heterocycles scaffold: a review. Adv. Biol. Res. 5, 120–144 (2011). j g p y 3. Dua, R., Shrivastava, S., Sonwane, S. K. & Srivastava, S. K. Pharmacological significance of synthetic heterocycles scaffold: a review Adv. Biol. Res. 5, 120–144 (2011). g y pp y ( ) 5. Frank, E. & Schneider, G. Synthesis of sex hormone-derived modified steroids possessing antiproliferative activity. J. Steroid Biochem. Mol. Biol. 137, 301–315 (2013).h 5. Frank, E. & Schneider, G. Synthesis of sex hormone-derived modified steroids possessing antiproliferative activity. J. Steroid Biochem. Mol. Biol. 137, 301–315 (2013).h 6. Lavie, D., Glotter, E. & Shvo, Y. 1371. Constituents of Withania somnifera Dun. Part IV. The structure of withaferin A. J. Chem. Soc 7517–7531 (1965).f 7. Devi, P. U., Sharada, A. C. & Solomon, F. E. In vivo growth inhibitory and radiosensitizing effects of withaferin A on mouse Ehrlich ascites carcinoma. Cancer Lett. 95, 189–193 (1995). , ( ) 8. Jayaprakasam, B., Zhang, Y., Seeram, N. P. & Nair, M. G. Growth inhibition of human tumor cell lines by withanolides from Withania somnifera leaves. Life Sci. 74, 125–132 (2003). f ( ) 9. Tangallapally, R. P. et al. Discovery of novel isoxazolines as anti-tuberculosis agents. Bioorg. Med. Chem. Lett. 17, 6638–6642 (2 9. Tangallapally, R. P. et al. Discovery of novel isoxazolines as anti-tuberculosis agents. Bioorg. Med. Chem. Lett. 17, 6638–6642 (2007). 10. JÃger, V., Buss, V. & Schwab, W. 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Cancer Prev. 20, 185 (2015). 26 Suman S et al Withaferin A suppress AKT induced tumor growth in colorectal cancer cells Oncotarget 7 13854 13864 (2016) , K , g y k g p y J 20, 185 (2015). 26. www.nature.com/scientificreports/ White solid powder; 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 8.3 Hz, 2H), 5.00 (d, J = 10.7 Hz, 1H), 4.43–4.28 (m, 4H), 3.50 (s, 1H), 2.65 (s, 1H), 2.54–2.45 (m, 1H), 2.12–2.07 (m, 1H), 2.03 (s, 3H), 2.00–1.92 (m, 3H), 1.66 (dd, J = 16.8, 5.9 Hz, 4H), 1.31 (s, 3H), 1.24 (s, 5H), 1.16–1.06 (m, 3H), 0.99 (d, J = 6.6 Hz, 3H), 0.85 (dd, J = 16.2, 9.3 Hz, 2H), 0.65 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 203.20 (s), 167.36 (s), 153.79 (s), 153.22 (s), 132.74 (s), 132.42 (s), 127.74 (s), 126.32 (dd, J = 7.4, 3.6 Hz), 126.10 (s), 122.75 (s), 84.01 (s), 78.95 (s), 74.16 (s), 62.87 (s), 59.51 (s), 58.56 (s), 57.87 (s), Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 16 www.nature.com/scientificreports/ 96, 164–171 (2017). p ( ) 45. 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Dual modulation of Ras-Mnk and PI3K-AKT-mTOR pathways: A Novel c-FLIP inhibitory mechanism of 3-AWA mediated translational attenuation through dephosphorylation of eIF4E. Sci. Rep. 6 (2016).f g y p 43. Lombardi, D., Lacombe, M. L. & Paggi, M. G. nm 23: Unraveling its biological function in cell differentiation. J. Cell. Physiol. 182, 144–149 (2000).f 44. Rasool, R. U. et al. Differential regulation of NM23-H1 under hypoxic and serum starvation conditions in metastatic cancer cells and its implication in EMT. Eur. J. Cell Biol. Author Contributions Conceive the idea: F.R., D.N., A.G., D.M.; Medicinal chemistry, synthesis and characterization of molecules: F.R., N.H.; Anticancer activity, mechanistic study and in vivo study: D.N., A.K., M.M.F., A.G.; Provide raw materials and synthesis of WA: S.K.Y., C.B., N.K.S.; Writing of manuscript: F.R., D.N.; Supervision, data interpretation: D.M., A.G. Acknowledgements g The work was supported by the institutional internal grant BSC-0108 and MLP-6002 with institutional publication number IIIM/2158/2017. We thank Dr. R.A. Vishwakarma, Director, IIIM, Jammu for his support to accomplish this research work. The authors also acknowledge Council of Scientific and Industrial Research (CSIR) and Department of Biotechnology (DBT), Govt. of India for providing fellowship to the research scholars. g The work was supported by the institutional internal grant BSC-0108 and MLP-6002 with institutional publication number IIIM/2158/2017. We thank Dr. R.A. Vishwakarma, Director, IIIM, Jammu for his support to accomplish this research work. The authors also acknowledge Council of Scientific and Industrial Research (CSIR) and Department of Biotechnology (DBT), Govt. of India for providing fellowship to the research scholars. References Suman, S. et al. Withaferin-A suppress AKT induced tumor growth in colorectal cancer cells. 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EMBO J. 21, 2180–2188 (2 Scientific Reports \| 7: 13749 \| DOI:10.1038/s41598-017-13664-x 17 www.nature.com/scientificreports/ Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-13664-x. Competing Interests: The authors declare that they have no competing interests. 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 an 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/W4231064615	https://www.researchsquare.com/article/rs-451/v3.pdf	English	null	A prospective randomized double-blind trial of the efficacy of a bilateral lumbar erector spinae block on the 24h morphine consumption after posterior lumbar interbody fusion surgery	Research Square (Research Square)	2,019	cc-by	4,905	A prospective randomized double-blind trial of the efficacy of a bilateral lumbar erector spinae block on the 24h morphine consumption after posterior lumbar interbody fusion surgery margaretha breebaart (  margaretha.breebaart@uza.be ) David Van Aken AZ Klina Olivier De Fré Universitair Ziekenhuis Antwerpen Luc Sermeus Universitair Ziekenhuis Antwerpen Niels Kamerling Universitair Ziekenhuis Antwerpen Jozef Michielsen Universitair Ziekenhuis Antwerpen Lars De Jong AZ Klina Ella Roelant Universitair Ziekenhuis Antwerpen Vera Saldien Universitair Ziekenhuis Antwerpen Barbara Versyck Catharina Ziekenhuis DOI: https://doi.org/10.21203/rs.2.452/v3 Page 1/13 Page 1/13 Version of Record: A version of this preprint was published on July 17th, 2019. See the published version at https://doi.org/10.1186/s13063-019-3541-y. Page 2/13 Abstract Background: Spine surgery is associated with considerable postoperative pain and can be challenging to treat. A loco-regional technique suitable for spine surgery should cover the dorsal root of the spinal nerves at the levels where surgery is performed. The erector spinae block is a loco-regional technique with promising results that was recently described at the thoracic level. There are no randomized trials of this technique on a lumbar level. This study tests the hypothesis that the 24hour postoperative morphine consumption is significantly lower in patients undergoing posterior lumbar inter-body fusion surgery with a lumbar erector spinae (LUMBES) block when compared to a sham block. Methods: This prospective randomized double-blind multicentre study will randomly allocate 80 adult patients undergoing elective posterior lumbar inter-body fusion surgery during general anaesthesia to one of two groups as follows: 1. bilateral erector spinae block (20 mL 0.25% levobupivacaine) or 2. bilateral sham block (20 ml NaCl 0.9%). Our primary endpoint is the 24-hour postoperative morphine consumption. Secondary endpoints include: 72-hour morphine consumption, intraoperative sufentanil dosage, postoperative pain scores at regular time intervals both at rest and during movement, time to first post-operative mobilization and the Quality of Recovery 40 score. Discussion: The LUMBES trial is a pragmatic clinical study that will provide evidence of whether a bilateral lumbar erector spinae block is effective in reducing 24-hour postoperative morphine consumption in patients undergoing lumbar inter-body fusion surgery. If this hypothesis is confirmed, this finding could contribute to more widespread implementation of this technique. Objectives and purpose In this prospective, randomized, double-blind placebo controlled clinical trial we will investigate the effect of bilateral erector spinae block (20 mL levobupivacaine 0.25%) on the 24-hour postoperative morphine consumption when compared to a sham block (20 ml of NaCl 0.9%) following posterior lumbar inter-body fusion surgery. Secondary objectives include the effect of the erector spinae block (ESB) on the numeric rating scale (NRS) pain scores in rest at fixed time points: at the time of inclusion, in the post anaesthesia care unit (PACU, [T0 = arrival in PACU, T+15min, T+30min]) and on the ward (twice daily- morning and evening until postoperative day 3). Other secondary objectives are the effect of an ESB on the NRS pain scores during defined movement (mobilization to chair) at 24 hours, 48 hours and 72 hours; time to first mobilization to chair (hours); time to first walk of 20 meters (hours); the required sufentanil dose during surgery (micrograms), total morphine consumption during the first 72 hours postoperatively (milligrams) and the Quality of Recovery 40 score (QoR-40) at day 1 and 3. Background information Patients undergoing spine surgery often fear postoperative pain, which can be a source of considerable preoperative distress. Many of these patients are already diagnosed as so-called chronic pain patients requiring high doses of narcotics and other analgesics. In spine surgery postoperative pain can often be severe and difficult to treat, certainly if a one-dimensional approach is used to achieve pain control [1, 2] . Many caregivers are reluctant to prescribe liberal doses of opioids to achieve adequate analgesia as this may be associated with side effects such as respiratory depression, sedation and nausea. Many techniques have been combined in order to decrease opioid consumption after spinal surgery e.g. epidural catheters, spinal and epidural morphine or local infiltration[3] . The introduction of ultrasound has allowed the performance of plane blocks and other techniques such as root blocks and facet infiltrations without the use of unreliable ‘pop-techniques’ or x-ray. A loco-regional technique suitable for back surgery should cover the innervation of the relevant vertebrae and paravertebral muscles and include the dorsal roots of the spinal nerves at this level[4] .Dorsal ramus blocks have been shown to be feasible in the treatment of chronic back pain [5] . Recently a series of case reports has been described in which bilateral block of the lumbar dorsal ramus nerve resulted in a positive effect on pain scores and morphine consumption after spine surgery [6] . To our knowledge, however, no RCT’s have yet been performed to study the effect on pain scores and opioid Page 3/13 consumption. Furthermore, there are promising results for postoperative analgesia with a new plane block, the erector spinae block, which has recently been described as a safe and simple technique for neuropathic and acute postoperative pain at the thoracic level. [7, 8] . consumption. Furthermore, there are promising results for postoperative analgesia with a new plane block, the erector spinae block, which has recently been described as a safe and simple technique for neuropathic and acute postoperative pain at the thoracic level. [7, 8] . Rationale Theoretically, an infiltration between the erector spinae muscle and the transverse process provides anesthesia of the dorsal ramus at the same vertebral level. Since the local anaesthetic is injected into a plane, the solution can spread both caudally and cranially via the thoracolumbar fascia, resulting in anaesthesia of the dorsal ramus of the spinal nerves above and below the injected level. The erector spinae block has been described at the thoracic level with promising results. We performed a small feasibility trial in which we found that the erector spinae block could easily be performed without major inconvenience for the patients (clinicaltrials.gov NCT0321453). In this proposed trial we aim to determine the effect of a lumbar erector spinae block on pain after back surgery, expressed as morphine consumption during the first 24 hours postoperatively. Study design and registration Study design and registration This is an investigator-initiated prospective randomized double-blind multicentre trial. This is an investigator-initiated prospective randomized double-blind multi The study is being performed in accordance with the Declaration of Helsinki (Fortaleza, Brazil, October 2013) and Good Clinical Practice guidelines. The study has been approved by the ethics committee at Antwerp University Hospital, Wilrijk, Belgium and the AZ Klina Hospital, Brasschaat, Belgium (reference: B300201837508 ) The trial has been prospectively registered at www.clinicaltrails.gov (reference:NCT03825198) and will be monitored by the clinical trial centre of the Antwerp University Hospital. Randomization Patients will be assigned consecutive numbers upon inclusion in the study. These numbers are 1:1 randomly allocated to the ESB or the sham group using a web-based randomisation system QMinim. Qminim uses stratified randomisation, stratification will be done according to site, gender and levels of surgery. In Qminim a minimisation procedure is used to randomize the patients to ensure a similar distribution of the stratifying arms. Online randomization will be carried out by an independent anaesthetist who will also prepare the medication. Participation Page 4/13 Page 4/13 Patients scheduled for elective 1or 2 level posterior lumbar inter-body fusion surgery in the AZ KLINA Hospital and the Antwerp University Hospital will be asked for informed consent by a member of the anaesthesiology department. Recruitment will occur during the preoperative consultation and will open on 15th of February 2019 until the required number of patients have been included. Inclusion criteria are as follows: (1) American Society of Anaesthesiologist (ASA) physical status of 1-3, (2) Age: 18 - 75 years, (3) normal liver and renal function. Exclusion criteria are as follows: (1) Body Mass Index (BMI) < 20 or BMI > 35, (2) allergy to one or more medications used in the study including epinephrine, levobupivacaine, dexamethasone, propofol, sufentanil, rocuronium, ketorolac, morphine, ketamine, dehydrobenzperidol, ondansetron, alizapride (3) chronic strong opioid use (>3 administrations per week), (4) contraindications to a regional anaesthetic technique, (4) contraindications to one or more of the study medications, (5) patient refusal and/or no informed consent. Blinding All investigators, staff and patients will be blinded to the treatment groups. The study medication will be prepared by an anaesthesiologist who is not involved in the study or in the care of the patient. Both solutions and syringes will appear identical. Unmasking will only occur after statistical analysis has been completed, unless if medically indicated. Interventional treatment Unmasking will only occur after statistical analysis has been completed, unless if medically indicated. Unmasking will only occur after statistical analysis has been completed, u Medication The ESB study medication will be 20 ml levobupivacaine 0.25% (Chirocaine, AbbVie). The preparing 20 ml of NaCl 0.9% (B. Braun) Primary endpoint The primary endpoint is the morphine consumption during the first 24 hours postoperatively in milligram and will be determined from the PCIA pump. Interventional treatment Patients will be extubated in the operating theatre and admitted to the PACU. General anaesthesia will then be induced in a standardized way with propofol 2-3mg/kg, sufentanil 15µg and rocuronium 0.5mg/kg. After tracheal intubation, anaesthesia will be maintained with sevoflurane and intraoperative analgesia provided with sufentanil. The dosages of these agents will be determined at the discretion of the attending anaesthesiologist. At the end of surgery, patients will receive acetaminophen 1g IV, ketorolac 0.5 mg/kg IV (max. 30 mg) and a morphine loading dose (0.1 mg/kg) IV to manage postoperative pain. Patients will be extubated in the operating theatre and admitted to the PACU. Postoperative nausea and vomiting prophylaxis will be administered with dexamethasone 5mg IV just before induction of general anaesthesia. If required, this will be supplemented by ondansetron 4mg IV and further with alizapride 50mg IV as rescue. Postoperative nausea and vomiting prophylaxis will be administered with dexamethasone 5mg IV just before induction of general anaesthesia. If required, this will be supplemented by ondansetron 4mg IV and further with alizapride 50mg IV as rescue. Postoperative pain in the PACU and on the ward will be treated with regular doses of acetaminophen 1g IV around the clock (4 times daily) and by a patient controlled intravenous analgesia (PCIA) pump containing morphine at a concentration of 1 mg/mL and dehydrobenzperidol 0.05mg/ml. The PCIA pump will be programmed as follows: no continuous infusion, a bolus dose of 1.5mg morphine, a lock-out interval of 8 minutes and an hourly limit of 7.5 mg. If pain management on the PACU is inadequate, defined as a Numeric Rating Scale (NRS) pain score > 3 ( 0 [no pain] – 10 [worst imaginable pain]) additional boluses of 1mg morphine IV will be administered by the PACU nurses with a total additional dose of morphine limited to 0.15 mg/kg. If pain management with morphine remains inadequate, an IV ketamine (Ketalar, Pfizer) bolus (0.2mg/kg) will be administered. Interventional treatment All patients will receive a bilateral ESB. The blocks will be performed by experts in the field of ultrasound guided regional anaesthesia. The blocks will be performed preoperatively in a separate block room with ultrasound, after obtaining IV access and application of standard ASA monitoring. The blocks will be placed as described by Chin et al. 6 and modified for the lumbar level. The patient will be placed in the Page 5/13 lateral or sitting position. A curved array probe or a high frequency linear probe, depending on the BMI of the patient, will be placed in longitudinal alignment, 2-3 cm lateral to the vertebral column. The transverse processes of the vertebrae at the level of surgery, the erector spinae muscle and the psoas muscle will be identified. In case of two-level surgery, the transverse process of the upper level will be considered as the target. A 5 or 8 cm 22G ultrasound needle will be inserted with an in-plane technique in a cephalad to caudal direction until bone contact with the top of the transverse process is reached. After slight retraction of the needle, 20 mL of the study medication will be injected behind the erector spinae muscle. The same procedure will be repeated on the contralateral side. lateral or sitting position. A curved array probe or a high frequency linear pr the patient, will be placed in longitudinal alignment, 2-3 cm lateral to the ve processes of the vertebrae at the level of surgery, the erector spinae muscle identified. In case of two-level surgery, the transverse process of the upper l target. A 5 or 8 cm 22G ultrasound needle will be inserted with an in-plane t caudal direction until bone contact with the top of the transverse process is retraction of the needle, 20 mL of the study medication will be injected beh The same procedure will be repeated on the contralateral side. General anaesthesia will then be induced in a standardized way with propofol 2-3mg/kg, sufentanil 15µg and rocuronium 0.5mg/kg. After tracheal intubation, anaesthesia will be maintained with sevoflurane and intraoperative analgesia provided with sufentanil. The dosages of these agents will be determined at the discretion of the attending anaesthesiologist. At the end of surgery, patients will receive acetaminophen 1g IV, ketorolac 0.5 mg/kg IV (max. 30 mg) and a morphine loading dose (0.1 mg/kg) IV to manage postoperative pain. Discomfort during puncture Allergy for the disinfectant or levobupivacaine (very rare 1:10.000- 1:100.000) Infection at the skin, needle trajectory or point of injection (very rare). The clinical presentation can be variable, e.g. redness at the puncture site or in extreme cases an intramuscular abscess. Therefore, the ESB will be performed under strict sterile conditions using a sterile gown, gloves and mask, and a sterile field. Bleeding: very rare with the use of an ultrasound-guided technique. When bleeding occurs, this will be noted by the surgeon. Neural damage: very rare since the target of the puncture is a muscular plane and not the nerve root or nerve ramus itself Neural damage: very rare since the target of the puncture is a muscular plane and not the nerve root or nerve ramus itself Local anaesthetic systemic toxicity: since the doses are substantial there is a clinically significant risk for local anaesthetic systemic toxicity, as with any existing plane block. It can immediately be treated with intralipid. For this reason, the patient will be monitored during and after the placement of the erector spinae block until the start of surgery. Intralipid should be available in any medical environment where regional anaesthesia is performed. Secondary endpoints As secondary endpoint, the total morphine consumption in mg, during the first 72 postoperative hours, will be extracted out of the PCIA pump. Pain scores at rest will be assessed with the NRS (0=no pain, 10 = worst imaginable pain) and tested at regular time intervals: at the time of inclusion, in the post anaesthesia care unit (PACU, [T0 = arrival in PACU, T+15min, T+30min]) and on the ward (twice daily- morning and evening until postoperative day 3). Pain scores during defined movement (first moving to a chair and sitting upright) will be registered. Time to first mobilization to a chair (in hours since T0) and Page 6/13 time to first walk of twenty meters (in hours since T0) will be noted in the patients’ study diary. The Quality of Recovery 40 score (QoR-40) will be calculated from the responses to a standard questionnaire at postoperative day 1 and day 3. The QoR-40 is a widely used and extensively validated measure of quality of recovery. It is a 40-item questionnaire on quality of recovery from anesthesia that has been shown to measure health status after surgery[9, 10] . Tertiary endpoints Other endpoints include preoperative expected NRS pain score, postoperative nausea and vomiting score according to hospital protocol, number of administered postoperative anti-emetics, time to first meal and time to first defecation. All block complications or adverse events will be registered. Summary of known and potential risks The erector spinae block is a plane block where a substantial dose of local anaesthetic is used. As this technique has only recently been described, limited evidence is available regarding the potential risks of the block. The potential risks described below relate to the known risks of a plane block, facet infiltration and intramuscular injection: Discomfort during puncture Sample size Our sample size calculation is based on data from a randomised controlled trial comparing the effect of systemic infused lidocaine with placebo, on the 24h morphine requirement in posterior lumbar arthrodesis[11] . We considered a 25% reduction in PCA morphine consumption as clinically relevant. To calculate the sample size, we assumed a mean of 51 mg morphine with standard deviation 19 mg (mean morphine consumption for the placebo group of the above-mentioned trial), a type 1 failure risk of 5% and a type 2 failure risk of 20%. Thirty-five patients are be required in each group to detect a 25% reduction in morphine equivalent over 24 hours. The sample size calculation was based on an independent samples t- test. We plan to include 80 patients in total to compensate for potential dropouts and uncertainty in predicting the actual standard deviation. Data collection Patients’ demographic data will be collected at the inclusion assessment (height, weight, age, sex and ASA classification). The attending anaesthesiologist will collect data with regard to the anaesthesia and surgical procedure. Nurses will collect the data in the PACU. When transferred to the orthopaedic ward, the Page 7/13 Acute Pain Service Team will score the Quality of Recovery 40 survey (QoR-40) daily, adjust analgesia when necessary and systematically screen for side effects. Ward nurses will assess NRS pain scores in the morning and the evening on postoperative day 1-3. Morphine PCA consumption will electronically be registered by the PCA pump, all other data will be registered by nurses of the PACU, ward pain department or trial nurses. All medication can be retrieved from the patient data management systems. Acute Pain Service Team will score the Quality of Recovery 40 survey (QoR-40) daily, adjust analgesia when necessary and systematically screen for side effects. Ward nurses will assess NRS pain scores in the morning and the evening on postoperative day 1-3. Morphine PCA consumption will electronically be registered by the PCA pump, all other data will be registered by nurses of the PACU, ward pain department or trial nurses. All medication can be retrieved from the patient data management systems. Complications will be assessed on the day of discharge. During the 72 hours of the trial, data will be registered on paper. After termination of the trial (72 hours after surgery) the data will be directly registered in the software program Open Clinica. Complications will be assessed on the day of discharge. During the 72 hours of the trial, data will be registered on paper. After termination of the trial (72 hours after surgery) the data will be directly registered in the software program Open Clinica. An independent trial monitor from the clinical trial center (CTC) at Antwerp University Hospital will conduct a follow up on the GCP performance of the trial in both study locations. All data will be published anonymously. Patient characteristics and baseline comparisons Demographic and other baseline characteristics will be summarized by treatment group. For categorical variables, frequencies and percentages will be reported. Where values are missing, percentages will be calculated for the available cases, and the denominator will be mentioned. Continuous variables will be summarized as mean with standard deviation or median with interquartile range as appropriate. Comparisons of demographic and baseline characteristics between the treatment groups will be conducted to assess the effectiveness of randomization. For categorical variables the chi-squared test or Fisher exact test (when numbers are low) will be used. For continuous variables, a t-test or Mann-Whitney U test will be used as appropriate. The following baseline information prior to randomization will be collected: age, sex, BMI, ASA physical status, indication for surgery, preoperative pain (NRS) and use of analgesics. Analysis of the endpoints Page 8/13 SPSS software version 21 (SPSS, Chicago, IL, USA) or 3.3.2 will be used for statistical analysis. The primary endpoint will be analysed using an independent samples t-test intention-to-treat population (in Page 8/13 Page 8/13 Page 8/13 case of normality). To evaluate the sensitivity of the results of the primary outcome analysis, a linear regression will be used to model the cumulative morphine consumption during the first 24h after surgery with treatment as predictor and taking into account possible confounders. A linear mixed model will be used to model the cumulative morphine consumption over time with subject as random effect. This model allows correction for confounders and adding a random intercept for site. From this model the difference in morphine consumption at the different time points can be estimated. To compare the continuous outcomes (intraoperative sufentanil dosage, required morphine dose, pain scores, Quality of Recovery 40 score, nausea and vomiting score, number of administered postoperative anti-emetics) at different time points we will use an independent samples t-test if they are normally distributed or a Mann Whitney U test if otherwise. We will also fit a linear regression model for these outcomes, which makes it possible to correct for confounders. A linear mixed model will be studied for the continuous outcomes measured over time. The time to the different events of interest (first mobilization to a chair, first walk of 20 meters, first meal and first defecation) will be studied in a time-to-event analysis comparing the two treatment arms. If required, we will use a Cox proportional hazard model to adjust for other variables. The trial’s results will be submitted to a peer-reviewed journal regardless of the outcome. The trial’s results will be submitted to a peer-reviewed journal regardless of the outcome. Discussion Posterior spine surgery ranks amongst the most painful surgical procedures and can be challenging to treat. High doses of opioids are often prescribed [1, 2] . Musculoskeletal postoperative pain in posterior approach spine surgery arises from iatrogenic mechanical damage, intraoperative retraction, partial devascularisation and denervation of bone, ligaments, muscles, intervertebral disks and zygapophysial joints. In addition, neuropathic pain arises from compression and damage to nerve roots exiting the spinal canal and sometimes damage to the spinal cord itself[12] . In order to reduce opioid use, loco- regional and local anaesthesia were introduced. In spine surgery loco-regional techniques were limited to epidural catheters, spinal and epidural morphine. These techniques have side effects and are not routinely used. Local anaesthetic wound infiltration is often performed with unfortunately short-lived effect[3] . The loco-regional technique used in this type of surgery should aim to anaesthetise the dorsal root of the spinal nerves at the appropriate operative level[4] . Dorsal ramus blocks have been shown to be feasible in the treatment of chronic pain[5] . Recently, a series of case reports has been described where a bilateral block of the lumbar dorsal ramus nerve showed improved pain scores and reduced morphine consumption after spine surgery[6] . Also, there are promising results with the erector spinae block, which has recently been described as a safe and simple technique for neuropathic and acute Page 9/13 Page 9/13 postoperative pain at the thoracic level[8] . Furthermore, ESB has been shown to effectively control postoperative pain in patients undergoing breast surgery. However, no comparative data for the lumbar level is available[13] . postoperative pain at the thoracic level[8] . Furthermore, ESB has been shown to effectively control postoperative pain in patients undergoing breast surgery. However, no comparative data for the lumbar level is available[13] . This study will provide clinical evidence on the efficacy of the lumbar erector spinae block in reducing postoperative opioid consumption for posterior lumbar inter-body fusion surgery. If the LUMBES trial demonstrates efficacy, the findings will provide high-quality evidence to support the implementation of this technique in clinical practice. Furthermore, it might trigger studies from other researchers to test our outcomes in their practice. Potential benefits of the lumbar erector spinae block include the ease of performance with clear landmarks for ultrasound anatomy. Trial status This document is based on version 8 (02/02/2019) of the original protocol. We anticipate randomizing the first patient on 15th of March 2019 and plan to complete the study in February 2020. Informed consent @ ethic approval The study is being performed in accordance with the Declaration of Helsinki (Fortaleza, Brazil, October 2013) and Good Clinical Practice guidelines. The study has been approved by the ethics committee of Antwerp University Hospital, Wilrijk, Belgium and the AZ Klina Hospital, Brasschaat, Belgium (reference: B300201837508) Informed consent will be obtained from all study participants. Discussion The technique is inherently safe, as the target site for injection is a muscular plane and there is practically no risk for mechanical nerve contact. Other benefits include the possible reduction in perioperative opioid consumption. The ESB performed in patients under anticoagulant therapy or with coagulopathies [8] . Furthermore, hemodynamic instability due to sympathetic blockade, as with epidural and spinal anaesthesia, occurs rarely. Possible risks consist primarily of local anaesthetic systemic toxicity. Since substantial doses are considered necessary, there is a clinically significant risk for Local Anaesthetic Systemic Toxicity (LAST), as with any high-volume fascial block. For this reason, patients need to be monitored according to ASRA (American Society of Regional Anesthesia) guidelines with Intralipid available at all times [14] . Consent for publication The document does not contain personal data and consent for publication is not applicable Availability of data The document does not contain personal data and consent for publication Availability of data Availability of data Page 10/13 Funding Funding of the study will be departmental and will be sponsored by using a Belgian Association of Regional Anaesthesia (BARA) research grant. Funding of the study will be departmental and will be sponsored by using a Belgian Association of Regional Anaesthesia (BARA) research grant. Page 10/13 Page 10/13 The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request and will only be accessible to personnel involved in the trial. Competing interests All authors declare that they have no competing interests. Abbreviations ASA: American Society of Anaesthesiologists PCA: Patient Controlled Intravenous Analgesia QoR-40: Quality of Recovery 40 score Authors’ contributions Margaretha Breebaart, David Van Aken, Olivier de Fre, and Barbara Verysck drafted the protocol and study design, read and approved the final manuscript Ella Roelant supported the drafting of the statistical analysis plan. Ella Roelant supported the drafting of the statistical analysis plan. Luc Sermeus, Lars de Jong, Vera Saldien, Niels Kamerling and Jozef Michielsen read and approved the final manuscript Luc Sermeus, Lars de Jong, Vera Saldien, Niels Kamerling and Jozef Michielsen read and approved the final manuscript References 1. Lai LT, Ortiz-Cardona JR, Bendo AA. Perioperative pain management in the neurosurgical patient. Anesthesiol Clin. 2012;30(2):347-67. 1. Lai LT, Ortiz-Cardona JR, Bendo AA. Perioperative pain management in the neurosurgical patient. Anesthesiol Clin. 2012;30(2):347-67. Page 11/13 Page 11/13 Page 11/13 2. Nielsen RV, Fomsgaard JS, Dahl JB, Mathiesen O. Insufficient pain management after spine surgery. Dan Med J. 2014;61(5):A4835. 3. Benyahia NM, Verster A, Saldien V, Breebaart M, Sermeus L, Vercauteren M. Regional anaesthesia and postoperative analgesia techniques for spine surgery - a review. Rom J Anaesth Intensive Care. 2015;22(1):25-33. 4. Linqiu Zhou CDS, Zhenhai Shao. The Anatomy of Dorsal Ramus Nerves and Its Implications in Lower Back Pain. Neuroscience & Medicine. 2012;3(2):10. 5. Miyakoshi N, Shimada Y, Kasukawa Y, Saito H, Kodama H, Itoi E. Total dorsal ramus block for the treatment of chronic low back pain: a preliminary study. Joint Bone Spine. 2007;74(3):270-4. 6. Al-Alami A, Abou El Ezz A, Kassab F. Ultrasound Guided Dorsal Ramus Nerve Block for Reduction of Postoperative Pain in Patients Undergoing Lumbar Spine Surgery: A Case Series Imaging Study. Middle East J Anaesthesiol. 2015;23(2):251-6. 7. Forero M, Adhikary SD, Lopez H, Tsui C, Chin KJ. The Erector Spinae Plane Block: A Novel Analgesic Technique in Thoracic Neuropathic Pain. Reg Anesth Pain Med. 2016;41(5):621-7. 8. Tsui BCH, Fonseca A, Munshey F, McFadyen G, Caruso TJ. The erector spinae plane (ESP) block: A pooled review of 242 cases. J Clin Anesth. 2019;53:29-34. 9. Gornall BF, Myles PS, Smith CL, Burke JA, Leslie K, Pereira MJ, et al. Measurement of quality of recovery using the QoR-40: a quantitative systematic review. Br J Anaesth. 2013;111(2):161-9. 10. Myles PS, Weitkamp B, Jones K, Melick J, Hensen S. Validity and reliability of a postoperative quality of recovery score: the QoR-40. Br J Anaesth. 2000;84(1):11-5. 11. Dewinter G, Moens P, Fieuws S, Vanaudenaerde B, Van de Velde M, Rex S. Systemic lidocaine fails to improve postoperative morphine consumption, postoperative recovery and quality of life in patients undergoing posterior spinal arthrodesis. A double-blind, randomized, placebo-controlled trial. Br J Anaesth. 2017;118(4):576-85. 12. Sharma S, Balireddy RK, Vorenkamp KE, Durieux ME. Beyond opioid patient-controlled analgesia: a systematic review of analgesia after major spine surgery. Reg Anesth Pain Med. 2012;37(1):79-98. 13. Gurkan Y, Aksu C, Kus A, Yorukoglu UH, Kilic CT. Ultrasound guided erector spinae plane block reduces postoperative opioid consumption following breast surgery: A randomized controlled study. J Clin Anesth. 2018;50:65-8. 14. Neal JM, Brull R, Horn JL, Liu SS, McCartney CJ, Perlas A, et al. Page 11/13 The Second American Society of Regional Anesthesia and Pain Medicine Evidence-Based Medicine Assessment of Ultrasound-Guided R i l A th i E ti S R A th P i M d 2016 41(2) 181 94 Regional Anesthesia: Executive Summary. Reg Anesth Pain Med. 2016;41(2):181-94. Page 12/13 Page 12/13 Page 12/13 Page 12/13 Page 12/13 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. supplement1.doc supplement2.doc Page 13/13
https://openalex.org/W4200087771	https://www.iiste.org/Journals/index.php/JETP/article/download/57658/59542	English	null	Analysis of the Effect of Distributed Generation on Loss Reduction in Electrical Distribution Network	Deleted Journal	2,021	cc-by	6,669	Abstract Distribution network is said to be the most visual part of the electric production and the most observed by the utilities for investment, maintenance and operation. The system have been operated under stressed conditions due to limited structure and increasing day to day requirement of power consumption, which have a significant economic and social impact on the system. Due to the system high resistance to impendence ratio, large amount of power loss occur in the network. This loss is the most severity factors affecting the power quality delivered to the end users and depend on power network expansion and load complexity. Among the support methods available for power loss minimization in distribution network, strategic allocation of Distributed Generation (DG) in distribution system is widely considered a viable option. DGs are electrical sources connected to the power network located to consumer’s side but very small when compared with the centralized power plant. They can be in form of wind, mini-hydro, photovoltaic and fuel-based system such as fuel cells and micro-turbines. Therefore, in this study, different approaches for power loss minimization in electrical distribution system with the incorporation of DG by various researchers were reviewed. These approaches have become powerful tools to overcome the problem of power loss minimization in distribution system. p y Keywords: Distribution System, Power Loss. Distributed Generation, Power Consumption, Photovoltaic System, Centralized Power Plant. DOI: 10.7176/JETP/11-6-02 DOI: 10.7176/JETP/11-6-02 Publication date: November 30th 2021 Publication date: November 30th 2021 Publication date: November 30th 2021 Analysis of the Effect of Distributed Generation on Loss Reduction in Electrical Distribution Network Ganiyu Adedayo Ajenikoko, Adebayo Wasiu Eboda, Abass Balogun, Adenle Musibau Shittu, Yusuf Mukhtar Department of Electronic and Electrical Engineering, Ladoke Akintola University of Technology, P.M.B, 4000, Ogbomoso, Nigeria I. Introduction The performance of distribution system is the ultimate issues bothering the power distribution system operator. This is essential in order to fulfill load demands, which increase tremendously. The process in enhancing the efficiency of the distribution network is beset by high real power losses especially in the conventional radial distribution system [5]. This has brought a difficult task to power operators in sustaining a reliable network economically. Distribution system loss reductions constitute a significant part of the total electrical power deterioration. However, despite this identified problem, the demand for power is constantly increasing on daily basis. Therefore, in order to gain a better performance of electricity supply service, there is the need to minimize power loss in distribution system [1-4]. Electric utilities operators are concerned with maintaining distribution system losses within the specified limit due to increase in population, unscheduled loading and other economic and ecological restrictions. Hence, the main achievement in power system operations is the power loss minimization in distribution system. Distribution power losses are chiefly affected by types of connected loads, voltage and current conditions in the system, number and characteristics of electrical device. Therefore, the quest to appreciably reduce the power loss is the emergent desire of power system engineers [6], [9], [12]. Distributed Generation (DG) is an electrical resources connected directly to the distributed network. The DGs include; turbines, Photo-voltaic (PV), fuel cells, and storage devices. Hence; integration of DG into the distribution network results in the highest loss reduction, improved voltage profile, improving system reliability and security. Thus,The DG has become an efficient and clean alternative for the traditional electric energy sources and recent technologies are making DGs economically feasible [8], [17]. DG is more economical than running a power line to rural area because it provides back-up power during outages for facilities requiring uninterrupted service, provide higher power quality for electronic equipment and aids network stability when employed using fast response equipment for a secured distribution system [10-11], [42]. www.iiste.org www.iiste.org Journal of Energy Technologies and Policy Journal of Energy Technologies and Policy ISSN 2224-3232 (Paper) ISSN 2225-0573 (Online) Vol.11, No.6, 2021 a. Power Distribution Network a. Power Distribution Network Distribution network is the last stage in the supplying of electric power in order to provide power to individual consumer premises [13]. Distribution substations lower the transmission voltage to medium voltage with the use of transformers. Distribution lines connect the medium voltage to distribution transformers located near the individual customer’s premises while the transformers adjust the voltage to the household utilization voltage through secondary distribution lines [17], [27]. n practice, power distribution system can be radial or ring with a single or multiple alternate sources. A radi bution system is a separate feeder which radiates from a lone sub-station but feed the distributor at other en 3 Journal of Energy Technologies and Policy ISSN 2224-3232 (Paper) ISSN 2225-0573 (Online) Vol.11, No.6, 2021 The network is a distribution network where power is been transferred from the main branch to the sub-branch and splits out from the sub-branches to the load as shown in Figure 1 [14]. The radial structure indicates no loops in the networks and each bus is connected to the source through one path [16] [19]. The network is a distribution network where power is been transferred from the main branch to the sub-branch and splits out from the sub-branches to the load as shown in Figure 1 [14]. The radial structure indicates no loops in the networks and each bus is connected to the source through one path [16] [19]. Generally, the cost of the construction of the ring main system is higher compared with the radial system. Also, more switches and conductors are essential in ring main system construction. Thus, the system is not ideal for low voltage level and also have high construction cost. Thus, due to these factors the radial system is widely used in distribution network [15], [18], [20]. used in distribution network [15], [18], [20]. Figure 1: A Radial Distribution System b. Losses in Distribution System Distribution system power losses are the major problem in power sector and occur as a result of distributing electric energy end users. This may lead to performance of distribution system to become inefficient [11]. Power losses in distribution system are either real loss or reactive loss. The real power loss occurs due to the line resistance while reactive power loss is due to the reactive elements. a. Power Distribution Network The real power loss reduction draws more attention due to its significant effect on reducing the efficiency of distribution power to the end users. However, reactive power needs to be maintained to ensure transfer of real power at sufficient voltage level to customers. These losses have turn into the most concerned issue for power system engineers because it minimizes the capability of electric energy transfer to the consumer [21], [25]. Factors that influence power losses are: failure in maintaining the voltage profile across networks due to the fl f i l ti t f t t it hi h i i t d lt t i th l l used in distribution network [15], [18], [20]. Figure 1: A Radial Distribution System b. Losses in Distribution System b. Losses in Distribution System Distribution system power losses are the major problem in power sector and occur as a result of distributing electric energy end users. This may lead to performance of distribution system to become inefficient [11]. Power losses in distribution system are either real loss or reactive loss. The real power loss occurs due to the line resistance while reactive power loss is due to the reactive elements. The real power loss reduction draws more attention due to its significant effect on reducing the efficiency of distribution power to the end users. However, reactive power needs to be maintained to ensure transfer of real power at sufficient voltage level to customers. These losses have turn into the most concerned issue for power system engineers because it minimizes the capability of electric energy transfer to the consumer [21], [25]. y Distribution system power losses are the major problem in power sector and occur as a result of distributing electric energy end users. This may lead to performance of distribution system to become inefficient [11]. Power losses in distribution system are either real loss or reactive loss. The real power loss occurs due to the line resistance while reactive power loss is due to the reactive elements. The real power loss reduction draws more attention due to its significant effect on reducing the efficiency of distribution power to the end users. However, reactive power needs to be maintained to ensure transfer of real power at sufficient voltage level to customers. a. Power Distribution Network These losses have turn into the most concerned issue for power system engineers because it minimizes the capability of electric energy transfer to the consumer [21], [25]. Factors that influence power losses are: failure in maintaining the voltage profile across networks due to the flow of circulating current, power factor at unity which increases in current and resultant in the real power losses and voltage regulation which reduces both maximum demand and energy losses [10],[30]. Abbagane et al. (2010) analyzed that 70 % of power losses occurred in the distribution system [1]. Therefore, improving the capability of electric power delivery and this has forced the power engineers to be able to reduce the power loss in the distribution network. In this case, changing environment of electric network design and operation has necessitated the need to consider active distribution network with DG incorporation. The inclusion of DR in distribution network improve the voltage profile and reduce power loss in distribution network.[24], [26]. c. Distributed Generation c. Distributed Generation In addition, interconnection of DG into distribution system offers major benefits which can be classified as follows [34-35], [37]: In addition, interconnection of DG into distribution system offers major benefits which can be classified as follows [34-35], [37]: i. Technical Benefits: DG can provide benefits such as; voltage magnitude improvement, minimization of line losses, power quality adjustment and enhancement in system reliability and security. ii. Economic Benefits: These include; belated the facilities investments for upgrading, operational and maintenance costs minimization of some DG technologies, enhanced productivity, reduced health care costs, reduced fuel costs, associated costs and lower operating costs. ii. Economic Benefits: These include; belated the facilities investments for upgrading, operational and maintenance costs minimization of some DG technologies, enhanced productivity, reduced health care costs, reduced fuel costs, associated costs and lower operating costs. p g iii. Environmental Benefits: DG offer an environmentally friendly source of electrical power by reduced pollutants and encourage the use of renewable energy based generation. iii. Environmental Benefits: DG offer an environmentally friendly source of electrical power by reduced pollutants and encourage the use of renewable energy based generation. More so, the effect of DG in the operation of power network nowadays has become eminent. DGs are reduced characterized as electric sources joined precisely to the distribution network or on the customer side. Higher penetration levels of DG modified the conventional power flows as the power injected at any point on the feeder with DG units [38-39]. Table 1: Options for Small Scale DG Type Size Range (kW) Electrical Efficiency (%) Applications Biomass energy 5-10000 40-50 Co-generation and grid Support Fuel Cells 1-1000 41-66 Co-generation and grid support Micro Turbines 30-5000 20-35 Stand-by power, reliability, power quality and cogeneration Photovoltaic Arrays 1-100 5-15 Base load, peak shaving Reciprocating Engines 5-7000 25-45 Backup power, base load, grid support and peak Shaving Stirling Engines 1-25 12-20 Vehicles, Refrigeration Aircraft, Space Wind systems Several kW-5000 20-40 Remote power, grid Support II. Approaches for Power Distribution Loss Minimization with DG Deependra et al (2007) [10] used Genetic Algorithm (GA) for optimal integration of Distributed Generation (DG) at diverse loading conditions in electric power system in order to reduced system loss. The strategy was implemented on IEEE 16, 37 and 75-bus power systems. The paper concluded that during light load conditions, the optimal placements of DG difference at minimum loss but very close during heavy load periods. c. Distributed Generation Nowadays, one of the alternative solutions to traditional power system operation in order to meet electric energy demands is Distributed Generation (DG). DG is one of the current techniques in decentralized power systems to supplement the increase in power demand. The DG also called decentralized generation; dispersed generation or embedded generation is considered as source of electrical energy connected directly to the distribution network [23]. According to Kansal et al. (2011), DG is defined as a small generating units of 30 MW or less, sited near customer sites to meet provide to support the economic operation of the distribution network [21]. DG impact on distribution system has attracted much attention due to its capacity to correct the flaws caused by its improper installation at feeders, such as those in system losses, reduction in power quality and improvement 4 Journal of Energy Technologies and Policy ISSN 2224-3232 (Paper) ISSN 2225-0573 (Online) Vol.11, No.6, 2021 of voltage at the feeders [3-4], [28], [42]. DG includes renewable; wind, solar, and biomass, and nonrenewable energies; micro-turbines, gas turbines and fuel cells. However, the environmental benefits of renewable DG energies outweigh that of non-renewable DG energies [28], [42]. of voltage at the feeders [3-4], [28], [42]. DG includes renewable; wind, solar, and biomass, and nonrenewable energies; micro-turbines, gas turbines and fuel cells. However, the environmental benefits of renewable DG energies outweigh that of non-renewable DG energies [28], [42]. g g g In addition, installing DG on power network has influences on the network parameters. These impacts can be positive or negative [27], [29]. The formal includes increasing the power quality, improving the voltage profile and deferring the necessity of improving the capacity of substations [4]. While the later includes injection of voltage with harmonics and protection coordination. Therefore, there is the need for new network planning that can guarantee the changes in the distribution- load configuration for technical constraints and penetration levels, in such a way that the benefit is maximized. These limitations must be resolved before introducing DG as an alternative option [31], [36] The DG technologies which are available at present are depicted in Table 1. There are other technologies besides the listed ones [6], [26], [28], [32-33]. c. Distributed Generation Sedighizadeh and Rezazadeh (2008) [37] used Genetic Algorithm (GA) for power loss reduction as well as for voltage profile improvement of electric distribution network with optimal integration of Distribution Generation (DG). The algorithm was implemented on Tehran electricity distribution system and simulated using both MATLAB and ETAP software respectively. The simulation result showed an improvement in system voltage profile and loss reduction indices. Subrahmanyam and Radhakrishna (2009) [40] used Voltage Index (VI) analysis to investigated the best placement and sizing of Distributed Generation (DG) in three-phase Unbalanced Radial Distribution System (URDS) aimed at power loss minimization and system voltage profile improvement. The results of simulation were tested on IEEE 25 and 37- bus unbalanced radial distribution system. Abbagana et al. (2010) [1] used Differential Evolution (DE) for optimal location and size of Distributed Generation (DG) in order to reduce the power losses and improve the voltage magnitude of distribution network. The algorithm was implemented on IEEE 33 bus radial distribution network and simulation was done using MATPOWER and MATLAB software. The results revealed that the system loss was reduced to the barest minimum with the installation of DG. The study concluded that proper placement and sizing of DG in power system have a significant impact on system loss reduction as well as voltage profile improvement. Manjunatha and Vittal (2010) [25] employed a heuristic technique; Network Performance Enhancement 5 Journal of Energy Technologies and Policy ISSN 2224-3232 (Paper) ISSN 2225-0573 (Online) Vol.11, No.6, 2021 Journal of Energy Technologies and Policy w.iiste.org Index (NPEI) for provision of Distributed Generation (DG) source in a distribution network. The developed approach was applied and tested on IEEE 33 and 90-bus power system respectively. The results obtained showed that the technique gave better and economical solution for system improvement. Index (NPEI) for provision of Distributed Generation (DG) source in a distribution network. The developed approach was applied and tested on IEEE 33 and 90-bus power system respectively. The results obtained showed that the technique gave better and economical solution for system improvement. q g y p Amanifar and Hamedani (2011) [6] presented a heuristic optimization technique; Particle Swarm Optimization (PSO) and sensitivity analysis for Distributed Generation (DG) placement aimed at loss reduction, Third Harmonic Distortion (THD) reduction and voltage profile improvement in distribution network. The approach was tested on IEEE 15-bus radial distribution network. The results showed the best position of DG with minimum economic cost. c. Distributed Generation This indicated that PSO have effectiveness to search optimum point and size of DGs on power system. Kansal et al. (2011) [21] employed Particle Swarm Optimization (PSO) to find the optimal location and sizing of Distributed Generation (DG) in the radial distribution system for active power losses minimization and enhancement of voltage magnitude. The technique was implemented on IEEE 33-bus power system and the obtained results were compared with the exhaustive load flows technique. The results revealed that the optimal placement of DG by PSO improved the system voltage magnitude as well as appreciably reduced the system real power loss. p Mohammad and Akbari (2011) [29] used Particle Swarm Optimization (PSO) for optimal location of Distributed Generators (DG) in the radial distribution systems to reduce the real power losses and to improve the reliability of the power system. The algorithm was implemented on a IEEE 12 bus power system and simulated in a MATLAB environment. The results presented were compared with different approaches and the results revealed that the algorithm out-performed the other methods in terms of computational efficiency. Satish et al. (2011) [36] employed Particle Swarm Optimization (PSO) to find the optimal placement and sitting of Distributed Generation (DG) in distribution networks in order to reduce the real power losses as well as enhanced the system voltage profile. The technique was implemented on IEEE 33-bus power system and the obtained results were compared with the load flows technique. The result showed that by taking the voltage limits of the system into consideration, the optimal placement of DG by PSO technique minimized the real power loss. Chandrasekhar et al. (2012) [9] presented Voltage Index (VI) analysis for optimal placement and siting of Distributed Generators (DGs) for system loss minimization and tested on 38-bus distribution systems. Wind and solar systems were modeled as constant power factor and variable reactive power model respectively. The indices of the total system were related with and without considering the DGs limiting busses. Ghamgeen and Rashed (2012) [12] analyzed optimal location and setting of Thyristor Control Series Compensator (TCSC) for power loss reduction using Differential Evolution (DE) and Genetic Algorithm (GA) by comparing their performances. The simulations were carried out on a IEEE 5-bus and 14-bus power system. The obtained results indicated that DE was easy to use, fast and robust compared to GA. c. Distributed Generation The author concluded that when comparing the results with Immune algorithm method, the results showed that Harmony search algorithm gave the same real power loss and voltage with less distributed generator size than Immune algorithm method. reduction. The methods were tested on IEEE 33 and 69-bus radial distribution network. The author concluded that when comparing the results with Immune algorithm method, the results showed that Harmony search algorithm gave the same real power loss and voltage with less distributed generator size than Immune algorithm method. Syed and Shah (2012) [41] investigated the effect of Distributed Generators (DG) under occurrences of faults on electrical distribution network. The simulations were considered on different types of wind turbines. The results showed how voltage dips were cause by different line faults which affected the network circuit breakers coordination with the inclusion of DG. Aida and Azah (2013) [2] presented Gravitational Search Algorithm (GSA) for suitable placement of Distribution Generation (DG) in distribution network. The performance of the algorithm was compared with Particle Swarm Optimization (PSO) and Evolutionary Programming (EP). The algorithm was implemented on 69- bus distribution network. The obtained results indicated that GSA performance better in terms of the best fitness and convergence rate. It also revealed that the GSA was effective for optimum sitting of DGs in power network. Behnam (2013) [8] implemented a Particle Swarm Optimization (PSO) and Weighted Coefficient Method (WCM) for optimization of Distributed Generation (DG) in distribution network aimed at network power quality and reliability improvement. The simulation was implemented on IEEE 12-bus power system. Newton-Raphson (NR) algorithm was employed in each island. However, when DGs or load point voltage constraints are not satisfies, the island would be shut down. However, the method used was not guaranteed to be optimal. Gummadi and Obulesh (2013) [13] employed Sensitivity Analysis (SA) for power loss reduction in distribution feeder in terms of Distributed Generator (DG) size. The technique was verified using MATLAB and the results showed that the inclusion of DG in the distribution network provided an effective solution for power loss reduction as well as improving the voltage profile in the network. Haruna and Sanusi (2013) [15] employed Ranked Evolutionary Particle Swarm Optimization (REPSO) for allocation of Distributed Generator (DG) in distribution network for improving the voltage profile and network losses reduction. The effectiveness of the algorithm was demonstrated on 69-bus power system. c. Distributed Generation The obtained results showed that installation of DG in the system could improve the system voltage profile and at the same time reduced the network losses. Julius and Nicodemus (2013) [20] presented Genetic Algorithm Immune Particle Swarm Optimization (GA- IPSO) for optimal siting and size a multi-type DG on IEEE 57-bus power system aimed at network losses reduction and voltage magnitude improvement. The obtained results revealed that the network losses reduced to minimum and the voltage magnitude in the network improved from its bus voltage deviation with inclusion DG. Manafi et al. (2013) [24] employed Particle Swarm Optimization (PSO) and Differential Evolution (DE) algorithms for optimal location of Distributed Generations in distribution network in order to minimize real power losses. Simulation result was implemented on IEEE 33 and 69-bus power systems. It was observed that both PSO and DE techniques gave suitable results. Engy et al. (2014) [11] presented Combined Heat and Power (CHP) for optimal sizing and siting of Distributed Generators (DG) for distribution cost maximization. The algorithm was simulated in MATLAB environment and implemented on IEEE 37-bus feeder. Obtained results showed the effectiveness of the CHP for optimal selection of DGs size and siting in order to maximize the total cost in the network. Hamid et al. (2014) [14] presented an algorithm for the optimal quantity and siting of DG and capacitors simultaneously in a distribution network. The models were evaluated on IEEE-33 bus power system. The paper showed that the network simultaneous operated at optimal level with expansion of DG units in the network. Jegadeesan and keerthana (2014) [19] implemented an Artificial Bee Colony (ABC) for optimal size of DG unit’s and proper placement in distribution network to minimize the total network loss. Algorithm was tested on IEEE 15 and 34 bus power systems. Obtained results clearly explained that the optimal placement and siting of DG minimized the network losses. Kavitha and Muthamil (2014) [22] used Hybrid Genetic Algorithm and Particle Swarm Optimization Search (HGAPSO) for Distributed Generators (DG) placement in distribution network. The analysis was implemented on IEEE 18 bus system. The results of indicated that HGAPSO provided best fitness, high convergence rate and also demonstrated the capability of the technique for average (THD) and network loss minimization. Nasser and Gbolamreza (2014) [32] analyzed the optimal placement methods for sizing and location of Distributed Generation (DG) in distribution network. c. Distributed Generation Hossein and Morteza (2012) [16] presented an optimal sizing and placement of Distributed Generation (DG) considering different load models using Improved Harmony Search (IHS) algorithm. The algorithm was implemented on 69-bus distribution systems. The paper concluded that, the IHS algorithm had good capability for most of the continuous optimization problems. The obtained results showed a better performance compared to other approaches. Jasrul and Mohd (2012) [18] implemented Iterative Particle Swarm Optimization (IPSO) algorithm for sizing DG units and its performance were related with two other optimization technique; Artificial Immune System (AIS) and Evolutionary Programming (EP). The method was implemented on 69 bus distribution systems with 4 units of DG that operated in PV mode. The results showed that, IPSO gave better results compared to EP and AIS in terms of power loss reduction and voltage profile, Lieven et al. (2012) [23] investigated the effect of converter connected DG units on harmonic losses in the distribution system. The research introduced numerous indices to quantify the DG benefits. The most important indices were the line reduction and transformer losses, improved system voltage profile, enhanced power quality, reduced environmental impacts and relieved transmission and distribution congestion. The obtained results indicated a decrease in power losses per phase when the DG unit was connected to the most loaded phase. Mohit et al. (2012) [31] used Ladder Load Flow (LLF) algorithm to investigate the effect of multiple Distributed Generation (DG) distribution system for the minimization of power loss. The method was tested on a 3-phase radial distribution feeder with laterals and sub-lateral generations. An approximation formula was used to minimize the number of required solution. Slaven et al. (2012) [38] employed Genetic Algorithms (GA) to determine the capacity of Distributed Generation (DG) in power system. The obtained results based on minimal losses showed that GA had a fast convergence towards a set of optimal solutions. Thus, a substantial increase of distributed production in low and medium voltage networks result in decentralizing power systems as well as favoring renewable energy sources to decrease greenhouse effect. g Srinivasa and Nageswara (2012) [39] employed Immune Algorithm (IA) method and Harmony Search Algorithm (HSA for optimal location of multiple Distributed Generator (DG) in distribution network for loss 6 Journal of Energy Technologies and Policy ISSN 2224-3232 (Paper) ISSN 2225-0573 (Online) Vol.11, No.6, 2021 ww.iiste.org reduction. The methods were tested on IEEE 33 and 69-bus radial distribution network. c. Distributed Generation The algorithms were implemented on IEEE 33 bus reconfigured power system and simulated in MATLAB environment with multiple DG units. The results showed that the efficiency of the algorithms used improved the voltage profile and reduced the power losses. Ramakanth and Vinod (2014) [34] presented an Improved Teaching-Learning Based Optimization (ITLBO) algorithm for optimal DG placement in electrical distribution network. The algorithm was implemented on IEEE- 33 and 69-bus distribution network and simulated in MATLAB environment. The simulation results revealed the effectiveness of the ITLBO. Rashmi and Surya (2014) [35] presented Genetic Algorithm (GA) for the optimal siting and sizing buted Generator (DG) in distribution system. The algorithm was simulated in MATLAB environment an 7 Journal of Energy Technologies and Policy Journal of Energy Technologies and Policy ISSN 2224-3232 (Paper) ISSN 2225-0573 (Online) Vol.11, No.6, 2021 www.iiste.org ww.iiste.org implemented in a IEEE 16 bus power system. The results verified the analytical approaches. implemented in a IEEE 16 bus power system. The results verified the analytical approaches. Attia (2015) [7] used a Backtracking Search Optimization Algorithm (BSOA) for assigning multiple DGs in distribution networks. Loss sensitivity factors and bus voltages were employed based on fuzzy rules to identify the initial DG’s placement. The method was validated with different DG sizes and complexities through many radial distribution networks. The results showed that application of BSOA could efficiently generate high-quality solutions for DG placement. Ajenikoko and Eboda (2015a) [3] analyzed the impact of Dispersed Generation (DG) on optimization of power export using Line Loss Reduction Index (LLRI) based on computer simulation of hourly loads. The simulation was implemented on the selected feeders of Kaduna distribution system. The results showed that with the line application of loss reduction index, the losses were reduced and optimization of power export was achieved when DG are integrating into the distribution network, Ajenikoko and Eboda (2015b) [4] discussed the impact of embedded generation on network voltage collapse on distribution network using Voltage Stability Index (VSI). Simulation was carried out by reconfiguring the distribution network of Afe Babalola University (ABUAD), Ado-Ekiti. The results showed that after the reconfiguration of the network, the maximum active power, reactive power and voltage connected loads on selected transformer increased respectively. IV. Conclusion A comprehensive analysis of the various techniques employed for power loss minimization in distribution system with integration of Distributed Generation (DG) had been reviewed and presented in this study. The review showed that with incorporation of DG in distribution network, the total power losses along distribution lines are reduced and this enhanced the performance of the distribution system infrastructure. The review also showed that incorporation of DG into the distribution system had technical benefits that complement the distribution system performance through minimization of power losses, thus improving electrical energy delivered to the consumers. III.Review Analysis In this study, 34 research papers were reviewed with sixteen different approaches used for power loss minimization with incorporation of DG on distribution system for nine (9) years 2007-2015. Each technique tried to mitigate the power loss minimization problem with various objectives and constraints. Some objectives such as cost minimization and voltage magnitude improvement were common in most of them. Most of the reviewed paper focused on implementing the techniques on standard networks while only few researchers applied the techniques to practical network. Figure 2 shows the relationship between numbers of paper reviewed and reviewed years. From the Figure, 34 papers were reviewed within nine (9) years. One (1) paper each was reviewed for 2007, 2008 and 2009 respectively. Two papers were reviewed in 2010 while four (4) papers were reviewed in 2011. Year 2012 recorded highest number of reviewed papers with nine (9) papers while six (6) papers were reviewed in year 2013. In addition, seven (7) and three (3) papers were reviewed in Years 2014 and 2015 respectively. Figure 3 indicated the relationship between different approaches for DG implementation on distribution system and number of researchers. It was observed from the Figure that one (1) researcher each employed Backtracking Search Optimization Algorithm (BSOA), Firefly Algorithm (FA), Genetic Algorithm Immune Particle Swarm Optimization (GAIPSO), Hybrid Genetic Algorithm Particle Swarm Optimization Search (HGAPSO), Immune Algorithm and Harmony Search Algorithm (IA-HAS), Improved Harmony Search Algorithm (IHSA), Improved Teaching–Learning Based Optimization (ITLBO), Line Loss Reduction Index (LLRI), Network Performance Enhancement Index (NPEI), Ranked Evolutionary Particle Swarm Optimization (REPSO) and Gravitational Search Algorithm (GSA) for power loss minimization with incorporation of DG. Three researchers employed Deferential Evolution (DE) for power loss minimization while four (4) researchers each employed Genetic Algorithm (GA) and Sensitivity Analysis (SA) approaches respectively. Five researchers employed Voltage Stability Index (VSI) while seven (7) researchers employed Particle Swarm Optimization (PSO). Figure 2: Relationship between numbers of Paper Reviewed and Reviewed Years Figure 2: Relationship between numbers of Paper Reviewed and Reviewed Years 8 Journal of Energy Technologies and Policy ISSN 2224-3232 (Paper) ISSN 2225-0573 (Online) Vol.11, No.6, 2021 Vol.11, No.6, 2021 Figure 3: Relationship between different Approaches and Number of Researchers Figure 3: Relationship between different Approaches and Number of Researchers VI. References [1] Abbagana, M., Bakare, G. A. and Mustapha, I. (2010). Optimal placement and sizing of a distributed generator in a power distribution system using differential evolution. Proceedings of the 1st International Technology, Education and Environment Conference (c) African Society for Scientific Research, Human Resource Management Academic Research Society: 536-549. [2] Aida F.A and Azah M. (2013): “Optimal sizing and placement of distributed generation in distribution system considering losses using gravitational search algorithm” Przegląd Elektrotechniczny, http: //www.iiste.org. Vol. 5, No. 3, Pp. 132-136. [3] Ajenikoko G.A and Eboda A.W (2015a): “Impact of dispersed generation on optimization of power exports” International Journal of Engineering Research and Applications, Vol. 5, No. 5, Pp. 61 – 67. [4] Ajenikoko G.A and Eboda A.W (2015b): “Impact of embedded generation on power distribution system voltage collapse” European Journal of Computer Science and Information Technology,Vol. 3, No. 4, Pp. 33 – 42. [5] Ali Kadhem A. (2006): “Load flow method for radial system with distribution generation” A Lecture Note of Technical College, Najaf, Iran. www.google.com. Vol. 2, No. 4, Pp. 1-15. [6] Amanifar O.M.E and Hamedani G. 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(2013): “Optimal allocation and sizing of distributed generation for power loss reduction using modified PSO for radial distribution systems” Journal of Energy Technologies and Policy, Vol.3, No.3, Pp. 1 - 9. p [16] Hossein N and Morteza N. (2012): “Load model effect assessment on optimal distributed generation sizing and allocation using improved harmony search algorithm” Research Journal of Applied Sciences, Engineering and Technology, Vol.2, No. 7, Pp. 832- 837. [17] Ibrahim T.A. (2009): “Optimal sizing of capacitor banks and distributed generation in distorted distribution networks by genetic algorithms” International Conference on Electricity Distribution, Vol. 4, No. 4, Pp. 8- 11. [18] Jasrul J.J and Mohd W.M. (2012): “Distributed generator sizing: an iteration particle swarm optimization approach” IASTED International Conference Power and Energy System, Vol. 5, No. 9, Pp. 439 – 444. [19] Jegadeesan M and Keerthana V. 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https://openalex.org/W4307453841	https://www.biorxiv.org/content/biorxiv/early/2022/10/21/2022.10.20.513032.full.pdf	English	null	Categorising update mechanisms for graph-structured metapopulations	bioRxiv (Cold Spring Harbor Laboratory)	2,022	cc-by	15,105	. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint Abstract The structure of a population strongly inﬂuences its evolutionary dynamics. In var- ious settings ranging from biology to social systems, individuals tend to interact more often with those present in their proximity and rarely with those far away. A common approach to model the structure of a population is Evolutionary Graph Theory. In this framework, each graph node is occupied by a reproducing individ- ual. The links connect these individuals to their neighbours. The oﬀspring can be placed on neighbouring nodes, replacing the neighbours – or the progeny of its neighbours can replace a node during the course of ongoing evolutionary dynamics. Extending this theory by replacing single individuals with subpopulations at nodes yields a graph-structured metapopulation. The dynamics between the diﬀerent lo- cal subpopulations is set by an update mechanism. There are many such update mechanisms. Here, we classify update mechanisms for structured metapopulations, which allows to ﬁnd commonalities between past work and illustrate directions for further research and current gaps of investigation. Sedigheh Yagoobi, Nikhil Sharma and Arne Traulsen Sedigheh Yagoobi, Nikhil Sharma and Arne Traulsen Department of Evolutionary Theory, Max Planck Institute for Evolutionary Biology, August-Thienemann Str.1, 124306 Pl¨on, Germany. Department of Evolutionary Theory, Max Planck Institute for Evolutionary Biology, August-Thienemann Str.1, 124306 Pl¨on, Germany. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint 1 Introduction The spatial structure of a population has a considerable impact on the evolutionary dynamics of a population. One of the most popular theories for studying the eﬀect of underlying structure on the evolution of a population is Evolutionary Graph Theory [1], where graphs represent spatial structures. The nodes of a graph represent individuals, and links indicate individual’s neighbours. A link determines where an individual can place their oﬀspring. An update mechanism describes which individuals produce oﬀspring and how this oﬀspring is placed. Here, we focus on 1 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint ﬁxed structures, but the framework can be extended to the case where a spatial structure itself varies over time [2–5]. An extension of evolutionary graph theory is given by graph-structured metapop- ulations in which the nodes indicate subpopulations, and the links indicate the mi- gration between subpopulations. Given that fragmented habitats are ubiquitous in ecology, metapopulations, and their evolution and ecology have been extensively studied [6–8]. However, there are few studies on graph-structured metapopulations with more complex graphs [9–11] and thus, this issue calls for further investigation. Depending on the system, diﬀerent update mechanisms have been applied to de- scribe the dynamics. In general, evolutionary dynamics are not robust to the choice of update mechanisms [5, 12, 13]. An important factor that changes the system’s dynamics is how selection acts. Depending on the update mechanism, selection can be global or local. This will aﬀect the evolutionary dynamics dramatically. The two most important events that govern the dynamics of the population are birth and death. The order in which birth and death occur, which often determines if selection is local or global, has a high impact on the fate of the population [14]. We aim to categorize the diﬀerent update mechanisms for the evolution of structured metapopulations to facilitate future work. First, we recall the update mechanisms on graphs of individuals, and then we generalize them to graphs of subpopulations. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint 2 Update mechanisms in graphs of individuals In graphs of individuals, three main events inﬂuence the evolution of a population: birth, mutation, and death. In the long run, a mutation-selection balance is reached [15–18]. Here, we focus on the low mutation regime in which the system reaches ﬁxation or extinction of the mutant before the next mutation occurs. In that case, the population is typically homogeneous, and mutations reach ﬁxation one after another. In the low mutation regime, the two quantities of most interest are the ﬁxation probability and the average time to ﬁxation. If the ﬁxation probability for advantageous mutants on a graph is higher than the ﬁxation probability of the complete graph (and vice versa for disadvantageous mutations), the graph is called an ampliﬁer of selection. On the other hand, if the ﬁxation probability for advantageous mutants on a graph is less than the ﬁxation probability on the complete graph (and vice versa for disadvantageous mutations), this graph is called a suppressor of selection. These notions have been ﬁrst intro- duced in [1]. The order of birth and death events and how selection acts upon them can substantially inﬂuence the population’s fate. We use the following scheme to diﬀer- entiate between the update mechanisms. For birth, we use b if birth is random, i.e., a birth-giving individual is chosen randomly, and B if the birth-giving individual is chosen with probability proportional to its selection parameter for birth. Similarly, we represent the death event by D if the individual is selected for death with a prob- ability associated with its selection parameter for death and by d if the individual dies at random. 2 Figure 1: Diﬀerent update schemes for graph of individuals. We consider a random population structure of size eight with ﬁve wild-type individuals (in blue) and three mutant individuals (in red). Neighbours are connected via links. Individual encircled with solid black circle represents the birth giving parent, whereas, the individual encircled with black dashed circle is the one chosen for death. The population size remains constant throughout the dynamics with oﬀspring replacing dead individuals. 2 Update mechanisms in graphs of individuals Assuming that the selection parameter for birth of a mutant individual is r = 2 (1 for the wild type) and that the selection parameter for death of the mutant is t = 1/2 (1 for the wild type), the probabilities that the transition shown in the ﬁgure takes place, is diﬀerent for the diﬀerent update mechanisms shown in Tab. 2.1. For example, in the case of BD, the probability to choose this particular mutant individual for birth is 2 3·2+5·1 = 2 11. The probability to choose this particular wild type neighbor for death is 1 1·1/2+2·1 = 2 5, which leads to a probability 4 55 ≃0.072. Similarly, we ﬁnd: Bd: 2 3·2+5·1 · 1 3 ≃0.60. bD: 1 8 · 1 1·1/2+2·1 = 0.05. bd: 1 8 · 1 3 ≃0.042. DB: 1 3·1/2+5·1 · 2 1·2+2·1 ≃0.077. Db: 1 3·1/2+5·1 · 1 3 ≃0.51. dB: 1 8 · 2 1·2+2·1 ≃0.062. db: 1 8 · 1 3 ≃0.042. Figure 1: Diﬀerent update schemes for graph of individuals. We consider a random population structure of size eight with ﬁve wild-type individuals (in blue) and three mutant individuals (in red). Neighbours are connected via links. Individual encircled with solid black circle represents the birth giving parent, whereas, the individual encircled with black dashed circle is the one chosen for death. The population size remains constant throughout the dynamics with oﬀspring replacing dead individuals. Assuming that the selection parameter for birth of a mutant individual is r = 2 (1 for the wild type) and that the selection parameter for death of the mutant is t = 1/2 (1 for the wild type), the probabilities that the transition shown in the ﬁgure takes place, is diﬀerent for the diﬀerent update mechanisms shown in Tab. 2.1. For example, in the case of BD, the probability to choose this particular mutant individual for birth is 2 3·2+5·1 = 2 11. The probability to choose this particular wild type neighbor for death is 1 1·1/2+2·1 = 2 5, which leads to a probability 4 55 ≃0.072. Similarly, we ﬁnd: Bd: 2 3·2+5·1 · 1 3 ≃0.60. bD: 1 8 · 1 1·1/2+2·1 = 0.05. bd: 1 8 · 1 3 ≃0.042. DB: 1 3·1/2+5·1 · 2 1·2+2·1 ≃0.077. Db: 1 3·1/2+5·1 · 1 3 ≃0.51. dB: 1 8 · 2 1·2+2·1 ≃0.062. db: 1 8 · 1 3 ≃0.042. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint 2 Update mechanisms in graphs of individuals Figure 1: Diﬀerent update schemes for graph of individuals. We consider a random population structure of size eight with ﬁve wild-type individuals (in blue) and three mutant individuals (in red). Neighbours are connected via links. Individual encircled with solid black circle represents the birth giving parent, whereas, the individual encircled with black dashed circle is the one chosen for death. The population size remains constant throughout the dynamics with oﬀspring replacing dead individuals. Assuming that the selection parameter for birth of a mutant individual is r = 2 (1 for the wild type) and that the selection parameter for death of the mutant is t = 1/2 (1 for the wild type), the probabilities that the transition shown in the ﬁgure takes place, is diﬀerent for the diﬀerent update mechanisms shown in Tab. 2.1. For example, in the case of BD, the probability to choose this particular mutant individual for birth is 2 3·2+5·1 = 2 11. The probability to choose this particular wild type neighbor for death is 1 1·1/2+2·1 = 2 5, which leads to a probability 4 55 ≃0.072. Similarly, we ﬁnd: Bd: 2 3·2+5·1 · 1 3 ≃0.60. bD: 1 8 · 1 1·1/2+2·1 = 0.05. bd: 1 8 · 1 3 ≃0.042. DB: 1 3·1/2+5·1 · 2 1·2+2·1 ≃0.077. Db: 1 3·1/2+5·1 · 1 3 ≃0.51. dB: 1 8 · 2 1·2+2·1 ≃0.062. db: 1 8 · 1 3 ≃0.042. 3 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint Accordingly, the possible update mechanisms are BD, Bd, bD, bd, DB, Db, dB, and db, where the order shows which event is ﬁrst, see Table 2.1. In all these update mechanisms, the ﬁrst event is global, meaning that the individual is selected from the whole population. In contrast, the second event is local because the individual is selected from a subset of the population in the neighbourhood of the ﬁrst individual. As an example, applying the update mechanism Bd in a well-mixed population of size N consisting of two types of individuals, N −n wild-type and n mutants, with mutants having a relative selection parameter for birth r with respect to wild- types. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint 2 Update mechanisms in graphs of individuals CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint Update mechanism Comment References BD - [14, 26, 35] Bd Most popular update mechanism [1, 14, 20, 21, 26, 40–42] bD - [14, 26] bd Equivalent to a completely neutral model [14, 34] DB - [14, 26] Db Biased voter model [14, 24, 26, 32, 36] dB Biased voter model [13, 14, 26, 28, 29, 40–42] db Equivalent to a completely neutral model [14, 34] Table 2.1: Update mechanisms in graphs of individuals. In these update mech- anisms, birth and death change the state of the population. The ﬁrst event is global and the second event is local. Among the 8 update mechanisms for graphs of individuals, bd and db are identical, describing a system where natural selection has no role in the evolution of the population [14, 34]. In the update mechanisms DB and BD selection acts both on death and birth [14, 26, 35], but they are not equivalent in general. Intuitively, it is expected that the ﬁxation probability of an advantageous mutant is higher in the presence of update mechanisms DB and BD than in the other update mechanisms. The general transition probabilities for these 8 update mechanisms are given in Appendix A. In the Fig. 1, the transition probability for a speciﬁc example under the 8 update mechanisms is given. In addition to the above update mechanisms, there are other update mechanisms in which, instead of two individuals (one for birth, one for death), one edge is selected [24, 36, 37]. Then an individual dies at one end, the other gives birth, and the oﬀspring ﬁlls the neighbouring empty spot. In this update mechanism, selection can act on death and birth events or not. Here, we will not consider this kind of update mechanism. 2 Update mechanisms in graphs of individuals To increase the number of mutants in the population, one mutant is selected for reproduction (global event). Then, from the neighbours of the mutant, one wild-type is selected for death (local event). The oﬀspring ﬁlls the empty spot of the dead individual. The probability of increasing the number of mutants by one is then T n+ Bd = rn rn + N −n \| {z } birth of mutant N −n N −1 \| {z } death of wild-type . (2.1) (2.1) birth of mutant death of wild-type birth of mutant death of wild-type Similarly, the probability of decreasing the number of mutants by one is T n− Bd = N −n rn + N −n \| {z } birth of wild-type n N −1 \| {z } death of mutant . (2.2) (2.2) Using the recursive relation for the ﬁxation probability [19, 20], the ﬁxation proba- bility starting from n mutants is φ(n) = 1 + Pn−1 i=1 Qi j=1 T j+ T j− 1 + PN−1 i=1 Qi j=1 T j+ T j− = 1 − 1 rn 1 − 1 rN (2.3) (2.3) The update rule Bd has been vastly explored in both structured and well-mixed populations [1, 21–26]. For small populations under the update mechanism Bd, most small structures are ampliﬁers of selection, and only a minimal fraction of structures suppress selection [5]. In larger populations, in the weak selection regime, there are quite a lot of graphs that suppress selection [27]. Furthermore, some structures have the same ﬁxation probability as the complete graph. This is the case when the total weight of incoming edges to a node is homogeneous through the entire graph, as shown in the “isothermal theorem” [1, 20]. Similarly, many studies are dedicated to dB updating [5, 13, 14, 22, 23, 26, 28– 31]. Contrary to Bd, only a small fraction of undirected graphs under the update mechanism dB amplify selection and the majority suppress selection [5]. Another popular update mechanism is Db which is the same as the score-dependent viability model introduced in [32], this is equivalent to the voter model in statistical physics [24, 25, 33]. In [26], it is shown that the dynamics of a lattice of an integer dimension under update mechanisms Bd and Db are equivalent. It is illustrated however, that the dynamics of this lattice under update mechanisms dB and bD are fundamentally diﬀerent. 4 . 3 Categorising update mechanisms for graphs of subpopulations In most of the potential applications of evolutionary graph theory, including bio- logical and social systems, individuals tend to interact locally in subpopulations, with some rare interactions with other groups of individuals. This is because the populations are segregated for various reasons, and they are geographically distant. Individuals in the same geographical area compete over resources or provide common goods. However, there is an occasional migration to and from other geographical areas. In [10], it has been shown that for a particular update mechanism, the results of evolutionary graph theory [1] do not carry over into graphs of subpopulations. For example, a star-structured metapopulation does not always amplify selection – in some migration regimes, it suppresses selection. It will be interesting to see if this is the case for other update mechanisms. In a model that retains the local population’s size ﬁxed, birth and death either happen in the same subpopulation, or the ﬁrst event can be followed by an indi- vidual’s migration to or from another subpopulation. Having this in mind, we can categorize update mechanisms into two groups: • A set of update rules where there is always the possibility that the ﬁrst event is accompanied by migration (Fig. 3 A-B). • A set of updates where birth and death events are always in the same sub- population and migration happens independently from birth and death, (see Fig. 3A & C). • A set of updates where birth and death events are always in the same sub- population and migration happens independently from birth and death, (see Fig. 3A & C). We refer to the former category as update mechanisms with coupled migration and the latter as update mechanisms with uncoupled migration. The order of events (birth and death) in each of these classes and how selection acts upon them might aﬀect the dynamics considerably. In both categories of update mechanisms, selection for the ﬁrst event can act on both patch and individual levels, i.e., one ﬁrst selects a patch and then an individual from the chosen patch. Selection on the second event can act both on the patch and individual levels in the update mechanisms with coupled migration. However, in the update mechanisms with uncoupled migration, selection in the second event always acts on the individual level since the second event happens in the same patch as the ﬁrst event. 2 Update mechanisms in graphs of individuals For more information about the comparison of diﬀerent update mechanisms on various graphs with diﬀerent features, we refer to the following references: In [36] the authors ask when the ﬁxation probability in an evolutionary graph equals the ﬁxation probability in a Moran process. A Moran process [38] is equivalent to the update mechanism Bd in a complete graph. In [39], the authors investigate the evolutionary game dynamics on the star graph in the presence of diﬀerent update mechanisms. They show that the evolutionary dynamics of heterogeneous graphs is not robust under the choice of update mechanism. In [37] the eﬀect of the directionality of a graph on its evolutionary dynamics for diﬀerent update mechanisms is investigated. It has been shown that regardless of the update mechanism, the directionality always suppresses selection. In this manuscript, we extend the above update mechanisms to graphs of subpopulations where each node comprises a well-mixed subpopulation. The links indicate the migration between the patches (see Fig. 2). 5 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint 3 Categorising update mechanisms for graphs of subpopulations We code the update mechanisms as follows: the ﬁrst letter stands for migration to show if it is coupled (M) or uncoupled (m). If selection on an event acts both at the patch and individual levels, we allocate two labels to it. Otherwise, we assign one label to the event. The order of letters, except for the letter for migration, indicates the order of events. In addition, if selection is associated with selection parameters, we assign a capital letter and, otherwise, a lowercase letter. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint 3.1 Update mechanisms with coupled migration In this class of update mechanisms, the ﬁrst event is directly coupled to migration. The ﬁrst event can be birth or death. If the birth occurs ﬁrst, one of the patches is 6 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint Figure 2: Graph of subpopulations. Each node in the graph includes a well-mixed subpopulation and each link indicates migration between two subpopulations. Figure 2: Graph of subpopulations. Each node in the graph includes a well-mixed subpopulation and each link indicates migration between two subpopulations. selected randomly proportional to the size of the patch (b) or randomly proportional to the sum of the selection parameters for birth of that patch (B). Next, an individual from the patch is selected purely randomly (b) or randomly proportional to its selection parameter for birth (B) to produce an identical oﬀspring. The oﬀspring can either stay and substitute one of the individuals in its patch (Fig. 3A) or migrate to one of the neighbouring patches and replace one of the individuals there (Fig. 3B). ) If the oﬀspring stays in its own patch, one of the individuals is chosen for death purely at random (d) or proportional to its selection parameter for death (D) (a common choice is the inverse of the selection parameter for birth). If the oﬀspring migrates to a neighbouring patch, an individual in an adjacent patch is selected in two stages. First, among the neighbouring patches, one patch is selected randomly proportional to the size of the patch (d) or randomly proportional to the collective selection parameter for death of the patch (D). Finally, one of its individuals is selected for death uniformly at random (d) or randomly proportional to its selection parameter for death (D). As an example, consider the update mechanism MbBDd. In this update mech- anism (i) ﬁrst, a random patch is selected, (ii) then, from the random patch, one individual is selected proportional to its selection parameter for birth to produce an oﬀspring (iii) next, with a certain probability, the oﬀspring will migrate to one of the adjacent patches or remain in its innate patch. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint 3.1 Update mechanisms with coupled migration In the former case, one of the neighbouring patches is selected at random as a function of its collective selection parameter for death. (iv) Finally, in the selected patch, which can be either an adjacent patch or the innate patch, one individual dies uniformly at random, and the oﬀspring ﬁlls its empty spot. Based on this procedure, there are 16 diﬀerent update mechanisms, with birth being the ﬁrst event; see table 3.1. Similarly, if the ﬁrst event is death, there are 7 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint A B C Figure 3: Update mechanisms in a graph-structured metapopulation. The population consists of two types of individuals, wild-types (blue) and mutants (red). The individual marked with the solid black line gives birth and the individual marked with black dashed line is selected for death. (A) Birth-death or death-birth in one patch without migration: This includes birth-death or death-birth in a coupled update mechanism without migration as well as an uncoupled update mechanism in which both death and birth happen in the same subpopulation. (B) A coupled update mechanism with migration: if birth is coupled with migration after each birth the newborn migrates to an adjacent patch and replace one of its individual. If death is coupled to migration a death in one patch is followed by birth of an individual in one of the adjacent patch where newborn occupies the place of dead individual. (C) Migration in an uncoupled update mechanism: migration happens independently from birth or death and it only exchanges the position of two individuals from diﬀerent patches. This process is completely random. A B C A B Figure 3: Update mechanisms in a graph-structured metapopulation. The population consists of two types of individuals, wild-types (blue) and mutants (red). The individual marked with the solid black line gives birth and the individual marked with black dashed line is selected for death. (A) Birth-death or death-birth in one patch without migration: This includes birth-death or death-birth in a coupled update mechanism without migration as well as an uncoupled update mechanism in which both death and birth happen in the same subpopulation. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint 3.1 Update mechanisms with coupled migration (B) A coupled update mechanism with migration: if birth is coupled with migration after each birth the newborn migrates to an adjacent patch and replace one of its individual. If death is coupled to migration a death in one patch is followed by birth of an individual in one of the adjacent patch where newborn occupies the place of dead individual. (C) Migration in an uncoupled update mechanism: migration happens independently from birth or death and it only exchanges the position of two individuals from diﬀerent patches. This process is completely random. Figure 3: Update mechanisms in a graph-structured metapopulation. The 8 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint 16 diﬀerent update mechanisms; see table 3.2. So far, only a few of these mecha- nisms have been studied in detail. For example, MBBdd is adopted in [9, 10, 43]. Not all of the observations made in graphs of individuals with the Bd update rule carry over to a graph of subpopulations when the update rule is MBBdd [10, 43]. In fact, in the graph of subpopulations, the dynamics and, in particular, the fate of advantageous mutants are highly dependent on the pattern of migration, local population size, and the graph structure itself. Also, applying MddBB in the graph of subpopulations reduces the chance of advantageous mutants compared to the equivalent well-mixed population with the update mechanism dB [43]. Further- more, employing MddBB in the star of islands in which many subpopulations are connected only via a central subpopulation, it is shown that it is the relative size of the local population in the leaves and the center that determines whether the star of islands is an ampliﬁer, reducer or transient ampliﬁer of selection [13]. In general, in this class of update mechanisms, intuition suggests that the more selection is associated with the selection parameters, the more likely a beneﬁcial mutant will spread through the population. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint 3.2 Equivalence to weighted graphs In the mechanisms with coupled migration, some update mechanisms reduce to simpler ones in a weighted graph of individuals, where the weights of the links that connect individuals in the same subpopulation are diﬀerent from the weights of the links that connect individuals in diﬀerent subpopulations (see Fig. 4). For instance, for the update rule MDDBB, ﬁrst, a patch is selected randomly proportional to its collective selection parameter for death. Afterwards, within the patch, an individual is chosen for death randomly proportional to its selection parameter for death. This is equivalent to selecting one individual from the whole population with a probability proportional to its selection parameter for death. Similarly, selection at birth both at the patch and individual levels is equivalent to selecting an individual for birth proportional to its selection parameter for birth from the whole population (for more details go to appendix C ). Hence, MDDBB on the graph-structured metapopulations can be treated as a DB on the equivalent graph of individuals with weighted links between individuals such that the weights of links that connect the individuals belonging to one patch diﬀer from the ones that connect individuals from diﬀerent patches. In fact, in the coupled update mechanisms whenever selection on each of the birth and death events both on the patch level and individual level is either purely random or ran- domly proportional to selection parameters (BB or bb, and DD or dd), the update mechanism reduces to an update mechanism in an equivalent graph of individuals as mentioned in Tabs. 3.1, 3.2. 9 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint Figure 4: Equivalence of an update mechanism in a graph of subpopulation to an update mechanism in a graph of individuals. In a coupled update mech- anism whenever selection on each of the birth and death events both on the patch and individual levels is either purely random or randomly proportional to selection parameters, the update mechanism is equivalent to an update mechanism in an associated weighted graph of individuals. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint 3.2 Equivalence to weighted graphs In this weighted graph, the weights of the links that connect local individuals are diﬀerent from the weights of the links that connect individuals in neighbouring subpopulations and depend on the migration probability as well as local population sizes. Figure 4: Equivalence of an update mechanism in a graph of subpopulation to an update mechanism in a graph of individuals. In a coupled update mech- anism whenever selection on each of the birth and death events both on the patch and individual levels is either purely random or randomly proportional to selection parameters, the update mechanism is equivalent to an update mechanism in an associated weighted graph of individuals. In this weighted graph, the weights of the links that connect local individuals are diﬀerent from the weights of the links that connect individuals in neighbouring subpopulations and depend on the migration probability as well as local population sizes. 10 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint Update mechanism Comments References MBBDD Equivalent to BD in a graph of individuals - MBBDd - - MBBdD - - MBBdd Equivalent to Bd in a graph of individuals [9, 10, 43] MBbDD - - MBbDd - - MBbdD - - MBbdd - [9] MbBDD - - MbBDd - - MbBdD - - MbBdd - - MbbDD Equivalent to bD in a graph of individuals - MbbDd - - MbbdD - - Mbbdd Equivalent to a completely neutral model - Table 3.1: Birth-death processes with migration coupled to reproduction. In all these update mechanisms in graph-structured metapopulations, the individual producing oﬀspring is identiﬁed ﬁrst and the individual to be removed afterwards. In both steps, we can select for the patch and for the individual separately, leading to 16 such update mechanisms. 3.2 Equivalence to weighted graphs Update mechanism Comments References MBBDD Equivalent to BD in a graph of individuals - MBBDd - - MBBdD - - MBBdd Equivalent to Bd in a graph of individuals [9, 10, 43] MBbDD - - MBbDd - - MBbdD - - MBbdd - [9] MbBDD - - MbBDd - - MbBdD - - MbBdd - - MbbDD Equivalent to bD in a graph of individuals - MbbDd - - MbbdD - - Mbbdd Equivalent to a completely neutral model - Table 3.1: Birth-death processes with migration coupled to reproduction. In all these update mechanisms in graph-structured metapopulations, the individual producing oﬀspring is identiﬁed ﬁrst and the individual to be removed afterwards. In both steps, we can select for the patch and for the individual separately, leading to 16 such update mechanisms. Update mechanism Comments References MBBDD Equivalent to BD in a graph of individuals - MBBDd - - MBBdD - - MBBdd Equivalent to Bd in a graph of individuals [9, 10, 43] MBbDD - - MBbDd - - MBbdD - - MBbdd - [9] MbBDD - - MbBDd - - MbBdD - - MbBdd - - MbbDD Equivalent to bD in a graph of individuals - MbbDd - - MbbdD - - Mbbdd Equivalent to a completely neutral model - Table 3.1: Birth-death processes with migration coupled to reproduction. In all these update mechanisms in graph-structured metapopulations, the individual producing oﬀspring is identiﬁed ﬁrst and the individual to be removed afterwards. In both steps, we can select for the patch and for the individual separately, leading to 16 such update mechanisms. Table 3.1: Birth-death processes with migration coupled to reproduction. In all these update mechanisms in graph-structured metapopulations, the individual producing oﬀspring is identiﬁed ﬁrst and the individual to be removed afterwards. In both steps, we can select for the patch and for the individual separately, leading to 16 such update mechanisms. 11 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint 3.2 Equivalence to weighted graphs ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint Update mechanism Comments References MDDBB Equivalent to DB in a graph of individuals - MDDBb - - MDDbB - - MDDbb Equivalent to Db in a graph of individuals - MDdBB - - MDdBb - - MDdbB - - MDdbb - - MdDBB - - MdDBb - - MdDbB - - MdDbb - - MddBB Equivalent to dB in a graph of individuals [13, 43] MddBb - - MddbB - - Mddbb Equivalent to a completely neutral model - Table 3.2: Death-birth processes with migration coupled to death. In all these update mechanisms in graph-structured metapopulations, the individual being removed is identiﬁed ﬁrst and the individual producing oﬀspring afterwards. Again, there are 16 such update mechanisms. Update mechanism Comments References MDDBB Equivalent to DB in a graph of individuals - MDDBb - - MDDbB - - MDDbb Equivalent to Db in a graph of individuals - MDdBB - - MDdBb - - MDdbB - - MDdbb - - MdDBB - - MdDBb - - MdDbB - - MdDbb - - MddBB Equivalent to dB in a graph of individuals [13, 43] MddBb - - MddbB - - Mddbb Equivalent to a completely neutral model - Table 3.2: Death-birth processes with migration coupled to death. In all these update mechanisms in graph-structured metapopulations, the individual being removed is identiﬁed ﬁrst and the individual producing oﬀspring afterwards. Again, there are 16 such update mechanisms. Table 3.2: Death-birth processes with migration coupled to death. In all these update mechanisms in graph-structured metapopulations, the individual being removed is identiﬁed ﬁrst and the individual producing oﬀspring afterwards. Again, there are 16 such update mechanisms. 12 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint 3.3 Update mechanisms with uncoupled migration While this appears to be a natural choice in many contexts, it makes the comparison to classical models of Evolutionary Graph Theory more challenging. Similarly, when death happens ﬁrst, there are eight other update mechanisms. In the update mechanisms mbbd and mddb where selection is not active in either of the events, the update mechanism is identical to the neutral model i.e. bd. In many popular models of the population genetics literature, migration is as- sumed to be independent from birth and death [11, 44–51]. However, most of these studies use the Wright-Fisher model as the local update mechanism and are thus not directly equivalent to the models more popular in evolutionary graph theory. If we do not require a strictly constant population size, one can envision many addi- tional update mechanisms, such as the migration of a single individual to another patch. While this appears to be a natural choice in many contexts, it makes the comparison to classical models of Evolutionary Graph Theory more challenging. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint 3.3 Update mechanisms with uncoupled migration Migration is said to be uncoupled if birth and death events take place within a single patch (Fig. 3A), and migration independently disperses the individuals such that the population size in each patch remains constant (Fig. 3C). In this scenario, we can model migration as follows: with a certain probability, two random individuals from two random connected patches exchange their positions. In this way, the local population size will remain constant. The ﬁrst event can be birth or death. If birth happens ﬁrst, one of the patches is selected randomly (b) or proportional to its selection parameter for birth (B). From the chosen patch, one individual is selected for birth uniformly at random (b) or randomly proportional to its selection parameter for birth(B) and produces an identical oﬀspring. Once an individual is selected for birth, one individual is selected for death purely random (d) or randomly proportional to a its selection parameter for death (D) from the same patch. The new oﬀspring replaces the empty spot of dead individual. In this category, there are eight diﬀerent update mechanisms. As an example, let us consider mbBD. Here, ﬁrst, a random patch is selected, and in the random patch, an individual is selected randomly proportional to its selection parameter for birth to reproduce. After that, one of the individuals from the same patch is chosen randomly proportional to its selection parameter for death to die. Then, the oﬀspring will ﬁll the empty spot. In this example, there is no patch selection for the second event (death). Hence the second event is indicated by a single letter, D. single letter, D. Similarly, when death happens ﬁrst, there are eight other update mechanisms. In the update mechanisms mbbd and mddb where selection is not active in either of the events, the update mechanism is identical to the neutral model i.e. bd. In many popular models of the population genetics literature, migration is as- sumed to be independent from birth and death [11, 44–51]. However, most of these studies use the Wright-Fisher model as the local update mechanism and are thus not directly equivalent to the models more popular in evolutionary graph theory. If we do not require a strictly constant population size, one can envision many addi- tional update mechanisms, such as the migration of a single individual to another patch. 4 Discussion Evolutionary Graph Theory is a mathematical framework that has been used to think of the role of population structure in evolutionary dynamics. More recently, empirical scientists have become interested in this framework, but in most of the systems in their focus, the nodes are subpopulations and not individuals. Here, we have classiﬁed diﬀerent classes of update mechanisms on such graph structured metapopulations. We focus on update mechanisms that are natural extensions of the update mechanisms typically used in evolutionary graph theory for graphs of individuals. 13 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint Our classiﬁcation is based on three factors: (i) First, if migration is coupled to reproduction or not. (ii) Second, the order of birth and death events. (iii) Third, how selection acts on the growth and survival of the population. Each of these update mechanisms can result in diﬀerent dynamics — using diﬀerent update mechanisms can aﬀect not only the ﬁxation probability and ﬁxation time of newly arising mutations but also other features of the dynamics such as the long term diversity. y The ﬁxation probability in the graphs of individuals under Bd when selection for birth is proportional to a selection parameter and Db when selection for death is proportional to the inverse of the selection parameter for birth are equivalent in undirected regular graphs [24] and they both follow the isothermal theorem [1]. For more details, see A.1. In addition, in a fully connected graph of individuals, where all individuals are equivalent, and every node in the graph includes a self- loop, meaning that the individual selected for birth can die or the individual selected for death can give birth, Bd is equivalent to dB, bD is equivalent to Db, and BD is equivalent to DB. It is worth-mentioning that in an update mechanism where death is followed by birth, having self-loops in the graphs makes only limited sense if we think of the actual physical death of individuals. However, it is sensible if we think of it in the social setting where death and birth are interpreted as imitating one’s idea or sticking to your own [5]. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint 4 Discussion Furthermore, in a fully-connected graph including self-loops, if selection for death equals the inverse of selection for birth, then the update mechanisms Bd, dB, bD, and Db and their corresponding ﬁxation probability is the same as the ﬁxation probability of the well-mixed population under the update mechanism Bd [36]. However, in this condition, the ﬁxation probability of an arbitrary graph under BD and DB is higher than the ﬁxation probability of the corresponding well-mixed population under Bd. In a system where the individuals with a higher selection parameter for birth have a lower selection parameter for death, the more the birth and death are associated with the selection parameters, the ﬁtter individuals have a higher probability to take over the population. This implies that the ﬁxation probability under BD is higher than the ﬁxation probability under Bd and bD. Also the ﬁxation probability under DB is higher than the ﬁxation probability under Db and dB. In addition, the ﬁxation probability of a beneﬁcial mutant in an arbitrary graph under an update mechanism in which selection is global is more than or equal to its ﬁxation probability under an update mechanism in which selection is local [14]. Equality holds for a well- mixed population which includes self-loops meaning that every individual can also replace itself. In a fully connected graph of subpopulations, where the migration rate is symmetric, and the population is homogeneously distributed among the subpopulations, the ﬁxation probability of the update mechanisms such as MBBdd that reduce to update mechanisms in a graph of individuals is the same as the ﬁxation probability of the equivalent well-mixed population under update mechanism Bd [10]. It is interesting to see how the ﬁxation probability of an arbitrary graph under diﬀerent update mechanisms changes, as it is known from graphs of individuals that the choice of the update mechanism can be crucial [5]. In the update mech- anisms where migration is coupled with death or birth, the ﬁxation probability of 14 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint an advantageous mutant in an arbitrary graph is higher under some update mech- anisms compared to others. 4 Discussion As it is shown in Appendix B, if we have two update mechanisms that are exactly the same except that the individual selection for birth or death in one is purely random and in the other is random but associated with selection parameters, the ﬁxation probability of an advantageous mutant under the latter update mechanism is higher than the corresponding ﬁxation probability under the former update mechanism. For example, the update mechanism MBBDD does better in ﬁxing the advantageous mutant than MBbDD. In addition, one intuitively expects that if selection on the patch level is associated with collective selection pa- rameter, the beneﬁcial mutant has a higher chance of being ﬁxed. Nevertheless, it is not straightforward to prove this. In Appendix B we show this in more detail. Similarly, as it is shown in Appendix B, for the update mechanisms with un- coupled migration, if we have two update mechanisms that are only diﬀerent in individual selection on birth or death, the ﬁxation probability of an advantageous mutant under the update mechanism in which individual selection is purely random is smaller than the corresponding ﬁxation probability under the update mechanism in which individual selection is associated with selection parameters. For instance, the ﬁxation probability of an advantageous mutant under mBBD is higher than the corresponding ﬁxation probability under mBBd in an arbitrary graph. In the low migration regime, the dynamics of the system under update mecha- nisms with coupled migration are approximately the same as the dynamics of the system under update mechanisms with uncoupled migration, if selection of patches for birth and death is not associated with the collective selection parameters. This is because in the low migration regime, in both classes, migration is very slow such that it only takes place when the population in each patch is homogeneous. This way, in both classes, migration links homogeneous populations, and it does not change the local dynamics. In addition, it is interesting to see under which of the coupled or uncoupled update mechanisms, the beneﬁcial mutant has a higher chance of taking over the whole population. Intuitively, under the coupled update mechanism, migration helps to spread beneﬁcial mutants, whereas under uncoupled migration, exchanges the individual between the patches occur randomly and independent of the selection parameters. We hope that this paper paves the way for future works on the evolutionary dy- namics of graph-structured metapopulations. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint A The transition probabilities for the graph of indi- viduals Assume a connected graph of individuals where wij is the weight of the link con- necting node i to j. The population consists of two types, wild-type A and mutant B. The variable si indicates the status of node i, si = 0 if it is occupied by a wild- type, and si = 1 if it is occupied by a mutant. The selection parameter for the birth of the mutant with respect to the wild-type is r, and the selection parameter for the death of the mutant with respect to the wild-type is t. In an update mechanism where birth is global and death is local, one individual is selected for birth, and then one of its neighbours is chosen for death. The oﬀspring will replace the empty spot. Based on this model, there are two possible transitions: increasing and de- creasing the number of mutants by one. The probability T n+ birth−death of increasing the number of mutants, n = P i si, by one is creasing the number of mutants by one. The probability T n+ birth−death of increasing the number of mutants, n = P i si, by one is T n+ birth−death = X i rsi r P k sk + P k(1 −sk) \| {z } birth P j wij(1 −sj) t P l wilsl + P l wil(1 −sl) \| {z } death . (A.1) (A.1) This equation is the summation over all the possibilities that the number of mutants increases. The probability T n− birth−death of decreasing the number of mutants n by one is T n− birth−death = X i 1 −si r P k sk + P k(1 −sk) \| {z } birth t P j wijsj t P l wilsl + P l wil(1 −sl) \| {z } death . (A.2) (A.2) When death is global and birth is local, an individual is selected for death, and then from its neighbour, one individual is selected for birth. 4 Discussion Here, we only classify possible update mechanisms on metapopulations and do not analyze them in detail. However, the update mechanism is a crucial ingredient of evolutionary graph theory and better understanding how it aﬀects evolutionary dynamics in structured metapopulations will be necessary to move the ﬁeld forward. 15 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint (i) In the above equations if r ̸= 1 and t ̸= 1 there are selection pressures both on the birth and the death. This corresponds to the update mechanism BD in which birth is global and death is local, and the update mechanism DB in which death is global and death is local. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint A The transition probabilities for the graph of indi- viduals In this case the transition probabilities are T n+ death−birth = X i 1 −si t P k sk + P k(1 −sk) \| {z } death r P j wijsj r P l wilsl + P l wil(1 −sl) \| {z } birth (A.3) T n− death−birth = X i tsi t P k sk + P k(1 −sk) \| {z } death P j wij(1 −sj) r P l wilsl + P l wil(1 −sl) \| {z } birth (A.4) (A.3) (A.4) Based on the values of r and t, the update mechanisms are categorized as follows: Based on the values of r and t, the update mechanisms are categorized as follows: 16 (ii) If r ̸= 1 and t = 1 birth-death corresponds to update mechanism Bd and death-birth corresponds to dB. (iii) If r = 1 and t ̸= 1, birth-death correspond to bD and death-birth correspond to Db. (iii) If r = 1 and t ̸= 1, birth-death correspond to bD and death-birth correspond to Db. (iv) If r = 1 and t = 1 birth-death corresponds to bd and death-birth corresponds to db. A.1 Equivalence of Bd and Db in undirected regular graphs Using the equations in Appendix A.1, the transition probabilities of an arbitrary graph under update mechanisms Bd are T n+ Bd = X i rsi r P k sk + P ( 1 −sk) \| {z } birth P j wij(1 −sj) P l wil \| {z } death (A.5) T n+ Bd = X i rsi r P k sk + P ( 1 −sk) \| {z } birth P j wij(1 −sj) P l wil \| {z } death (A.5) T n− Bd = X i 1 −si r P k sk + P k(1 −sk) \| {z } birth P j wijsj P l wil \| {z } death (A.6) (A.5) T n− Bd = X i 1 −si r P k sk + P k(1 −sk) \| {z } birth P j wijsj P l wil \| {z } death (A.6) (A.6) The transition probabilities of an arbitrary graph under update mechanisms Db are The transition probabilities of an arbitrary graph under update mechanisms Db are T n+ Db = X i 1 −si t P k sk + P k(1 −sk) \| {z } death P j wijsj P l wil \| {z } birth (A.7) T n− Db = X i tsi t P k sk + P k(1 −sk) \| {z } death P j wij(1 −sj) P l wil \| {z } birth (A.8) T n+ Db = X i 1 −si t P k sk + P k(1 −sk) \| {z } death P j wijsj P l wil \| {z } birth (A.7) (A.7) {z death T n− Db = X i tsi t P k sk + P k(1 −sk) \| {z } death P j wij(1 −sj) P l wil \| {z } birth (A.8) (A.8) {z death In a regular graph P l wil is identical for all the nodes. Thus we can set it as P l wil = α. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint A.1 Equivalence of Bd and Db in undirected regular graphs As a result, the transition probabilities for update mechanism Bd simplify to T n+ Bd = r α(rn + N −n) X i,j wijsi(1 −sj), (A.9) T n− Bd = 1 α(rn + N −n) X i,j wij(1 −si)sj (A.10) (A.9) (A.9) and T n− Bd = 1 α(rn + N −n) X i,j wij(1 −si)sj (A.10) (A.10) Therefore, T n− Bd T n+ Bd = 1/r and as a result the ﬁxation probability is φ(n) = 1−1/rn 1−1/rN . Similarly, the transition probabilities for the update mechanism Db simplify T n+ Db = 1 α(tn + N −n) X i,j wij(1 −si)sj (A.11) (A.11) 17 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint T n− Db = t α(tn + N −n) X i,j wijsi(1 −sj). (A.12) (A.12) Since T n− Db T n+ Db = t, the ﬁxation probability is φ(n) = 1−tn 1−tN . If we set t = 1/r the ﬁxation probability is the same as the ﬁxation probability of the equivalent well- mixed population under the update mechanism Bd. Since T n− Db T n+ Db = t, the ﬁxation probability is φ(n) = 1−tn 1−tN . If we set t = 1/r the ﬁxation probability is the same as the ﬁxation probability of the equivalent well- mixed population under the update mechanism Bd. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint B Comparing the ﬁxation probability in structured metapopulations under diﬀerent update mecha- (B.3) The transition probabilities for the update mechanism MBbDD are Parameter Description n Total number of mutants: P i ni N Total population size: P i Ni Ni Population size in patch i ni Number of mutants in patch i r Selection parameter for birth of the mutant t Selection parameter for death of the mutant λ Migration probability wij Weight of the link from patch i to patch j Table B.1: Parameters for graph of subpopulations Parameter Description n Total number of mutants: P i ni N Total population size: P i Ni Ni Population size in patch i ni Number of mutants in patch i r Selection parameter for birth of the mutant t Selection parameter for death of the mutant λ Migration probability wij Weight of the link from patch i to patch j Table B.1: Parameters for graph of subpopulations T n− MBBDD = X i rni + Ni −ni rn + N −n \| {z } selection patch birth Ni −ni rni + Ni −ni \| {z } birth of individual · λ X j wij tnj + Nj −nj P k wik(tnk + Nk −nk) \| {z } choosing a patch to migrate to tnj tnj + Nj −nj \| {z } death of individual +(1 −λ) tni tni + Ni −ni \| {z } death of individual in parental patch ! . (B 3) T n− MBBDD = X i rni + Ni −ni rn + N −n \| {z } selection patch birth Ni −ni rni + Ni −ni \| {z } birth of individual · λ X j wij tnj + Nj −nj P k wik(tnk + Nk −nk) \| {z } choosing a patch to migrate to tnj tnj + Nj −nj \| {z } death of individual +(1 −λ) tni tni + Ni −ni \| {z } death of individual in parental patch ! . (B 3) death of individual in parental patch death of individual in parental patch {z choosing a patch to migrate to (B.3) The transition probabilities for the update mechanism MBbDD are T n+ MBbDD = X i rni + Ni −ni rn + N −n ni Ni · λ X j wij tnj + Nj −nj P k wik(tnk + Nk −nk) Nj −nj tnj + Nj −nj + (1 −λ) Ni −ni tni + Ni −ni ! B Comparing the ﬁxation probability in structured metapopulations under diﬀerent update mecha- CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint Parameter Description n Total number of mutants: P i ni N Total population size: P i Ni Ni Population size in patch i ni Number of mutants in patch i r Selection parameter for birth of the mutant t Selection parameter for death of the mutant λ Migration probability wij Weight of the link from patch i to patch j Table B.1: Parameters for graph of subpopulations T n− MBBDD = X i rni + Ni −ni rn + N −n \| {z } selection patch birth Ni −ni rni + Ni −ni \| {z } birth of individual · λ X j wij tnj + Nj −nj P k wik(tnk + Nk −nk) \| {z } choosing a patch to migrate to tnj tnj + Nj −nj \| {z } death of individual +(1 −λ) tni tni + Ni −ni \| {z } death of individual in parental patch ! . B Comparing the ﬁxation probability in structured metapopulations under diﬀerent update mecha- This appendix discusses why some update mechanisms ﬁx beneﬁcial mutants with higher probability. Assume that we have two types of individuals, mutants, and wild- types. The transition probabilities of increasing and decreasing the total number of mutants n are given by T n+ and T n−, respectively. Here, we use a mean-ﬁeld approximation and assume that the transition probabilities are the summation of the transition probabilities for all the possible conﬁgurations for a speciﬁc number of mutants n. The other parameters are described in Tab.B.1. If we start with a single randomly placed mutant in a pure wild-type population, the average ﬁxation probability of a mutant [20] is given by φ(1) = 1 1 + PN−1 i=1 Qi j=1 T j− T j+ . (B.1) (B.1) Therefore, in order to investigate how the ﬁxation probabilities in diﬀerent update rules vary, it is suﬃcient to compare the transition probabilities. By comparing the transition probabilities, we can see that for both coupled and uncoupled update mechanisms, if two update mechanisms only diﬀer in individual-level selection for either birth or death, the ﬁxation probability of the beneﬁcial mutant under the update mechanism in which individual-selection is associated with selection param- eter is higher than the one in which individual-selection is purely random. Here, we compare the transition probabilities of update mechanisms MBBDD, MBbDD, MBBDd. The transition probabilities of increasing and decreasing the total number of mutants, n = P i ni update mechanism MBBDD are T n+ MBBDD = X i rni + Ni −ni rn + N −n \| {z } birth patch rni rni + Ni −ni \| {z } birth of individual · λ X j wij tnj + Nj −nj P k wik(tnk + Nk −nk) \| {z } choosing a patch to migrate to Nj −nj tnj + Nj −nj \| {z } death of individual +(1 −λ) Ni −ni tni + Ni −ni \| {z } death of individual in parental patch ! ( ) {z death of individual in parental patch {z choosing a patch to migrate to (B.2) 18 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint . B Comparing the ﬁxation probability in structured metapopulations under diﬀerent update mecha- (B.4) T n− MBbDD = X i rni + Ni −ni rn + N −n Ni −ni Ni · λ X j wij tnj + Nj −nj P k wik(tnk + Nk −nk) tnj tnj + Nj −nj + (1 −λ) tni tni + Ni −ni ! . (B.5) T n+ MBbDD = X i rni + Ni −ni rn + N −n ni Ni · λ X j wij tnj + Nj −nj P k wik(tnk + Nk −nk) Nj −nj tnj + Nj −nj + (1 −λ) Ni −ni tni + Ni −ni ! (B.4) T n− MBbDD = X i rni + Ni −ni rn + N −n Ni −ni Ni · λ X j wij tnj + Nj −nj P k wik(tnk + Nk −nk) tnj tnj + Nj −nj + (1 −λ) tni tni + Ni −ni ! . (B.5) (B.5) 19 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint The transition probabilities for the update mechanism MBBDd are T n+ MBBDd = X i rni + Ni −ni rn + N −n rni rni + Ni −ni · λ X j wij tnj + Nj −nj P k wik(tnk + Nk −nk) Nj −nj Nj + (1 −λ)Ni −ni Ni ! j ! (B.6) ! (B.6) (B.6) T n− MBBDd = X i rni + Ni −ni rn + N −n Ni −ni rni + Ni −ni · λ X j wij tnj + Nj −nj P k wik(tnk + Nk −nk) nj Nj + (1 −λ) ni Ni ! . . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint B Comparing the ﬁxation probability in structured metapopulations under diﬀerent update mecha- (B.7) (B.7) Comparing Eqs. B.2 and B.4, all the terms are the same except the second term which is the probability of choosing a mutant in patch i for birth. Since rni rni + Ni −ni > ni Ni , for beneﬁcial mutants, r > 1, except for ni = Ni and ni = 0 where right hand side and left are equal, that implies T n+ MBBDD > T n+ MBbDD. Also if we compare Eqs. B.3 and B.5 since Also if we compare Eqs. B.3 and B.5 since Ni −ni rni + Ni −ni < Ni −ni Ni , for beneﬁcial mutants, r > 1, except for ni = Ni and ni = 0 where right hand side and left hand side of the above statement are equal then T n− MBBDD < T n− MBbDD. As a result, T n− MBBDD T n+ MBBDD < T n− MBbDD T n+ MBbDD for all 1 < n < N −1 The ratio T n− T n+ appears in the denominator of Eq. B.1. This implies that the ﬁxation probability of an advantageous mutant under MBBDD is higher than the corre- sponding ﬁxation probability under MBbDD for an arbitrary graph, φMBBDD > φMBbDD. Similarly, we can show that the ﬁxation probability of a deleterious mutant under MBBDD is lower than the corresponding ﬁxation probability under MBbDD for an arbitrary graph, φMBBDD < φMBbDD. 20 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint In addition, comparing Eqs. B.2 and B.6, since In addition, comparing Eqs. B.2 and B.6, since Nj −nj tnj + Nj −nj > Nj −nj Nj , for the beneﬁcial mutants, t < 1, we have for the beneﬁcial mutants, t < 1, we have T n+ MBBDD > T n+ MBBDd. B Comparing the ﬁxation probability in structured metapopulations under diﬀerent update mecha- Also by comparing Eqs.B.3 and B.7, we have Also by comparing Eqs.B.3 and B.7, we have T n− MBBDD < T n− MBBDd because dnj dnj + Nj −nj < nj Nj , (B.8) (B.8) for all 1 < nj < Nj −1 and for nj = Nj both sides are equal. In conclusion, the ﬁxation probability of an advantageous mutant in an arbitrary graph under MBBDd is less than the corresponding ﬁxation probability under MBBDD. On the other hand, for deleterious mutants, φMBBDd > φMBBDD. Furthermore, we can show in a similar way as above that for beneﬁcial mu- tants that the ﬁxation probability, φ of an arbitrary graph under update mechanism MBBDD, MBbDD, and MBbDd has the following relationship with each other, φMBBDD > φMBbDD > φMBbDd, φMBBDD > φMBbDD > φMBbDd, and similarly the relation between the ﬁxation probability of a beneﬁcial mutant for an arbitrary graph under update mechanisms MBBDD, MBBDd, and MBbDd is φMBBDD > φMBBDd > φMBbDd. φMBBDD > φMBBDd > φMBbDd. In the above expressions, one cannot simply state that which of the φMBBDd and φMBbDD is higher. The relation between these two values might be dependent on the graph structure. In the above expressions, one cannot simply state that which of the φMBBDd and φMBbDD is higher. The relation between these two values might be dependent on the graph structure. On the other hand, the relation between the ﬁxation probabilities, φ, of a dele- terious mutant in an arbitrary graph under the update mechanisms, MBBDD, MBbDD, and MBbDd is φMBBDD < φMBbDD < φMBbDd, and similarly, we can simply show that the ﬁxation probabilities of a deleterious mutant in an arbitrary graph under the update mechanisms MBBDD, MBBDd, and MBbDd has the following relationship, φMBBDD < φMBBDd < φMBbDd. (B.9) (B.9) . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint φMBBDD < φMBBDd < φMBbDd. As we see from these equations, for an arbitrary graph, the more an update mech- anism is associated with selection parameters, the more the ﬁxation probability of 21 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint the advantageous mutants and the less the ﬁxation probability of the deleterious mutants. Comparing the transition probabilities of a beneﬁcial mutant under two update mechanisms that only diﬀer in patch level selection for either birth or death is not straightforward. Intuitively we expect that the update mechanism in which patch level selection is associated with the collective selection parameter of the patch has a higher ﬁxation probability. In the following, we show why one cannot easily infer from the transition probabilities which one is higher. Let us compare the transition probabilities for increasing the population size by one under the update mechanisms MBBDD and MbBDD. T n+ MbBDD = X i Ni N \|{z} birth patch rni rni + Ni −ni \| {z } birth of individual · λ X j wij tnj + Nj −nj P k wik(tnk + Nk −nk) \| {z } choosing a patch to migrate to Nj −nj tnj + Nj −nj \| {z } death of individual +(1 −λ) Ni −ni tni + Ni −ni \| {z } death of individual in parental patch ! (B.10) {z choosing a patch to migrate to (B.10) Comparing the equations B.2 and B.10 the only diﬀerence is the term for the birth patch. T n+ MBBDD > T n+ MbBDD if for all the i values Comparing the equations B.2 and B.10 the only diﬀerence is the term for the birth patch. T n+ MBBDD > T n+ MbBDD if for all the i values rni + Ni −ni rn + N −n ≥Ni N . (B.11) (B.11) The above relation holds if and only if ni Ni ≥n N . (B.12) (B.12) However, the above relation does not always hold, and it depends on the conﬁg- uration of the population. Hence, it is not easy to compare equations B.2 and B.10. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint φMBBDD < φMBBDd < φMBbDd. (B.16) From Eqs. B.13 and B.15, we can see that for beneﬁcial mutants, t < 1, From Eqs. B.13 and B.15, we can see that for beneﬁcial mutants, t < 1, T n+ mBBD > T n+ mBBd, because for 1 < ni < Ni we have because for 1 < ni < Ni we have Ni −ni dni + Ni −ni > Ni −ni Ni . Analogously, for the beneﬁcial mutants we have (B.17) T n− mBBD < T n− mBBd. (B.17) This suggests that the ﬁxation probability of an advantageous mutant under mBBD is higher than the corresponding ﬁxation probability under mBBd, φmBBD > φmBBd. Similarly we can see that for a beneﬁcial mutant φmBbd < φmBbD < φmBBD. (B.18) (B.18) On the other hand, for the deleterious mutants we have an opposite relation between the ﬁxation probabilities: On the other hand, for the deleterious mutants we have an opposite relation between the ﬁxation probabilities: φmBbd > φmBbD > φmBBD. (B.19) (B.19) φMBBDD < φMBBDd < φMBbDd. Similarly, by comparing the transition probabilities for uncoupled update mech- anisms, we can see that the ﬁxation probability of an advantageous mutant under update mechanisms in which individual-selection is associated with selection param- eters is higher than the corresponding ﬁxation probability under update mechanisms in which individual-selection is independent of selection parameters. The transition probabilities of this update mechanism only include the non-migrative terms because the migrative term does not change the number of mutants in the whole population. The transition probabilities for the update mechanism mBBD are T n+ mBBD = (1 −λ) X i rni + Ni −ni rn + N −n rni rni + Ni −ni Ni −ni tni + Ni −ni , (B.13) T n− mBBD = (1 −λ) X i rni + Ni −ni rn + N −n Ni −ni rni + Ni −ni tni tni + Ni −ni , (B.14) T n+ mBBD = (1 −λ) X i rni + Ni −ni rn + N −n rni rni + Ni −ni Ni −ni tni + Ni −ni , (B.13) (B.13) T n− mBBD = (1 −λ) X i rni + Ni −ni rn + N −n Ni −ni rni + Ni −ni tni tni + Ni −ni , (B.14) T n− mBBD = (1 −λ) X i rni + Ni −ni rn + N −n Ni −ni rni + Ni −ni tni tni + Ni −ni , (B.14) (B.14) 22 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint and the transition probabilities for the update mechanism mBBd are T n+ mBBd = (1 −λ) X i rni + Ni −ni rn + N −n rni rni + Ni −ni Ni −ni Ni , (B.15) T n− mBBd = (1 −λ) X i rni + Ni −ni rn + N −n Ni −ni rni + Ni −ni ni Ni . (B.16) T n+ mBBd = (1 −λ) X i rni + Ni −ni rn + N −n rni rni + Ni −ni Ni −ni Ni , (B.15) (B.15) T n− mBBd = (1 −λ) X i rni + Ni −ni rn + N −n Ni −ni rni + Ni −ni ni Ni . . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint C Reduction of update mechanisms on graph-structured metapopulations to update mechanisms on graph of individuals In the mechanisms with coupled migration, some update mechanisms reduce to simpler ones in a weighted graph of individuals, where the weights of the links that connect individuals locally are diﬀerent from the weights of the links that connect individuals in adjacent subpopulations (see Fig. 4). In order for an update mechanism to reduce to a simper update mechanism, selection for birth and death should be either associated with selection parameter or not for both patch and individual levels. As an example MBBdd reduces to Bd. This can be easily shown by transition probabilities; In order to increase the number of mutants by one through selecting one mutant from the patch i consists of two parts; ﬁrst selecting a mutant from patch i with probability rni + Ni −ni rn + N −n r rni + Ni −ni . 23 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.20.513032 doi: bioRxiv preprint This probability is simpliﬁed to r rn+N−n which is equivalent to the probability of selecting one mutant from the whole population regardless of the collective selec- tion parameter of the patches. The second part is choosing one of the wild-type neighbours of the selected mutant for death. The neighbour could be either selected from the parental patch with probability This probability is simpliﬁed to r rn+N−n which is equivalent to the probability of selecting one mutant from the whole population regardless of the collective selec- tion parameter of the patches. The second part is choosing one of the wild-type neighbours of the selected mutant for death. The neighbour could be either selected from the parental patch with probability (1 −λ)Ni −ni Ni , or from the neighbouring patches with probability λ X j wij Nj −nj P k wikNk The above two equations for the death probability of a wild-type imply that a graph of patches can be reduced to a graph of individuals. C Reduction of update mechanisms on graph-structured metapopulations to update mechanisms on graph of individuals In the equivalent graph of individuals the weight of the link between each two individuals within the patch i is 1−λ Ni if we take into account self-loops and the weight of the link from an individual from patch i to an individual from patch j is λwij P j wijNj . Therefore, the update mechanism MBBdd on a graph of sub-populations is equivalent to the update mechanism Bd on a graph of individuals in which the weight of the links that connect local individuals diﬀer from the weight of links that connect individuals in diﬀerent patches. The weight of the links depends on the migration probability as well as the local population sizes. . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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https://openalex.org/W3184437506	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0255154&type=printable	English	null	Can we predict the severe course of COVID-19 - a systematic review and meta-analysis of indicators of clinical outcome?	PloS one	2,021	cc-by	10,277	PLOS ONE PLOS ONE RESEARCH ARTICLE Background COVID-19 has been reported in over 40million people globally with variable clinical out- comes. In this systematic review and meta-analysis, we assessed demographic, laboratory and clinical indicators as predictors for severe courses of COVID-19. Editor: Chiara Lazzeri, Azienda Ospedaliero Universitaria Careggi, ITALY Received: January 1, 2021 Accepted: July 10, 2021 Published: July 29, 2021 Editor: Chiara Lazzeri, Azienda Ospedaliero Universitaria Careggi, ITALY Received: January 1, 2021 Accepted: July 10, 2021 Published: July 29, 2021 Methods This systematic review was registered at PROSPERO under CRD42020177154. We sys- tematically searched multiple databases (PubMed, Web of Science Core Collection, MedR- vix and bioRvix) for publications from December 2019 to May 31st 2020. Random-effects meta-analyses were used to calculate pooled odds ratios and differences of medians between (1) patients admitted to ICU versus non-ICU patients and (2) patients who died ver- sus those who survived. We adapted an existing Cochrane risk-of-bias assessment tool for outcome studies. 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.0255154 Abstract Citation: Katzenschlager S, Zimmer AJ, Gottschalk C, Grafeneder J, Schmitz S, Kraker S, et al. (2021) Can we predict the severe course of COVID-19 - a systematic review and meta-analysis of indicators of clinical outcome? PLoS ONE 16(7): e0255154. https://doi.org/10.1371/journal. pone.0255154 Can we predict the severe course of COVID-19 - a systematic review and meta-analysis of indicators of clinical outcome? Stephan KatzenschlagerID1☯, Alexandra J. ZimmerID2☯, Claudius Gottschalk3, Ju¨rgen GrafenederID4, Stephani SchmitzID3, Sara Kraker3, Marlene Ganslmeier3, Amelie Muth3, Alexander Seitel5, Lena Maier-HeinID5, Andrea Benedetti2, Jan LarmannID1, Markus A. Weigand1, Sean McGrathID6‡, Claudia M. DenkingerID3,7‡* Jurgen GrafenederID , Stephani SchmitzID , Sara Kraker , Marlene Ganslmeier , Amelie Muth3, Alexander Seitel5, Lena Maier-HeinID5, Andrea Benedetti2, Jan LarmannID1, Markus A. Weigand1, Sean McGrathID6‡, Claudia M. DenkingerID3,7‡* g ID , p ID , , , Amelie Muth3, Alexander Seitel5, Lena Maier-HeinID5, Andrea Benedetti2, Jan LarmannID1, Markus A. Weigand1, Sean McGrathID6‡, Claudia M. DenkingerID3,7‡* 1 Department of Anesthesiology, Heidelberg University Hospital, Heidelberg, Germany, 2 Departments of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, Canada, 3 Division of Tropical Medicine, Center for Infectious Diseases, Heidelberg University Hospital, Heidelberg, Germany, 4 Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria, 5 Division of Computer Assisted Medical Interventions, German Cancer Research Center (DKFZ), Heidelberg, Germany, 6 Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA, United States of America, 7 German Center for Infection Research (DZIF), partner site Heidelberg, Heidelberg, Germany a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. ‡ These authors also contributed equally to this work. * Claudia.Denkinger@uni-heidelberg.de ☯These authors contributed equally to this work. ‡ These authors also contributed equally to this work. * Claudia.Denkinger@uni-heidelberg.de Discussion This comprehensive meta-analysis found age, cerebrovascular disease, CRP, LDH and cTnI to be the most important risk-factors that predict severe COVID-19 outcomes and will inform clinical scores to support early decision-making. Results Copyright: © 2021 Katzenschlager 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. Of 6,702 unique citations, we included 88 articles with 69,762 patients. There was concern for bias across all articles included. Age was strongly associated with mortality with a differ- ence of medians (DoM) of 13.15 years (95% confidence interval (CI) 11.37 to 14.94) between those who died and those who survived. We found a clinically relevant difference between non-survivors and survivors for C-reactive protein (CRP; DoM 69.10 mg/L, CI 50.43 to 87.77), lactate dehydrogenase (LDH; DoM 189.49 U/L, CI 155.00 to 223.98), Data Availability Statement: All relevant data are uploaded to Zenodo and publicly accessible via the PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 1 / 19 PLOS ONE Meta-analysis: Risk factors of COVID-19 cardiac troponin I (cTnI; DoM 21.88 pg/mL, CI 9.78 to 33.99) and D-Dimer (DoM 1.29mg/L, CI 0.9 to 1.69). Furthermore, cerebrovascular disease was the co-morbidity most strongly associated with mortality (Odds Ratio 3.45, CI 2.42 to 4.91) and ICU admission (Odds Ratio 5.88, CI 2.35 to 14.73). following URL: https://zenodo.org/record/ 5102836#.YPH8vehKg2x. following URL: https://zenodo.org/record/ 5102836#.YPH8vehKg2x. cardiac troponin I (cTnI; DoM 21.88 pg/mL, CI 9.78 to 33.99) and D-Dimer (DoM 1.29mg/L, CI 0.9 to 1.69). Furthermore, cerebrovascular disease was the co-morbidity most strongly associated with mortality (Odds Ratio 3.45, CI 2.42 to 4.91) and ICU admission (Odds Ratio 5.88, CI 2.35 to 14.73). following URL: https://zenodo.org/record/ 5102836#.YPH8vehKg2x. Funding: SM acknowledges support from the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE1745303, National Library Of Medicine of the National Institutes of Health under Award Number T32LM012411, and Fonds de recherche du Que´bec-Nature et technologies B1X research scholarship. CMD acknowledges the support of the Heidelberg University Hospital. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author (s) and do not necessarily reflect the views of the funding agencies. Methods This trial was registered at PROSPERO on April 4th, 2020 (Registration number: CRD42020177154). The PRISMA checklist is provided in the supplementary S2 in S1 This trial was registered at PROSPERO on April 4th, 2020 (Registration number: CRD42020177154). The PRISMA checklist is provided in the supplementary S2 in S1 File. This trial was registered at PROSPERO on April 4 , 2020 (Registration number: CRD42020177154). The PRISMA checklist is provided in the supplementary S2 in S1 File. Introduction Competing interests: I have read the journal’s policy and none of the authors of this manuscript have a competing interest. Coronavirus disease (COVID-19) was declared a pandemic by the World Health Organization (WHO) on March 11th, 2020 [1]. As of October 31st 2020 approximately 46 million people were infected with this virus [2]. The outcomes of COVID-19 vary from completely asymp- tomatic to hospitalization, ICU admission and death [3, 4]. Several studies aimed to identify possible risk factors for a severe outcome. Studies investi- gated demographic risk factors and found advanced age to be the strongest predictor of a severe course [5–7]. However, age alone does not explain the variability in the severity of dis- ease with sufficient granularity [8]. Symptoms on presentation associated with severe disease include dyspnea, fever, cough, and fatigue [6, 9, 10]. Several co-morbidities have been identi- fied as risk factors, including cardiovascular disease, obesity, chronic respiratory disease, dia- betes, cerebrovascular disease, chronic renal failure and cancer [7, 11–18]. The effect of other co-morbidities on disease outcome remain less clear: e.g. hypertension being associated with a decreased risk [7, 19] for death in some and an increased risk [20] in other publications. Simi- larly, data on past and current smoking are inconsistent in respect to the association with dis- ease severity [21–25]. Biomarkers predicting severe disease include different markers of inflammation and acute phase reaction (e.g. CRP, procalcitonin (PCT), white blood cells (WBC), lymphopenia, interleukin 6 (IL-6)) [26, 27]. Increased D-dimer levels, as a marker for coagulation and thrombosis, were found to be elevated in non-survivors, whereas other coagu- lation markers failed to show statistical and clinical difference [13, 28–30]. Markers indicating cardiac damage, such as cardiac troponin I or T and N terminal pro B type natriuretic peptide (NT-proBNP) were also associated with severe disease and mortality [31]. This systematic review aims, to our knowledge for the first time, to comprehensively evalu- ate demographic, clinical and laboratory indicators for their association with severe COVID- 19 and death. PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 Assessment of study quality To analyze risk of bias in individual studies, we evaluated the studies using an approach adapted from an existing Cochrane tool by Higgins et al. [33] for systematic reviews that assessed indicators of outcomes. Specifically, we analyzed three areas: 1) case definition and severity definition; 2) patient data availability and exclusions and; 3) selection bias and applica- bility. We rated the risk of bias in low, intermediate and high risks of bias. Search strategy Medline [PubMed] and Web of Science Core Collection as well as preprint databases (bioRxiv and medRxiv) were searched. The exact search terms were developed with an experienced medical librarian (GG) using combinations of subject headings (when applicable) and text- words for the concepts without language restrictions. The full search strategy used for PubMed is presented in the supplementary S1 in S1 File. The results of the search term were imported into the bibliography manager Zotero (Version 5.0.92) for further processing. Study screening and data extraction Study selection was done by three authors (SK, CG and JG) initially in parallel for five ran- domly selected papers and after alignment in the selection was guaranteed, it was done inde- pendently by each of the reviewers. Article title and abstracts were screened for eligibility in English or German language, followed by a full-text review for those eligible. A structured electronic data extraction form was developed (AS, LMH, SO, LAS and BP), piloted on five randomly selected papers and then used to extract information from included studies. Six reviewers (SK, CG, SS, SaK, MG and AM) performed data extraction in duplicate for the first five randomly selected papers to ensure alignment and then independently, with concerns being discussed jointly. For continuous indicators we extracted means and standard deviation as well as medians, first quartiles and third quartiles if available. The comprehensive list of data items that were collected is presented in the supplementary S12 in S1 File. Through- out screening and extraction, disagreements were discussed until consensus was reached, and a senior author (CMD) was consulted when necessary. Given the concern for reporting of the same patients in different publications [32] leading to a bias in the data, we excluded papers which included patients from the same hospital with an overlapping inclusion date. Furthermore, we excluded data from 23 articles (peer-reviewed and preprint), because the reported laboratory values with the reported units were obviously incorrect (supplementary S3 in S1 File), unless we were able to clarify the issue with the authors of the respective paper directly. Eligibility criteria Studies eligible for inclusion provided data on demographic, clinical and/or laboratory risk factors for the following outcomes: hospitalization, intubation, ICU admission, and/or death. Laboratory values and vital parameters taken at hospital admission were considered. Cross- sectional studies, cohort studies, randomized and non-randomized controlled trials were 2 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 PLOS ONE Meta-analysis: Risk factors of COVID-19 included. No specific restrictions were placed in terms of demographic and clinical character- istics of the population being studied. The search was conducted on July 29th, the search date was set from December 1st 2019 to May 31st 2020. A full list of data items screened for in the studies is available in the supplementary S12 in S1 File. These data items were chosen, on the one hand, according to the information available in the existing literature and, on the other hand, in order to identify risk factors at hospital admission. Statistical analysis We grouped indicators into binary and continuous indicators across five categories: (1) demo- graphics, (2) symptoms, (3) co-morbidities, (4) laboratory and (5) clinical course/treatment. 3 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 PLOS ONE Meta-analysis: Risk factors of COVID-19 We analyzed all available indicators between (1) hospitalized and non-hospitalized patients, (2) ICU-admitted patients and non-ICU admitted patients, (3) intubated and non-intubated patients, and (4) patients who died and patients who survived. Most data were available for ICU admission and death. Thus, we focus on these comparison groups in the main paper and present data on hospitalization and intubation in the supplement. Clinical significance was determined by expert consensus with clinicians. A predefined rule (e.g. 10% above normal range) across biomarkers or vital parameters is not possible as it is different for every marker and the unit chosen. Meta-analyses were only performed when there were at least 4 primary studies reporting adequate summary data. As the continuous indicators were often skewed and were summa- rized by medians in most primary studies, we meta-analyzed the difference of medians across groups for continuous indicators. Specifically, we pooled the difference of medians in a ran- dom effects meta-analysis using the Quantile Estimation (QE) approach proposed by McGrath et al. [34]. In secondary analyses, median value of indicators in each comparison group were pooled using the same approach. The QE approach estimates the variance of the (difference of) medians in studies that report the sample median and first and third quartiles of the outcome. When studies report sample means and standard deviations of the outcome, this approach estimates the (difference of) medians and its variance. Then, the standard inverse-variance approach is applied to obtain a pooled estimate of the population (difference of) medians. The population difference of medi- ans can be interpreted as the difference between the median value of the indicator in one group (e.g., those who survived) and the median value of the indicator in the other group (e.g., those who died). For binary indicators, the pooled odds ratios (OR) and associated 95% confidence intervals (CI) were estimated in a random effects meta-analysis. For both binary and continuous indica- tors, the restricted maximum likelihood (REML) approach was used to estimate between- study heterogeneity. When REML failed to converge for a continuous indicator, we used the DerSimmonian and Laird (DL) estimator for all analyses involving this indicator. Statistical analysis For all analyses, between-study heterogeneity was assessed by the I2 statistic. The presence of small-study effects was visually assessed in funnel plots. Analyses were performed in R (ver- sion 4.0.2) with package ‘metamedian’ [35] and in Stata (Version 16.1). The code is publicly available on GitHub (https://github.com/stmcg/covid-ma). PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 Results The search resulted in 6,702 articles, of which 3,733 were excluded because they did not present primary data (e.g. guidelines, recommendations, letter to the editors or corre- spondences, study protocols, modeling), 792 were case reports, 465 focused on patients younger than 18 years and 381 were systematic reviews. In total, 88 articles were included (Fig 1). The majority of studies (52) were conducted in China, 21 in Europe, 12 in the USA, two in Iran, one in South Korea. Most studies were retrospective cohorts (n = 84) and four had a prospective study design. All studies were in English. Data on mortality were reported in 64 studies, data on ICU admission were available in 26 studies (two stud- ies reported both and patients were counted twice). In total, data from 69,762 patients were meta-analyzed, of whom 5,311 died and 57,321 survived and 2,112 provided data on ICU admission while 5,018 did not require ICU admission. We were not able to perform a meta-analysis for all indicators (supplementary S12 in S1 File) extracted from the publica- tions. Meta-analysis for all eligible indicators for each outcome is listed in the supplemen- tary section (S5–S8 in S1 File). 4 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 PLOS ONE Meta-analysis: Risk factors of COVID-19 Fig 1. PRISMA flow diagram. h //d i /10 1371/j l 0255154 001 Fig 1. PRISMA flow diagram. https://doi.org/10.1371/journal.pone.0255154.g001 https://doi.org/10.1371/journal.pone.0255154.g001 Study quality The findings on study quality can be found in Fig 2. When considering the case and severity definition of COVID-19, almost 50% of studies were considered low risk of bias, while only 9.1% had a high risk. In contrast, many studies were identified to have high concerns for bias in respect to patient selection and generalizability of findings (36.4% high risk, 9.1% low risk). In more than a third of studies, we had high concern that the full data on patients were not PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 5 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 PLOS ONE Meta-analysis: Risk factors of COVID-19 available and inappropriate exclusion might have occurred (35.2%). The full explanation of the Fig 2. Risk of bias assessment. https://doi.org/10.1371/journal.pone.0255154.g002 Fig 2. Risk of bias assessment. https://doi.org/10.1371/journal.pone.0255154.g002 Fig 2. Risk of bias assessment. https://doi.org/10.1371/journal.pone.0255154.g002 available and inappropriate exclusion might have occurred (35.2%). The full explanation of the risk of bias assessment and the assessment of each paper individually is available in the supple- ment S4 in S1 File. Overall, high- or intermediate risk of bias for at least one category was found in almost three fourth (73.8%) of studies. No study scored low risk in all three categories. available and inappropriate exclusion might have occurred (35.2%). The full explanation of the risk of bias assessment and the assessment of each paper individually is available in the supple- ment S4 in S1 File. Overall, high- or intermediate risk of bias for at least one category was found in almost three fourth (73.8%) of studies. No study scored low risk in all three categories. ICU admission ICU = Intensive care unit, OR = odds ratio, CI = confidence interval, COPD = chronic obstructive pulmonary disease, ART = anti-retroviral treatment, NIV = non-invasive ventilation. htt //d i /10 1371/j l 0255154 003 Fig 3. Pooled odds ratios among ICU vs. non ICU groups. ICU = Intensive care unit, OR = odds ratio, CI = confidence interval, COPD = chronic obstructive pulmonary disease, ART = anti-retroviral treatment, NIV = non-invasive ventilation. Fig 3. Pooled odds ratios among ICU vs. non ICU groups. ICU = Intensive care unit, OR = odds ratio, CI = confidence interval, COPD = chronic obstructive pulmonary disease, ART = anti-retroviral treatment, NIV = non-invasive ventilation. https://doi.org/10.1371/journal.pone.0255154.g003 https://doi.org/10.1371/journal.pone.0255154.g003 https://doi.org/10.1371/journal.pone.0255154.g003 patients admitted to an ICU, but the absolute elevation over those in non-ICU patients were small and of questionable clinical relevance (Table 1). Patients developing acute kidney failure, as a complication at any stage, had the highest risk for ICU admission (OR 15.69, CI 11.22 to 21.90). Patients developing acute kidney failure, as a complication at any stage, had the highest risk for ICU admission (OR 15.69, CI 11.22 to 21.90). ICU admission Fig 3 and Table 1 show the pooled odds ratios (OR) and differences of medians (DoM), respec- tively, for ICU admission for the different indicators in the five categories: demographic, symptoms, comorbidities, laboratory and clinical values [12, 13, 29, 36–57]. Patients requiring ICU admission had a median age of 65 years (CI 62.27 to 66.16). Those not requiring ICU admission were significantly younger with a median age of 59 years (CI 55.93 to 61.86) with a DoM of 4.63 years (CI 1.43 to 7.82) (Table 1). We were not able to per- form a subgroup analysis of different age groups as data provided by primary studies was insufficient. Of the many possible symptoms of COVID-19, we found dyspnea (OR 5.34, CI 2.77 to 10.28) and fatigue (OR 1.63, CI 1.20 to 2.22) to be significantly associated with ICU admission. In terms of co-morbidities, patients admitted to the ICU were more likely to suffer from cere- brovascular disease (OR 5.88, CI 2.35 to 14.73), hypertension (OR 1.62 CI 1.24 to 2.12), diabe- tes (OR 1.58, CI 1.29 to 1.93) and chronic kidney disease (OR 1.48, CI 1.08 to 2.03). In contrast, cardiovascular diseases (OR 1.50, CI 0.99 to 2.28), chronic obstructive pulmonary disease (COPD) (OR 1.39, CI 0.90 to 2.16), chronic lung disease (OR 1.06, CI 0.89 to 1.25) and smoking (OR 1.00, CI 0.77 to 1.29) were not associated with ICU admission. Few laboratory values showed differences between patients that required ICU admission and those who did not (Table 1). D-dimer failed to show a statistically significant difference (DoM 0.3 mg/L, CI -0.2 to 0.81). We found a clinically relevant elevation of CRP and cardiac Troponin I (cTnI) in patients requiring ICU admission, although cTnI failed to be statistically significant (DoM for CRP 56.41 mg/L, CI 39.8 to 73.02 and DoM for cTnI 19.27 pg/mL, CI -4.13 to 42.68). A clinically significant reduction in lymphocytes was also observed (DoM -0.34, CI -0.39 to -0.29). Leukocytes, neutrophiles and LDH were also significantly higher in PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 6 / 19 PLOS ONE Meta-analysis: Risk factors of COVID-19 Fig 3. Pooled odds ratios among ICU vs. non ICU groups. ICU = Intensive care unit, OR = odds ratio, CI = confidence interval, COPD = chronic obstructive pulmonary disease, ART = anti-retroviral treatment, NIV = non-invasive ventilation. https://doi.org/10.1371/journal.pone.0255154.g003 Fig 3. Pooled odds ratios among ICU vs. non ICU groups. Mortality Fig 4 and Table 2 show the pooled odds ratios and differences of medians, respectively, for mortality for symptoms, comorbidities, laboratory and clinical values [11, 16, 28, 58–110]. Patients who died had a median age of 71 years (CI 69.3 to 71.61) compared to survivors with a median age of 58 years (CI 55.03 to 59.4) for a DoM of 13.15 years (CI 11.37 to 14.94] (Table 2). Again, dyspnea was the symptom that differentiated markedly between survivors and non-survivors (OR 3.69, CI 2.54 to 5.36). Also, fatigue was more frequently observed in those who died (OR 1.48, CI 1.15 to 1.89). Regarding vital parameters at admission, patients who died presented with a median peripheral oxygen saturation (SpO2) on room air of 89% (CI 87.32 to 90.91) to the hospital, while those who survived had 95% (CI 94.59 to 96.63) (DoM -6.33%, CI -8.14 to -4.52). Patients who died were more likely to suffer from cardiovascular disease (OR 3.93, CI 2.91 to 5.30), cerebrovascular disease (OR 3.45, CI 2.42 to 4.91), chronic lung disease (OR 3.12, CI 2.17 to 4.49), COPD (OR 2.54, CI 1.87 to 3.44; Fig 4) and hypertension (OR 2.49, CI 2.11 to 2.94). Current and former smokers had an increased risk of mortality (OR 1.36, CI 1.10 to 1.67). Patients with chronic kidney disease (CKD) (OR 2.36, CI 1.89 to 2.94), diabetes (OR PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 7 / 19 PLOS ONE Meta-analysis: Risk factors of COVID-19 Table 1. Summary of the meta-analysis results for continuous indicators comparing those who were admitted to the ICU and those who were not. Indicator N. Mortality Studies Pooled DoM [95% CI] I2 Demographics Age (years) 22 4.63 [1.43, 7.82] 89.89 Clinical Values Respiratory Rate (per min) 5 3.15 [0.11, 6.19] 79.27 Laboratory Values Hemoglobin (g/L) 7 -5.97 [-11.78, -0.16] 56.12 Leukocyte (109/L) 15 1.2 [0.54, 1.85] 62.23 Lymphocyte (109/L) 19 -0.26 [-0.34, -0.17] 75.34 Neutrophil (109/L) 14 2.67 [1.43, 3.91] 89.14 Platelets (109/L) 17 -10.4 [-20.83, 0.04] 32.66 APTT (sec) 7 0.38 [-1.2, 1.95] 49.45 D-dimer (mg/L) 14 0.30 [-0.20, 0.81] 83.97 Prothrombin (sec) 7 0.48 [0.2, 0.76] 0.00 ALAT (U/L) 15 4.37 [2.11, 6.64] 16.17 Albumin (g/L) 5 -6.05 [-8.75, -3.35] 79.38 ASAT (U/L) 13 11.77 [7.24, 16.3] 64.91 LDH (U/L) 12 140.4 [81.04, 199.76] 86.32 BUN (mmol/L) 7 1.9 [1.34, 2.45] 0.00 Creatinine (μmol/L) 16 9.41 [5.18, 13.63] 40.23 CRP (mg/L) 10 56.41 [39.8, 73.02] 76.56 PCT (ng/mL) 6 0.08 [-0.01, 0.16] 88.76 CK (U/L) 9 33.57 [1.76, 65.38] 55.08 CK-MB (U/L) 4 2.47 [0.67, 4.26] 0.00 cTnI (pg/mL) 6 19.27 [-4.13, 42.68] 96.82 Indicates that the DL approach was used to estimate between-study heterogeneity. APTT = activated partial thrombin time; ALAT = Alanine transaminase; ASAT = Aspartate transaminase; LDH = Lactate dehydrogenase; BUN = Blood urea nitrogen; CRP = C-reactive protein; PCT = Procalcitonin CK = Creatine kinase; CK-MB = Creatine kinase–myocardial band; TnI = cardiac Troponin I. https://doi.org/10.1371/journal.pone.0255154.t001 Table 1. Summary of the meta-analysis results for continuous indicators comparing those who were admitted to the ICU and those who were not. https://doi.org/10.1371/journal.pone.0255154.t001 2.14, CI 1.82 to 2.52) and cancer (OR 2.08, CI 1.55 to 2.77) also had an increased odds of mor- tality. Co-morbidities not associated with increased odds of mortality were asthma, liver dis- ease, digestive system disease and immunosuppressive therapy (Fig 4). Clinically relevant elevations outside the normal laboratory range in patients who died compared to those who survived were observed in two markers of inflammation: CRP was elevated by 69.1mg/L (CI 50.43 to 87.77) and IL-6 by 31.19 pg/mL (CI 11.96 to 50.41). Furthermore, clinically significant elevations were observed in cTnI by 21.88pg/mL (CI 9.78 to 33.99) and D-dimer by 1.29mg/L (CI 0.9 to 1.69), while lymphocytes were significantly lower: -0.34x109/L (CI -0.39 to -0.29). Other makers (hemoglobin, leukocytes, neutrophils, platelets, international normalized ratio (INR), Prothrombin, alanine transaminase (ALAT), aspartate transaminase (ASAT), Albumin, LDH, blood urea nitrogen (BUN), Creatinine, PCT, BNP, CK and creatine kinase myocardial band (CK-MB)) were also significantly elevated in those who died. PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 Indicates that the DL approach was used to estimate between-study heterogeneity. APTT = activated partial thrombin time; ALAT = Alanine transaminase; ASAT = Aspartate transaminase; LDH = Lactate dehydrogenase; BUN = Blood urea nitrogen; CRP = C-reactive protein; PCT = Procalcitonin CK = Creatine kinase; CK-MB = Creatine kinase–myocardial band; TnI = cardiac Troponin I. https://doi.org/10.1371/journal.pone.0255154.t001 Mortality OR = odds ratio, CI = confidence interval, COPD = chronic obstructive pulmonary disease, ART = anti-retroviral treatment, ECMO = extracorporeal membrane oxygenation, NIV = non-invasive ventilation. Fig 4. Pooled odds ratios among mortality vs. survived groups. OR = odds ratio, CI = confidence interval, COPD = chronic obstructive pulmonary disease, ART = anti-retroviral treatment, ECMO = extracorporeal membrane oxygenation, NIV = non-invasive ventilation. https://doi.org/10.1371/journal.pone.0255154.g004 https://doi.org/10.1371/journal.pone.0255154.g004 As a clinical complication, acute kidney injury showed the highest overall odds ratio for mortality (OR 20.87, CI 9.21 to 47.32), followed by requiring non-invasive ventilation (NIV) (OR 7.38, CI 4.25 to 12.82). Fig 5 shows pooled median estimates along with their normal laboratory ranges for selected number of indicators among patients who died, patients who survived, ICU-admitted patients, and non-ICU admitted patients. Pooled difference of medians estimates for all indicators are available in the supplementary files (S5 for mortality in S1 File, S6 for ICU admission in S1 File, S7 for intubation and hospitalization in S8 in S1 File). After removing large outliers in a sensitivity analyses for CRP and D-dimer, results did not change substantially (results available in the supplementary S9 in S1 File). Funnel plots showed no substantial asymmetry suggesting small-study effects except for data assessing acute kidney injury (supplementary S10 for mor- tality and S11 for ICU admission in S1 File). Mortality However, the absolute dif- ference compared to those who survived was small and thus likely not clinically relevant. For leukocytes, neutrophils, platelets, prothrombin, ALAT, ASAT, BUN, Creatinine, CK and CK-MB the point estimates even stayed within the normal laboratory range. 2.14, CI 1.82 to 2.52) and cancer (OR 2.08, CI 1.55 to 2.77) also had an increased odds of mor- tality. Co-morbidities not associated with increased odds of mortality were asthma, liver dis- ease, digestive system disease and immunosuppressive therapy (Fig 4). Clinically relevant elevations outside the normal laboratory range in patients who died compared to those who survived were observed in two markers of inflammation: CRP was elevated by 69.1mg/L (CI 50.43 to 87.77) and IL-6 by 31.19 pg/mL (CI 11.96 to 50.41). Furthermore, clinically significant elevations were observed in cTnI by 21.88pg/mL (CI 9.78 to 33.99) and D-dimer by 1.29mg/L (CI 0.9 to 1.69), while lymphocytes were significantly lower: -0.34x109/L (CI -0.39 to -0.29). Other makers (hemoglobin, leukocytes, neutrophils, platelets, international normalized ratio (INR), Prothrombin, alanine transaminase (ALAT), aspartate transaminase (ASAT), Albumin, LDH, blood urea nitrogen (BUN), Creatinine, PCT, BNP, CK and creatine kinase myocardial band (CK-MB)) were also significantly elevated in those who died. However, the absolute dif- ference compared to those who survived was small and thus likely not clinically relevant. For leukocytes, neutrophils, platelets, prothrombin, ALAT, ASAT, BUN, Creatinine, CK and CK-MB the point estimates even stayed within the normal laboratory range. 8 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 PLOS ONE Meta-analysis: Risk factors of COVID-19 Fig 4. Pooled odds ratios among mortality vs. survived groups. OR = odds ratio, CI = confidence interval, COPD = chronic obstructive pulmonary disease, ART = anti-retroviral treatment, ECMO = extracorporeal membrane oxygenation, NIV = non-invasive ventilation. 4 Pooled odds ratios among mortality vs survived groups OR odds ratio CI confidence interval COPD chronic obstructive pulmonary disease Fig 4. Pooled odds ratios among mortality vs. survived groups. OR = odds ratio, CI = confidence interval, COPD = chronic obstructive pulmonary disease, ART = anti-retroviral treatment, ECMO = extracorporeal membrane oxygenation, NIV = non-invasive ventilation. Fig 4. Pooled odds ratios among mortality vs. survived groups. OR = odds ratio, CI = confidence interval, COPD = chronic obstructive pulmonary disease, ART = anti-retroviral treatment, ECMO = extracorporeal membrane oxygenation, NIV = non-invasive ventilation. https://doi.org/10.1371/journal.pone.0255154.g004 Fig 4. Pooled odds ratios among mortality vs. survived groups. https://doi.org/10.1371/journal.pone.0255154.g004 Discussion In this comprehensive systematic review and meta-analysis, we corroborate known markers of severe disease for COVID-19 and shed light on further indicators, whose significance was indeterminate to date. With respect to co-morbidities, we identified cardiovascular disease, which includes chronic heart disease and coronary artery disease (OR 3.93), chronic lung disease (OR 3.12) and COPD (OR 2.54) as strong risk factors of mortality among COVID-19 patients but not for ICU admission. Only cerebrovascular disease was strongly associated with an increased risk of both ICU admission and death (almost six- and three-fold higher ratio for ICU admission and PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 9 / 19 PLOS ONE Meta-analysis: Risk factors of COVID-19 Table 2. Summary of the meta-analysis results for continuous indicators comparing those who died and those who survived. Indicator N. Studies Pooled DoM [95% CI] I2 Demographics Age (years) 52 13.15 [11.37, 14.94] 86.74 Clinical Values SpO2—without O2 (%) 15 -6.33 [-8.14, -4.52] 81.77 Respiratory Rate (per min) 15 3.41 [2.26, 4.55] 62.32 Laboratory Values Hemoglobin (g/L) 18 -2.66 [-5.12, -0.2] 43.36 Leukocyte (109/L) 37 2.79 [2.23, 3.35] 70.35 Lymphocyte (109/L) 38 -0.34 [-0.39, -0.29] 70.03 Neutrophil (109/L) 25 3.26 [2.56, 3.95] 82.2 Platelets (109/L) 30 -31.94 [-41.11, -22.77] 58.13 APTT (sec) 16 0.59 [-0.51, 1.69] 61.88 D-Dimer (mg/L) 30 1.29 [0.90, 1.69] 81.53 Fibrinogen (g/L) 7 0.01 [-0.12, 0.15] 0.00 INR 7 0.06 [0.01, 0.12] 63.31 Prothrombin (sec) 25 0.91 [0.67, 1.14] 54.65 ALAT (U/L) 34 4.43 [2.41, 6.46] 26.64 Albumin (g/L) 21 -4.64 [-5.83, -3.45] 85.16 ASAT (U/L) 27 13.35 [10.54, 16.15] 42.83 LDH (U/L) 23 189.49 [155, 223.98] 75.03 BUN (mmol/L) 17 2.77 [2.07, 3.46] 66.77 Creatinine (μmol/L) 29 15.3 [10.3, 20.29] 61.63 CRP (mg/L) 34 69.1 [50.43, 87.77] 95.99 IL-6 (pg/mL) 11 31.19 [11.96, 50.41] 99.75 PCT (ng/mL) 18 0.16 [0.1, 0.22] 68.09 BNP (pg/mL) 7 405.26 [116.51, 694.02] 95.81 CK (U/L) 18 64.09 [29.04, 99.13] 81.47 CK-MB (U/L) 9 3.66 [1.19, 6.14] 67.12 cTnI (pg/mL) 13 21.88 [9.78, 33.99] 75.17 Table 2. Summary of the meta-analysis results for continuous indicators comparing those who died and those who survived. https://doi.org/10.1371/journal.pone.0255154.t002 death, respectively). Overall, the finding that cerebrovascular disease is associated with poor outcomes is in line with the more recent data highlighting the importance of delirium and an overall depressed mental state in severe COVID-19 [111–113]. Indicates that the DL approach was used to estimate between-study heterogeneity. SpO2 = Oxygen saturation; APTT = activated partial thrombin time; INR = Internationalized normalized ratio; ALAT = Alanine transaminase; ASAT = Aspartate transaminase; LDH = Lactate dehydrogenase; BUN = Blood urea nitrogen; CRP = C-reactive protein; IL-6 = Interleukin-6; BNP = brain natriuretic peptide; PCT = Procalcitonin CK = creatine kinase; CK-MB = creatine kinase–myocardial band; TnI = cardiac Troponin I. https://doi.org/10.1371/journal.pone.0255154.t002 Discussion Our findings found chronic kidney disease, diabetes [18] and COPD/chronic lung disease to be risk factors, however, asso- ciations are less strong than those for cardiovascular or cerebrovascular disease [111]. Evidence from previous studies regarding the risk associated with hypertension has been inconclusive. Our work identifies hypertension as a clear risk factor for ICU admission (OR 1.62, CI 1.24 to 2.12) and death (OR 2.49, CI 2.11 to 2.94) [7, 19, 20]. Similarly, while prior data were inconclu- sive with respect to the influence of smoking for severe COVID-19 [3, 22–25], our meta-analy- sis shows the increased risk of mortality among smokers (OR 1.36, CI 1.10 to 1.67). However, PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 10 / 19 PLOS ONE our data did not allow for meta-regression to assess whether this effect was independent of the k d h h l d l h d h ld Fig 5. Pooled median estimates of selected indicators along with their normal laboratory ranges among patients who died, patients who survived, ICU-admitted patients, and non-ICU admitted patients. ICU = Intensive care unit, CRP = C-reactive protein, LDH = Lactate dehydrogenase. https://doi.org/10.1371/journal.pone.0255154.g005 ONE Meta-analysis: Risk factors of COVID-19 Meta-analysis: Risk factors of COVID-19 Fig 5. Pooled median estimates of selected indicators along with their normal laboratory ranges among patients who died, patients who survived, ICU-admitted patients, and non-ICU admitted patients. ICU = Intensive care unit, CRP = C-reactive protein, LDH = Lactate dehydrogenase. https://doi.org/10.1371/journal.pone.0255154.g005 https://doi.org/10.1371/journal.pone.0255154.g005 our data did not allow for meta-regression to assess whether this effect was independent of the risk associated with chronic lung disease. In line with some recent studies on asthma, we could not find an increased risk for mortality [114] in our meta-analysis (OR 0.88, CI 0.58 to 1.35). CRP was the only laboratory marker that was associated with a higher risk of ICU admis- sion (DoM 56.41 mg/L) and death (DoM 69.1 mg/L), while D-dimer elevation was only PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 11 / 19 PLOS ONE Meta-analysis: Risk factors of COVID-19 significantly associated with death (DoM 1.29 mg/L), but not with ICU admission. Although the median elevation of cTnI was clinically relevant both in those who were admitted to the ICU (DoM 19.27 pg/mL) and those who died (DoM 21.88 pg/mL), only in those who died was the difference statistically significant. We were able to identify clinically relevant lymphopenia as a marker. Discussion Lymphopenia was a marker that was used early on for triage purposes to predict disease severity [115] and our findings on increased odds for mortality corroborate the systematic review results on this topic published by Huang and Pranata [116]. For categorical variables, OR was used to measure the association between the outcome (mortality, ICU admission, hospitalization, intubation) and the risk factor/biomarker of inter- est. We decided to use the OR instead of the relative risk (RR) given the nature of the research question and the data that were available to us. For example, calculating the risk of ICU among people with fever compared to the risk of ICU among people without a fever is not as informa- tive as computing the odds ratio of ICU admission between fever and non-fever patients. We acknowledge, however, when interpreting the results that the ORs are more extreme (further from the null) than the RRs whenever there is a non-null association. In line with previous reviews, we acknowledge risk factors such as age, dyspnea, smoking, diabetes, hypertension and cardiovascular disease to be associated with increased odds for mortality. On the one hand we could not find an increased odds for asthma, whereas ‘respiratory dis- eases’ [3] had an increased OR in other reviews. On the other hand, we find COPD to be a strong risk factor for mortality. This can be explained by different data extractions and pooling of similar diseases. Nevertheless, this suggests that lung diseases are associated with an increased risk of a severe course. PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 Strengths and limitations of this study Our study provides a comprehensive review of the data from both pre-print and peer-reviewed sources with a broad geographic distribution and assesses the different categories of risk fac- tors from symptoms, co-morbidities and laboratory values to clinical complications. Correlat- ing the indicators to the two clinical outcomes death and ICU admission, has both strengths and limitations. While ICU admission is a clinical decision, it is, especially early on in a new disease, sometimes a measure of precaution. This might weaken the association of indicators with clinical outcomes. At the same time, when capacity of ICU beds is exhausted, triage deci- sions might have been made based on age and co-morbidities to not admit to the ICU, thus strengthening an association of an indicator beyond what would be expected under routine conditions. We also assessed the association with hospitalization and intubation (see supple- ment), but here confounding factors seemed to be even more pronounced, and data are further limited. Confounding factors that lead to this conclusion are for example different health care systems across the globe with or without the possibility of self-care in less severe cases instead of hospital admission or the change in the approach regarding early intubation from the first wave towards a more conservative approach with novel methods such as ‘awake proning’. In addition, with improving care and novel therapies certain associations might be less pro- nounced. We did not observe improved survival with antiviral therapy in the studies included, suggesting that this effect might not yet have occurred in the time frame of studies included here. We have not assessed radiological findings as these would likely correlate with clinical signs and symptoms, as well as changes in laboratory parameters. In contrast to other system- atic reviews, we did not focus on the course of the disease (e.g. critical, severe), but rather on the outcome [3]. Furthermore risk factors were assessed for each outcome individually [17]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 12 / 19 PLOS ONE Meta-analysis: Risk factors of COVID-19 Additional limitations primarily relate to data quality of the included studies. Our quality assessment of studies clearly indicated that substantial bias was present across studies. Primar- ily the selection bias as suggested for example by the high case fatality rate (e.g. Zhou et al. [11], 28.3%, Chen et al. [117] 11.1%, and Huang et al. S1 PRISMA checklist. S1 File. Supplementary file with supporting information. This contains supporting informa- tion for all outcomes with summary forest plots for ‘intubation’ and ‘hospitalization’, risk of bias assessment and funnel plots. (DOCX) Acknowledgments We thank Genevieve Gore for her help with the search terms. We thank Sinan Onogur, Laura Aguilera Saiz, and Bu¨nyamin Pekdemir for their help setting up the data extraction tool. Strengths and limitations of this study [12] 14.6%) is likely to have impacted our results and prospective data collection to confirm findings of these studies is important [118]. However, an analysis of quality was performed at the study level. Conceivably a high- quality study will contribute more high quality data on the risk-factor outcome association, but this cannot be ascertained. In addition, 13 studies were still in preprint at the time of extraction. Furthermore, we found a large number of studies (n = 21, list available in supple- mentary S3 in S1 File) to include laboratory values that were clinically out of range, which sug- gests that despite peer-review in some of them, the rush of publication in this pandemic impacted the quality of reporting [119]. Conclusion Our data on mortality and ICU admission corroborate most of the proposed indicators of clin- ical outcomes, clarifies the strength of association and highlights additional indicators. In addi- tion, this systematic review highlights the limitations of the studies published and calls for better quality in prospective collections. Author Contributions Conceptualization: Stephan Katzenschlager, Claudius Gottschalk, Ju¨rgen Grafeneder, Alexan- der Seitel, Andrea Benedetti, Sean McGrath, Claudia M. Denkinger. Conceptualization: Stephan Katzenschlager, Claudius Gottschalk, Ju¨rgen Grafeneder, Alexan- der Seitel, Andrea Benedetti, Sean McGrath, Claudia M. Denkinger. Data curation: Stephan Katzenschlager, Claudius Gottschalk, Ju¨rgen Grafeneder, Stephani Schmitz, Sara Kraker, Marlene Ganslmeier, Amelie Muth, Alexander Seitel, Lena Maier- Hein, Sean McGrath, Claudia M. Denkinger. Formal analysis: Stephan Katzenschlager, Alexandra J. Zimmer, Stephani Schmitz, Sara Kra- ker, Marlene Ganslmeier, Amelie Muth, Sean McGrath, Claudia M. Denkinger. Investigation: Claudia M. Denkinger. Methodology: Stephan Katzenschlager, Alexandra J. Zimmer, Claudius Gottschalk, Ju¨rgen Grafeneder, Alexander Seitel, Lena Maier-Hein, Andrea Benedetti, Sean McGrath, Claudia M. Denkinger. Project administration: Stephan Katzenschlager, Claudia M. Denkinger. 13 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0255154 July 29, 2021 PLOS ONE Meta-analysis: Risk factors of COVID-19 oftware: Alexandra J. Zimmer, Lena Maier-Hein, Sean McGrath, Claudia M. Denkinger. Software: Alexandra J. Zimmer, Lena Maier-Hein, Sean McGrath, Claudia M. Denkinger. Supervision: Stephan Katzenschlager, Claudia M. Denkinger. Validation: Stephan Katzenschlager, Alexandra J. Zimmer, Claudia M. Denkinger. Visualization: Stephan Katzenschlager, Alexandra J. Zimmer, Claudia M. Denkinger. Writing – original draft: Stephan Katzenschlager, Alexandra J. Zimmer, Sean McGrath, Clau- dia M. Denkinger. Writing – review & editing: Stephan Katzenschlager, Alexandra J. 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https://openalex.org/W2170450331	https://bmcevolbiol.biomedcentral.com/track/pdf/10.1186/s12862-015-0462-6	English	null	Host-parasite coevolution in populations of constant and variable size	BMC evolutionary biology	2,015	cc-by	12,689	© 2015 Song et al. 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. Song et al. BMC Evolutionary Biology (2015) 15:212 DOI 10.1186/s12862-015-0462-6 Song et al. BMC Evolutionary Biology (2015) 15:212 DOI 10.1186/s12862-015-0462-6 Open Access Abstract Background: The matching-allele and gene-for-gene models are widely used in mathematical approaches that study the dynamics of host-parasite interactions. Agrawal and Lively (Evolutionary Ecology Research 4:79–90, 2002) captured these two models in a single framework and numerically explored the associated time discrete dynamics of allele frequencies. Results: Here, we present a detailed analytical investigation of this unifying framework in continuous time and provide a generalization. We extend the model to take into account changing population sizes, which result from the antagonistic nature of the interaction and follow the Lotka-Volterra equations. Under this extension, the population dynamics become most complex as the model moves away from pure matching-allele and becomes more gene-for-gene-like. While the population densities oscillate with a single oscillation frequency in the pure matching-allele model, a second oscillation frequency arises under gene-for-gene-like conditions. These observations hold for general interaction parameters and allow to infer generic patterns of the dynamics. Conclusion: Our results suggest that experimentally inferred dynamical patterns of host-parasite coevolution should typically be much more complex than the popular illustrations of Red Queen dynamics. A single parasite that infects more than one host can substantially alter the cyclic dynamics. Keywords: Matching-allele, Gene-for-gene, Lotka-Volterra equation, Replicator dynamics. Red Queen hypothesis, Stability analysis Keywords: Matching-allele, Gene-for-gene, Lotka-Volterra equation, Replicator dynamics. Red Queen hypothesis, Stability analysis Host-parasite coevolution in populations of constant and variable size Yixian Song1, Chaitanya S Gokhale2, Andrei Papkou3, Hinrich Schulenburg3 and Arne Traulsen1* Correspondence: traulsen@evolbio.mpg.de 1Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306, Plön, Germany Full list of author information is available at the end of the article Background incorporated by taking into account the current under- standing of resistance-infectivity patterns in biological systems. The gene-for-gene (GfG) model was proposed by Flor [7] to capture disease resistance patterns in plants. Here, a host individual carrying a resistance gene can rec- ognize parasites harboring the corresponding avirulence product and trigger a defense response averting the infec- tion [8]. Inspired by self-nonself recognition in immune systems [9], the matching-allele (MA) model was intro- duced to reflect host-pathogen interactions in animals. In this case, parasites carrying a certain allele can only invade host individuals with the corresponding allele. By com- bining predictive power of mathematical modeling and their connection to empirical data, these models success- fully served to understand key evolutionary problems. To mention only the most important examples, these mod- els were used to assess the Red Queen hypothesis for the evolution of sexual reproduction [10], the maintenance of genetic diversity by parasite-mediated selection [11], and The antagonistic interaction between hosts and their parasites are of particular interest in ecology and evo- lution, because they are ubiquitous and usually associ- ated with high selection pressure that affects numerous life history traits. Because of the negative effect of par- asites on host fitness, the study of these interactions is of central importance in biomedical [1, 2], agricul- tural [3, 4] and species conservation research [5, 6]. The exact dynamics of the two coevolving populations are usually evaluated with the help of mathematical mod- els. Among these, models including an explicit genetic description of host-parasite interaction, such as gene- for-gene (GfG) and matching-allele (MA) models, are particularly widespread. Genetic interaction is usually Correspondence: traulsen@evolbio.mpg.de 1Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306, Plön, Germany Full list of author information is available at the end of the article Song et al. BMC Evolutionary Biology (2015) 15:212 Page 2 of 15 the role of the cost of resistance/virulence in coevolution [12, 13]. constant as well as changing population size, as there are biological examples for both of them. Agrawal and Lively [14] developed a general model that interpolates between a pure matching-allele model and a pure gene-for-gene model, as a single parameter is tuned between 0 and 1. This model was introduced for haplotypes of two loci with mutation and recombina- tion. Variance in host and parasite allele frequency was plotted as an evaluation of the time discrete dynamics. Model and Results We consider haploid hosts and parasites with two alleles on a single locus. Hence, there are two host types and two parasite types that are denoted by H1, H2, P1, and P2, respectively. In the simplest case, each parasite type can only infect the corresponding host type. Hence, no host/parasite type is superior to the other. This case cor- responds to the matching-allele model, which under the assumption of constant population density is equivalent to the evolutionary game of matching pennies [21, 22]. In a GfG model, the virulent parasite P2 can potentially infect both hosts, the one with susceptible allele H1 and the one with resistance allele H2. Yet, the avirulent para- site P1 can only infect the susceptible host H1, as the host H2 with the resistance allele can prevent infection by P1. Thus, there is an advantage to the virulent parasite and the resistant host. To maintain the different types in the population, intrinsic costs of virulence and resistance have been suggested [23]. In this paper, we take the existing and currently widely applied modeling approach [14] and develop a new mathe- matical framework that allows for an analytical characteri- zation of the involved dynamics. Based on this framework, the aim of our study is to improve our understanding of host-parasite coevolution by investigating both the impact of different types of interactions and the consequence of interaction-dependent population size changes. We sim- plify the model of Agrawal and Lively and focus on a single locus to keep interaction among loci from inter- fering with the conclusion, in particular the differences between the GfG model [16] and the MA model [17]. We use the assumptions of Agrawal and Lively [14] inspired by Parker [13] to connect the two popular models by a single parameter, but also provide an alternative, linear inter- polation in the discussion. To enhance clarity, we focus on a system with two host genotypes and two parasite genotypes and use their interaction to characterize the evolutionary dynamics of the two populations involved in this reaction. In addition, we depart from the usual assumption of constant population size and apply the Lotka-Volterra equations to acknowledge inter-dependent population dynamics during host-parasite coevolution. To compare the dynamics with a model assuming constant population density, we apply the Replicator Dynamics [18–21] with the same interaction matrix between hosts and parasites. Background The highly dynamical aspects of matching-allele models were observed across most of the MA-GfG continuum. Agrawal and Lively showed that cyclic dynamics of host and parasite genotypes is observed not only in the MA model, but also in all the intermediate models and in the GfG model. This finding indicates that the cyclic Red Queen dynamics does not hinge upon the use of a par- ticular model for host parasite interactions. However, this study was computational and only performed for partic- ular parameter sets due to the complexity of the model. Instead of tackling the dynamics from an analytical per- spective to allow for general statements for all parameter sets, subsequent theoretical approaches have increased complexity of the assumed interaction in order to increase the biological realism, for instance by defining a multi- locus model that deals with various combinations of MA loci and GfG loci [15]. We conducted a linear stability analysis at the interior fixed point of the resulting nonlinear dynamical system, which indicates critical differences in dynamical patterns between the models of host-parasite coevolution. Either with constant or with changing population size, the popu- lation densities oscillate with a single frequency in a pure MA model. In a model deviating from MA, a second oscil- lation frequency arises with changing population density, but not with constant population size. Model and Results While the dynamics between the two mod- els is different, it seems to be crucial to understand both Figure 1 illustrates the fitness of the two parasites on each host for the MA and the GfG model and also for two intermediate cases, where the parasite P2 can “partially” infect the host H1. We simplified the model of Agrawal and Lively [14] by regarding only one locus. The interactions between hosts and parasites can be expressed with two matrices (corresponding to a bi-matrix game in evolutionary game theory). We are interested in the role of population size changes, determined by the birth and death rates of host and parasite. We assume that the interaction between hosts and parasites affects the death rates of hosts and the birth rates of parasites. The birth rate of hosts and the death rates of parasites are chosen either as constant parameters (such that the population size changes) or in a way that ensures that the total population sizes of hosts and parasites remain constant, see below. For the parasite, we assume that the interactions with the hosts increase birth rates according to the matrix Mp = H1 H2 P1 σ 0 P2 α(1 −ακ)σ (1 −ακ)σ . (1) Mp = H1 H2 P1 σ 0 P2 α(1 −ακ)σ (1 −ακ)σ . (1) (1) Song et al. BMC Evolutionary Biology (2015) 15:212 Page 3 of 15 Fig. 1 Illustration of parasite fitness. Fitness of avirulent parasite P1 and virulent parasite P2 on the two hosts H1 and H2 for the matching-allele model (α = 0, top), the gene-for-gene model (α = 1, bottom), and two intermediate models (α = 1/3 and α = 2/3). Gray areas represent the fitness reduction for P2 due to the cost of virulence κ = 1/2, which is ακσ in H2 (Eq. (1b)), hence, σ/2 in GfG model. In H1 the fitness reduction for P2 due to the cost of virulence is α2κσ Fig. 1 Illustration of parasite fitness. Fitness of avirulent parasite P1 and virulent parasite P2 on the two hosts H1 and H2 for the matching-allele model (α = 0, top), the gene-for-gene model (α = 1, bottom), and two intermediate models (α = 1/3 and α = 2/3). Gray areas represent the fitness reduction for P2 due to the cost of virulence κ = 1/2, which is ακσ in H2 (Eq. Model and Results (1b)), hence, σ/2 in GfG model. In H1 the fitness reduction for P2 due to the cost of virulence is α2κσ For example, the birth rate of parasite P1 in interac- tions with H1 is σ, while it is 0 when P1 interacts with H2 (see Appendix A.3 for a generalization of these assump- tions). The maximum virulence of the parasite is given by σ. The parameter κ describes the cost for the para- site virulence, as usually assumed in the GfG model. This model interpolates between the MA and the GfG model as the parameter α is varied between 0 (MA) and 1 (GfG). From the interaction matrix Eq. (1), the birth rates for the parasites are given by We assume a large population size and focus on the change in population densities. The population densities of the two host and two parasite types are given by h1, h2, p1, and p2, respectively. The population dynamics of the hosts and parasites are driven by their respective birth and death rates and can be captured by a set of differential equations ˙h1 = h1(bh + dH1) (5a) ˙h2 = h2(bh + dH2) ˙p1 = p1(bP1 −dp) (5b) ˙p2 = p2(bP2 −dp) , (5a) bP1 =Mp 11h1 + Mp 12h2 = σ h1 (2) bP2 =Mp 21h1 + Mp 22h2 = α(1 −ακ)σ h1 + (1 −ακ)σ h2 (2) For the host, we assume that the interactions increase the death rate according to the matrix where bh is the birth rate of both hosts, and dp is the death rate of both parasites. We will choose the host birth rate bh and parasite death rate dp in two dis- tinct ways. Our first approach assumes constant values for bh and dp, which leads to a host/parasite population that is changing in size. This corresponds to the com- petitive Lotka-Volterra dynamics. The second approach focuses on relative abundances of host and parasite alleles and implies a normalization of the two popula- tion sizes. This corresponds to the Replicator Dynamics for an evolutionary role game between hosts and para- sites, which implies two constant population sizes in our context. where bh is the birth rate of both hosts, and dp is the death rate of both parasites. We will choose the host birth rate bh and parasite death rate dp in two dis- tinct ways. Stability of boundary fixed points To obtain a model of constant population size that is comparable to the one described above, we retain the interaction matrices and adjust the host birth rate and par- asite death rate to maintain the population size. Requiring constant h1 + h2 and constant p1 + p2 implies ˙h1 + ˙h2 = 0 as well as ˙p1 + ˙p2 = 0. This leads to The fixed points of the system are the points where all population sizes remain constant in time, ˙h1 = ˙h2 = ˙p1 = ˙p2 = 0. The position of the fixed points and their stability change with changing parameters. For the Lotka-Volterra dynamics, a trivial fixed point is (h1, h2, p1, p2) = (0, 0, 0, 0) where both the hosts and parasites are absent, cf. Eq. (6). Additionally, extinction of one host and the associated parasite leads to two further fixed points, (h1, h2, p1, p2) = ( dp σ , 0, bh σ , 0) and (h1, h2, p1, p2) = (0, dp σ(1−ακ), 0, bh αγ (1−σ)+σ(1−ακ(1−αγ ))). In gene-for-gene-like models, α > 0, the susceptible host H1 and the virulent P2 can coexist in the absence of H2 and P1, (h1, h2, p1, p2) = ( dp ασ(1−ακ), 0, 0, bh ασ(1−ακ)). The oppo- site case, coexistence between H2 and P1 in the absence of H1 and P2 is not possible, as our host-parasite inter- action model assumes that the birth rate of P1 is zero in the absence of H1. A linear stability analysis of the Lotka- Volterra model shows that all boundary fixed points are unstable for αγ < σ. That is, if the cost of resistance αγ (which is scaled by the amount of GfG influence) is less than the maximum host fitness reduction caused by infection σ, then all host and parasite types will coexist. bh = −h1dH1 + h2dH2 h1 + h2 (7a) dp = p1bP1 + p2bP2 p1 + p2 . (7b) (7a) (7b) The normalization h1 + h2 = 1 implies that a single equation for h1 is sufficient to describe the dynamics for the host. Similarly, due to the normalization p1 + p2 = 1 the parasite dynamics are fully captured by tracking p1. Inserting the dynamical host birth and parasite death rates in the dynamical system Eq. Stability of boundary fixed points (2), the equations become identical to the Replicator Dynamics (RD) [19, 21, 24], ˙h1 = h1(1 −h1)(dH1 −dH2) (8a) ˙p1 = p1(1 −p1)(bP1 −bP2) . (8b) (8a) (8b) (8a) (8b) The Replicator Dynamic system, Eq. (8), has four fixed points at the boundaries, each is reflecting fixation of one host and one parasite: (h1, p1) = (0, 0), (h1, p1) = (1, 0), (h1, p1) = (0, 1), (h1, p1) = (1, 1). A linear stability analysis reveals that all these fixed points are unstable. While the death rates of the host still depend on the para- sites and the birth rates of the parasites still depend on the hosts, the dynamics of this system is in general less com- plex than in the case of changing population size, as it is only two-dimensional. Changing population size induced by interactions use a linear stability analysis of the unique interior fixed point to infer the dynamical patterns arising in this system [16, 25]. Finally, we also assess constants of motion. With constant host birth rate bh and parasite death rate dp, inserting the host parasite interactions Eqs. (2) and (6) into the dynamical system Eq. (5) leads to Numerical solution of the dynamics ˙h1 = h1 (bh −p1σ −p2α(1 −ακ)σ) (6a) ˙h2 = h2 (bh −p1αγ −p2 ((1 −αγ )(1 −ακ)σ + αγ )) ˙p1 = p1(σh1 −dp) (6b) ˙p2 = p2 σ (h1α(1 −ακ) + h2(1 −ακ)) −dp . (6a) To illustrate the differences in the population dynamics described in Eqs. (6) and (8), we show numerical solutions side by side in Fig. 2. The dynamics in models with constant host and para- site population sizes resemble the common Red Queen pattern. Under changing population sizes the system is uncoupled into two independent host-parasite pairs in a pure MA model. As the model deviates from the MA model with increasing α, the dynamics becomes more complex, since the four population densities of the types P1, P2, H1, and H2 are coupled. This model, which we analyze in detail below, results in changes in the population sizes of both hosts and para- sites. In particular, the changes are caused by the antago- nistic interactions between the hosts and the parasite - as a consequence of the Lotka-Volterra relationship. Model and Results Our first approach assumes constant values for bh and dp, which leads to a host/parasite population that is changing in size. This corresponds to the com- petitive Lotka-Volterra dynamics. The second approach focuses on relative abundances of host and parasite alleles and implies a normalization of the two popula- tion sizes. This corresponds to the Replicator Dynamics for an evolutionary role game between hosts and para- sites, which implies two constant population sizes in our context. Mh = P1 P2 H1 −σ −α(1 −ακ)σ H2 −αγ (1 −αγ )(1 −(1 −ακ)σ) −1 , (3) (3) where the parameter γ describes the cost for the host resistance. This leads to the host death rates dH1 = Mh 11p1 + Mh 12p2 = −σ p1 −α(1 −ακ)σ p2 dH2 = Mh 21p1 + Mh 22p2 (4) = −αγ p1 + ((1 −αγ )(1 −(1 −ακ)σ) −1) p2 dH1 = Mh 11p1 + Mh 12p2 = −σ p1 −α(1 −ακ)σ p2 dH2 = Mh 21p1 + Mh 22p2 (4) (4) = −αγ p1 + ((1 −αγ )(1 −(1 −ακ)σ) −1) p2 Song et al. BMC Evolutionary Biology (2015) 15:212 Page 4 of 15 Population dynamics In addition to the boundary fixed points, the system has a unique fixed point in the interior. In the Lotka-Volterra system, we obtain a non-trivial fixed point of the four dimensional dynamical system described in Eq. (6) when αγ < σ. This fixed point, where all types coexist, is given by To obtain first information about the population dynam- ics, we calculated the trajectories of the system numer- ically for a particular set of parameters. In addition, we identify the fixed points of the differential equations and study their stability to gain insight into the coevolutionary dynamics for all parameter sets. More specifically, we can Song et al. BMC Evolutionary Biology (2015) 15:212 Page 5 of 15 Fig. 2 Example of population dynamics based on the Lotka-Volterra equations (left) and the Replicator Dynamics (right). While the dynamics on the right side resembles the common Red Queen pattern, the left side is more complex. In a pure matching-allele model (top), the plot on the left shows two independent sets of Lotka-Volterra dynamics, one for H1 and P1 (blue and red solid lines, correspondingly) and a second one for H2 and P2 (blue and red dotted lines). As the model deviates from MA model with increasing α (rows 2–4) more complicated dynamics arise, since the four population densities of H1, H2, P1, and P2 are coupled (parameters γ = 0.005, κ = 0.5, and σ = 0.01 for both Lotka-Volterra and Replicator Dynamics. Host birth rate bh = 1.5 and parasite death rate dp = 1.0 in the Lotka-Volterra case. Initial population densities h1 = p1 = 150, h2 = p2 = 50) Fig. 2 Example of population dynamics based on the Lotka-Volterra equations (left) and the Replicator Dynamics (right). While the dynamics on the right side resembles the common Red Queen pattern, the left side is more complex. In a pure matching-allele model (top), the plot on the left shows two independent sets of Lotka-Volterra dynamics, one for H1 and P1 (blue and red solid lines, correspondingly) and a second one for H2 and P2 (blue and red dotted lines). As the model deviates from MA model with increasing α (rows 2–4) more complicated dynamics arise, since the four population densities of H1, H2, P1, and P2 are coupled (parameters γ = 0.005, κ = 0.5, and σ = 0.01 for both Lotka-Volterra and Replicator Dynamics. Population dynamics BMC Evolutionary Biology (2015) 15:212 Page 6 of 15 h1 −p1 plane (dark green solid lines both for LV and RD equations) and the he initial conditions. The black rectangle represent a special set of initial ectories. With Replicator Dynamics the h1 −p1 trajectory is a closed circle. nitial conditions fulfill Eq. (25) (black lines). For the closed circles (black in % above the corresponding fixed point, while the parasite population n trajectories with LV resemble tori instead of closed circles, an implication t as α increases from 0 to 1, the trajectories are plotted all in the same Fig. 3 Trajectories close to the interior fixed points (black points) on the h1 −p1 plane (dark green solid lines both for LV and RD equations) and the h2 −p2 plane (light green dashed lines LV only). The black crosses mark the initial conditions. The black rectangle represent a special set of initial condition while the black solid/dashed lines show the corresponding trajectories. With Replicator Dynamics the h1 −p1 trajectory is a closed circle. With Lotka-Volterra dynamics, the trajectories are closed circle when the initial conditions fulfill Eq. (25) (black lines). For the closed circles (black in LV and green in RD) the initial host population densities, h1 and h2 are 5 % above the corresponding fixed point, while the parasite population densities are 5 % beneath the fixed point. Except for α = 0 (MA) the green trajectories with LV resemble tori instead of closed circles, an implication for two oscillation frequencies. To show the shift of the interior fixed point as α increases from 0 to 1, the trajectories are plotted all in the same coordinate system at the bottom h1 −p1 plane (dark green solid lines both for LV and RD equations) and the he initial conditions. The black rectangle represent a special set of initial ectories. With Replicator Dynamics the h1 −p1 trajectory is a closed circle. initial conditions fulfill Eq. (25) (black lines). For the closed circles (black in % above the corresponding fixed point, while the parasite population n trajectories with LV resemble tori instead of closed circles, an implication t as α increases from 0 to 1, the trajectories are plotted all in the same Fig. Population dynamics Host birth rate bh = 1.5 and parasite death rate dp = 1.0 in the Lotka-Volterra case. Initial population densities h1 = p1 = 150, h2 = p2 = 50) h∗ 1 = 1 σ dp (9a) h∗ 2 = 1 σ 1 −α(1 −ακ) 1 −ακ dp p∗ 1 = 1 σ σ(1 −α)(1 −ακ) + αγ (1 −σ(1 −ακ) σ(1 −αγ )(1 −ακ) + αγ (1 −α(1 −ακ))bh (9b) p∗ 2 = 1 σ (σ −αγ ) σ(1 −αγ )(1 −ακ) + αγ (1 −α(1 −ακ))bh . without real parts. This means there are two distinct oscillation frequencies in the system, 1 2π bhdp and m 2π bhdp , (10) where m = √σ(1 −α)(1 −ακ) + αγ (1 −σ(1 −ακ))√1 −α(1 −ακ) √σ√σ(1 −αγ )(1 −ακ) + αγ (1 −α(1 −ακ)) × √σ −αγ (11) 1 2π bhdp and m 2π bhdp , (10) h (10) m = √σ(1 −α)(1 −ακ) + αγ (1 −σ(1 −ακ))√1 −α(1 −ακ) √σ√σ(1 −αγ )(1 −ακ) + αγ (1 −α(1 −ακ)) × √σ −αγ × √σ αγ (11) (11) measures the ratio between the two oscillation frequen- cies. This ratio decreases when we move away from the MA interaction model. For α ≪1 we find For αγ > σ, the resistant host is always disadvantageous because of the high cost of the resistance allele (γ ). Con- sequently, extinction of H2 and P2 then becomes a stable fixed point. For αγ < σ, h∗ 1 and h∗ 2 increase linearly with parasites’ death rate dp, while p∗ 1 and p∗ 2 increase lin- early with hosts’ birth rate bh. A linear stability analysis of the interior fixed point (see Appendix A.1 for details) shows that the equilibrium is neutrally stable. Close to the interior fixed point, the system exhibits undamped oscillations. More specifically, the four eigenvalues of the Jacobi-matrix are two distinct pairs of complex conjugates m ≈1 −α 1 + γ 2σ . (12) (12) In particular, for the MA model both oscillation frequen- cies collapse into a single one. However, all solutions for α > 0 exhibit both of the frequencies (Fig. 3). For the Replicator Dynamics system, in which the popu- lation size is constant, the non-trivial fixed point of Eq. (8) is given by Song et al. Population dynamics 3 Trajectories close to the interior fixed points (black points) on the h1 −p1 plane (dark green solid lines both for LV and RD equations) and the h2 −p2 plane (light green dashed lines LV only). The black crosses mark the initial conditions. The black rectangle represent a special set of initial condition while the black solid/dashed lines show the corresponding trajectories. With Replicator Dynamics the h1 −p1 trajectory is a closed circle. With Lotka-Volterra dynamics, the trajectories are closed circle when the initial conditions fulfill Eq. (25) (black lines). For the closed circles (black in LV and green in RD) the initial host population densities, h1 and h2 are 5 % above the corresponding fixed point, while the parasite population densities are 5 % beneath the fixed point. Except for α = 0 (MA) the green trajectories with LV resemble tori instead of closed circles, an implication for two oscillation frequencies. To show the shift of the interior fixed point as α increases from 0 to 1, the trajectories are plotted all in the same coordinate system at the bottom Page 7 of 15 Song et al. BMC Evolutionary Biology (2015) 15:212 Song et al. BMC Evolutionary Biology (2015) 15:212 Page 7 of 15 h∗ 1 = 1 −ακ 2 −ακ −α(1 −ακ) (13a) p∗ 1 = αγ (1 −σ(1 −ακ)) + σ(1 −α)(1 −ακ) σ((1 −αγ (1 −ακ)) + (1 −α)(1 −ακ)) . (13b) h∗ 1 = 1 −ακ 2 −ακ −α(1 −ακ) p∗ 1 = αγ (1 −σ(1 −ακ)) + σ(1 −α)(1 −ακ) σ((1 −αγ (1 −ακ)) + (1 −α)(1 −ακ)) . (13a) −ακ)) + σ(1 −α)(1 −ακ) 1 −ακ)) + (1 −α)(1 −ακ)) . (13b) (13b) A linear stability analysis shows that the interior fixed point is again neutrally stable, as the two eigenvalues are a pair of purely imaginary, complex conjugated numbers when αγ < σ (see Appendix A.2 for details). Hence, there is only one characteristic oscillation frequency of the dynamical system at the fixed point, A linear stability analysis shows that the interior fixed point is again neutrally stable, as the two eigenvalues are a pair of purely imaginary, complex conjugated numbers when αγ < σ (see Appendix A.2 for details). Population dynamics Hence, there is only one characteristic oscillation frequency of the dynamical system at the fixed point, l = √(1 −ακ)(1 −α(1 −ακ))(αγ (1 −σ(1 −ακ)) + (1 −α)σ(1 −ακ)) √(1 + (1 −α)(1 −ακ))(1 −αγ (1 −ακ) + (1 −α)(1 −ακ)) √σ −αγ 2π (14) (14) l has a maximum value, σ/(4π), in the pure matching-allele model (α = 0). Close to the matching-allele model, α ≪1 the oscillation frequency decreases with increasing α as l has a maximum value, σ/(4π), in the pure matching-allele model (α = 0). Close to the matching-allele model, α ≪1 the oscillation frequency decreases with increasing α as l ≈σ 4π − α 16π (2 + γ + 2κ)σ . (15) + γ + 2κ)σ . (15) l ≈σ 4π − α 16π (2 + γ + 2κ)σ . (15) The solutions around the fixed point exhibit the oscillation frequency described by Eq. (14). The trajectories are closed circles as shown on the right side of Fig. 3. The solutions around the fixed point exhibit the oscillation frequency described by Eq. (14). The trajectories are closed circles as shown on the right side of Fig. 3. Disentangling evolutionary and ecological dynamics To clarify the ecological effect on the dynamics, particularly at the interior fixed point, we derive the dynamics of the host and parasite population sizes, h = h1 + h2 and p = p1 + p2, and the relative abundance of H1 and P1 in the population, x = h1/h and y = p1/p, from Eq. (2). According to Eq. (2) the differential equations for the population sizes of hosts h and parasites p are ˙h = h(p f(x, y) + b) (16a) ˙p = p(h g(x, y) −d) (16b) where ˙h = h(p f(x, y) + b) ˙p = p(h g(x, y) −d) ˙h = h(p f(x, y) + b) (16a) (16b) where f (x, y) = Mh 11xy + Mh 12x(1 −y) + Mh 21(1 −x)y + Mh 22(1 −x)(1 −y) (17) g(x, y) = Mp 11yx + Mp 12y(1 −x) + Mp 21(1 −y)x + Mp 22(1 −y)(1 −x), (17) general interaction matrices. The differential equations for relative abundances of H1 and P1 are ˙x = px(1 −x)((Mh 11 −Mh 21)y + (Mh 12 −Mh 22)(1 −y)) (18a) ˙y = hy(1 −y)((Mp 11 −Mp 21)x + (Mp 12 −Mp 22)(1 −x)) , (18b) (18a) (18b) If f (x, y) and g(x, y) are constant in time Eq. (16) yield simple Lotka-Volterra dynamics, while Eq. (18) result in Replicator Dynamics with rescaled time if the population sizes are kept constant. f f (x, y) and g(x, y) are constant in time Eq. (16) yield simple Lotka-Volterra dynamics, while Eq. (18) result in Replicator Dynamics with rescaled time if the population sizes are kept constant. At the interior fixed point one of the oscillation frequencies, bhdp/(2π), results solely from Lotka-Volterra dynamics. The other oscillation frequency At the interior fixed point one of the oscillation frequencies, bhdp/(2π), results solely from Lotka-Volterra dynamics. The other oscillation frequency bhdp 2π m = bhdp 2π (Mh 11 −Mh 21)(Mh 12 −Mh 22)(Mp 11 −Mp 21)(Mp 12 −Mp 22) (Mh 11Mh 22 −Mh 12Mh 21) (Mp 11Mp 22 −Mp 12Mp 21) (19) (19) (see Eq. Short overview Host-parasite interactions are acknowledged as a driving evolutionary force promoting biological diversity and sex- ual reproduction [10, 11], with the MA and GfG model being the most popular models to describe the genetic interaction for coevolving hosts and parasites [26–32]. Despite a number of important insights provided within their framework, the generality of findings often suffers from the complexity of the models employed and, as a consequence, the difficulty to fully understand them analytically [33]. Constants of motion The system with constant population size has a constant of motion (Eq. (10.22) in [21]) given by L = + Mh 12 −Mh 22 ln p1 + Mh 21 −Mh 11 ln(1 −p1) − Mp 12 −Mp 22 ln h1 − Mp 21 −Mp 11 ln(1 −h1) = + (αγ (1 −σ(1 −ακ)) + (1 −α)σ(1 −ακ)) ln p1 + (σ −αγ ) ln(1 −p1) + (1 −ακ)σ ln h1 + (1 −α(1 −ακ))σ ln(1 −h1). In this study, we present a very general yet parsimonious model of host-parasite coevolution spanning from MA to GfG with either constant or interaction-driven changing population size. Derived analytical solutions revealed that the coevolution dynamics differs qualitatively between the models with constant and changing population sizes. Apart from the pure MA situation, the well known Red Queen dynamics with closed trajectories is only observed in models with constant population size. This implies that the patterns of host-parasite dynamics to be expected in real biological systems can be much more intricate than suggested by the most popular theoretical models. (23) (23) Due to ˙L = 0, we obtain sustained oscillations for any ini- tial condition, even far away from the interior fixed point Eq. (13) The case of changing population size is more intri- cate. In the case of a matching allele model α = 0, the two equations decouple and we have two indepen- dent Lotka-Volterra systems with sustained oscillations, characterized by the two constants of motion L1 = bh ln p1 −σp1 + dp ln h1 −σh1 (24a) L2 = bh ln p2 −σp2 + dp ln h2 −σh2 . (24b) (24a) (24b) Disentangling evolutionary and ecological dynamics (36) in Appendix A.3) is the product of the oscillation frequency with constant population size l = (Mh 11 −Mh 21)(Mh 22 −Mh 12)(Mp 11 −Mp 21)(Mp 22 −Mp 12) 2π (Mh 11 + Mh 22 −Mh 12 −Mh 21)(Mp 12 + Mp 21 −Mp 11 −Mp 22) (20) l = (Mh 11 −Mh 21)(Mh 22 −Mh 12)(Mp 11 −Mp 21)(Mp 22 −Mp 12) 2π (Mh 11 + Mh 22 −Mh 12 −Mh 21)(Mp 12 + Mp 21 −Mp 11 −Mp 22) (20) (20) (see Eq. (40) in Appendix A.3) and the geometric mean of host and parasite population size h∗· p∗, i.e., Eq. (40) in Appendix A.3) and the geometric mean of host and parasite population size h∗· p∗, i.e (see Eq. (40) in Appendix A.3) and the geometric mean of host and parasite population size m bhdp 2π = l h∗· p∗, (21) Song et al. BMC Evolutionary Biology (2015) 15:212 Page 8 of 15 Page 8 of 15 with Note that with the condition Eq. (25a) the ratio p1/p2 remains constant and with the condition Eq. (25b), the ratio h1/h2 remains constant. This shows that the nature of the dynamics in this case does not only depend on the choice of parameters, but also on the initial state of the system, which in principle leads to a fur- ther complication for the corresponding experimental systems. h∗= d(Mp 11 −Mp 12 −Mp 21 + Mp 22) Mp 11Mp 22 −Mp 12Mp 21 (22a) p∗= b(Mh 12 + Mh 21 −Mh 11 −Mh 22) Mh 11Mh 22 −Mh 12Mh 21 (22b) (22b) (calculated from Eq. (33) in Appendix A.3). Thus, one of the oscillations results purely from ecological interac- tions, while the other one arises from the combination of ecology and evolution in our system. Generality of results h l To test the generality of our findings we additionally ana- lyzed the interaction matrix suggested by Parker [13] (Eq. (37)). There, a factor that denotes the fitness reduc- tion of the avirulent parasite encountering the resistant host and an advantage of the virulent parasite meeting the resistant host are assumed in addition. These two parameters together with the costs of resistance and vir- ulence determine whether the model is MA or GfG. Again we obtain two distinct oscillation frequencies for the population dynamics with changing population sizes in GfG-like models (the ratio is shown in Eq. (38) in the Appendix A.3). Despite the convincing biological relevance of the inter- action matrix elements in [14], they do not change mono- tonically on the MA-GfG continuum, e.g., with a cost of virulence κ > 0.5, Mp 21 in Eq. (1) first increases then decreases as α increases from 0 to 1. As an alternative interpolation, we therefore also considered interaction matrices that describe a linear transition from MA to GfG model, such that Mh = P1 P2 H1 −σ −α(1 −κ)σ H2 −αγ −αγ −(1 −ακ)σ (27a) Mp = H1 H2 P1 σ 0 P2 α(1 −κ)σ (1 −ακ)σ . (27b) (27a) Mp = H1 H2 P1 σ 0 P2 α(1 −κ)σ (1 −ακ)σ . (27b) (27b) Impact of eco-evo feedback in genetically explicit models Impact of eco-evo feedback in genetically explicit models In the last two decades it has been realized that evo- lutionary changes can be faster than previously thought and, thus, occurring on the same time-scale as ecolog- ical interactions, especially in case of coevolving hosts and parasites [35–38]. Population dynamics can influence the pace of coevolution via so called eco-evolutionary feedbacks, or even give rise to a new type of coevolu- tionary dynamics as we showed in our study. Interestingly enough, a comprehensive part of the theoretical stud- ies on eco-evolutionary feedbacks is conducted within the framework of evolutionary game theory and adap- tive dynamics [21, 39]. In contrast to our model, these approaches usually do not include an explicit definition of genetic interaction between the species, which lim- its their application for interpreting patterns of genetic variability in natural populations [40]. Rapid changes in genetic composition may lead to perturbation in host demography and disease dynamics, as was observed for the myxoma virus epidemic in Australian populations of European rabbit [41]. Genetic adaptation can improve overall population fitness and "buffer" the unfavorable impact of pathogens (evolutionary rescue) [42]. However, population perturbations may constrain adaptability, for example, via enhancing inbreeding, affecting trait heri- tabilities and disturbing allele composition irrespective of natural selection [43–46]. Thus, models accounting simul- taneously for the genetic basis of host-parasite interaction and associated population dynamics may be necessary to fully understand ongoing coevolution among species and the effect it would have on genetic diversity. We are aware of only a few such models [29, 47–51], and most of them confirm that ecological parameters can have a very strong effect on coevolution. Main results and analytical solution While we do not find a constant of motion for the gen- eral case of α > 0, particular initial conditions can lead to invariants. If the initial condition fulfills Our study is based on a simplification of the model sug- gested by Agrawal and Lively [14] that explores a con- tinuum between the MA and GfG models. We study the model in the context of haplotypes with a single locus, but relax the restriction to constant population size. With a coevolutionary system of two host and two parasite types we achieved an analytical characteriza- tion across the entire parameter space. To study eco- logical effects caused by the victim-exploiter interaction [34] between hosts and parasites, we consider models with changing population size aside of models with con- stant population size. Under the assumption of constant population size, the dynamics in MA and GfG models appear to be very similar, both showing sustained oscilla- tions with only one oscillation frequency. Yet, introducing changing population size according to the Lotka-Volterra equations, we obtain distinct patterns of the population h1 h2 = Mp 22 −Mp 12 Mp 11 −Mp 21 and (25a) p1 p2 = Mh 22 −Mh 12 Mh 11 −Mh 21 (25b) (25a) (25b) which corresponds to a two-dimensional subspace, then there are two constants that remain invariant over time, L1 = bh ln p1 + Mh 11p1 + Mh 12p2 + dp ln h1 −Mp 11h1 −Mp 12h2 (26a) L2 = bh ln p2 + Mh 21p1 + Mh 22p2 + dp ln h2 −Mp 21h1 −Mp 22h2. (26b) L1 = bh ln p1 + Mh 11p1 + Mh 12p2 + dp ln h1 −Mp 11h1 −Mp 12h2 (26a) (26a) L2 = bh ln p2 + Mh 21p1 + Mh 22p2 + dp ln h2 −Mp 21h1 −Mp 22h2. (26b) −Mp 21h1 −Mp 22h2. (26b) (26b) Song et al. BMC Evolutionary Biology (2015) 15:212 Page 9 of 15 Lively, or us are only a small subset of the possible mod- els for host-parasite interaction. An observation that will hold for any such model is that as long as the population sizes are kept constant, the population dynamics follows a closed circle with a single oscillation frequency. How- ever, with changing population size a second oscillation frequency arises when the model become GfG-like, which can lead to much more intricate dynamics. Main results and analytical solution For a pure MA model or an inverse MA model (where the diago- nal instead of the off-diagonal matrix elements are zero), there still is only one oscillation frequency (see Eq. (36) in Appendix A.3). dynamics. For changing population sizes, a single oscil- lation frequency is present only in the MA model. An additional oscillation frequency arises for all other points on the MA-GfG continuum in that case. In other words, changing population size leads to a much more complex dynamics in GfG-like models, but not in the pure MA model. In the stochastic model analyzed in [29], the analysis of allele fixation time for the MA model revealed that Lotka-Volterra dynamics in combination with the associ- ated stochastic effects quickly break down the Red Queen circle. As the dynamics in GfG-like models take a com- pletely different nature with changing population size, the influence of Lotka-Volterra dynamics on the Red Queen circle is yet unclear and remains to be assessed in more detail in the future, especially as our current analysis did not take stochastic effects into account. Implications for maintenance of genetic diversity This is especially true for the GfG model, as a parasite with the virulent allele would be quickly fixed, unless having a cost of virulence [3, 12, 56]. In addition to the cost, other factors have been examined for their potential role in maintaining variation, including epidemi- ological feedback [51, 57], spatial structure [48, 49, 58, 59], genetic drift [60], diffuse multi-species coevolution [61], models with multiple alleles and multiple loci [16, 60, 62]. Several studies proposed that multiple factors need to act jointly for long-term coexistence of multiple resisto- and infectotypes [33]. The view of a multifactorial basis of the maintenance of diversity creates an additional chal- lenge for theoretical and empirical studies to disentangle them. As opposed to that, Tellier [34] presented a sim- ple GfG framework showing that the general condition for stability is the presence of direct frequency-dependent selection (where fitness of an allele declines with increas- ing frequency of that allele itself). In this context, the distinction is made between direct frequency dependence and indirect frequency-dependent selection where fit- ness is mediated by the frequency of the corresponding antagonist. Direct frequency-dependent selection can be introduced in the model by incorporation of epidemiolog- ical or ecological factors ([32], Table 1). If we introduce a direct frequency-dependent element by applying com- petitive Lotka-Volterra equations or the concept of empty spaces [63] (implying the existence of a carrying capacity) into our model, the neutrally stable interior fixed point becomes stable. Instead of forming tori or moving along closed circles, the deterministic trajectory spirals inwards. In this case, the oscillation of allele frequencies lasts longer in stochastic simulations, hence the polymorphic state is more stable. dynamics for intermediate points on the MA-GfG con- tinuum, especially as experimental studies provide some examples of such types of interaction [64]. In contrast to [34] and many other studies [14, 16, 59], our model is implemented on a continuous time-scale and, there- fore, covers host and parasite systems with overlapping generations. Interestingly, it has been proposed that mod- els with discrete generations would favor coevolutionary cycling by synchronizing ecological and epidemiologi- cal processes [51], while in [34] the condition for stable cycling is more restrictive for discrete generations when compared to the continuous model. Conclusions In summary, we have shown that only a small and possibly biased subset of possible host-parasite interaction dynam- ics is captured by the mathematical models that assume fixed population size or particular genetics for the inter- action, such as the MA model. Even in a simple model that allows for a full analytical description, the dynamics can vary substantially between subsequent coevolution- ary cycles. We demonstrate analytically that the complex dynamics found for changing population sizes is not a result of choosing a particular interaction matrix. The complex pattern is not limited to the set of models con- sidered here, but rather a general property of models beyond fixed population size. Our findings highlight the importance of the interconnectedness between coevolu- tion and population dynamics, and its potential role in understanding the generation and maintenance of genetic variation. Our model provides a solid framework that can be extended to more realistic (i.e., usually more complex) scenarios in the future, including the specification of alter- native virulence components and their differential effect on host fitness, or consideration of population carrying capacity with its limiting effect on growth rate. The stability analysis derived the condition for coexis- tence αγ < σ, suggesting that departing from the GfG end of the continuum would increase a range of parameters at which the oscillation of allele frequencies is maintained. Therefore, patterns of "partial" infectivity by a virulent parasite are more likely to result in cycling dynamics com- pared to a pure GfG situation. Agrawal and Lively [14] came to the same conclusion by evaluating computational simulations. This reinforces the importance of exploring Implications for maintenance of genetic diversity Numerous field studies identified the presence of com- prehensive heritable variation in resistance-infectivity patterns for plant and animal populations and their respective pathogens, suggesting that coevolution acts to maintain genetic diversity [3, 11, 52–55]. However, already The analysis in Appendix A.4 shows that our conclu- sion also holds for the linear interpolation. One should keep in mind that both MA and GfG models and even the intermediate models proposed by Parker, Agrawal & Song et al. BMC Evolutionary Biology (2015) 15:212 Page 10 of 15 the first studies, which attempted to explain such vari- ation by cycling dynamics, encountered the problem of stability. This is especially true for the GfG model, as a parasite with the virulent allele would be quickly fixed, unless having a cost of virulence [3, 12, 56]. In addition to the cost, other factors have been examined for their potential role in maintaining variation, including epidemi- ological feedback [51, 57], spatial structure [48, 49, 58, 59], genetic drift [60], diffuse multi-species coevolution [61], models with multiple alleles and multiple loci [16, 60, 62]. Several studies proposed that multiple factors need to act jointly for long-term coexistence of multiple resisto- and infectotypes [33]. The view of a multifactorial basis of the maintenance of diversity creates an additional chal- lenge for theoretical and empirical studies to disentangle them. As opposed to that, Tellier [34] presented a sim- ple GfG framework showing that the general condition for stability is the presence of direct frequency-dependent selection (where fitness of an allele declines with increas- ing frequency of that allele itself). In this context, the distinction is made between direct frequency dependence and indirect frequency-dependent selection where fit- ness is mediated by the frequency of the corresponding antagonist. Direct frequency-dependent selection can be introduced in the model by incorporation of epidemiolog- ical or ecological factors ([32], Table 1). If we introduce a direct frequency-dependent element by applying com- petitive Lotka-Volterra equations or the concept of empty spaces [63] (implying the existence of a carrying capacity) into our model, the neutrally stable interior fixed point becomes stable. Instead of forming tori or moving along closed circles, the deterministic trajectory spirals inwards. In this case, the oscillation of allele frequencies lasts longer in stochastic simulations, hence the polymorphic state is more stable. the first studies, which attempted to explain such vari- ation by cycling dynamics, encountered the problem of stability. pp A.1 Stability of the interior fixed point in the Lotka-Volterra dynamics In order to analyse the system at the interior fixed point (h∗ 1, h∗ 2, p∗ 1, p∗ 2), we first linearise the system around this point. For general points (h1, h2, p1, p2), the linearised sys- tem is given by by the Jacobian matrix J(h1, h2, p1, p2) = ⎛ ⎜⎜⎜⎜⎜⎜⎜⎜⎜⎝ bh−p1σ−p2α(1−ακ)σ 0 −h1σ −h1α(1−ακ)σ 0 bh−p1αγ +p2((1−αγ )(1−(1−ακ)σ)−1) −h2αγ h2((1−αγ )(1−(1−ακ)σ)−1) p1σ 0 h1σ−dp 0 p2α(1−ακ)σ p2(1−ακ)σ 0 (h2(1−ακ)+h1α(1−ακ))σ−dp ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎟⎠ . 0 Song et al. BMC Evolutionary Biology (2015) 15:212 Page 11 of 15 At the interior fixed point (h∗ 1, h∗ 2, p∗ 1, p∗ 2), we have J(h∗ 1, h∗ 2, p∗ 1, p∗ 2) = ⎛ ⎜⎜⎜⎜⎜⎜⎜⎜⎜⎝ 0 0 −dp dpα(ακ−1) 0 0 dpαγ (α(ακ−1)+1) (ακ−1)σ dp(α(ακ−1)+1)(αγ +(αγ −1)(ακ−1)σ) (ακ−1)σ bh(αγ +(γ α+α−1)(ακ−1)σ) αγ (α(ακ−1)+1)+(αγ −1)(ακ−1)σ 0 0 0 bhα(1−ακ)(σ−αγ ) αγ (α(ακ−1)+1)+(αγ −1)(ακ−1)σ bh(1−ακ)(σ−αγ ) αγ (α(ακ−1)+1)+(αγ −1)(ακ−1)σ 0 0 ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎟⎠ . (28) The eigenvalues of this matrix determine linear stability at the fixed point [25]. If there is at least one eigenvalue with positive real part, the point would be unstable. If all eigenvalues have negative real parts, the point would be stable. In our case, the four eigenvalues are −dp dpα(ακ−1) dpαγ (α(ακ−1)+1) (ακ−1)σ dp(α(ακ−1)+1)(αγ +(αγ −1)(ακ−1)σ) (ακ−1)σ 0 0 σ 0 0 ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎟⎠ . (28) (28) The eigenvalues of this matrix determine linear stability at the fixed point [25]. If there is at least one eigenvalue with positive real part, the point would be unstable. If all eigenvalues have negative real parts, the point would be stable. In our case, the four eigenvalues are The eigenvalues of this matrix determine linear stability at the fixed point [25]. If there is at least one eigenvalue with positive real part, the point would be unstable. If all eigenvalues have negative real parts, the point would be stable. In our case, the four eigenvalues are The eigenvalues of this matrix determine linear stability at the fixed point [25]. If there is at least one eigenvalue with positive real part, the point would be unstable. If all eigenvalues have negative real parts, the point would be stable. A.2 Stability of the interior fixed point in the Replicator Dynamics For the system with constant population size, the Jacobian matrix in general is J(h1, p1) = ⎛ ⎜⎝ (1−2h1)(α(γ −σ(1−p1)(γ +(−γ α−α+1)κ+1))−2p1σ+σ) σh1(1−h1)(−κ(γ +1)α2+(γ +κ+1)α−2) σp1(1−p1)(−(1−α)κα−α+2) σ(1−2p1)(h1(−κ(1−α)α−α+2)+ακ−1) ⎞ ⎟⎠. (α(γ −σ(1−p1)(γ +(−γ α−α+1)κ+1))−2p1σ+σ) σh1(1−h1)(−κ(γ +1)α2+(γ +κ+1)α−2) σp1(1−p1)(−(1−α)κα−α+2) σ(1−2p1)(h1(−κ(1−α)α−α+2)+ακ−1) ⎞ ⎟⎠. At the interior fixed point (h∗ 1, p∗ 1), the matrix is given by J(h∗ 1, p∗ 1) = ⎛ ⎜⎜⎜⎜⎝ 0 (ακ−1)((γ +1)κα2−(γ +κ+1)α+2)(α(ακ−1)+1)σ ((α−1)κα−α+2)2 −((α−1)κα−α+2)(αγ −σ)(αγ +(γ α+α−1)(ακ−1)σ) ((γ +1)κα2−(γ +κ+1)α+2)2σ 0 ⎞ ⎟⎟⎟⎟⎠ . (30) The eigenvalues are 1,2 = ∓i √(1 −ακ)(1 −α(1 −ακ))(αγ (1 −σ(1 −ακ)) + (1 −α)σ(1 −ακ)) √(1 + (1 −α)(1 −ακ))(1 −αγ (1 −ακ) + (1 −α)(1 −ακ)) √σ −αγ . (31) F th i l l i i h th fi d i t i t l t (30) The eigenvalues are (31) For αγ < σ, the eigenvalues are purely imaginary, hence, the fixed point is a neutral center. For αγ < σ, the eigenvalues are purely imaginary, hence, the fixed point is a neutral center. pp A.1 Stability of the interior fixed point in the Lotka-Volterra dynamics In our case, the four eigenvalues are 1,2 = ±i bhdp and (29) 3,4 = ± bhdp √σ(1 −α)(1 −ακ) + αγ (1 −σ(1 −ακ))√1 −α(1 −ακ) √σ√σ(1 −αγ )(1 −ακ) + αγ (1 −α(1 −ακ)) √αγ −σ, (29) Except the term √αγ −σ, the remaining factors in in 3,4 are positive. For αγ > σ, allele H1 is always beneficial. Consequently, the fixed point is unstable as one of the eigenvalues 3 or 4 is positive. For αγ < σ, the fixed point is a center with neutral stability as all eigenvalues are purely imaginary. Only the case of αγ < σ is of further interest in this manuscript, as the result is straightforward in the opposite case. The Jacobian matrix at any defined point is J(h1, h2, p1, p2) = The Jacobian matrix at any defined point is J(h1, h2, p1, p2) = ⎛ ⎜⎜⎜⎜⎜⎜⎜⎜⎝ bh+Mh 11p1+Mh 12p2 0 h1Mh 11 h1Mh 12 0 bh+Mh 21p1+Mh 22p2 h2Mh 21 h2Mh 22 Mp 11p1 Mh 12p1 −dp+h1Mp 11+h2Mp 12 0 Mp 21p2 Mp 22p2 0 −dp+h1Mp 21+h2Mp 22 ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎠ ⎛ ⎜⎜⎜⎜⎜⎜⎜⎜⎝ bh+Mh 11p1+Mh 12p2 0 h1Mh 11 h1Mh 12 0 bh+Mh 21p1+Mh 22p2 h2Mh 21 h2Mh 22 Mp 11p1 Mh 12p1 −dp+h1Mp 11+h2Mp 12 0 Mp 21p2 Mp 22p2 0 −dp+h1Mp 21+h2Mp 22 ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎠ (34) At the interior fixed point (h∗ 1, h∗ 2, p∗ 1, p∗ 2), we now have J(h∗ 1, h∗ 2, p∗ 1, p∗ 2) = At the interior fixed point (h∗ 1, h∗ 2, p∗ 1, p∗ 2), we now have J(h∗ 1, h∗ 2, p∗ 1, p∗ 2) = ⎛ ⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎝ 0 0 Mh 11(Mp 12−Mp 22)dp Mp 12Mp 21−Mp 11Mp 22 Mh 12(Mp 12−Mp 22)dp Mp 12Mp 21−Mp 11Mp 22 0 0 Mh 21(Mp 11−Mp 21)dp Mp 11Mp 22−Mp 12Mp 21 Mh 22(Mp 11−Mp 21)dp Mp 11Mp 22−Mp 12Mp 21 Mp 11(Mh 12−Mh 22)bh Mh 11Mh 22−Mh 12Mh 21 Mp 12(Mh 12−Mh 22)bh Mh 11Mh 22−Mh 12Mh 21 0 0 Mp 21(Mh 21−Mh 11)bh Mh 11Mh 22−Mh 12Mh 21 Mp 22(Mh 21−Mh 11)bh Mh 11Mh 22−Mh 12Mh 21 0 0 ⎞ ⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎠ (35) There are four eigenvalues There are four eigenvalues 1,2 = ±i bhdp and (36) 3,4 = ±i bhdp (Mh 11 −Mh 21)(Mh 12 −Mh 22)(Mp 11 −Mp 21)(Mp 12 −Mp 22) (Mh 11Mh 22 −Mh 12Mh 21) (Mp 11Mp 22 −Mp 12Mp 21) . 1,2 = ±i bhdp and 3,4 = ±i bhdp (Mh 11 −Mh 21)(Mh 12 −Mh 22)(Mp 11 −Mp 21)(Mp 12 −Mp 22) (Mh 11Mh 22 −Mh 12Mh 21) (Mp 11Mp 22 −Mp 12Mp 21) . (36) It is often assumed that (i) Mh 11 < Mh 21 ≤0 (H2 is beneficial if there is only P1 in the population), (ii) Mh 22 < Mh 12 ≤0 (H1 is beneficial if there is only P2 in the population), (iii) Mp 11 > Mp 21 ≥0 (P1 is beneficial if there is only H1 in the population), and (iv) Mp 22 > Mp 12 ≥0 (P1 is beneficial if there is only H1 in the population). A.3 Stability of the interior fixed point for general interaction matrices The appearance of the second oscillation frequency at the interior fixed point in gene-for-gene-like models with chang- ing population sizes does not depend on the exact choice of the interaction matrices in Eq. (2). To show this, we recalculate the interior fixed point and apply linear stability analysis on interaction matrices of a general form, Mh = P1 P2 H1 Mh 11 Mh 12 H2 Mh 21 Mh 22 (32a) Mh = P1 P2 H1 Mh 11 Mh 12 H2 Mh 21 Mh 22 (32a) Song et al. BMC Evolutionary Biology (2015) 15:212 Song et al. BMC Evolutionary Biology (2015) 15:212 Page 12 of 15 Page 12 of 15 Mp = H1 H2 P1 Mp 11 Mp 12 P2 Mp 21 Mp 22 . (32b) Mp = H1 H2 P1 Mp 11 Mp 12 P2 Mp 21 Mp 22 . (32b) P1 M11 M12 P2 Mp 21 Mp 22 . he interior fixed point for our host parasite system with Lotka-Volterra dynamics (Eq. (2)) is then The interior fixed point for our host parasite system with Lotka-Volterra dynamics (Eq. (2)) is then h∗ 1 = Mp 12−Mp 22 Mp 12Mp 21−Mp 11Mp 22 dp (33a) h∗ 2 = Mp 21−Mp 11 Mp 12Mp 21−Mp 11Mp 22 dp p∗ 1 = Mh 12−Mh 22 Mh 11Mh 22−Mh 12Mh 21 bh (33b) p∗ 2 = Mh 21−Mh 11 Mh 11Mh 22−Mh 12Mh 21 bh . h∗ 1 = Mp 12−Mp 22 Mp 12Mp 21−Mp 11Mp 22 dp (33a) h∗ 2 = Mp 21−Mp 11 Mp 12Mp 21−Mp 11Mp 22 dp p∗ 1 = Mh 12−Mh 22 Mh 11Mh 22−Mh 12Mh 21 bh (33b) h h (33a) M11M22 M12M21 (33b) p∗ 2 = Mh 21−Mh 11 Mh 11Mh 22−Mh 12Mh 21 bh . (33b) The Jacobian matrix at any defined point is J(h1, h2, p1, p2) = The ratio between the two oscillation frequencies at the interior fixed point is where the notations a, c, k, t, and s in [13] are changed to α, γ , κ, τ, and σ, respectively. According to [13], the fitness of the “narrowly virulent pathogen” P1 is reduced by a factor τ by interacting with the resistant host H2; a fitness penalty κ (the cost of virulence) is inflicted on the “broadly virulent pathogen” P2 independent of which host it exploits; α the “advantage of adapted pathogens on resistant host” measures a special advantage of P2 on H2; a fitness penalty γ (the cost of resistance) is paid by the resistant host H2. When τ = κ = α = 1 and γ = 0 the fitnesses conform to the pattern of pure MA model. When τ = 1 and α = 0 the fitnesses revert to a pure GfG pattern. The ratio between the two oscillation frequencies at the interior fixed point is m = √κ√ασ + γ √α −κ + τ√στ −γ √σ√α −κτ + τ√σ(α −κτ + τ) + γ κ . m = √κ√ασ + γ √α −κ + τ√στ −γ √σ√α −κτ + τ√σ(α −κτ + τ) + γ κ . (38) (38) The ratio is 1 for pure MA model. With a set of parameter used in [13], α = 0.33, γ = 0, κ = 0.05, and σ = τ = 1 the ratio is about 0.1. The ratio is 1 for pure MA model. With a set of parameter used in [13], α = 0.33, γ = 0, κ = 0.05, and σ = τ = 1 the ratio is about 0.1. The same method can be applied for the system with constant population size. In that case, the interior fixed point expressed by the general interaction matrices elements is h∗ 1 = Mp 22−Mp 12 Mp 11+Mp 22−Mp 12−Mp 21 (39a) h∗ 1 = Mp 22−Mp 12 Mp 11+Mp 22−Mp 12−Mp 21 (39a) p∗ 1 = Mh 22−Mh 12 Mh 11+Mh 22−Mh 12−Mh 21 , (39b) h∗ 1 = Mp 22−Mp 12 Mp 11+Mp 22−Mp 12−Mp 21 (39a) ∗ Mh 22−Mh 12 (39b) (39a) p∗ 1 = Mh 22−Mh 12 Mh 11+Mh 22−Mh 12−Mh 21 , (39b) (39b) while h∗ 2 = 1 −h∗ 1 and p∗ 2 = 1 −p∗ 1. The Jacobian matrix at any defined point is J(h1, h2, p1, p2) = The eigenvalues of the Jacobian matrix at the interior fixed point are 1,2 = 0 and (40) 3,4 = ±i −(Mh 11 −Mh 21)(Mh 22 −Mh 12)(Mp 11 −Mp 21)(Mp 22 −Mp 12) (Mh 11 + Mh 22 −Mh 12 −Mh 21)(Mp 11 + Mp 22 −Mp 12 −Mp 21) while h∗ 2 = 1 −h∗ 1 and p∗ 2 = 1 −p∗ 1. The eigenvalues of the Jacobian matrix at the interior fixed point are 1,2 = 0 and (40) 3,4 = ±i −(Mh 11 −Mh 21)(Mh 22 −Mh 12)(Mp 11 −Mp 21)(Mp 22 −Mp 12) (Mh 11 + Mh 22 −Mh 12 −Mh 21)(Mp 11 + Mp 22 −Mp 12 −Mp 21) (40) Hence, there only is one oscillation frequency at the interior fixed point in models with constant population size, regardless of the specific assumption for the interaction matrices. Hence, there only is one oscillation frequency at the interior fixed point in models with constant population size, regardless of the specific assumption for the interaction matrices. The Jacobian matrix at any defined point is J(h1, h2, p1, p2) = With these minimal assumptions the eigenvalues are purely imaginary, i.e., the interior fixed point is a neutrally stable center. The Song et al. BMC Evolutionary Biology (2015) 15:212 Page 13 of 15 Song et al. BMC Evolutionary Biology (2015) 15:212 Page 13 of 15 Page 13 of 15 ratio between the eigenvalues, which determines the oscillation frequencies at the center, differs in different interaction models. For example, in Parker [13] the interaction matrices for haploid types are Mh = P1 P2 H1 −σ −(1 −κ)σ H2 −γ −σ(1 −τ) −σ(α −κ + 1) −γ Mh = P1 P2 H1 −σ −(1 −κ)σ H2 −γ −σ(1 −τ) −σ(α −κ + 1) −γ (37a) Mp i,j = H1 H2 P1 σ σ(1 −τ) P2 (1 −κ)σ σ(1 + α −κ) , (37b) Mh = P1 P2 H1 −σ −(1 −κ)σ H2 −γ −σ(1 −τ) −σ(α −κ + 1) −γ Mp i,j = H1 H2 P1 σ σ(1 −τ) P2 (1 −κ)σ σ(1 + α −κ) , Mh = P1 P2 H1 −σ −(1 −κ)σ H2 −γ −σ(1 −τ) −σ(α −κ + 1) −γ (37a) Mh = P1 P2 H1 −σ −(1 −κ)σ H2 −γ −σ(1 −τ) −σ(α −κ + 1) −γ (37a) H1 H2 P2 −(1 −κ)σ σ(α −κ + 1) −γ (37a) (37a) Mp i,j = H1 H2 P1 σ σ(1 −τ) P2 (1 −κ)σ σ(1 + α −κ) , Mp i,j = H1 H2 P1 σ σ(1 −τ) P2 (1 −κ)σ σ(1 + α −κ) , (37b) σ(1 −τ) κ)σ σ(1 + α −κ) , (37b) (37b) where the notations a, c, k, t, and s in [13] are changed to α, γ , κ, τ, and σ, respectively. According to [13], the fitness of the “narrowly virulent pathogen” P1 is reduced by a factor τ by interacting with the resistant host H2; a fitness penalty κ (the cost of virulence) is inflicted on the “broadly virulent pathogen” P2 independent of which host it exploits; α the “advantage of adapted pathogens on resistant host” measures a special advantage of P2 on H2; a fitness penalty γ (the cost of resistance) is paid by the resistant host H2. When τ = κ = α = 1 and γ = 0 the fitnesses conform to the pattern of pure MA model. When τ = 1 and α = 0 the fitnesses revert to a pure GfG pattern. Author details 1 1Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, 24306, Plön, Germany. 2New Zealand Institute for Advanced Study, Massey University, Auckland, New Zealand. 3Department of Evolutionary Ecology and Genetics, University of Kiel, Kiel, Germany. and the eigenvalues of the Jacobian matrix at this point are Competing interests Competing interests 17. Sardanyés J, Solé RV. Matching allele dynamics and coevolution in a minimal predator–prey replicator model. Phys Lett A. 2008;372:341–50. The authors declare that they have no competing interests. 18. Zeeman EC. Population dynamics from game theory. Lecture Notes Math. 1980;819:471–97. Received: 19 December 2014 Accepted: 21 August 2015 1,2 = ±i bhdp and 3,4 =±i bhdp √1 −α(1 −κ)√(σ −αγ )(αγ + (1 −α)σ) √σ√αγ (1 −α(1 −κ)) + σ(1 −ακ) . (43) A.4 Linear interpolation between MA and GfG models Alternatively to the models of [14] and [13], one could also use a linear interpolation between MA and gene-for-gene model, where the matrix elements linearly spans over the values of the two models as a single parameter α varies between 0 and 1 Mh = P1 P2 H1 −σ −α(1 −κ)σ H2 −αγ −αγ −(1 −ακ)σ (41a) Mp = H1 H2 P1 σ 0 P2 α(1 −κ)σ (1 −ακ)σ . (41b) Mh = P1 P2 H1 −σ −α(1 −κ)σ H2 −αγ −αγ −(1 −ακ)σ Mh = P1 P2 H1 −σ −α(1 −κ)σ H2 −αγ −αγ −(1 −ακ)σ (41a) H1 H2 (41a) Mp = H1 H2 P1 σ 0 P2 α(1 −κ)σ (1 −ακ)σ . (41b) Song et al. BMC Evolutionary Biology (2015) 15:212 Page 14 of 15 The fixed point with Lotka-Volterra dynamics is then The fixed point with Lotka-Volterra dynamics is then drafted the manuscript. AT conceived and supervised the study, developed the model, and drafted the manuscript. All authors read and approved the final manuscript. The fixed point with Lotka-Volterra dynamics is then h∗ 1 = 1 σ dp (42a) h∗ 2 = 1 −α(1 −κ) σ(1 −ακ) dp p∗ 1 = α(γ −σ) + σ σ(αγ (1 −α(1 −κ)) + σ(1 −ακ))bh (42b) p∗ 2 = σ −αγ σ(αγ (1 −α(1 −κ)) −σ(1 −ακ))bh , (42a) Acknowledgements g YS, CSG, and AT acknowledge generous funding by the Max Planck Society. AP was funded through grants of the German Science Foundation to HS (DFG grants SCHU 1415/8 and SCHU 1415/9 within the German priority program SPP1399 on host-parasite coevolution). AP was additionally supported by the International Max-Planck Research School (IMPRS) for Evolutionary Biology. g YS, CSG, and AT acknowledge generous funding by the Max Planck Society. AP was funded through grants of the German Science Foundation to HS (DFG grants SCHU 1415/8 and SCHU 1415/9 within the German priority program SPP1399 on host-parasite coevolution). AP was additionally supported by the International Max-Planck Research School (IMPRS) for Evolutionary Biology. (42b) References J Heredity. 2010;101(suppl 1):13–20. doi:10.1093/jhered/esq010. http://jhered.oxfordjournals.org/content/ 101/suppl_1/S13.full.pdf+html. 1,2 = 0 and 3,4 = ±i 1 −α + α2κ(1 −κ)√(σ −αγ )(αγ + (1 −α)σ) 2 −α 1,2 = 0 and 1,2 = 0 and 11. Lively CM, Apanius V. Genetic diversity in host-parasite interactions. In: Ecology of Infectious Diseases in Natural Populations. Cambridge, UK: Cambridge University Press; 1995. p. 421–49. 11. Lively CM, Apanius V. Genetic diversity in host-parasite interactions. In: Ecology of Infectious Diseases in Natural Populations. Cambridge, UK: Cambridge University Press; 1995. p. 421–49. (46) (46) 12. Leonard KJ. Selection pressures and plant pathogens. Ann NY Acad Sci. 1977;287:207–22. doi:10.1111/j.1749-6632.1977.tb34240.x. 12. Leonard KJ. Selection pressures and plant pathogens. Ann NY Acad Sci. 1977;287:207–22. doi:10.1111/j.1749-6632.1977.tb34240.x. Hence, there is only one oscillation frequency l = 3/(i2π) at the interior fixed point in models with con- stant population size. As long as αγ < σ, the oscillation frequency l decreases with α and increases with γ and σ, while l increases with κ until κ reaches the value 1/2, then l decreases as κ increases from 1/2 to 1. For α ≪1, 13. Parker MA. Pathogens and sex in plants. Evol Ecol. 1994;8(5):560–84. doi:10.1007/BF01238258. 13. Parker MA. Pathogens and sex in plants. Evol Ecol. 1994;8(5):560–84. doi:10.1007/BF01238258. 14. Agrawal A, Lively CM. Infection genetics: gene-for-gene versus matching-alleles models and all points in between. Evol Ecol Res. 2002;4:79–90. 14. Agrawal A, Lively CM. Infection genetics: gene-for-gene versus matching-alleles models and all points in between. Evol Ecol Res. 2002;4:79–90. 15. Agrawal AF, Lively CM. Modelling infection as a two-step process combining gene-for-gene and matching-allele genetics. Proc R Soc B: Bio Sci. 2003;270(1512):323–34. 15. Agrawal AF, Lively CM. Modelling infection as a two-step process combining gene-for-gene and matching-allele genetics. Proc R Soc B: Biol Sci. 2003;270(1512):323–34. l ≈1 2π σ 2 −ασ 4 . (47) (47) 16. Tellier A, Brown JKM. Polymorphism in multilocus host-paraiste coevolutionary interactions. Genetics. 2007;177:1777–90. References 1. Woolhouse MEJ, Webster JP, Domingo E, Charlesworth B, Levin BR. Biological and biomedical implications of the co-evolution of pathogens and their hosts. Nat Genet. 2002;32(4):569–77. 1. 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https://openalex.org/W2761415648	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0185712&type=printable	English	null	Estimating risk propagation between interacting firms on inter-firm complex network	PloS one	2,017	cc-by	8,221	Hayato Goto1, Hideki Takayasu2,3, Misako Takayasu1,2* Hayato Goto1, Hideki Takayasu2,3, Misako Takayasu1,2* 1 Department of Computational Intelligence and Systems Science, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259, Nagatsuta-cho, Yokohama 226-8502, Japan, 2 Institute of Innovative Research, Tokyo Institute of Technology, 4259, Nagatsuta-cho, Yokohama 226-8502, Japan, 3 Sony Computer Science Laboratories, 3-14-13 Higashigotanda, Shinagawa-ku, Tokyo 141-0022, Japan a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Editor: Tobias Preis, University of Warwick, UNITED KINGDOM Editor: Tobias Preis, University of Warwick, UNITED KINGDOM UNITED KINGDOM Received: March 23, 2017 Accepted: September 18, 2017 Published: October 3, 2017 Copyright: © 2017 Goto 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. Received: March 23, 2017 Accepted: September 18, 2017 Published: October 3, 2017 Copyright: © 2017 Goto 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. Abstract We derive a stochastic function of risk propagation empirically from comprehensive data of chain-reaction bankruptcy events in Japan from 2006 to 2015 over 5,000 pairs of firms. The probability is formulated by firm interaction between the pair of firms; it is proportional to the product of α-th power of the size of the first bankrupt firm and β-th power of that of the chain- reaction bankrupt firm. We confirm that α is positive and β is negative throughout the observ- ing period, meaning that the probability of cascading failure is higher between a larger first bankrupt firm and smaller trading firm. We additionally introduce a numerical model simulat- ing the whole ecosystem of firms and show that the interaction kernel is a key factor to express complexities of spreading bankruptcy risks on real ecosystems. RESEARCH ARTICLE OPEN ACCESS Citation: Goto H, Takayasu H, Takayasu M (2017) Estimating risk propagation between interacting firms on inter-firm complex network. PLoS ONE 12 (10): e0185712. https://doi.org/10.1371/journal. pone.0185712 * takayasu.m.aa@m.titech.ac.jp Estimating risk propagation between firms and scale-free property [27], accompanied by various scaling relations [28–34]. Fig 1(b) shows an example of an inter-firm business transaction network within Kyoto prefecture in 2015. Strategic International Collaborative Research Program (SICORP) on the topic of “ICT for a Resilient Society” by Japan and Israel. This research was partially supported by MEXT as “Exploratory Challenges on Post-K computer (Study on multilayered multiscale spacetime simulations for social and economical phenomena)” (Grant Recipient: MT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Coauthor Hideki Takayasu is employed by Sony Computer Science Laboratories. Sony Computer Science Laboratories provided support in the form of salaries for author HT, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. A representative cascading failure of a firm or financial ecosystem is a chain-reaction bank- ruptcy [35–43], which is defined as bankruptcy triggered by a proceeding default of a firm that trades or has other relations with the bankrupting firm [44]. For instance, Fujiwara et al.[39] conducted a study using a combination of data on a Japanese inter-firm transaction network and a bankruptcy list in 2006. They choose firms randomly from the business network with a probability of bankruptcy; however, the cluster size distribution of chain-reaction bankrupt- cies, where the size of the cluster is defined as the number of cascade-reaction bankrupt firms, as shown in Fig 1(c), 1(d) and 1(e), was not reproduced by the model. This indicated the addi- tional effect of spreading bankruptcy risks on the real ecosystem. Bardoscia et al.[42, 43] also investigated the effect as shock propagation on bank lending networks. They formulated a microscopic theory by iterating balance sheets and found that the network effects could amplify exogenous shocks in some conditions. As reported in those analyses, risk is spread on an economy based on the complex interplay between agents. In this study, we analyze comprehensive data of chain-reaction bankruptcy events in Japan from 2006 to 2015 and empirically derive a stochastic function of risk propagation. The proba- bility is formulated by firm interaction between a first bankruptcy firm and a chain-reaction bankruptcy firm. Introduction It has been generally recognized that social and economic networks can be captured as com- plex systems whose nodes are individual agents that interact [1, 2]. Indeed, not only econo- mists but also physicists have shown interest in the complexity underlying the systems and how they work [3–10]. Their contributions provide profound new insights into such areas of interest as cascading failures and resilience [11–20] using the lens of the science of complex systems [21–24]. These theoretical and empirical analyses deepen our fundamental insight into the systems’ dynamics to better predict the impact of economic or social crises, such as subprime mortgages in 2008 [2]. Data Availability Statement: The data underlying this study was provided from Teikoku Databank, Ltd. Interested and qualified researchers can request access to the data set in the same manner as the authors at the following URL: https://www. tdb.co.jp/service/mail_e/form.jsp. Funding: The authors appreciate Teikoku Databank, Ltd., Center for TDB Advanced Data Analysis and Modeling for providing both the data and financial support. This study is partially supported by the Grant-in-Aid for Scientific Research (B), Grant Number 26310207 and JST, Similar to other complex networks, business firm networks are complex ecosystems inter- acting with others in various ways. An inter-firm business transaction network is a typical example. This network, whose nodes are firms that link through business transactions from customers and providers, producing a money flow (opposite from a goods/service flow) as shown in Fig 1(a), has statistical complex properties [25], such as small world property [26] PLOS ONE \| https://doi.org/10.1371/journal.pone.0185712 October 3, 2017 1 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185712 October 3, 2017 The amount of risk propagation between firms is approximately propor- tional to the product of α-th power of the size of a customer firm and β-th power of that of a provider firm, which is very similar to the form of “money flow” from customers and providers [45]. This also indicates that each α and β of the interaction kernel is positive and negative, respectively. To further our understanding, we introduce a simulation-based network evolu- tion model [46, 47] using inter-firm business transaction networks from 2006 to 2015. The results numerically confirm that the interaction kernel works to reproduce the cluster size dis- tribution of chain-reaction bankruptcies in Japan. Competing interests: This study is financially supported by Teikoku Databank, Ltd. and coauthor HT is employed by Sony Computer Science Laboratories. There are no patents, products in development, or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. Materials and methods Empirical data analysis Chain-reaction bankruptcy. Business practices in Japan are unique. When building trust- worthy relationships or managing credit risk, the Japanese first tend to gather their business partners’ detailed corporate information. Then, professional third-party organizations are used to evaluate their partners’ credit status. Teikoku Databank Ltd (TDB) is one of the largest corporate research providers in Japan; it has been assessing the credit status of firms for 117 years. Their credit research reports include detailed information of the financial statements of firms, their history, business partners, management, and banking transactions. TDB also has detailed information about bankruptcies in Japan. For example, it includes bad debt, which is the total amount of accounts receivable that will not be collected, as a scale of bankruptcy. In this study, we analyze a Japanese firm database collected by TDB that includes chain-reaction bankruptcies. The list of chain-reaction bankruptcies in the database includes 5,245 pairs of 3,370 first bankruptcy firms and 5,245 chain-reaction bankruptcy firms, hereafter called f and c, respec- tively. According to the chain-reaction bankruptcy data from 2006 to 2015 in Table 1, there were 11,644 bankruptcies and 525 chain-reaction bankruptcies per year on average, respec- tively. There were over 10 chain-reaction firm clusters each year and a little difference in tail (Table 1). In addition, Fig 1(f) shows the distributions of a time delay from first bankruptcies f to next cascades c. About 40% of these events happened within a month and about 85% were within a year. 2 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185712 October 3, 2017 Estimating risk propagation between firms Fig 1. (a) A component of the inter-firm business transaction networks. The nodes are firms and links are business transactions from customers to providers, indicating a money flow. (b) An example of the inter-firm business transaction network within Kyoto prefecture in 2015. The nodes are firms in Kyoto prefecture and links are business transactions between the firms. The network has 18,391 nodes and 38,540 links and has typical complex network properties such as scale-free property and small world property. (c) A component of a cluster of chain-reaction bankruptcies. The cluster nodes are firms and links are risk propagations from first bankruptcies to chain-reaction bankruptcies. (d) A nine-size cluster of chain-reaction bankruptcies. Each circle and dotted arrow shows a bankrupt firm and the direction of risk propagation, respectively. The center number is annual sales (million yen) of a bankrupt firm. Materials and methods Empirical data analysis (d) A nine-size cluster of chain-reaction bankruptcies. Each circle and dotted arrow shows a bankrupt firm and the direction of risk propagation, respectively. The center number is annual sales (million yen) of a bankrupt firm. The top circle shows the first bankrupt firm and the other circles show chain-reaction bankruptcy firms. (e) Cluster size distribution of chain-reaction bankruptcies from 2006 to 2015 in log-log scale. As shown in (d), the size of the cluster is defined as the number of bankrupt firms of a cascade reaction. (f) Time delay distribution from first bankruptcies to next cascades. About 40% of these events happened within a month and about 85% were within a year. https://doi.org/10.1371/journal.pone.0185712.g001 Scaling relations. In earlier studies on national business networks and their transactions, it was found that there are scaling laws of continuing firms between the median of pairs of sales S, number of employees E, and number of business transactions k such that such that S / E1.3, S / k1.3, and E / k1.0 [33]. The TDB-supplied Japanese firm database also includes a bank- ruptcy list with each firm’s S, E, k, and bad debt D. Fig 2(a), 2(b) and 2(c) show the scaling rela- tions of bankrupt firms between the median of the pairs of S, E, and k in 2015 in log-log scale that are observed as S / E0.91.0, S / k1.01.1, and E / k1.01.1, respectively. The scaling rela- tions of bankrupt firms are different from that of continuing firms, the relation between employees E and the business transactions k is nearly the same, while sales S is nearly a linear function of E and k. Moreover, Fig 2(d), 2(e) and 2(f) show the scaling relations of bankrupt firms between the median of pairs of S, E, k, and D in log-log scale that are observed for D / S0.80.9, D / E0.80.9, and D / k0.70.9, respectively. The larger the firm, the larger its amount of bad debt, nearly proportionally. In the following discussion, we apply k as the measure of Scaling relations. In earlier studies on national business networks and their transactions, it was found that there are scaling laws of continuing firms between the median of pairs of sales S, number of employees E, and number of business transactions k such that such that S / E1.3, S / k1.3, and E / k1.0 [33]. Materials and methods Empirical data analysis The top circle shows the first bankrupt firm and the other circles show chain-reaction bankruptcy firms. (e) Cluster size distribution of chain-reaction bankruptcies from 2006 to 2015 in log-log scale. As shown in (d), the size of the cluster is defined as the number of bankrupt firms of a cascade reaction. (f) Time delay distribution from first bankruptcies to next cascades. About 40% of these events happened within a month and about 85% were within a Fig 1. (a) A component of the inter-firm business transaction networks. The nodes are firms and links are business transactions from customers to providers, indicating a money flow. (b) An example of the inter-firm business transaction network within Kyoto prefecture in 2015. The nodes are firms in Kyoto prefecture and links are business transactions between the firms. The network has 18,391 nodes and 38,540 links and has typical complex network properties such as scale-free property and small world property. (c) A component of a cluster of chain-reaction bankruptcies. The cluster nodes are firms and links are risk propagations from first bankruptcies to chain-reaction bankruptcies. (d) A nine-size cluster of chain-reaction bankruptcies. Each circle and dotted arrow shows a bankrupt firm and the direction of risk propagation, respectively. The center number is annual sales (million yen) of a bankrupt firm. The top circle shows the first bankrupt firm and the other circles show chain-reaction bankruptcy firms. (e) Cluster size distribution of chain-reaction bankruptcies from 2006 to 2015 in log-log scale. As shown in (d), the size of the cluster is defined as the number of bankrupt firms of a cascade reaction. (f) Time delay distribution from first bankruptcies to next cascades. About 40% of these events happened within a month and about 85% were within a year. Fig 1. (a) A component of the inter-firm business transaction networks. The nodes are firms and links are business transactions from customers to providers, indicating a money flow. (b) An example of the inter-firm business transaction network within Kyoto prefecture in 2015. The nodes are firms in Kyoto prefecture and links are business transactions between the firms. The network has 18,391 nodes and 38,540 links and has typical complex network properties such as scale-free property and small world property. (c) A component of a cluster of chain-reaction bankruptcies. The cluster nodes are firms and links are risk propagations from first bankruptcies to chain-reaction bankruptcies. PLOS ONE \| https://doi.org/10.1371/journal.pone.0185712 October 3, 2017 https://doi.org/10.1371/journal.pone.0185712.g001 Materials and methods Empirical data analysis The TDB-supplied Japanese firm database also includes a bank- ruptcy list with each firm’s S, E, k, and bad debt D. Fig 2(a), 2(b) and 2(c) show the scaling rela- tions of bankrupt firms between the median of the pairs of S, E, and k in 2015 in log-log scale that are observed as S / E0.91.0, S / k1.01.1, and E / k1.01.1, respectively. The scaling rela- tions of bankrupt firms are different from that of continuing firms the relation between 3 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185712 October 3, 2017 Estimating risk propagation between firms Table 1. Frequency of bankruptcies and chain-reaction bankruptcies per year from 2006 to 2015 in Japan. There are about 1% bankruptcies of the total number of firms and about 4% chain-reaction bankruptcies of the total number of bankruptcies per year on average. Table 1. Frequency of bankruptcies and chain-reaction bankruptcies per year from 2006 to 2015 in Japan. There are about 1% bankruptcies of the total number of firms and about 4% chain-reaction bankruptcies of the total number of bankruptcies per year on average. Year Number of firms Number of bankruptcy firms Number of chain-reaction bankruptcy firms Maximum cluster size of chain-reaction bankruptcies 2006 1,003,366 11,832 313 16 2007 1,004,275 12,610 785 9 2008 1,017,459 14,346 892 9 2009 1,035,113 13,973 782 13 2010 1,073,941 12,216 526 8 2011 1,114,309 11,788 494 6 2012 1,119,895 11,443 415 15 2013 1,124,316 10,499 383 20 2014 1,131,821 9,168 366 20 2015 1,136,179 8,560 289 16 Ave. 1,076,067 11,644 525 13 https://doi.org/10.1371/journal.pone.0185712.t001 https://doi.org/10.1371/journal.pone.0185712.t001 Fig 2. Scaling laws of median of bankrupt firms between pairs of (a) S and E, (b) S and k, (c) E and k, (d) D and S, (e) D and E, and (f) D and k in 2015 in log-log scale, respectively. There are scaling laws of continuing firms such that S / E1.3, S / k1.3, and E / k1.0, however, those of bankrupt firms are different than are observed as S / E0.91.0, S / k1.01.1, and E / k1.01.1. We note that these exponents are observed by the least squares method with coefficient of determination R2 0.9 and the error-bars show first and third quartile, respectively. htt //d i /10 1371/j l 0185712 002 Fig 2. Scaling laws of median of bankrupt firms between pairs of (a) S and E, (b) S and k, (c) E and k, (d) D and S, (e) D and E, and (f) D and k in 2015 in log-log scale, respectively. There are scaling laws of continuing firms such that S / E1.3, S / k1.3, and E / k1.0, however, those of bankrupt firms are different than are observed as S / E0.91.0, S / k1.01.1, and E / k1.0*1.1. We note that these exponents are observed by the least squares method with coefficient of determination R2 0.9 and the error-bars show first and third quartile, respectively. PLOS ONE \| https://doi.org/10.1371/journal.pone.0185712 October 3, 2017 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185712 October 3, 2017 4 / 12 Estimating risk propagation between firms Fig 3. (a) Frequency of chain-reaction bankruptcies A(kf, kc)p(kf)p(kc) from 2006 to 2015, where A(kf, kc) shows the interaction kernel between the number of business transactions for the first bankrupted firm kf and that of successive firm kc and p shows the probability density. First bankruptcy firms tend to be larger than their chain-reaction bankruptcy firms. (b) Probability of chain-reaction bankruptcies A(kf, kc) from 2006 to 2015. The probability of chain-reaction bankruptcies between larger firms is higher than between smaller ones. (c) A(kf, kc)’s scaling laws with respect to kf and kc, respectively. Green triangles and red squares show the relation between the median of A(kf, kc) and kc or kf with fixed kf or kc from 2006 to 2015 in log-log scale, respectively. Each dashed-line shows a slope of α (red) and β (green). The probability of cascading failure is higher between larger first bankruptcy firms and smaller trading firms. We note that these exponents are observed by the least squares method with coefficient of determination R2 0.9 and the error-bars show first and third quartile, respectively. Fig 3. (a) Frequency of chain-reaction bankruptcies A(kf, kc)p(kf)p(kc) from 2006 to 2015, where A(kf, kc) shows the interaction kernel between the number of business transactions for the first bankrupted firm kf and that of successive firm kc and p shows the probability density. First bankruptcy firms tend to be larger than their chain-reaction bankruptcy firms. (b) Probability of chain-reaction bankruptcies A(kf, kc) from 2006 to 2015. The probability of chain-reaction bankruptcies between larger firms is higher than between smaller ones. (c) A(kf, kc)’s scaling laws with respect to kf and kc, respectively. Green triangles and red squares show the relation between the median of A(kf, kc) and kc or kf with fixed kf or kc from 2006 to 2015 in log-log scale, respectively. Each dashed-line shows a slope of α (red) and β (green). The probability of cascading failure is higher between larger first bankruptcy firms and smaller trading firms. We note that these exponents are observed by the least squares method with coefficient of determination R2 0.9 and the error-bars show first and third quartile, respectively. https://doi.org/10.1371/journal.pone.0185712.g003 https://doi.org/10.1371/journal.pone.0185712.g003 firm size (it can be substituted by E or S, given that there are scaling relations among those quantities as described above). PLOS ONE \| https://doi.org/10.1371/journal.pone.0185712 October 3, 2017 Monte Carlo simulation It is expected that the empirically observed interaction kernel plays the role of the complexity underlying the firm ecosystem from the viewpoint of chain-reaction bankruptcies. In this sec- tion, we apply Eq 1 Aðkf; kcÞ / ka f kb c for numerical simulation and confirm whether the kernel can work for the reproduction of the cluster size distribution of chain-reaction bankruptcies as observed in Fig 1(e). Miura et al.[46] proposed a simple business network model in which money flow direction between a pair of firms is represented by a directed link connecting nodes, and they take into account the effects of new establishments, bankruptcies, and mergers and acquisitions (M&As) by creation of new nodes, removal of nodes, and aggregation of nodes together with links, respectively, as schematically shown in Fig 4(a). The model accu- rately reproduces basic statistical characteristics such as degree distribution of business net- work transactions. Additionally, by using a merger kernel estimated through an M&A data analysis, the model reproduces business network characteristics with the parameter set esti- mated by real firm data [47]. Here, we revise the model introducing a new effect of chain-reac- tion bankruptcy based on the empirically estimated kernel A(kf, kc) as follows; Step1 Start with N0 firms with real links given from the data of business transactions as shown in Table 1 (in Fig 5, N0 = 1,286,379 using the network data from 2009; in Fig 6, N0 depends on the year). Step1 Start with N0 firms with real links given from the data of business transactions as shown in Table 1 (in Fig 5, N0 = 1,286,379 using the network data from 2009; in Fig 6, N0 depends on the year). Step2 Choose one of the following three events stochastically. The occurrence probabilities of new establishments, M&As, bankruptcies, and chain-reaction bankruptcies are denoted by Step2 Choose one of the following three events stochastically. The occurrence probabilities of new establishments, M&As, bankruptcies, and chain-reaction bankruptcies are denoted by nts, M&As, bankruptcies, and chain-reaction bankruptcies in our simulation model. (b) Time esponds to the state of real business firm ecosystems in Japan [47]; the occurrence Fig 4. (a) The three basic processes for firms: new establishments, M&As, bankruptcies, and chain-reaction bankruptcies in our simulation model. (b) Time step flow chart of our simulation model. q Kernel of chain-reaction bankruptcies. We observe frequency of bankruptcies as a func- tion of link numbers kf and kc, the number of business transactions for the first bankrupted firm and that of the successive firm, respectively. For quantity measurement of firms, we focus on the number of business transactions k, although it can be substituted as E or S due to the scaling laws between them as mentioned previously. Fig 3(a) and 3(b) show the distributions of frequency of chain bankruptcy A(kf, kc)p(kf)p(kc) and the interaction kernel A(kf, kc), where p(kf) and p(kc) denote the probability densities of kf and kc, respectively. We note that A(kf, kc)p (kf)p(kc) > 1. Chain-reaction bankruptcies include the following characteristics: 1. First bankruptcy firms tend to be larger than their chain-reaction bankruptcy firms, as shown in red in Fig 3(a). 2. The probability of chain-reaction bankruptcies between larger firms is higher than between smaller ones, as shown in red in Fig 3(b). Result 1 suggests that smaller firms are likely not to bear the propagating risks when the first bankruptcy firm is larger. These results imply that a large firm’s bankruptcy is expected to spread a higher amount of bad debt than small cases. We note the functional form of A(kf, kc) from Fig 3(c) as follows; Aðkf; kcÞ / ka f kb c ð1Þ ð1Þ ð1Þ Here, (α, β) ’ (0.7 ± 0.1, −0.3 ± 0.1). Each α and β of the interaction kernel is positive and neg- ative, respectively, meaning that the probability of cascading failure is higher between larger first bankruptcy firms and smaller trading firms. Here, (α, β) ’ (0.7 ± 0.1, −0.3 ± 0.1). Each α and β of the interaction kernel is positive and neg- ative, respectively, meaning that the probability of cascading failure is higher between larger first bankruptcy firms and smaller trading firms. Here, (α, β) ’ (0.7 ± 0.1, −0.3 ± 0.1). Each α and β of the interaction kernel is positive and neg- ative, respectively, meaning that the probability of cascading failure is higher between larger first bankruptcy firms and smaller trading firms. 5 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185712 October 3, 2017 Estimating risk propagation between firms https://doi.org/10.1371/journal.pone.0185712.g004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185712 October 3, 2017 Estimating risk propagation between firms Fig 5. Cluster size distribution of chain-reaction bankruptcies in log-log plot. Black circles show the real distribution from 2006 to 2015. (a) Red triangles and green x-marks show the Monte Carlo simulation results with α = 0.7, β = −0.3 and α = β = 0.0 by 10 attempts, respectively. (b,c) Light blue squares and pink downward triangles show the (b) α = 0.7, β = −0.2 and α = 0.7, β = −0.4, and (c) α = 0.6, β = −0.3 and α = 0.8, β = −0.3 by 10 attempts, respectively. (d) Two-sample Kolmogorov-Smirnov statistical distribution (χ2) between real and simulated distribution. The smaller the χ2, the smaller the difference between real and simulated distribution. The cases that take into account of the interaction kernel of chain-reaction bankruptcies (red triangles) fit better with the real data even though the case that ignores the interaction kernel of chain-reaction bankruptcies (green x-marks) is similar to the previous study. h //d i /10 1371/j l 0185712 005 Fig 5. Cluster size distribution of chain-reaction bankruptcies in log-log plot. Black circles show the real distribution from 2006 to 2015. (a) Red triangles and green x-marks show the Monte Carlo simulation results with α = 0.7, β = −0.3 and α = β = 0.0 by 10 attempts, respectively. (b,c) Light blue squares and pink downward triangles show the (b) α = 0.7, β = −0.2 and α = 0.7, β = −0.4, and (c) α = 0.6, β = −0.3 and α = 0.8, β = −0.3 by 10 attempts, respectively. (d) Two-sample Kolmogorov-Smirnov statistical distribution (χ2) between real and simulated distribution. The smaller the χ2, the smaller the difference between real and simulated distribution. The cases that take into account of the interaction kernel of chain-reaction bankruptcies (red triangles) fit better with the real data even though the case that ignores the interaction kernel of chain-reaction bankruptcies (green x-marks) is similar to the previous study rn, rm, rb, and rc, respectively (rn: rm: rb: rc = 0.5: 0.15: 0.33: 0.02, which corresponds to the state of real business firm ecosystems in Japan [47]. In addition, rb and rc is estimated in Table 1). PLOS ONE \| https://doi.org/10.1371/journal.pone.0185712 October 3, 2017 Monte Carlo simulation Each parameter set corresponds to the state of real business firm ecosystems in Japan [47]; the occurrence probabilities rn: rm: rb: rc = 0.5: 0.15: 0.33: 0.02, the preferential attachment exponent for new comers λ = 1.0, and the merger kernel’s exponents α0 = 1.1, β0 = 0.7. Fig 4. (a) The three basic processes for firms: new establishments, M&As, bankruptcies, and chain-reaction bankruptcies in our simulation model. (b) Time step flow chart of our simulation model. Each parameter set corresponds to the state of real business firm ecosystems in Japan [47]; the occurrence probabilities rn: rm: rb: rc = 0.5: 0.15: 0.33: 0.02, the preferential attachment exponent for new comers λ = 1.0, and the merger kernel’s exponents α0 = 1.1, β0 = 0.7. Fig 4. (a) The three basic processes for firms: new establishments, M&As, bankruptcies, and chain-reaction bankruptcies in our simulation model. (b) Time step flow chart of our simulation model. Each parameter set corresponds to the state of real business firm ecosystems in Japan [47]; the occurrence probabilities rn: rm: rb: rc = 0.5: 0.15: 0.33: 0.02, the preferential attachment exponent for new comers λ = 1.0, and the merger kernel’s exponents α0 = 1.1, β0 = 0 7 https://doi.org/10.1371/journal.pone.0185712.g004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185712 October 3, 2017 6 / 12 Black circles show the real distribution for each year. Red triangles, orange crosses, and pink x-marks show the Monte Carlo simulation results of the best parameters set, the second best parameters set, and the third best parameters set by judging from the median of χ2 of each parameters set by 20 attempts, respectively. (a) α = 0.7, β = −0.3 in 2006. (b) α = 0.8, β = −0.3 in 2007. (c) α = 0.7, β = −0.2 in 2008. (d) α = 0.7, β = −0.4 in 2009. (e) α = 0.7, β = −0.3 in 2010. (f) α = 0.7, β = −0.2 in 2011. (g) α = 0.8, β = −0.3 in 2012. (h) α = 0.8, β = −0.2 in 2013. (i) α = 0.7, β = −0.2 in 2014. (j) α = 0.6, β = −0.2 in 2015. Each α and β of the interaction kernel is positive and negative, respectively, without time variant despite the fact that the number of bankruptcies in Japan has gradually decreased since 2008 in Table 1. https://doi org/10 1371/journal pone 0185712 g006 Chain-reaction Bankruptcies A randomly chosen firm is removed, along with all trans- action partners connected to this firm, following the interaction kernel of chain-reac- tion bankruptcies Aðkf ; kcÞ / ka f kb c to choose a first bankrupted f, which has already gone bankrupt, and chain-reaction bankruptcies c, which are directly connecting the first bankrupt firm. Step3 Repeat Step2 for T times (T = 25,000 in Figs 5 and 6, which corresponds to a year in accordance with average annual number of bankruptcy firms in Japan (Table 1) and it is also the typical time delay of failure cascade as already shown in Fig 1(f). Step3 Repeat Step2 for T times (T = 25,000 in Figs 5 and 6, which corresponds to a year in accordance with average annual number of bankruptcy firms in Japan (Table 1) and it is also the typical time delay of failure cascade as already shown in Fig 1(f). Step3 Repeat Step2 for T times (T = 25,000 in Figs 5 and 6, which corresponds to a year in accordance with average annual number of bankruptcy firms in Japan (Table 1) and it is also the typical time delay of failure cascade as already shown in Fig 1(f). We illustrate our algorithm by a flow chart in Fig 4(b). Estimating risk propagation between firms Fig 6. Cluster size distribution of chain-reaction bankruptcies in log-log plot. Black circles show the real distribution for each year. Red triangles, orange crosses, and pink x-marks show the Monte Carlo simulation results of the best parameters set, the second best parameters set, and the third best parameters set by judging from the median of χ2 of each parameters set by 20 attempts, respectively. (a) α = 0.7, β = −0.3 in 2006. (b) α = 0.8, β = −0.3 in 2007. (c) α = 0.7, β = −0.2 in 2008. (d) α = 0.7, β = −0.4 in 2009. (e) α = 0.7, β = −0.3 in 2010. (f) α = 0.7, β = −0.2 in 2011. (g) α = 0.8, β = −0.3 in 2012. (h) α = 0.8, β = −0.2 in 2013. (i) α = 0.7, β = −0.2 in 2014. (j) α = 0.6, β = −0.2 in 2015. Each α and β of the interaction kernel is positive and negative, respectively, without time variant despite the fact that the number of bankruptcies in Japan has gradually decreased since 2008 in Table 1. https://doi.org/10.1371/journal.pone.0185712.g006 Fig 6. Cluster size distribution of chain-reaction bankruptcies in log-log plot. Black circles show the real distribution for each year. Red triangles, orange crosses, and pink x-marks show the Monte Carlo simulation results of the best parameters set, the second best parameters set, and the third best parameters set by judging from the median of χ2 of each parameters set by 20 attempts, respectively. (a) α = 0.7, β = −0.3 in 2006. (b) α = 0.8, β = −0.3 in 2007. (c) α = 0.7, β = −0.2 in 2008. (d) α = 0.7, β = −0.4 in 2009. (e) α = 0.7, β = −0.3 in 2010. (f) α = 0.7, β = −0.2 in 2011. (g) α = 0.8, β = −0.3 in 2012. (h) α = 0.8, β = −0.2 in 2013. (i) α = 0.7, β = −0.2 in 2014. (j) α = 0.6, β = −0.2 in 2015. Each α and β of the interaction kernel is positive and negative, respectively, without time variant despite the fact that the number of bankruptcies in Japan has gradually decreased since 2008 in Table 1. https://doi.org/10.1371/journal.pone.0185712.g006 Fig 6. Cluster size distribution of chain-reaction bankruptcies in log-log plot. PLOS ONE \| https://doi.org/10.1371/journal.pone.0185712 October 3, 2017 New establishments A new firm having four transaction partners (two in-links and two out-links) is added; it is roughly consistent with the rate between the number of firms and the number of transaction partners [47]. In addition, each transaction partner is connected to a firm chosen randomly following the preferential attachment rule with exponent λ (λ = 1.0 corresponding to the state of real business firm ecosystems in Japan [46][47]). M&As A pair of firms are randomly chosen following the merger kernel Kðka; ktÞ / ka0 a kb0 t to choose an acquirer firm a and a target firm t. All transaction partners connected to the target firm t are also rewired to the acquirer firm a (α0 = 1.1, β0 = 0.7 corresponding to the state of real business firm ecosystems in Japan [47]). Bankruptcies A randomly chosen firm is removed along with all transaction links con- nected to this firm. This is because a firm’s lifetime follows an exponential distribution; it is roughly consistent with the simple assumption that a firm disappears randomly fol- lowing a Poisson process [46, 47]. Bankruptcies A randomly chosen firm is removed along with all transaction links con- nected to this firm. This is because a firm’s lifetime follows an exponential distribution; it is roughly consistent with the simple assumption that a firm disappears randomly fol- lowing a Poisson process [46, 47]. 7 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185712 October 3, 2017 Conclusions In this paper, we have shown the empirical and numerical analysis results of chain-reaction bankruptcies using a TDB Japanese firm database from 2006 to 2015. The main finding was that the kernel of chain-reaction bankruptcies was proportional to the product of α-th power of the size of first bankruptcy firms and β-th power of the size of chain-reaction bankruptcy firms similar to the case of money flow. We simultaneously found that α is positive and β is negative. It is obvious that the larger the first bankrupt firm and the smaller the trading firm, the higher the occurrence probability of cascading failure. Furthermore, we introduced simu- lations based on a network evolution model using inter-firm business transaction networks from 2006 to 2015 and showed that it reproduces the real cluster size distribution of chain- reaction bankruptcies. It shows that the interaction kernel is a key factor in expressing com- plexities of spreading bankruptcy risks on the real ecosystem. Moreover, we estimated that α is positive and β is negative throughout the whole period. This result suggests that the stability of firm ecosystems does not change from 2006 to 2015 despite the fact that the number of bank- ruptcies in Japan has gradually decreased since 2008. The year and parameters are (a) α = 0.7, β = −0.3 in 2006, (b) α = 0.8, β = −0.3 in 2007, (c) α = 0.7, β = −0.2 in 2008, (d) α = 0.7, β = −0.4 in 2009, (e) α = 0.7, β = −0.3 in 2010, (f) α = 0.7, β = −0.2 in 2011, (g) α = 0.8, β = −0.3 in 2012, (h) α = 0.8, β = −0.2 in 2013, (i) α = 0.7, β = −0.2 in 2014, and (j) α = 0.6, β = −0.2 in 2015. Each param- eter is estimated using the same method as in Fig 5. Black circles show the real distribution for each year. Red triangles, orange crosses, and pink x-marks show the Monte Carlo simulation results of the best parameters set, the second best parameters set, and the third best parameters set by judging from the median of χ2 of each parameters set by 20 attempts, respectively. It shows that α is positive and β is negative throughout the whole period. Acknowledgments We would like to express our appreciation to Center for TDB Advanced Data Analysis and Modeling, Tokyo Institute of Technology for providing the datasets. Author Contributions Conceptualization: Hideki Takayasu, Misako Takayasu. Data curation: Hayato Goto. To find the best set of parameters of α and β, we apply the two-sample Kolmogorov—Smirnov test [48], which measures the differ- ence between two distributions. The definition of the test statistic χ2 is 4D2 n1n2 n1þn2, where D is a maximum vertical deviation between two distributions and n1 and n2 are the number of sam- ples of those distributions. From Fig 5(d), we find that the simulated distribution comes closest to the real one with the parameter set α = 0.7 and β = −0.3, the case of red triangles in Fig 5(a). Fig 6 show the numerical analysis results of this simulation with different years to check the time dependency of the interaction kernel. The year and parameters are (a) α = 0.7, β = −0.3 in 2006, (b) α = 0.8, β = −0.3 in 2007, (c) α = 0.7, β = −0.2 in 2008, (d) α = 0.7, β = −0.4 in 2009, (e) α = 0.7, β = −0.3 in 2010, (f) α = 0.7, β = −0.2 in 2011, (g) α = 0.8, β = −0.3 in 2012, (h) α = 0.8, β = −0.2 in 2013, (i) α = 0.7, β = −0.2 in 2014, and (j) α = 0.6, β = −0.2 in 2015. Each param- eter is estimated using the same method as in Fig 5. Black circles show the real distribution for each year. Red triangles, orange crosses, and pink x-marks show the Monte Carlo simulation results of the best parameters set, the second best parameters set, and the third best parameters set by judging from the median of χ2 of each parameters set by 20 attempts, respectively. It shows that α is positive and β is negative throughout the whole period. Fig 6 show the numerical analysis results of this simulation with different years to check the time dependency of the interaction kernel. Fig 5 show the analysis results of this numerical simulation with varying parameters. Black circles show the real distribution of 8 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185712 October 3, 2017 Estimating risk propagation between firms chain-reaction bankruptcies accumulated from 2006 to 2015, which are shown in Fig 1(e). The parameters are (a) α = 0.7, β = −0.3 (red triangles), α = 0.0, β = 0.0 (green x-marks), (b) α = 0.7, β = −0.2 (light blue squares), α = 0.7, β = −0.4 (pink downward triangles), and (c) α = 0.6, β = −0.3 (light blue squares), α = 0.8, β = −0.3 (pink downward triangles), respectively. The simulation result that ignores the interaction kernel of chain-reaction bankruptcies (green x- marks) is similar to the previous study [39]; it clearly decays more quickly than the real cluster size distribution. On the other hand, the cases that take into account the interaction kernel of chain-reaction bankruptcy fit better with the real data. To find the best set of parameters of α and β, we apply the two-sample Kolmogorov—Smirnov test [48], which measures the differ- ence between two distributions. The definition of the test statistic χ2 is 4D2 n1n2 n1þn2, where D is a maximum vertical deviation between two distributions and n1 and n2 are the number of sam- ples of those distributions. From Fig 5(d), we find that the simulated distribution comes closest to the real one with the parameter set α = 0.7 and β = −0.3, the case of red triangles in Fig 5(a). chain-reaction bankruptcies accumulated from 2006 to 2015, which are shown in Fig 1(e). The parameters are (a) α = 0.7, β = −0.3 (red triangles), α = 0.0, β = 0.0 (green x-marks), (b) α = 0.7, β = −0.2 (light blue squares), α = 0.7, β = −0.4 (pink downward triangles), and (c) α = 0.6, β = −0.3 (light blue squares), α = 0.8, β = −0.3 (pink downward triangles), respectively. The simulation result that ignores the interaction kernel of chain-reaction bankruptcies (green x- marks) is similar to the previous study [39]; it clearly decays more quickly than the real cluster size distribution. On the other hand, the cases that take into account the interaction kernel of chain-reaction bankruptcy fit better with the real data. Author Contributions Conceptualization: Hideki Takayasu, Misako Takayasu. Data curation: Hayato Goto. 9 / 12 PLOS ONE \| https://doi.org/10.1371/journal.pone.0185712 October 3, 2017 Estimating risk propagation between firms Formal analysis: Hayato Goto, Hideki Takayasu, Misako Takayasu. Funding acquisition: Misako Takayasu. Investigation: Hayato Goto, Hideki Takayasu, Misako Takayasu. Methodology: Hayato Goto, Hideki Takayasu, Misako Takayasu. Project administration: Hideki Takayasu, Misako Takayasu. Resources: Misako Takayasu. Software: Hayato Goto. Supervision: Hideki Takayasu, Misako Takayasu. Validation: Hayato Goto, Hideki Takayasu, Misako Takayasu. Visualization: Hayato Goto. Writing – original draft: Hayato Goto, Hideki Takayasu, Misako Takayasu. Writing – review & editing: Hayato Goto, Hideki Takayasu, Misako Takayasu. Formal analysis: Hayato Goto, Hideki Takayasu, Misako Takayasu. Funding acquisition: Misako Takayasu. Investigation: Hayato Goto, Hideki Takayasu, Misako Takayasu. Methodology: Hayato Goto, Hideki Takayasu, Misako Takayasu. Methodology: Hayato Goto, Hideki Takayasu, Misako Takayasu. Project administration: Hideki Takayasu, Misako Takayasu. Software: Hayato Goto. Supervision: Hideki Takayasu, Misako Takayasu. Supervision: Hideki Takayasu, Misako Takayasu. Validation: Hayato Goto, Hideki Takayasu, Misako Takayasu. Validation: Hayato Goto, Hideki Takayasu, Misako Takayasu. Visualization: Hayato Goto. Writing – original draft: Hayato Goto, Hideki Takayasu, Misako Takayasu. Writing – original draft: Hayato Goto, Hideki Takayasu, Misako Takayasu. 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https://openalex.org/W2164839146	https://figshare.com/articles/journal_contribution/Supplementary_Figure_1_from_Phosphatidylserine-Targeting_Antibody_Induces_M1_Macrophage_Polarization_and_Promotes_Myeloid-Derived_Suppressor_Cell_Differentiation/22535986/1/files/39999277.pdf	Malagasy	null	Phosphatidylserine-Targeting Antibody Induces M1 Macrophage Polarization and Promotes Myeloid-Derived Suppressor Cell Differentiation	Cancer immunology research	2,013	cc-by	244	0.0 0.5 1.0 1.5 2.0 2.5 Tumor weight (g) 0.0 0.5 1.0 1.5 2.0 2.5 C44 2aG4 Doc 2aG4+Do Weeks 0 1 2 3 4 5 6 7 Tumor volume (cm3) A * * * * * * * * Treatment C44 2aG4 Doc 2aG4+Doc B Supplementary figure S1 0.0 0.5 1.0 1.5 2.0 2.5 Tumor weight (g) 0.0 0.5 1.0 1.5 2.0 2.5 C44 2aG4 Doc 2aG4+Do Weeks 0 1 2 3 4 5 6 7 Tumor volume (cm3) A * * * * * * * * Treatment C44 2aG4 Doc 2aG4+Doc B Supplementary figure S1 0.0 0.5 1.0 1.5 2.0 2.5 Tumor weight (g) 0.0 0.5 1.0 1.5 2.0 2.5 Tumor weight (g) 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 Tumor volume (cm3) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 W k C44 2aG4 Doc 2aG4+Doc C44 2aG4 Doc 2aG4+Doc Weeks 0 1 2 3 4 5 6 7 Tumor volume (cm3) * * * * * * * * * * * * * * * * Treatment Treatment C44 2aG4 Doc 2aG4+Doc A 0 B Tumor weight (g) 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 2.0 2.5 u o o u e (c ) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Weeks C44 2aG4 Doc 2aG4+Do Weeks * * * * * * * * Treatment Weeks
https://openalex.org/W1512614882	https://europepmc.org/articles/pmc5718660?pdf=render	English	null	Feasibility of using respiratory correlated mega voltage cone beam computed tomography to measure tumor motion	Journal of applied clinical medical physics	2,011	cc-by	6,312	a Corresponding author: Mingqing Chen, Department of Radiation Oncology, University of Iowa Hospitals and Clinics, 200 Hawkins Drive, Iowa City, IA, 52242, USA; phone: 319-353-8979; fax: 319-356-1530; email:mingqingchen@gmail.com Feasibility of using respiratory correlated mega voltage cone beam computed tomography to measure tumor motion Mingqing Chen,a R. Alfredo Siochi Department of Radiation Oncology, University of Iowa Hospitals and IA, 52242, USA mingqingchen@gmail.com Received 6 October, 2010; accepted January 10, 2011 Mingqing Chen,a R. Alfredo Siochi Department of Radiation Oncology, University of Iowa Hospitals and Clinics, Iowa City, IA, 52242, USA mingqingchen@gmail.com mingqingchen@gmail.com Received 6 October, 2010; accepted January 10, 2011 Received 6 October, 2010; accepted January 10, 2011 The purpose of this study was to test the feasibility of using respiratory correlated mega voltage cone-beam computed tomography (MVCBCT), taken during patient localization, to quantify the size and motion of lung tumors. An imaging phantom was constructed of a basswood frame embedded with six different-sized spherical pieces of paraffin wax. The Quasar respiratory motion phantom was programmed to move the imaging phantom using typical respiratory motion. The moving imag ing phantom was scanned using various MVCBCT imaging parameters, including two beam line types, two protocols with different ranges of rotation and different imaging doses. A static phantom was also imaged as a control. For all the 3D volu metric images, the contours of the six spherical inserts were measured manually. Compared with the nominal sphere diameter, the average relative error in the size of the respiratory correlated MVCBCT spheres ranged from 5.3% to 12.6% for the four largest spheres, ranging in size from 3.6 cc to 29 cc. Larger errors were recorded for the two smallest inserts. The average relative error in motion was 5.1% smaller than the programmed amplitude of 3.0 cm. We are able to conclude that it is feasible to use respiratory correlated MVCBCT to quantify tumor motion for lung cancer patients. PACS numbers: 87.19.Wx, 87.57.Q PACS numbers: 87.19.Wx, 87.57.Q Key words: respiratory correlated, MVCBCT, tumor motion, non-small-cell lung cancer JOURNAL OF APPLIED CLINICAL MEDICAL PHYSICS, VOLUME 12, NUMBER 2, spring 2011 JOURNAL OF APPLIED CLINICAL MEDICAL PHYSICS, VOLUME 12, NUMBER 2, spring 2011 Chen et al.: Respiratory correlated MVCBCT Chen et al.: Respiratory correlated MVCBCT Advances in megavoltage CBCT (MVCBCT)(6,7) imaging have made it clinically possible to perform patient localization prior to each treatment by registering the treatment CT to the planning CT. This process uses the treatment beam from a linear accelerator (linac) and an electronic portal imaging device (EPID) to capture projection images as the gantry rotates. These projection images are used for reconstruction by default, or in cine mode(8) where they could be exported into DICOM format for visualization and analysis. It would be useful to identify tumor positions in these projections to calibrate the strain gauge. However, the contrast in most of these projections is low and the tumor boundary is poorly defined (see Fig. 1). Tracking techniques that have been successfully applied to fluoroscopic images, such as methods based on template matching(9) or optical flow,(10) may not be robust enough for MVCBCT projection images. An alternative approach may be to use the diaphragm motion since it correlates well with tumor motion for most lung cancer patients.(11) In a preliminary study, we identified the ipsilat eral hemidiaphragm apex (IHDA) and the superior edge of the tumor in MVCBCT projections from 27 treatment fractions of one non-small-cell lung cancer (NSCLC) patient. The tumor position correlates well with the IHDA position in these projection images, with an averaged coefficient of determination of 0.95 for the linear fit. Moreover, the diaphragm edge between air and tissue is clearly visible in the projection images. Previous methods have been able to detect 2D IHDA positions in MVCBCT projection images semi-automatically, and convert them to 3D room coordinates using an interpolated ray trace algorithm.(12,13) However, performing the same task for the tumor is more difficult. i One could use the MVCBCT images to quantify the relationship between tumor and dia phragm motion. Applying respiratory correlated (RC) reconstruction by retrospectively sorting all the projection data according to diaphragm position would reduce blurring significantly, enabling clinicians to identify tumor and diaphragm boundaries directly on the images. We can then derive the diaphragm-to-tumor motion ratio (DTMR), which is based on the tumor centroid displacement and the IHDA displacement between full exhale (FE) and full inhale (FI) CBCT images. The strain gauge signal could be calibrated for tumor motion by using the Journal of Applied Clinical Medical Physics, Vol. 12, No. 2, Spring 2011 Fig. 1. I. Introduction Lung tumor motion is of great concern in radiotherapy. Among the respiratory compensation techniques presently used, respiratory gating based on external surrogates has the advantage of requiring no extra fluoroscopy imaging dose(1) or surgery to implant fiducials.(2,3) Our clinic uses a strain gauge to record respiratory phases and normalized amplitudes for 4DCT imaging. We analyze these images to determine the strain gauge phases to use for gating the treatment beam and limit the treated motion to less than 1 cm. However, since tumor motion may change, the gating phases determined from the 4DCT may not represent the same displacement at the time of treatment. Large residual tumor motion with a high target miss rate (of about 30%) has been reported when using abdominal surface motion for the gating signal.(4,5) The imprecision of using the abdominal surrogate alone can cause tumor underdose or normal tissue overdose. Calibrating these surrogates to tumor motion would provide a more accurate beam-on trigger, although this could require daily respiratory-correlated cone-beam (CB) CT imaging. 201 201 202 202 Chen et al.: Respiratory correlated MVCBCT Projection image from the MVCBCT raw dataset of a NSCLC patient. The tumor is visible in the right lung just above the diaphragm. Fig. 1. Projection image from the MVCBCT raw dataset of a NSCLC patient. The tumor is visible in the right lung just above the diaphragm. Journal of Applied Clinical Medical Physics, Vol. 12, No. 2, Spring 2011 Chen et al.: Respiratory correlated MVCBCT 203 203 DTMR and correlating the IHDA positions with the corresponding strain gauge signal recorded for each projection image. Results based on our previous study have shown the feasibility of quantifying DTMR and tumor volume changes for a large tumor by using RC MVCBCT.(14) However, RC MVCBCT has fewer projections; this causes severe view aliasing artifacts and degrades reconstructed image quality, potentially limiting its applicability to larger tumors and small displacements. DTMR and correlating the IHDA positions with the corresponding strain gauge signal recorded for each projection image. Results based on our previous study have shown the feasibility of quantifying DTMR and tumor volume changes for a large tumor by using RC MVCBCT.(14) However, RC MVCBCT has fewer projections; this causes severe view aliasing artifacts and degrades reconstructed image quality, potentially limiting its applicability to larger tumors and small displacements. Phantom tests are needed to determine these limitations, since no ground truth can be estab lished for patient images. At best, one can only hope to establish agreement among multiple dynamic imaging modalities such as Cine-MR and 4DCT. Hence, in order to quantify the errors in volume and motion determination, a phantom with spherical inserts was imaged to study the feasibility of using RC MVCBCT to quantify tumor motion and tumor volumes. The actual motion of the phantom and the size of the inserts are known and serve as ground truth. A.1 MVCBCT The Siemens MVision (Siemens Medical Systems, Oncology Care Systems, Concord, CA) uses an amorphous Si flat-panel EPID with 1024 × 1024 detector elements, each measuring 0.4 mm × 0.4 mm. The source-to-axis distance (SAD) is 100 cm, while the source-to-imager distance (SID) is 145 cm. The commercially available product uses a 6 MV treatment beam line (TBL), while a test system in our clinic uses a 4.2 MeV imaging beam line (IBL). The lower energy photons provide a better quality image for the same dose,(15-17) allowing us to determine if RC MVCBCT benefits from the new beam line. For both TBL and IBL modes, the standard protocols use a 200° arc from -90° to 110°, generating one projection image per degree. We also tested a TBL protocol with a full rotation (359° arc), so that we can evaluate whether the increased number of projections improves our ability to determine tumor sizes and motion from RC MVCBCT. We acquired 12 scans, six with the phantom at rest and six with the phantom in motion. The six scans used the three modes (200° IBL, 200° TBL, 359° TBL) at 5 and 10 MU. The phantom was also scanned at rest and in motion using 4D kVCT to compare our RC MVCBCT results against a clinical 4D system. Journal of Applied Clinical Medical Physics, Vol. 12, No. 2, Spring 2011 A.2 Phantom h i i h The imaging phantom consists of two symmetrical blocks of basswood, with a density of about 0.4g/cc to mimic lung tissue. Each block has six different sized hollow hemispheres measuring 3.81, 3.18, 2.54, 1.91, 0.95, and 0.48 cm in diameter, respectively. These hollow hemispheres were filled with paraffin wax, with a density of about 0.93g/cc to mimic lung tumors. The two halves were then carefully aligned to form a rectangular box embedded with six spherical pieces of paraffin wax. Figure 2 shows a picture of the phantom and a corresponding axial CT slice through the center of the phantom. The imaging phantom is placed on a cart attached to the quasar respiratory motion (QRM) phantom (Modus Medical Devices, Inc, London, ON, Canada) to simulate respiratory motion. The QRM phantom is programmed to move only in the superior-inferior (SI) direction, with position as a function of time, t, defined as: (1) ) /) ( ( cos ) ( 0 4 0 0 t t A z t z The motion amplitude A0 is 30 mm, which represents extreme tumor motion, and z0 is the minimum position. The period, τ, is 4 s to represent typical breathing. The image acquisition Journal of Applied Clinical Medical Physics, Vol. 12, No. 2, Spring 2011 204 Chen et al.: Respiratory correlated MVCBCT 204 Fig. 2. A picture of (a) the imaging phantom and (b) an axial slice through the center. Fig. 2. A picture of (a) the imaging phantom and (b) an axial slice through the center. Fig. 2. A picture of (a) the imaging phantom and (b) an axial slice through the center. Fig. 2. A picture of (a) the imaging phantom and (b) an axial slice through the center. started at a time t0 after the motion phantom started moving. The designed motion and size of the spherical inserts are used as ground truth for motion and volume quantification. started at a time t0 after the motion phantom started moving. The designed motion and size of the spherical inserts are used as ground truth for motion and volume quantification. A.3 Respiratory signal detection To detect the respiratory signal in the MVCBCT projection images, we developed an in-house platform using Microsoft Visual Basic 2005 (Microsoft Corp. Redmond, WA). An algorithm based on the interpolated ray trace method(13) confines the respiratory motion of an object into a bounded rectangle for each projection image. It requires a user to manually identify a Journal of Applied Clinical Medical Physics, Vol. 12, No. 2, Spring 2011 Chen et al.: Respiratory correlated MVCBCT 205 205 tracking point (such as the apex of a hemidiaphragm for the clinical case) in two FE frames and two FI frames. This step typically takes 20 seconds for the diaphragm apex. Applying trigonometric relationships among these four points allows us to determine a smaller region of interest where the motion is contained. Dynamic Hough transforms based on parabolas are used to create cost functions, while dynamic programming is used to minimize the cost func tions to find the trajectory of the diaphragm. (The complete details are given in References 12 though 14.) In this study, we used the center of the largest spherical insert as the tracking point. Using an algorithm based on a Hough transform for circle detection,(18) we are able to detect the largest spherical insert within the rectangular region. Parameter tuning and manual correc tion is performed when a detection error occurs. Since the errors are limited to a few frames, this correction step takes less than 20 seconds. (Future work involves using robust correlation to predict the required correction and eliminate the manual correction step.) Having identified the position of the center of the largest insert in each projection, the 3D position as a function of time can be approximated by a sequence of trigonometric relationships, as described for the interpolated ray trace algorithm.(13) A.4 Respiratory correlated reconstruction The craniocaudal motion of the center of the spherical insert is automatically rescaled into a relative motion range from 0 to 100, which correspond to the most superior and the most infe rior positions, respectively. In previous lung cancer patient studies, we applied the rescaling to the motion of the apex of the hemidiaphragm below the tumor.(12) 3D images at FE (0%) and FI (100%) are sufficient to quantify tumor displacement between full exhale and inhale states. Projections are sorted to these two respiratory states with a fixed amplitude interval. The size of the allowed amplitude interval is a compromise between view aliasing artifacts (the reduction of which requires more projections) and residual motion (the reduction of which requires fewer projections). The interval is set at 10%, which corresponds to 3.0 mm in our study. Hence, the FE image is reconstructed from projections with sphere positions of approximately 0 to 3 mm from full exhale position. For the FI image, the selected projections have sphere positions of approximately 27 to 30 mm inferior to the full exhale position. For a CBCT scan that acquires 200 projections of a phantom that moves according to Eq. (1), about 70 projections are included in the FE phase, while about 30 belong to the FI phase. Image reconstruction was performed using the Feldkamp, Davis, Kress (FDK) method.(19) For the MVCBCT imaging of a static phantom, a clinical FDK reconstruction system is used. The dimension of the 3D volumetric image is 256 × 256 × 274 (274 is in the craniocaudal direction), with a voxel spacing of 1.0 mm. For imaging of the moving phantom, an offline FDK algorithm is developed for the RC reconstruction. (The offline application uses the same algorithm as the online version, but it is used to reduce demand for the clinical imaging workstation that performs the online reconstruction.) The sorted projections are reconstructed into an image of 256 × 256 × 256, with a voxel spacing of 1.071 mm. The dimension of each 3D image for the 4DCT scan is 512 × 512 × 274 (274 in the craniocaudal direction), with a transverse slice thickness of 1.0 mm. Journal of Applied Clinical Medical Physics, Vol. 12, No. 2, Spring 2011 A.5 Quantification of motion and volume All the 3D MVCBCT and kVCT volumetric images were stored in DICOM format and im ported into the Philips Pinnacle (Philips Medical Systems, Andover, MA) treatment planning system. All spherical inserts in all the images were contoured. The volume and center of the contoured regions of interest were derived using Pinnacle’s measurement tools. The displace ments between volumes in the FE and FI images were computed from the difference of the centroid positions. p Previous studies have demonstrated that the display window center (WC) and window width (WW) significantly influence the apparent size of an object in CT imaging.(20,21) It was found that the WC should be half of the attenuation differences between the object and the Journal of Applied Clinical Medical Physics, Vol. 12, No. 2, Spring 2011 206 Chen et al.: Respiratory correlated MVCBCT 206 background in order to yield the correct size. In this study, we set the WC at half of the attenu ation difference between the spherical insert and the basswood frame. The WW is set as the attenuation difference. background in order to yield the correct size. In this study, we set the WC at half of the attenu ation difference between the spherical insert and the basswood frame. The WW is set as the attenuation difference. A.1 Image quality Figure 3 shows one coronal slice of the FE phase for kVCT and RC MVCBCT imaging for different protocols. Figure 4 compares the attenuation profiles of those images for the largest two spherical inserts. For the kVCT, the image intensity is distributed uniformly within each spherical insert and the CT number represents the material density well. For the other three RC MVCBCT images, the image intensity is no longer uniformly distributed, as noise occurs in both wax and basswood regions. There is some difference between the CT number in the RC MVCBCT and the CT number that corresponds to the actual density of the material. As expected, the uniformity within the sphere improves as the dose is increased from 5 to 10 MU (from upper right to lower left panel), and as one goes to a softer energy spectrum (from lower left, TBL, to lower right, IBL). The smallest insert is identifiable in images reconstructed using IBL or images reconstructed from 359 projections. Using a wider range of projections and IBL improves the imaging quality. Fig. 3. One coronal slice of the phantom imaged using kVCT (top-left), RC MVCBCT with 5 MU TBL (top-right), RC MVCBCT with 10 MU TBL (bottom-left), and RC MVCBCT with 10 MU IBL (bottom-right). Fig. 3. One coronal slice of the phantom imaged using kVCT (top-left), RC MVCBCT with 5 MU TBL (top-right), RC MVCBCT with 10 MU TBL (bottom-left), and RC MVCBCT with 10 MU IBL (bottom-right). Journal of Applied Clinical Medical Physics, Vol. 12, No. 2, Spring 2011 Chen et al.: Respiratory correlated MVCBCT 207 207 Fig. 4. Imaging profile of the two largest inserts in FE phase images using kVCT (top-left), RC MVCBCT with 5 MU TBL (top-right), RC MVCBCT with 10 MU TBL (bottom-left), and RC MVCBCT with 10 MU IBL (bottom-right) Fig. 4. Imaging profile of the two largest inserts in FE phase images using kVCT (top-left), RC MVCBCT with 5 MU TBL (top-right), RC MVCBCT with 10 MU TBL (bottom-left), and RC MVCBCT with 10 MU IBL (bottom-right) Journal of Applied Clinical Medical Physics, Vol. 12, No. 2, Spring 2011 B.2 Volume determination Relative error in volume as a function of the phantom insert diameter. Fig. 5. Relative error in volume as a function of the phantom insert diameter. We further reclassified the results based on different imaging parameters to study their influ ence on the accuracy of volume determination. Tables 1 to 4 show the averages and standard deviations of relative errors belonging to different subsets of imaging parameters, including different respiratory states (Table 1), number of projections (Table 2), imaging dose (Table 3) and source energy (Table 4). In Table 1, the relative error of static objects using standard MVCBCT is significantly smaller than that for FE or FI images using RC reconstruction. The error using static object MVCBCT data is even comparable to that of the kVCT for the four larger inserts. This is, in part, a con sequence of using fewer projections, as can be seen as well in Table 2 where the relative errors for a complete rotation (359°) are lower than those for a 200° arc. However, one would expect that since the FE phase has more projections (70) than the FI phase (40), the FE phase should have better accuracy. This seems to be true only for the smallest sphere, while for the larger spheres, the FI phase is slightly better. This could be a consequence of the actual residual mo tion in the reconstruction. Although the projection sorting algorithm used a 3 mm window, the actual residual motion could be slightly smaller for the FI phase than for the FE phase. In fact, for the FI phase, typically only two projections were selected per respiratory cycle. With fewer projections in the FI phase, the likelihood of spanning the entire 3 mm window is lower. It is very likely that more projections available in a wider range of angles for each phase may reduce the view aliasing artifacts for tumors, although the effect of these artifacts on volume determination for spherical objects seems to be less of an issue than residual motion. However, using a 359° rotation increases the image acquisition time. This presents a compromise between reducing setup time and finding a more accurate protocol. Tables 3 and 4 present counterintuitive results. One would expect higher imaging doses to produce better images and, hence, improved volume determination. Similarly, one would expect softer energies to yield lower volume errors due to improved image quality. B.2 Volume determination o u e dete at o We use the relative error to measure the accuracy of volume determination. The relative error is defined as the normalized difference with the nominal designed value: rror designed designed actual V V V / (2) Relative Error designed designed actual V V V / (2) Relative Error designed designed actual V V V / (2) where Vactual is the volume measured from the contours and Vdesigned is the nominal designed volume. Figure 5 compares the average and standard deviation of the relative error in volume where Vactual is the volume measured from the contours and Vdesigned is the nominal designed volume. Figure 5 compares the average and standard deviation of the relative error in volume for all the kVCT images and RC MVCBCT images. For the planning CT, the error is within 10% for all the inserts of different sizes. For RC MVCBCT, an inverse relationship between object size and relative error is present. Image degradation due to view aliasing artifacts and residual motion only affects the apparent size of the border region for large objects, but may affect the small object entirely. The image pixel dimension also affects smaller objects, since it is a larger fraction of the object’s diameter. The residual motion of 3 mm also has a greater effect on smaller objects. The average error of the four larger inserts is about 10%, but er rors increase significantly when the object diameter is less than 1 cm, indicating that volume measurement in RC MVCBCT is not suitable for small objects. For larger tumors, we have observed tumor volume reduction through a course of treatment using methods similar to what is described here (see Fig. 6 in Reference 14), so RC MVCBCT could provide tumor re sponse assessments for tumors larger than 2 cm in diameter, as verified by 4D kVCT imaging. (14) However, the case study was for a regular breather who provided a sufficient distribution of uniformly spaced projections for RC MVCBCT. Irregular breathers will most likely have fewer projections with irregular spacing and could induce more artifacts. Further studies with irregular breathing patterns programmed into the phantom would be needed to determine the limitations on these situations. Chen et al.: Respiratory correlated MVCBCT 208 208 Fig. 5. Relative error in volume as a function of the phantom insert diameter. Fig. 5. Journal of Applied Clinical Medical Physics, Vol. 12, No. 2, Spring 2011 B.2 Volume determination Average and standard deviation of relative volume error for 5 and 10 MU. Table 4. Average and standard deviation of relative volume error for different energies. Sphere Diameter (cm) kVCT IBL MVCBCT TBL MVCBCT 3.81 4.02%±1.04% 4.81%±0.85% 4.37%±3.02% 3.18 3.80%±1.10% 6.65%±3.79% 5.72%±3.71% 2.54 4.69%±2.94% 8.36%±5.64% 5.86%±5.13% 1.91 5.66%±2.28% 14.8%±9.76% 9.21%±7.55% 0.95 6.37%±3.89% 32.5%±20.1% 20.3%±17.6% 0.4 7.98%±2.76% 45.0%±62.8% 45.1%±37.3% Table 4. Average and standard deviation of relative volume error for different energies. B.2 Volume determination Figure 3 shows how image quality improves according to this expected pattern. Tables 3 and 4, however, show the opposite trend. The differences, however, are within the standard deviations. Within experimental error, they essentially produce the same result. It is possible that the amount of residual motion varies quite a bit due to the random starting phase for image acquisition, and this is just enough to affect the results. Chen et al.: Respiratory correlated MVCBCT 209 209 Table 1. Average and standard deviation of relative volume error for FE and FI phases. Sphere Diameter (cm) Static FE Phase FI Phase 3.81 3.02%±1.53% 5.81%±2.03% 4.74%±3.12% 3.18 2.60%±2.28% 8.42%±2.95% 7.08%±3.03% 2.54 3.08%±2.51% 10.6%±5.20% 6.43%±5.26% 1.91 7.95%±10.3% 15.8%±5.65% 9.52%±8.11% 0.95 8.11%±6.73% 33.6%±20.3% 31.4%±16.4% 0.48 16.7%±10.1% 27.6%±19.9% 100%±48.3% Table 2. Average and standard deviation of relative volume error for different arcs. Sphere Diameter (cm) 200° 359° 3.81 2.84%±1.70% 5.91%±3.40% 3.18 6.33%±3.81% 5.12%±3.87% 2.54 8.09%±6.43% 3.62%±2.14% 1.91 11.8%±9.47% 6.58%±4.38% 0.95 21.3%±18.8% 19.3%±18.1% 0.48 55.8%±48.8% 36.2%±25.7% Table 3. Average and standard deviation of relative volume error for 5 and 10 MU. Sphere Diameter (cm) 5 MU 10 MU 3.81 3.66%±2.06% 5.38%±2.69% 3.18 4.52%±3.15% 7.54%±3.64% 2.54 5.29%±4.33% 8.08%±6.01% 1.91 8.20%±5.91% 14.0%±10.0% 0.95 23.9%±18.0% 24.7%±20.7% 0.48 42.1%±52.8% 47.7%±41.8% Table 4. Average and standard deviation of relative volume error for different energies. Sphere Diameter (cm) kVCT IBL MVCBCT TBL MVCBCT 3.81 4.02%±1.04% 4.81%±0.85% 4.37%±3.02% 3.18 3.80%±1.10% 6.65%±3.79% 5.72%±3.71% 2.54 4.69%±2.94% 8.36%±5.64% 5.86%±5.13% 1.91 5.66%±2.28% 14.8%±9.76% 9.21%±7.55% 0.95 6.37%±3.89% 32.5%±20.1% 20.3%±17.6% 0.4 7.98%±2.76% 45.0%±62.8% 45.1%±37.3% Table 1. Average and standard deviation of relative volume error for FE and FI phases. Sphere Diameter (cm) Static FE Phase FI Phase 3.81 3.02%±1.53% 5.81%±2.03% 4.74%±3.12% 3.18 2.60%±2.28% 8.42%±2.95% 7.08%±3.03% 2.54 3.08%±2.51% 10.6%±5.20% 6.43%±5.26% 1.91 7.95%±10.3% 15.8%±5.65% 9.52%±8.11% 0.95 8.11%±6.73% 33.6%±20.3% 31.4%±16.4% 0.48 16.7%±10.1% 27.6%±19.9% 100%±48.3% Table 1. Average and standard deviation of relative volume error for FE and FI phases. Table 2. Average and standard deviation of relative volume error for different arcs. Sphere Diameter (cm) 200° 359° 3.81 2.84%±1.70% 5.91%±3.40% 3.18 6.33%±3.81% 5.12%±3.87% 2.54 8.09%±6.43% 3.62%±2.14% 1.91 11.8%±9.47% 6.58%±4.38% 0.95 21.3%±18.8% 19.3%±18.1% 0.48 55.8%±48.8% 36.2%±25.7% Table 2. Average and standard deviation of relative volume error for different arcs. Table 3. Average and standard deviation of relative volume error for 5 and 10 MU. Sphere Diameter (cm) 5 MU 10 MU 3.81 3.66%±2.06% 5.38%±2.69% 3.18 4.52%±3.15% 7.54%±3.64% 2.54 5.29%±4.33% 8.08%±6.01% 1.91 8.20%±5.91% 14.0%±10.0% 0.95 23.9%±18.0% 24.7%±20.7% 0.48 42.1%±52.8% 47.7%±41.8% Table 3. Journal of Applied Clinical Medical Physics, Vol. 12, No. 2, Spring 2011 C.3 Motion quantification C.3 Motion quantification i Similar to volume determination, we also use relative error to present the normalized accuracy of motion quantification, expressed as: Relative Error designed designed actual M M M / (3) Relative Error designed designed actual M M M / (3) (3) where M represents the displacement of the centroid between FE and FI respiratory states and the subscripts are consistent with those in Eq. (2). Figure 6 shows the average and standard deviation of the relative error of the motion of the six spherical inserts when using kVCT and RC MVCBCT. It should be noted that there is only one kVCT scan of a moving object. The measured displacement is very accurate for this kVCT scan. The three largest inserts have Journal of Applied Clinical Medical Physics, Vol. 12, No. 2, Spring 2011 Chen et al.: Respiratory correlated MVCBCT 210 210 exactly the same motion measurement as the nominal designed value of 30 mm. The error for the smallest of the three inserts is within 2%. For RC MVCBCT, the errors for the five larg est inserts are all about 5%. The error is slightly larger for the smallest insert at 6.8%. All the relative error of motion is within 10%, which correlates well with the 10% amplitude interval in amplitude-based projection sorting. Since the difference of motion quantification between different inserts is small, we present the average and standard deviation of the relative error in Table 5 by summarizing all the inserts belonging to the same type of RC MVCBCT scan. The nomenclature for the various imaging parameters is consistent with that of the previous section. The difference in relative error is very small (0.3%) between different imaging parameters, indicating that motion quantification is comparably more robust and insensitive to variation in manual contouring than volume de termination. It is feasible to quantify tumor motion amplitudes between FE and FI respiratory states by using RC MVCBCT, even for objects with a diameter of about 0.5 cm. It should be Fig. 6. Relative error in displacement as a function of the phantom insert diameter. Fig. 6. Relative error in displacement as a function of the phantom insert diameter. Table 5. Average and standard deviation of relative error of motion using kVCT and RC MVCBCT. Journal of Applied Clinical Medical Physics, Vol. 12, No. 2, Spring 2011 D.3 Application to patients Because the study used spheres, they may be less susceptible to view aliasing artifacts. Volume determination for tumors may be less accurate than what is noted here, but these studies at least establish a lower limit on tumor sizes that can be evaluated with RC MVCBCT. For motion assessment, however, since the centroid of the tumor is used, it will be less sensitive to the identification of the tumor edges. This could explain why the accuracy for motion assessment is more robust. This could carry over into patients as well, and manual identification of the tumor in projection images for a test patient(12,14) showed that the tumor displacements between full inhale and exhale, averaged over all imaged respiratory cycles, was within 3 to 5 mm of the value determined from RC MVCBCT. When one determines volumes and motion within a patient, they need to know whether their imaging methods are causing errors. Using spheres allows us to reduce the possible errors com ing from user variability in contouring, and instead allows us to determine possible errors that come from the reconstruction of fewer projections than what one would normally expect. Our previous feasibility study for a patient(14) had to rely on comparison of the RC MVCBCT results against the results from the 4D planning CT. While volumes can be compared for MVCBCT and 4DCT images taken on the same day, one can not compare the amplitude of motion from full exhale to full inhale since the respiratory motion for the two separate imaging sessions may be different. This is the best one can do for patient studies, since the true motion and volume of the tumor can not be established; even the 4D planning CT will contain residual motion and artifacts, and its usefulness in serving as ground truth is subject to these errors. The results in our phantom studies provide some lower bound on errors, since the error may be greater due to inaccuracies in contouring nonspherical objects and reconstruction errors arising from irregular breathing patterns. More work is needed to verify our results using phantoms with realistically-shaped tumors. With respect to imaging the IHDA in order to sort projections, one must carefully choose the CBCT isocenter. For very lateral lesions on the Siemens MVision system (27.4 cm FOV at isocenter), or for large patients, we often have to shift the patient about 5 cm medially to avoid collisions. D.3 Application to patients This has not caused problems for viewing the ipsilateral hemidiaphragm apex in all the protocols that we have used clinically. On other CBCT systems, a half-fan option is available, and this may present problems for finding the IHDA. To use the techniques described here for patients, one must carefully understand the limitations of their imaging devices and protocols. A protocol using an angular range that keeps the IHDA in view would be preferable. The methods presented here can also be applied to test these alternative protocols. C.3 Motion quantification Imaging Parameter Relative Error kVCT 0.61%±0.83% All RC MVCBCT 5.06%±2.14% IBL 5.00%±2.07% TBL 5.09%±2.22% 5MU 4.90%±2.21% 10MU 5.20%±2.13% TBL 200 4.94%±2.41% TBL 359 5.22%±2.13% Fig. 6. Relative error in displacement as a function of the phantom insert diameter. Table 5. Average and standard deviation of relative error of motion using kVCT and RC MVCBCT. Imaging Parameter Relative Error kVCT 0.61%±0.83% All RC MVCBCT 5.06%±2.14% IBL 5.00%±2.07% TBL 5.09%±2.22% 5MU 4.90%±2.21% 10MU 5.20%±2.13% TBL 200 4.94%±2.41% TBL 359 5.22%±2.13% Table 5. Average and standard deviation of relative error of motion using kVCT and RC MVCBCT. Table 5. Average and standard deviation of relative error of motion using kVCT and RC MVCBCT. Chen et al.: Respiratory correlated MVCBCT 211 211 noted that typical tumor motion amplitudes range from 1.0 cm to 2.5 cm, which is smaller than the phantom motion in this study. This gives us confidence to extend the practice of evaluating motion between the FE and FI respiratory states of 4DCT data to cases of RC MVCBCT taken immediately prior to treatment, to determine if the maximum motion is consistent with the one determined at the time of treatment planning. Journal of Applied Clinical Medical Physics, Vol. 12, No. 2, Spring 2011 References 1. Berbeco R, Mostafavi H, Sharp G, Jiang S. Tumor tracking in the absence of radiopaque markers. In: Yi B, Ahn S, Choi E, Ha S, editors. The 14th International Conference on the Use of Computers in Radiation Therapy. Seoul, Korea: Jeong Publishing; 2004. p. 433–36. g g p 2. Kothary N, Dieterich S, Louie J, Chang D, Hofmann L, Sze D. 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Phys Med Biol. 2009;54(13):4195–212. 14. Chen M, Siochi RA. A clinical feasibility study on respiratory sorted megavoltage cone beam ct. In: Brown M, Bruijne MD, Ding K, et al. editors. IV. Conclusions In this study, we have evaluated the feasibility of using RC MVCBCT to quantify object motion and size. The primary source of object boundary detection errors is the reconstruction error induced by missing projections. Better accuracy can be achieved for volume determination when the object is sufficiently large (a minimum diameter of 2 cm). For larger tumors, response as sessment in terms of volume reduction is feasible for regular breathers, at least until the tumor shrinks down to 2 cm, where a 4D kVCT would be needed for volume determination. Motion measurement results, on the other hand, are more robust. The relative error is within 10% for even the smallest object, and it is independent of energy, dose and protocol. 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W3012213213.txt	https://bmcplantbiol.biomedcentral.com/track/pdf/10.1186/s12870-020-2322-9	en		Identification of the regulatory networks and hub genes controlling alfalfa floral pigmentation variation using RNA-sequencing analysis	BMC plant biology	2,020	cc-by	9,188	Duan et al. BMC Plant Biology (2020) 20:110 https://doi.org/10.1186/s12870-020-2322-9 RESEARCH ARTICLE Open Access Identification of the regulatory networks and hub genes controlling alfalfa floral pigmentation variation using RNAsequencing analysis Hui-Rong Duan1, Li-Rong Wang2, Guang-Xin Cui1, Xue-Hui Zhou1, Xiao-Rong Duan3 and Hong-Shan Yang1* Abstract Background: To understand the gene expression networks controlling flower color formation in alfalfa, flowers anthocyanins were identified using two materials with contrasting flower colors, namely Defu and Zhongtian No. 3, and transcriptome analyses of PacBio full-length sequencing combined with RNA sequencing were performed, across four flower developmental stages. Results: Malvidin and petunidin glycoside derivatives were the major anthocyanins in the flowers of Defu, which were lacking in the flowers of Zhongtian No. 3. The two transcriptomic datasets provided a comprehensive and systems-level view on the dynamic gene expression networks underpinning alfalfa flower color formation. By weighted gene coexpression network analyses, we identified candidate genes and hub genes from the modules closely related to floral developmental stages. PAL, 4CL, CHS, CHR, F3’H, DFR, and UFGT were enriched in the important modules. Additionally, PAL6, PAL9, 4CL18, CHS2, 4 and 8 were identified as hub genes. Thus, a hypothesis explaining the lack of purple color in the flower of Zhongtian No. 3 was proposed. Conclusions: These analyses identified a large number of potential key regulators controlling flower color pigmentation, thereby providing new insights into the molecular networks underlying alfalfa flower development. Keywords: PacBio Iso-Seq, Transcriptome, Floral pigmentation, Alfalfa, Cream color, Hub gene Background Flower color is an important horticultural trait of higher plants [1]. Variation in flower color can fulfill an important ecological function by attracting pollinator’s visitation and influencing reproductive success in flowering plants [2], can protect the plant and its reproductive organs from UV damage, pests, and pathogens [3, 4], and has been of paramount importance in plant evolution [5, 6]. Furthermore, flower color is associated with the * Correspondence: yanghsh123@126.com 1 Lanzhou Institute of Husbandry and Pharmaceutical Science, Chinese Academy of Agricultural Sciences, Lanzhou, China Full list of author information is available at the end of the article agronomic characters of plants directly or indirectly, and classical breeding methods have been extensively used to develop cultivars with flowers varying in color [7]. Three species of the genus Medicago L. are the most typical representatives of meadow ecosystems in the central part of European Russia: alfalfa (M. sativa L.), yellow lucerne (M. falcata L.), and black medic (M. lupulina L.), which are widely cultivated and grow easily in the wild [8–10]. The obvious differences in these species are their morphological features, among which flower color is the main trait used to distinguish them [11–13]. Understanding the differences in the growth period, botanical characteristics, agronomic characteristics, quality, © The Author(s). 2020 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://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Duan et al. BMC Plant Biology (2020) 20:110 and photosynthetic characteristics of different alfalfa germplasm materials associated with flower color would have great significance in alfalfa breeding [14, 15]. Of the above-mentioned Medicago species, purpleflowered alfalfa is the most productive perennial legume with high biomass productivity, an excellent nutritional profile, and adequate persistence [16, 17]. Yellow lucerne, which has yellow flowers, is closely related to alfalfa and exhibits better cold tolerance than alfalfa [18, 19]. Furthermore, the wild plants of M. varia with multiple flower color variations possess potential resistance to biotic and abiotic stressors [20]. The availability of abundant floral pigment mutants in Medicago species provides an ideal system for investigating the relationship between flower color and the stress resistance of alfalfa. Understanding the molecular mechanisms of flower color formation in alfalfa and identifying related key genes would contribute to the construction of an alfalfa core germplasm. Flavonoids, carotenoids, and betalains are the three major floral pigments [21, 22]. Flavonoids, especially anthocyanidins, contribute to the pigmentation of flowers in plants [23, 24]. In the process of flower blooming, a somatic mutation from the recessive white to the pigmented revertant allele occurs, and flower variegation is inevitably the result of the differential expression of regulatory genes [25, 26]. To date, flower color-associated genes have been identified in many ornamental plants and in numerous studies, such as grape hyacinth, Camellia nitidissima, Erysimum cheiri, and Matthiola incana [27–29]. Using the crucial genes related to flower color formation to create new plant variety with special flower color, is circumvented by genetic engineering, while conventional breeding methods may be difficult to obtain the phenotype accurately [30]. For example, expression of the F3’5’H (flavonoid-3′, 5′-hydroxylase) gene in Rosa hybrida resulted in a transgenic rose variety with a novel bluish flower color not achieved by hybridization breeding [31]. By transferring antisense CHS (chalcone synthase) gene, a new petunia variety with white color was successfully obtained [32]. Although in many important ornamental crops, flower colors modification are already realized by molecular breeding, alfalfa varieties with special flower colors are often selected by natural selection for lacking the molecular mechanism of flower color formation. RNA sequencing (RNA-Seq) technology has provided unique insights into the molecular characteristics of non-model organisms without a reference genome, and a series of genes involved in flavonoid pigment biosynthesis and carotenoid biosynthesis have been systematically analyzed [1, 33, 34]. However, the limitations of short-read sequencing lead to a number of computational challenges and hamper transcript reconstruction and the detection of splice events [35]. Chao et al. [36] found that, the PacBio Iso-Seq (isoform sequencing) platform could refine the data of short-read sequencing, Page 2 of 17 including cataloging and quantifying transcripts and searching more alternatively spliced events. Here, we used PacBio Iso-Seq combined RNA-Seq to identify specific genes related to flower color variation in two alfalfa materials with different flower colors. The dataset provides a comprehensive and system-level overview of the dynamic gene expression networks and their potential roles in controlling flower pigmentation. Using weighted gene coexpression network analysis (WGCNA), we identified modules of co-expressed genes and candidate hub genes for alfalfa with different flower colors. This work provides important insights into the molecular networks underlying alfalfa with cream flower pigmentation. Methods Plant material High quality seeds of alfalfa cultivar Defu (C) were sent to the space by the “Shenzhou 3” recoverable spacecraft that flew in the space for 7 days (March 25th to 31th 2002). 1/3 of these space exposed seeds were planted alongside the control C in Xiguoyuan of Lanzhou city in 2009, a single plant with cream flower color was found and its seeds were collected individually. After planting the seeds in Qinwangchuan of Lanzhou city in 2010 isolatedly, 29 plants from the F1 generation possessed cream flower color. The seeds were collected, mixed and planted for another three generations, a mutant line with a cream flower color from F4 generation was confirmed in 2014. Compared to the control C, the mutant line exhibited stable cream flower color in the blooming period, which was named as “Zhongtian No. 3” (M). The original seeds of M were conserved in Lanzhou Institute of Husbandry and Pharmaceutical Science, Chinese Academy of Agricultural Sciences. The alfalfa cultivar C and M were planted in the Dawashan experimental station (36°02′20′′ N, 103°44′36′′ E, 1697 H) of Lanzhou, Gansu, China in April 22th 2018. All seedlings of the same age were cultivated on homogenous loessal soil under the same management practices (soil management, irrigation, fertilization, and disease control). The petals of C and M were collected from four different development stages. The four stages were defined according to qualitative observations of the floral organs: S1 (the stage of the floret separating and the calyx packaging the petals), S2 (the stage of the petals appearing between the calyx lobes, with the length of the petals not exceeding more than 2 mm of the calyx), S3 (the stage where the petals exceed the calyx by 2 mm or more, the keel is still wrapped by the vexil, and during which the petals were just beginning to accumulate pigmentation), and S4 (the stage where the floret was in full bloom, with fully pigmented petals) (Fig. 1a). The four stages were assessed simultaneously for the indefinite inflorescence of alfalfa. Samples were harvested at the same time of day (9–11 AM) on July 4, 2018. Representative floral organs in each stage from three Duan et al. BMC Plant Biology (2020) 20:110 Page 3 of 17 Fig. 1 Phenotypes and anthocyanins compounds of the alfalfa materials. a Phenotypes of the different flower development stages from Defu and Zhongtian No. 3. b Anthocyanin compound contents in the peels of the two cultivars in S4. C, Defu; M, Zhongtian No. 3. Error bars indicate SEs different plants were combined to form a sample, and three biological replicates were used for each floral development stage. All the samples in each stage endowed the same characteristics both of size and flower color, which were prepared for anthocyanin contents measurement and Illumina sequencing. Tissues of the leaves, shoots, stems, roots, flowers from the four different developmental stages above, and the young fruits from three C plants, were collected and pooled together in approximately equivalent weights. The mixed sample from 9 different tissues was then prepared for PacBio full-length sequencing. The samples were immediately frozen in liquid nitrogen and stored at − 80 °C until use. High-performance liquid chromatography analysis (HPLC) of anthocyanins For anthocyanin extraction, fresh petal tissue was obtained from the fully-opened alfalfa flower in C-S4 and M-S4. Briefly, 0.5 g tissue from each sample was grounded in 1 mL of 98% methanol containing 1.6% formic acid at 4 °C. After 30 min of ultrasonic extraction, samples were centrifuged for 10 min at 12000 g, following with the supernatants were transferred to fresh tubes and the residual was extracted again. The supernatants were then combined and filtered through 0.45 mm nylon filters (Millipore). The standard substances included delphinidin 3-O-glucoside, cyanidin 3-O-glucoside, pelargonidin 3-O-glucoside, Duan et al. BMC Plant Biology (2020) 20:110 peonidin 3-O-glucoside, malvidin 3-O-glucoside, and petunidin 3-O-glucoside (ZZBIO Co., Ltd., Shanghai). According to the method of Tripathi et al. [24], 10 μL of the extract was analyzed using HPLC (Rigol L-3000, China). Mean values and standard errors (SEs) were obtained from three biological replicates. RNA quantification and assessment of quality Total RNA was extracted using a mirVana miRNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA). RNA degradation and contamination were assessed on 1% agarose gels. The RNA quantity and quality were determined using a NanoDrop 2000 instrument (Thermo Fisher Scientific, Waltham, MA, USA), and RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). PacBio Iso-Seq library preparation and sequencing The sequencing library of 1 μg total RNA from the mixed sample of C was performed using the SMRTbell™ Template Prep Kit 1.0-SPv3 (Pacific Biosciences, Menlo Park, CA, USA). The amount and concentration of the final library was verified with a Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). The size and purity of the library was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Following the Sequel Binding Kit 2.0 (Pacific Bioscience, USA) instruction for primer annealing and polymerase binding, the magbead-loaded SMRTbell template was performed on a PacBio Sequel instrument at Shanghai Oe Biotech Co., Ltd. (Shanghai, China). Page 4 of 17 the software GMAP (http://www.molecularevolution.org/ software/genomics/gmap). Afterward, redundant isoforms were then removed to generate a high-quality transcript dataset using the program TOFU (http://github.com/ PacificBiosciences/cDNA_primer/) with an identify value of 0.85. The integrity of the transcript dataset was evaluated using the software BUSCO (v3.0.1) (https://busco.ezlab.org/). All identified non-redundant transcripts were searched by BLASTX (E-value ≤1e-5) against the protein databases of Non-redundant (NR), SWISS-PROT, and Kyoto Encyclopedia of Genes and Genomes (KEGG), and the putative coding sequences (CDS) were confirmed from the highest ranked proteins. Furthermore, the CDS of the unmatched transcripts were predicted by the package ESTScan. The non-redundant transcripts were compared to the PlantTFDB (http://planttfdb.cbi.pku.edu.cn/index.php) and the AnimalTFDB (http://bioinfo.life.hust.edu.cn/ AnimalTFDB/) databases using BLAST to obtain the annotation information of the transcription factors (TFs). The software AStalavista [37] was used to detect alternative splicing events in the sample. Transcripts with lengths greater than 200 bp were selected as lncRNA candidates, from which the open reading frames (ORFs) greater than 300 bp were filtered out. Putative proteincoding RNAs were filtered out using a minimum exon length and number threshold. LncRNAs were further screened using four computational approaches, including CPC2, CNCI, Pfam and PLEK. Illumina data analysis Illumina transcriptome library preparation and sequencing The triplicate biological samples of two materials at the four stages yielded 24 non directional cDNA libraries (CS1, C-S2, C-S3, C-S4, M-S1, M-S2, M-S3 and M-S4), which were obtained from 4 μg of total RNA. The size and purity of the libraries were tested with an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The final libraries were generated using an Illumina HiSeq™ XTen instrument at Shanghai Oe biotech co., ltd. (Shanghai, China) PacBio data analysis After the quality control of Isoseq (https://github.com/ PacificBiosciences/IsoSeq_SA3nUP/wiki#datapub), including generation of circular consensus sequences (CCS), classification, and cluster analysis, high-quality consensus isoforms and low quality isoforms were recognized from the original subreads. Error correction of the high and low quality combined isoforms was conducted using the RNASeq data with the software LoRDEC. The corrected isoforms were compared with the reference genome using Twenty-four independent cDNA libraries of flowers for C and M at different developmental stages were constructed according to a tag-based digital gene expression (DGE) system protocol. After removing low quality tags, including tags with unknown nucleotide “Ns”, empty tags, and tags with only one copy number, the clean tags were mapped to our transcriptome reference database. For the analysis of gene expression, the number of clean tags for each gene was calculated and normalized to FPKM (Fragments Per Kilobase of transcript per Million mapped reads). A P-value ≤0.05 in multiple tests and an absolute log2 fold change value ≥2 were used as thresholds for determining significant differences in gene expression. Weighted gene co-expression network analysis The R package WGCNA was used to identify the modules of highly correlated genes based on the normalized expression matrix data [38]. The R package was used to filter the genes based on genes expression and variance (standard deviation ≤0.5). A total of 16,581 genes were ultimately remained. By conducting the function pickSoftThreshold, the soft threshold value of the correlation matrix was Duan et al. BMC Plant Biology (2020) 20:110 Page 5 of 17 Table 1 PacBio Iso-Seq output statistics Item Total number Total base (bp) Min length Max length Mean length Subreads 14328236 25008789438 50 106281 1745.419983 High quality isoforms 16340 33239138 336 8595 2034.218972 Low quality isoforms 124655 252521297 116 14650 2025.761478 Non-redundant isoforms 33899 72758476 156 14671 2146.331042 selected as 16, and the correlation coefficient was 0.83. The topological overlap (TO) matrix was generated by the TOM similarity algorithm, and then transcripts were hierarchically clustered with Hybrid Tree Cut algorithm 60 [29]. The first principal component was represented by the module eigengene. Real-time quantitative (RT-q) PCR validation Twelve selected DEGs involved in flavonoid synthesis were determined by RT-qPCR. Total RNA was extracted from the 24 samples (in triplicate) as described above. First-strand cDNA was synthesized from 0.1 μg of total RNA by the manufacturer’s instruction (Vazyme, R223– 01). The reactions were performed using a QuantiFast® SYBR® Green PCR Kit (Qiagen, Germany), and RTqPCR was carried out on an Applied Biosystems QuantStudio™ 5 platform (Thermo Fisher Scientific, Waltham, MA, USA). The primers were designed with the Primer premier 5.0 software and synthesized by TsingKe Biological Technology Co., Ltd. (Xi’an, China) (Table S1). Rer1 (JZ818481) was used as an internal standard [39]. The relative expression levels of genes were calculated using the 2−ΔΔCt method [40]. > 99%). Most of the corrected isoforms (98.52%) were mapped to the Medicago genome (M. truncatula Mt4.0v2) using GMAP, and TOFU processing yielded 33,908 non-redundant isoforms (Table 1). The nonredundant transcript isoforms were used in subsequent analyses. We compared the 33,908 isoforms against the Medicago genome set (Mt4.0v2), and 7784 (23%) new isoforms of annotated genes (ratio coverage < 50%) were obtained using MatchAnnot software (https://github. com/TomSkelly/MatchAnnot), and 513 novel isoforms were obtained that did not overlap with any annotated genes. To determine if the 513 novel isoforms were present in other plants, we conducted BLASTX searches against Swiss-Prot (E-value ≤ e− 10, see Methods). In total, 309 (60.23%) of these isoforms were annotated in the Swiss-Prot database, and the remaining isoforms were unannotated (Table S2). The numbers of isoforms distributed across the five main alternative splicing events were analyzed. IR (intron retention) was the most represented, accounting for 27.5% of alternative splicing transcripts (Fig. 2). MXE Statistical analysis All RT-qPCR data were expressed as means ± SE (n = 3). Results Quantification of anthocyanidins We quantified six anthocyanidins (delphinidin, cyanidin, pelargonidin, peonidin, malvidin, and petunidin) known to be involved in color development. Two high contents of malvidin and petunidin were detected in C-S4, the contents of which were 7.0 μg/g fresh weight (FW) and 2.5 μg/g FW, respectively. Otherwise, no color anthocyanidins were detected in the cream flowers of M-S4 (Fig. 1b). Sequencing and analysis of the floral transcriptome using the PacBio Iso-Seq platform To identify transcripts that are as long as possible, the transcriptome of the mixed sample from different tissues of C (see Methods for details) were sequenced by the Iso-Seq system, yielding 14.33 million subreads. After the quality control of Isoseq, 140,995 isoforms were obtained, including 16,340 high-quality isoforms (accuracy Fig. 2 Alternative splicing events from the Iso-Seq. IR, intron retention. A3SS, alternative 3ˊ splice sites. ES, exon skipping/ inclusion. A5SS, alternative 5ˊ splice sites. MXE, mutually exclusive exons Duan et al. BMC Plant Biology (2020) 20:110 (mutually exclusive exons) were being the least, accounting for 1.9% of alternative splicing transcripts (Fig. 2). By filtering and excluding transcripts with an ORF of more than 300 bp, 143 lncRNAs were finally obtained. The lncRNAs exhibited a wide length range from 202 bp to 2733 bp, and most of which (72%) were shorter than 700 bp. The average length of the lncRNAs (682 bp) was much shorter than the average length of all 33,908 isoforms (2146 bp). Sequencing and analysis of the floral transcriptome using the Illumina platform For performance comparison and validation purposes, we also independently generated standard short read RNA-Seq data on the Illumina HiSeq™ XTen sequencing platform. Four floral organs from different developmental stages were sampled from both varieties. To this end, identification of DEGs from different floral organs could contribute to the understanding of the differential control of flower pigmentation. RNA-Seq analysis was performed on the samples described above with three biological replicates for each. When compared to the PacBio transcript isoforms by BLASTN (coverage ≥0.85, e-value ≤1e-20, pairwise identity ≥90%, min bit score ≥ 100), 36% of the transcript contigs (29,662 contigs) exhibited similarity to 99% of the PacBio transcript isoforms (33,518 isoforms). There were 64% of the transcript contigs (53,870) and 1% of PacBio transcript isoforms (381 isoforms) that were unique to each of the datasets (Fig. 3). Transcripts with normalized reads lower than 0.5 FPKM were removed from the analysis. In total, 28,365, 28,242, 28,088, and 28,185 transcripts were found to be expressed in C-S1, C-S2, C-S3, and C-S4, respectively. Similarly, 27,810, 27,726, 27,711, and 27,878 transcripts were identified in the samples from the respective stages of M. The numbers of expressed transcripts distributed Page 6 of 17 in the 0.5–1 FPKM range, 1–10 FPKM range and ≥ 10 FPKM range are indicated in Fig. 4a. Principal component analysis (PCA) revealed that the 24 samples could be clearly assigned to eight groups as C-S1, C-S2, C-S3 C-S4, M-S1, M-S2, M-S3 and M-S4 (Fig. 4b). The samples of C and M from the same stage exhibited a distant clustering relationship, suggesting that the overall transcriptome profile is evidently different for C and M at each developmental stage (Fig. 4b). DEGs during the flower developments of alfalfa materials with purple and cream flower The differences in gene expression were analyzed by comparing the four different floral development stages, using the thresholds of false discovery rate (FDR) value < 0.05 and fold change > 2. In total, 2591, 1925 and 3771 DEGs were identified between C-S2 vs C-S1, C-S3 vs CS2, C-S4 vs C-S3, respectively (Fig. 5a). Similarly, 3282, 1490 and 3868 DEGs were identified between M-S2 vs M-S1, M-S3 vs M-S2, M-S4 vs M-S3, respectively (Fig. 5b). Contrasting S2 with S1, the down-regulated unigenes of C and M were similar to the up-regulated unigenes. Differently, the up-regulated unigenes were dominant between S3 vs S2, as well as between S4 vs S3 in both C and M. In order to analyze the flower color formation differences in C and M, we compared the DEGs of C and M in the same flower development stage. In total, 4052, 4355, 3293, and 4181 DEGs were identified between MS1 vs C-S1, M-S2 vs C-S2, M-S3 vs C-S3, and M-S4 vs C-S4, respectively. Furthermore, 1693, 1707, 1511, and 2092 DEGs were up-regulated, respectively (Fig. 6). To identify the metabolic pathways related to flavonoid biosynthesis that were enriched, an analysis of KEGG pathway was conducted by comparing different flowering stages in C and M. With the flower blooming, the enriched pathways related to flavonoid biosynthesis increased evidently. Especially, between M-S4 vs C-S4, flavone and flavonol biosynthesis (ko00944), flavonoid biosynthesis (ko00941) and phenylpropanoid biosynthesis (ko00940) were enriched on the top 5 KEGG pathways (Figure S1), implying the crucial flower color formation stage. Transcriptional profiles of the genes related to flavonoid biosynthesis Fig. 3 Comparison of isoforms from the PacBio Iso-Seq data and contigs from the RNA-Seq data To determine the key genes involved in flavonoid biosynthesis, the genes with FPKM values lower than 5 were excluded. Phenylalanine ammonia-lyase (PAL, 15 isoforms), 4-coumarate: coenzyme A ligase (4CL, 27 isoforms), CHS (15 isoforms), chalcone isomerase (CHI, 3 isoforms), flavanone 3-hydroxylase (F3H) / flavonol synthesis (FLS) (3 isoforms), flavonoid 3′-monooxygenase Duan et al. BMC Plant Biology (2020) 20:110 Page 7 of 17 Fig. 4 Global gene expression statistics in different floral development stages. a Numbers of detected transcripts in each sample. b Principal components analysis (PCA) of the RNA-Seq data (F3′H, 5 isoforms), F3′5′H (1 isoform), dihydroflavonol 4-reductase (DFR, 5 isoforms), anthocyanidin synthase (ANS, 4 isoforms), and UDP-glucose: flavonoid 3-Oglucosyltransferase (UFGT, 23 isoforms) were identified (Table S3). The expression pattern of the total of 101 isoforms (encoding 11 enzymes) was displayed in the heatmap, and the isoforms showed different changes during flower development in both C and M (Fig. 7). Among these DEGs, most PAL genes showed downregulated expression changes in C, but up-regulated expression patterns in M. In general, the FPKM values of many PALs were significantly higher in C than M (Fig. 7). It is possible that these PALs may be crucial in the formation of flower colors. Most genes encoding 4CLs, CHSs, CHIs, FLS/F3Hs, F3’Hs, F3’5’Hs, ANSs, and UFGTs exhibited similar expression patterns in both C and M with flower blooming However, the FPKM values differed greatly between C and M, indicating differential expression abundance in C and M. Additionally, we found 4 DFRs with different expression changes in C and M (particularly DFR1 and DFR2), the FPKM values of which were evidently higher in C than M, implying Duan et al. BMC Plant Biology (2020) 20:110 Page 8 of 17 them. From WGCNA, 18 co-expression modules were constructed, of which, the grey 60 module was the largest module, consisting of 2520 unigenes, whereas the darkseagreen 4 module was the smallest, consisting of only 56 unigenes. The distribution of isoforms in each module (labeled with different colors) and module-trait correlation relationships is shown in Fig. 9. A number of modules displayed a close relationship with different stages. The most important modules of our concern were the modules enriched in the C or M group, especially in S4 of C and M, which could help to distinguish the flower color phenotype. The modules of interest were thus selected according to the criteria \|r\| > 0.5 and P < 0.05, and were further annotated by KEGG and GO analysis. The module of skyblue 3 displayed a close relationship with M-S4. In the skyblue 3 module, many pathways related to color formation were enriched (P < 0.01). Among them, flavonoid biosynthesis (ko00941) and phenylpropanoid biosynthesis (ko00940) were the top 2 pathways (Table S4). Furthermore, the modules of bisque 4 and turquoise exhibited a close relationship with M or C, the enriched pathways (P < 0.01) of which were summarized in Table S4. Candidates responsible for the loss of purple color in alfalfa with cream-colored flower Fig. 5 Number of DEGs between the different floral development stages. a DEGs of alfalfa cultivar C. b DEGs of alfalfa cultivar M. C, Defu; M, Zhongtian No. 3 their potential functions in color formation in different flowers (Fig. 7). Gene co-expression network analysis based on flower pigments To reveal the regulatory network correlated with the changes in the successive developmental stages across the two varieties, we constructed the co-expression modules analysis by WGCNA (Fig. 8). Co-expression networks were constructed on the basis of pairwise correlations of gene expression across all samples. Modules were defined as clusters of highly interconnected genes, and genes within the same cluster have high correlation coefficients among The expression patterns of 23 candidate genes according to the closed modules are indicated in Table 2. In summary, all 9 PALs were down-regulated during the flower ripening process in C, while in M-S4, they remained stable or declined initially and then increased. Additionally, their relative expression levels in S1-S3 of C were significantly higher than in M. Importantly, PAL6 and PAL9 were identified as candidate hub genes for the module of bisque 4. 4CL18 and 4CL22 were enriched in the module of bisque 4, and 4CL18 was identified as a candidate hub gene for this module. The much higher expression levels of 4CL18 in S1-S3 of C, which were evidently higher than M, were suggestive of a particularly important role for 4CL18 in the pathway. Four CHSs were enriched in the module of skyblue 3, in which, CHS2, CHS4, and CHS8 were identified as candidate hub genes. They possessed the same expression changes in different stages of C and M, and in the M-S4, the relative expression levels of CHS2, CHS4, and CHS8 were 2.1-, 1.3-, and 2.5-fold higher than in CS4. We also searched 3 CHRs enriched in these important modules, and found that the expression change patterns of CHR1, CHR2, and CHR3 were consistent with the enriched CHSs. Furthermore, F3’H4, DFR1, DFR2, UFGT22, and UFGT23 were enriched in these modules. In S1 and S2, the expression levels of F3’H4 were 1.2- and 2.0- fold higher in C than in M. With flower development in C, DFR1 was up-regulated and peaked at S3, however, DFR1 exhibited almost no expression in M. DFR2 was up- Duan et al. BMC Plant Biology (2020) 20:110 Page 9 of 17 Fig. 6 Comparison of the DEGs between the two cultivars. C, Defu; M, Zhongtian No. 3 Fig. 7 Expression heatmap of the DEGs of flavonoid biosynthesis. The expression of DEGs is displayed as log10 (FPKM+ 1). PAL, phenylalanine ammonia-lyase; 4CL, 4-coumarate: coenzyme A ligase; CHS, chalcone synthase; CHI, chalcone isomerase; FLS, flavonol synthesis; F3H, flavanone 3hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′5′-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; UFGT, UDP-glucose: flavonoid 3-O-glucosyltransferase Duan et al. BMC Plant Biology (2020) 20:110 Page 10 of 17 Fig. 8 Gene co-expression modules detected by WGCNA. The clustering dendrogram of the genes across all the samples exhibits dissimilarity based on topological overlap, together with the original module colors (dynamic tree cut) and assigned merged module colors (merged dynamic) regulated and peaked at S3 in C, however, it exhibited low expression abundance and remained stable in M. The expression levels of DFR1 and DFR2 were evidently higher in all of the stages of C than M. Higher expression levels in C were also found in UFGT22 and UFGT23 (Table 2). To further confirm these results and verify the expression of the above genes in the C and M, RT-qPCR was performed to analyze the expression patterns of 12 genes (Fig. 10). Most genes exhibited similar expression patterns between the RT-qPCR and RNA-Seq data, which confirmed the reliability of the RNA-Seq data. Discussion Anthocyanin identification from the peels of two different materials Color mutants are widely used in horticultural and other crops, especially those that are commonly propagated vegetatively, such as most fruit trees [41, 42]. Purple color in the flower petals of alfalfa (M. sativa L., M. falcata L. and their hybrids) is due to the presence of sap-soluble anthocyanins [43]. The floral anthocyanins of alfalfa have been widely studied. Lesins [44] identified alfalfa flower with three pigments as glycosides of petunidin, malvidin and delphinidin. Furthermore, Cooper and Elliott [45] identified alfalfa flower with three anthocyanins as 3,5-diglucosides of petunidin, malvidin and delphinidin. Differently, using HPLC, we only found that malvidin 3-O-glucoside and petunidin 3-O-glucoside in the purple flower of C, while no color pigment was detectable in the cream flowers of M (Fig. 1). The results suggest that the drastic differences in anthocyanin accumulation are a result of cultivar and genetic specificity. PacBio full-length sequencing extends the alfalfa annotation and increases the accuracy of transcript quantification Due to technical limitations, the reference genome of alfalfa is not presently available. Our current knowledge on the alfalfa transcriptome is mainly based on RNA-Seq gene expression data. Thus, the alfalfa transcriptome has not been fully characterized due to the lack of full-length cDNA. In this work, we used PacBio third-generation technology to annotate the sequences of the C cultivar, and analyzed the DEGs in different flower development stages of C and M using Illumina sequencing platform. We obtained 140,995 isoforms, including 513 novel isoforms. After comparison in Swiss-Prot, 204 new isoforms specific to alfalfa, but with unknown functions, were identified and Duan et al. BMC Plant Biology (2020) 20:110 Page 11 of 17 Fig. 9 Module-trait associations using WGCNA. Each row corresponds to a module eigengene and each column to a stage. Each cell contains the corresponding correlation and P-value. The table is color-coded by correlation, according to the color legend would be useful in future studies (Table S2). In transcriptome studies of populus, maize, and sorghum by single-molecule long-read sequencing, 59,977 (69%), 62,547 (57%) and 11,342 (41%) new isoforms were identified, respectively [36]. Due to species divergence, we only identified 23% new isoforms. However, our data demonstrated that PacBio full-length sequencing could provide a more comprehensive set of isoforms than next-generation sequencing. Through a genome-based reconstruction strategy, using the Medicago genome (M. truncatula Mt4.0v2) as a reference, the mapping ratio of the corrected isoforms by PacBio full-length sequencing was 98.52%. Unfortunately, the mapping ratio of the clean reads by RNA-Seq was less than 50% (data not shown). We also compared the match ratio of the isoforms and contigs, from which we found that 99% of the isoforms (33,518) could be matched to known unigenes, indicating that the results Duan et al. BMC Plant Biology (2020) 20:110 Page 12 of 17 Table 2 FPKM value statistics of 23 candidate genes in the closed modules Gene name Isoform ID FPKM value C-S1 C-S2 C-S3 C-S4 PAL1 PB.11849.10\|chr7:40942885–40960253(+)\|i2_LQ_samplef2cfa8\|c97668/f1p0/2837 35.55 35.96 23.56 14.21 13.01 13.55 13.41 27.42 PAL2 PB.11849.12\|chr7:40942885–40959874(+)\|i2_LQ_samplef2cfa8\|c71112/f1p0/2392 11.03 12.11 7.05 7.75 6.82 5.44 7.74 12.71 PAL3 PB.11849.14\|chr7:40942885–40960512(+)\|i3_LQ_samplef2cfa8\|c5006/f1p3/3081 15.97 14.52 9.46 10.16 7.08 5.73 7.73 12.41 PAL4 PB.11849.16\|chr7:40942887–40959833(+)\|i2_LQ_samplef2cfa8\|c94554/f1p1/2514 21.12 12.71 8.35 4.72 2.90 4.04 3.68 PAL6 PB.11849.2\|chr7:40942885–40959392(+)\|i1_LQ_samplef2cfa8\|c131710/f1p63/1911 153.74 108.77 68.88 52.63 89.36 40.94 35.96 53.15 PAL7 PB.11849.3\|chr7:40942885–40959926(+)\|i2_HQ_samplef2cfa8\|c113826/f130p0/ 2449 43.81 38.00 33.44 24.79 26.81 24.34 26.89 49.16 PAL8 PB.11849.4\|chr7:40942885–40945992(+)\|i2_LQ_samplef2cfa8\|c4595/f1p3/2499 92.78 61.43 41.47 35.18 52.91 30.09 20.42 28.56 PAL9 PB.11849.8\|chr7:40942885–40959918(+)\|i2_LQ_samplef2cfa8\|c27489/f1p1/2504 149.34 116.95 84.52 61.45 99.89 56.62 49.55 91.97 PAL15 PB.9841.1\|chr5:43212802–43217702(−)\|i2_HQ_samplef2cfa8\|c6525/f8p0/2317 7.40 2.93 6.06 4.26 M-S1 M-S2 M-S3 M-S4 7.52 6.86 4.36 3.01 4.45 4CL18 PB.5838.1\|chr4:349590–353192(+)\|i1_HQ_samplef2cfa8\|c237238/f40p8/1909 83.07 62.39 41.54 28.99 30.55 16.48 15.56 51.90 4CL22 PB.8087.5\|chr4:53453111–53459491(+)\|i1_LQ_samplef2cfa8\|c10179/f1p0/1987 3.41 3.27 0.51 5.77 0.46 0.20 0.36 1.02 CHS1 PB.10727.1\|chr7:5288756–5290374(−)\|i1_LQ_samplef2cfa8\|c23258/f3p20/1452 10.24 2.46 2.49 8.30 4.75 2.61 1.14 8.10 CHS2 PB.10728.1\|chr7:5301940–5316126(+)\|i1_LQ_samplef2cfa8\|c190118/f2p13/1386 34.31 6.70 4.45 31.23 15.62 6.70 4.04 66.58 CHS4 PB.10728.3\|chr7:5301944–5316192(+)\|i1_HQ_samplef2cfa8\|c217277/f2p10/1333 15.81 1.35 2.35 18.09 9.13 3.34 2.27 23.23 CHS8 PB.1696.1\|chr1:44128070–44142309(+)\|i1_LQ_samplef2cfa8\|c11658/f1p17/1523 35.79 9.14 7.26 22.11 21.65 8.89 6.83 55.98 CHR1 PB.9832.2\|chr5:42889648–42891090(+)\|i1_LQ_samplef2cfa8\|c117495/f1p6/1258 22.31 5.33 3.37 24.62 20.12 6.06 3.31 50.52 CHR2 PB.9833.6\|chr5:42874302–42875653(−)\|i1_LQ_samplef2cfa8\|c203391/f1p6/1151 6.33 1.70 1.97 6.82 5.77 1.81 21.22 2.06 CHR3 PB.9833.7\|chr5:42883325–42884800(−)\|i1_LQ_samplef2cfa8\|c126525/f1p6/1270 14.73 2.13 3.79 9.10 10.79 2.13 0.68 30.23 F3’H4 PB.7478.2\|chr4:42392721–42394930(−)\|i1_HQ_samplef2cfa8\|c1984/f8p1/1981 9.37 4.63 5.21 10.27 11.26 9.17 4.75 10.66 DFR1 PB.339.2\|chr1:7156508–7160534(−)\|i1_HQ_samplef2cfa8\|c21297/f2p0/1255 18.91 75.03 123.51 36.44 16.53 3.76 DFR2 PB.340.1\|chr1:7164081–7167125(−)\|i1_LQ_samplef2cfa8\|c4738/f1p0/1273 15.51 31.71 45.29 1.94 1.33 22.74 9.27 7.72 8.19 15.33 3.04 4.97 14.76 UFGT22 PB.11876.1\|chr7:41535946–41537368(+)\|i1_HQ_samplef2cfa8\|c67068/f2p2/1422 19.33 29.03 29.83 50.50 4.40 UFGT23 PB.11878.1\|chr7:41563371–41564959(+)\|i1_LQ_samplef2cfa8\|c116694/f1p0/1538 32.07 41.14 42.39 51.69 16.29 18.70 15.55 34.02 of the long-read RNA sequencing were more integrated and accurate. Comparison of the genes related to the biosynthesis of flavonoids in different alfalfa materials Flavonoids are among the most important pigments in the petals of many plants [22, 46]. Anthocyanins are end products of the flavonoid biosynthetic pathway, and generate the widest spectrum of colors, ranging from pale yellow to blue-purple [47]. Our results demonstrated that the color difference between the purple and cream flowers of alfalfa is due to the loss of the flower anthocyanins malvidin and petunidin (Fig. 1). The shift from purple to cream requires a blockage of the anthocyanin biosynthetic pathway, which probably occurs in some reactions before malvidin and petunidin are formed. Therefore, the abundance of the candidate genes was compared in the C and M transcriptomes to identify the key genes of cream color metabolism. Most of the isoforms related to flavonoids synthesis, including PALs, 4CLs, CHIs, DFRs, ANSs and UFGTs, showed large-scale higher transcription expression in C with purple flowers than in M with cream flowers, particularly for the first three stages (Fig. 2), indicating that the mutation- induced change in expression by these genes might occur far earlier than the emergence of the phenotype. In the process of flavonoid biosynthesis, CHS catalyzes the first reaction step and help synthesizing the intermediate chalcone, which is extremely important for all classes of flavonoids [48]. So the function restrain of CHS reactions are always accompanied with the elimination of not only anthocyanin biosynthesis, but also other flavonoids compounds [49]. The mutation of a single CHS enzyme led to white flower lines in grape hyacinth [31], petunia [50], Silene littorea [33] and arctic mustard flower [51]. Conversely, in our study, we found that CHSs showed higher expression in M-S4 than C-S4 (Fig. 10). Interestingly, coumaroyl-CoA can be transformed into isoliquiritigenin (an important product for the isoflavone biosynthesis pathway) by the co-function of CHS and CHR [52, 53]. Upon further data analysis, we found that the expression patterns of CHRs were similar to CHSs (Fig. 10). We thus speculated that the higher abundance of CHSs participated in another branching point in flavonoid biosynthesis, being the intermediates in the production of isoflavone biosynthesis, and CHS and CHR in M-S4 might be crucial for the biosynthesis of isoliquiritigenin. Duan et al. BMC Plant Biology (2020) 20:110 Page 13 of 17 Fig. 10 Expression profiles of 12 candidate genes and RT-qPCR validation. EF1a is used as the internal control. The error bars represent the SEs of the RT-qPCR data (n = 3). “r” represents the Pearson correlation coefficient. Pearson’s correlations between the RNA-Seq data and RT-qPCR data are calculated using the log2 fold change and the relative expression level. a PAL6; b PAL9; c CHS2; d CHS4; e CHR1; f CHR2; g CHR3; h F3’H4; i DFR1; j DFR2; k UFGT22; l UFGT23 F3H, F3’H and F3’5’H play critical roles in the flavonoid biosynthetic pathway, they catalyze the hydroxylation of flavonoids including dihydrokaempferol, dihydroquercetin, and dihydromyricetin, which are necessary for anthocyanin biosynthesis [28, 54]. Additionally, the intermediates dihydroflavonols is the main precursor of the coloured anthocyanins production through DFR, and the colourless flavonols production through FLS [55]. So the substrate competition of dihydroflavonols will result in the reverse expression regulation of FLS and DFR, accompanied by the different accumulation of flavonols and anthocyanin, respectively [55]. In our study, much higher expression of FLS/F3Hs, F3’Hs, and F3’5’H was found in most stages of M than C. This was accompanied with the higher expression of DFR Duan et al. BMC Plant Biology (2020) 20:110 in C, but at a very low level from S2 to S4 of M (Fig. 10). A similar observation was found by Lou et al. [28], who concluded that DFR might be the target gene for the loss of blue pigmentation (delphinidin) in white grape hyacinth. Thus, the higher expression of FLS/F3Hs, F3’Hs, and F3’5’H might increase the production of other flavonoid compounds, such as dihydroquercetin, dihydrokaempferol, dihydromyricetin. Myricetin and kaempferol in M, and the down-regulated DFR might partially block the synthesis of anthocyanins, thereby eliminating the process of purple pigmentation. The purple flower ripening of C suggested that the fundamental transcriptional regulation of the genes from the upstream PAL to the end UFGT might play important roles in the accumulation of flavonoid intermediates and flower color formation. Hub genes related to flower formation were identified by WGCNA The cream-colored Zhongtian No. 3 alfalfa represents a color mutation, as the purple Defu alfalfa is the wild-type. Understanding the changes in the cream flower phenotype as a mutant of the wild-type could elucidate the mechanisms of the alfalfa flower pigmentation. Any functional loss of key enzymes in the flavonoid biosynthetic pathway could lead to a cream color mutation, including via transcript abundance changes in genes, and branching changes in flavone products [56, 57]. A novel finding from this study was that, by performing WGNCA, we identified floral developmental stage-specific gene modules (Figs. 8 and 9). To this end, 9 PALs, 2 4CLs, 4 CHSs, 3 CHRs, Page 14 of 17 F3’H4, 2 DFRs, and 2 UFGTs were highly associated in modules with close relationships to the M4 or M group. They all possessed evident differences in transcript abundance in C and M, indicating their important roles in floral formation variation. It was worth noting that, the above genes were not the genes with the highest expression levels, implying that the high expression genes were not necessary for distinguishing different flower colors [29]. Thus, the WGCNA analyses in this study provided a useful approach for selecting important genes related to the specific phenotypes. Du et al. [58] identified hub genes operating in the seed coat network in the early seed maturation stage by WGCNA analysis. Similar WGCNA analysis was used in golden camellia to identify unigenes correlated with flower color, and CHS, F3H, ANS and FLS were found to play critical roles in regulating the formation of flavonols and anthocyanidins [29]. The 6 hub genes were upstream of the flavonoid biosynthesis pathway, implying that the cream flower pigmentation of M was mainly blocked upstream. The decreased expression of PAL6, PAL9, and 4CL8, whether in C or M, is in line with the results in fig [57]. Wang et al. [57] found that the decreased expression of PALs and 4CLs affected the cinnamic acid content in the “Purple Peel” mature fruit peel. We speculated that the decreased expression of PAL6, PAL9, and 4CL8 might also affect the cinnamic acid content in the petals both in C and M. The elevated expression of CHSs in M-S4 might play crucial roles in the biosynthesis of other flavones, such as isoflavone, which is also a crucial factor in the color formation of different flowers in alfalfa. Fig. 11 A referred model for the process of anthocyanin synthesis in the purple flowers of C and cream flowers of M. The crucial isoform IDs are indicated at the side of each gene. Upstream of M, PAL and 4CL are suppressed, and an increasing branch of isoflavone biosynthesis regulated by CHS and CHR is dominant. Furthermore, the up-regulation of F3H/FLS, F3’H, and F3’5’H causes an increase in other flavonoid compounds, such as myricetin and kaempferol, further reducing the anthocyanin synthesis. Finally, the low expression level of DFR accompanied with the low abundance of UFGT might disrupt the anthocyanin synthesis, leading to the formation of the cream color Duan et al. BMC Plant Biology (2020) 20:110 Based on the above results, different flavonoid biosynthesis pathways in purple- and cream-colored alfalfa were inferred (Fig. 11). Briefly, compared to C, the flavonoid biosynthesis of M is blocked upstream, by PAL and 4CL, following which a branch of isoflavone biosynthesis regulated by CHS and CHR is dominant, completing the anthocyanin synthesis pathway. Additionally, the up-regulation of F3H/FLS, F3’H, and F3’5’H causes an increase in other flavonoid compounds, such as myricetin and kaempferol, further reducing anthocyanin synthesis. Finally, the low expression level of DFR accompanied with the low abundance of UFGT might disrupt the anthocyanin synthesis, leading to the formation of the cream color. Conclusions The mechanisms of anthocyanin and flavonoid pathways in the purple flower of Defu and cream flower of Zhongtian No. 3 were analyzed by HPLC, transcriptome analysis and RT-qPCR. Malvidin and petunidin glycoside derivatives were the major anthocyanins in the flowers of Defu, which were lacking in the flowers of Zhongtian No. 3. The PacBio long-read RNA sequencing was more integrated and accurate than RNA-Seq. A new hypothesis is proposed for the lack of purple phenotype in the alfalfa flowers, a series of candidate genes might be cofunctioned through flavonoid biosynthesis blocking, the competition of other flavonoid compounds formation, anthocyanin synthesis blocking, and so on. Further research is required to fully elucidate these processes. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12870-020-2322-9. Additional file 1: Figure S1. The significantly enriched KEGG pathway of DEGs between M-S4 and C-S4. Additional file 2: Table S1. Primers for the RT-qPCR. Additional file 3: Table S2. Isoforms statistics of the 513 new isoforms. A total of 309 new isoforms were annotated in the Swiss-Prot database, and the remaining 204 isoforms were unannotated. Page 15 of 17 Million mapped reads; FW: Fresh weight; HPLC: High-performance liquid chromatography analysis; IR: Intron retention; Iso-Seq: Isoform sequencing; KEGG: Kyoto Encyclopedia of Genes and Genomes; M: Zhongtian No. 3; M. falcata L.: Medicago falcata L.;; M. lupulina L.: Medicago lupulina L.; M. sativa L.: Medicago sativa L.; M. truncatula: Medicago truncatula; M. varia: Medicago varia; MXE: Mutually exclusive exons; NR: The protein databases of Nonredundant; ORFs: Open reading frames; PAL: Phenylalanine ammonia-lyase; PCA: Principal component analysis; RIN: RNA Integrity Number; RNA-Seq: RNA sequencing; RT-qPCR: Real-time quantitative PCR; SEs: Standard errors; TFs: Transcription factors; TO: Topological overlap; UFGT: UDP-glucose: flavonoid 3-O-glucosyltransferase; WGCNA: Weighted gene co-expression network analysis Acknowledgements Not applicable. Authors’ contributions D. H. R. and Y. H. S. designed the experiments. D. H. R., Y. H. S. and Z. X. H. performed the experiments. D. H. R., C. G. X., and D. X. R. analyzed transcriptome data. D. H. R. wrote the paper. W. L. R. and Y. H. S. revised this paper. All authors have read and approved the manuscript. Funding This work was supported by the National Natural Science Foundation of China (grant No. 31700338 and 31860118), the Fundamental Research Funds for the Central Public-interest Scientific Institution (1610322019012 and 1610322019013), and the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences (CAASASTIP-2019LIHPS-08). The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data in writing the manuscript. Availability of data and materials All raw sequence data have been submitted to the Sequence Read Archive (SRA) database under accession number PRJNA565675. The addresses are as follows: https://submit.ncbi.nlm.nih.gov/subs/sra. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details Lanzhou Institute of Husbandry and Pharmaceutical Science, Chinese Academy of Agricultural Sciences, Lanzhou, China. 2College of Ecological Environment and Resources, Qinghai Nationalities University, Xining, China. 3 Shanxi Electric Power Research Institute, State Grid Corporation of China, Taiyuan, China. 1 Received: 15 November 2019 Accepted: 28 February 2020 Additional file 4: Table S3. Isoforms ID of the genes on the heatmap related to the flavonoid synthesis. Additional file 5: Table S4. Enriched module information in all the stages of M, specifically M-S4. The module of skyblue3 displays a close relationship with M-S4, and the modules of bisque4 and turquoise exhibit a close relationship with M. The enriched pathways related to flower color formation of each module are summarized. Abbreviations 4CL: 4-coumarate: coenzyme A ligase; A3SS: Alternative 3ˊ splice sites; A5SS: Alternative 5ˊ splice sites; ANS: Anthocyanidin synthase; C: Defu; CCS: Circular consensus sequences; CDS: The putative coding sequences; CHI: Chalcone isomerase; CHR: Chalcone reductase; CHS: Chalcone synthase; DFR: Dihydroflavonol 4-reductase; DGE: Digital gene expression; ES: Exon skipping/inclusion; F3′5′H: Flavonoid-3′, 5′-hydroxylase; F3′H: Flavonoid 3′monooxygenase; F3H: Flavanone 3-hydroxylase; FDR: False discovery rate; FLS: Flavonol synthesis; FPKM: Fragments Per Kilobase of transcript per References 1. Gao LX, Yang HX, Liu HF, Yang J, Hu YH. Extensive transcriptome changes underlying the flower color intensity variation in Paeonia ostii. Front Plant Sci. 2016;6:1205. 2. Meng YY, Wang ZY, Wang YQ, Wang CN, Zhu BT, Liu H, et al. The MYB activator WHITE PETAL1 associates with MtTT8 and MtWD40-1 to regulate carotenoid-derived flower pigmentation in Medicago truncatula. 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https://openalex.org/W2247530666	https://peerj.com/articles/1574.pdf	English	null	Structure and stability of recombinant bovine odorant-binding protein: II. Unfolding of the monomeric forms	PeerJ	2,016	cc-by	10,845	ABSTRACT In a family of monomeric odorant-binding proteins (OBPs), bovine OBP (bOBP), that lacks conserved disulfide bond found in other OBPs, occupies unique niche because of its ability to form domain-swapped dimers. In this study, we analyzed conformational stabilities of the recombinant bOBP and its monomeric variants, the bOBP-Gly121+ mutant containing an additional glycine residue after the residue 121 of the bOBP, and the GCC-bOBP mutant obtained from the bOBP-Gly121+ form by introduction of the Trp64Cys/His155Cys double mutation to restore the canonical disulfide bond. We also analyzed the effect of the natural ligand binding on the conformational stabilities of these bOBP variants. Our data are consistent with the conclusion that the unfolding-refolding pathways of the recombinant bOBP and its mutant monomeric forms bOBP-Gly121+ and GCC-bOBP are similar and do not depend on the oligomeric status of the protein. This clearly shows that the information on the unfolding-refolding mechanism is encoded in the structure of the bOBP monomers. However, the process of the bOBP unfolding is significantly complicated by the formation of the domain-swapped dimer, and the rates of the unfolding-refolding reactions essentially depend on the conditions in which the protein is located. Submitted 26 October 2015 Accepted 16 December 2015 Published 18 April 2016 Corresponding authors Vladimir N. Uversky, vuversky@health.usf.edu Konstantin K. Turoverov, kkt@incras.ru Academic editor Pedro Silva Additional Information and Declarations can be found on page 19 DOI 10.7717/peerj.1574 Copyright 2016 Stepanenko et al. Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS Submitted 26 October 2015 Accepted 16 December 2015 Published 18 April 2016 Corresponding authors Vladimir N. Uversky, vuversky@health.usf.edu Konstantin K. Turoverov, kkt@incras.ru Academic editor Pedro Silva Additional Information and Declarations can be found on page 19 DOI 10.7717/peerj.1574 Submitted 26 October 2015 Accepted 16 December 2015 Published 18 April 2016 Corresponding authors Vladimir N. Uversky, vuversky@health.usf.edu Konstantin K. Turoverov, kkt@incras.ru Academic editor Pedro Silva Additional Information and Declarations can be found on page 19 DOI 10.7717/peerj.1574 Subjects Biochemistry, Bioinformatics, Biophysics, Molecular Biology Keywords Odorant-binding protein, Ligand binding, Disulfide bond, Domain swapping, Unfolding-refolding reaction, Conformational stability How to cite this article Stepanenko et al. (2016), Structure and stability of recombinant bovine odorant-binding protein: II. Unfolding of the monomeric forms. PeerJ 4:e1574; DOI 10.7717/peerj.1574 Structure and stability of recombinant bovine odorant-binding protein: II. Unfolding of the monomeric forms Olga V. Stepanenko1, Denis O. Roginskii1, Olesya V. Stepanenko1, Irina M. Kuznetsova1, Vladimir N. Uversky1,2 and Konstantin K. Turoverov1,3 1 Laboratory of Structural Dynamics, Stability and Folding of Proteins, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia 3 Peter the Great St. Petersburg Polytechnic University, St. Petersburg, Russia 2 Department of Molecular Medicine and USF Health Byrd Alzheimer’s Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL, United States INTRODUCTION Copyright 2016 Stepanenko et al. Odorant binding proteins (OBPs) are important components of olfactory apparatus in vertebrates where they play a specific role in olfaction by interacting directly with odorants (Xu et al., 2005). OBPs constitute a class of small extracellular proteins in the chemosensory systems of most terrestrial species ranging from drosophila to human. In mammals, OBPs are found at high concentrations (∼10 mM) in nasal mucosa of Distributed under Creative Commons CC-BY 4.0 Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS cow (Bignetti et al., 1985; Pelosi, Baldaccini & Pisanelli, 1982), rat (Pevsner et al., 1985), rabbit (Dal Monte et al., 1991), pig (Dal Monte et al., 1991), dog (D’Auria et al., 2006), and humans (Briand et al., 2002). Although they bind different kinds of small and hydrophobic odorant molecules (typically with the affinities in the micromolar range, their inability to discriminate different chemical classes of these molecules suggests that OBPs cannot serve as olfactory receptors (Boudjelal, Sivaprasadarao & Findlay, 1996). The precise biological functions of mammalian OBPs are not known as of yet, but it was hypothesized that these proteins can be involved in transport of hydrophobic odorants across the aqueous mucus layer to access the olfactory receptors, or might be involved in the termination of the olfactory signal by removing odorants from the receptors after their stimulation (Bignetti et al., 1987; Pevsner & Snyder, 1990). OBPs constitute a sub-class of lipocalins, which are small extracellular proteins found in gram negative bacteria, plants, invertebrates, and vertebrates. Although lipocalins are known to share limited regions of sequence homology, they do have a common tertiary structure architecture (Flower, North & Sansom, 2000; Grzyb, Latowski & Strzalka, 2006). The characteristic structural signature of the lipocalin family is a β-barrel composed by a 9-stranded anti-parallel β-sheet with an α-helical segment at the C-terminus (Flower, North & Sansom, 2000). The internal cavity of the lipocalin β-barrel is the binding site that can interact with the odorant molecules belonging to different chemical classes (Vincent et al., 2000). Bovine OBP (bOBP) was the first OBP for which crystal structure was solved, and the analysis of this structure revealed that bOBP exists as a domain- swapped dimer (Bianchet et al., 1996; Tegoni et al., 1996). This was in contrast to struc- tures of other lipocalins, including the porcine OBP (pOBP) (Spinelli et al., 1998), which are monomeric proteins. Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 Fluorescence spectroscopy Fluorescence experiments were performed using a Cary Eclipse spectrofluorometer (Varian, Australia) with microcells FLR (10 × 10 mm; Varian, Australia). Fluorescence lifetime were measured using a ‘‘home built’’ spectrofluorometer with a nanosecond impulse (Stepanenko et al., 2014a; Stepanenko et al., 2012; Turoverov et al., 1998) as well as micro-cells (101.016-QS 5 × 5 mm; Hellma, Germany). Tryptophan fluorescence in the protein was excited at the long-wave absorption spectrum edge (λex = 297 nm), wherein the tyrosine residue contribution to the bulk protein fluorescence is negligible (Stepanenko et al., 2015). The fluorescence spectra position and form were characterized using the parameter A = I320/I365, wherein I320 and I365 are the fluorescence intensities at the emission wavelengths 320 and 365 nm, respectively (Turoverov & Kuznetsova, 2003). The values for parameter A and the fluorescence spectrum were corrected for instrument sensitivity. The tryptophan fluorescence anisotropy was calculated using the equation: r = (I V V −GI V H )/(I V V +2GI V H ), wherein I V V and I V H are the vertical and horizontal fluorescence intensity components upon excitement by vertically polarized light. G is the relationship between the fluorescence intensity vertical and horizontal components upon excitement by horizontally polarized light (G = I H V /I H H ), λem = 365 nm (Turoverov et al., 1998). The fluorescence intensity for the fluorescent dye ANS was recorded at λem = 480 nm (λex = 365 nm). Gene expression and protein puriﬁcation The plasmids pT7-7-bOBP which encodes bOBP and its mutant forms with a poly- histidine tag were used to transform Escherichia coli BL21(DE3) host (Invitrogen) (Stepanenko et al., 2014c). The protein expression was induced by incubating the cells with 0.3 mM of isopropyl-beta-D-1-thiogalactopyranoside (IPTG; Fluka, Switzerland) for 24 h at 37 ◦C. The recombinant protein was purified with Ni+-agarose packed in HisGraviTrap columns (GE Healthcare, Sweden). The protein purity was determined through SDS- PAGE in 15% polyacrylamide gel (Laemmli, 1970). Materials Materials GdnHCl (Nacalai Tesque, Japan), 1-octen-3-ol (OCT; Sigma-Aldrich, USA) and ANS (ammonium salt of 8-anilinonaphtalene-1-sulfonic acid; Fluka, Switzerland) were used without further purification. The protein concentration was 0.1–0.2 mg/ml. The OCT concentration was 10 mM. The experiments were performed in 20 mM Na-phosphate- buffered solution at pH 7.8. INTRODUCTION The ability of bOBP to form domain-swapped dimers was explained by the absence of a glycine residue at the hinge region linking the β-barrel to the α-helix, and by the lack of the disulfide bridge which is present in all lipocalin sequences identified so far (Ramoni et al., 2002). This work is dedicated to the analysis of the peculiarities of the GdnHCl-induced unfolding—refolding reactions of the recombinant bOBP and its monomeric mutants, bOBP-Gly121+ and bOBP-Gly121+/W64C/H155C (GCC-bOBP). It continues a series of articles dedicated to the analysis of the effect of the environment (including the presence of crowding agents) on structural properties and conformational stability of bOBP. The mutant protein bOBP-Gly121+ contains an extra glycine residue introduced after the bOBP residue 121. This substitution was shown to promote monomerization of the bOBP (Stepanenko et al., 2016) likely via increasing the mobility of the loop connecting α-helix and 8th β-strand of the β-barrel. Substitutions of the residues Trp64 and His156 to cysteines in bOBP-Gly121+ generate a mutant form GCC-bOBP, which is expected to have stable monomeric structure due to the restoration of the disulfide bond typically seeing in other OBPs (Ramoni et al., 2008). We also investigated the role of the natural ligand in the stabilization of protein structure and looked at how the ligand binding affected the folding-unfolding reaction of these proteins. Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 2/23 2/23 Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 Fitting of denaturation curves The equilibrium dependences of the fluorescence intensity at 320 nm on the GdnHCl concentration were fit using a two-state model: S = SN +SUKN−U 1+KN−U , (1) KN−U = exp −1G0 N−U +mN−U[D] RT ! , (2) KN−U = FU/FN = (1−FN)/FN, (3) (1) (2) (3) taking into account (4) (5) SN = aN +bN[D], (4) SU = aU +bU[D], (5) U = aU +bU[D], (5) (5) where S is the fluorescence intensity at the measured GdnHCl concentration; [D] is the guanidine concentration; m is the linear dependence of 1GN−U on the denaturant concentration; 1G0 N−U is the free energy of unfolding at 0 M denaturant; FN and FU are the fractions of native and unfolded molecules, respectively; and SN and SU are the signal of the native and unfolded states, respectively; aN, bN, aU and bU are constants needed to fit linear dependences of the SN and SU signals on the GdnHCl concentration. Fitting was performed using a nonlinear regression with Sigma Plot. To evaluate conformational stability of the studied proteins we took into account that the formation of the native dimeric state of bOBP occurred at moderate GdnHCl concentration is followed by full protein unfolding while conformational perturbations of bOBP at low denaturant concentrations were not attributed to the unfolding of the protein globule (see ‘‘Results and Discussion’’ section). As bOBP unfolding is fully reversible the transition from native to unfolded state of the protein was used to calculate 1GN−U value. Conformational stability of the bOBP mutant forms was evaluated similarly taking into account that both mutant proteins unfold through the same scheme as bOBP does (see ‘‘Results and Discussion’’ section). GdnHCl-induced unfolding Protein unfolding was initiated by manually mixing the protein solution (40 µL) with a buffer solution (510 µL) so that the GdnHCl concentration was varied from 0.0 to 4.0– 5.0 M in the absence or presence of a natural ligand, 1-Octen-3-ol (OCT). The GdnHCl concentration was determined by the refraction coefficient using an Abbe refractometer (LOMO, Russia; (Pace, 1986)). The dependences of different fluorescent characteristics Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 3/23 of the studied proteins on GdnHCl were recorded following protein incubation in a solution with the appropriate denaturant concentration at 4 ◦C for different times (see in the text). The protein refolding was initiated by diluting the pre-denatured protein (in 3.0 M GdnHCl, 40 µL) with the buffer or denaturant solutions at various concentrations (510 µL). The spectrofluorimeter was equipped with a thermostat that holds the temperature constant at 23 ◦C. Fitting of denaturation curves Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 Gel ﬁltration experiments We performed gel filtration experiments for recombinant bOBPwt and its mutant forms in a buffered solution and with addition of GdnHCl using a Superdex-75 PC 3.2/30 column (GE Healthcare, Sweden) and an AKTApurifier system (GE Healthcare, Sweden). The column was equilibrated with the buffered solution or GdnHCl at the desired concentration, and 10 µl of the protein solution prepared under the same conditions was loaded on the pre-equilibrated column. The change in hydrodynamic dimensions for the studied proteins was evaluated as a change in the protein elution volume. Multiple proteins with known molecular masses (aprotinin (6.5 kDa), ribonuclease (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumin (43 kDa) and conalbumin (75 kDa), which are chromatography standards from GE Healthcare) were used to generate the calibration curve. Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 Circular dichroism measurements The CD spectra were generated using a Jasco-810 spectropolarimeter (Jasco, Japan). Far- UV CD spectra were recorded in a 1-mm path length cell from 260 nm to 190 nm with a 0.1 nm step size. For the spectra, we generated 3 scans on average. The CD spectra for the appropriate buffer solution were recorded and subtracted from the protein spectra. Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 4/23 RESULTS AND DISCUSSION Flexibility profiles were obtained using the FlexPred software available at http://kiharalab.org/flexPred/ (Jamroz, Kolinski & Kihara, 2012), whereas intrinsic disorder propensity was evaluated using the PONDR R⃝VSL2 algorithm (Peng et al., 2005). Figure 1 Analysis of the 3D structure of bOBP. Crystal 3D structures of natural bOBP (A) and monomeric mutant form GCC-bOBP (B). The individual subunits in the bOBP are in gray and orange. In the GCC-bOBP short α-helical segment that followed by the 9th β-strand and the disordered C-terminal region of the protein are drawn in orange. The tryptophan residues are indicated in red and blue in the different subunits of bOBP and in blue in GCC-bOBP. The Gly 121+ residue which donates the increased mobility of the loop connecting α-helix and 8th β-strand of the β-barrel and promotes the formation of a monomeric fold of the mutant protein bOBP-Gly121+ is in green. Two cysteines residues Cys 64 and Cys 156 in GCC-bOBP, which are believed to stabilize monomeric structure due to the disulfide bond formation are in yellow. The drawing of bOBP and GCC-bOBP was generated based on the 1OBP (Tegoni et al., 1996) and 2HLV files (Ramoni et al., 2008) from PDB (Dutta et al., 2009) using the graphic software VMD (Hsin et al., 2008) and Raster3D (Merritt & Bacon, 1977). Plot (C) represents the results of the multiple structural alignment of bOBP (PDB ID: 1OBP, blue structure), GCC-bOBP (PDB ID: 2HLV, red structure), and naturally monomeric pOBP (PDB ID: 1A3Y, green structure) using the MultiProt algorithm (http://bioinfo3d.cs.tau.ac.il/MultiProt/) (Shatsky, Nussinov & Wolfson, 2004). The drawing was generated using the graphic software VMD (Hsin et al., 2008). Plot (D) compares flexibility profiles obtained from crystal structures of bOBP (PDB ID: 1OBP, black and red lines for the chains A and B), naturally monomeric pOBP (PDB ID: 1A3Y, green and yellow lines for the chains A and B) and monomeric mutant GCC-bOBP (PDB ID: 2HLV, blue line) with the intrinsic disorder propensity of the bOBP (UniProt ID: P07435, pink dashed line). Flexibility profiles were obtained using the FlexPred software available at http://kiharalab.org/flexPred/ (Jamroz, Kolinski & Kihara, 2012), whereas intrinsic disorder propensity was evaluated using the PONDR R⃝VSL2 algorithm (Peng et al., 2005). Figure 1 Analysis of the 3D structure of bOBP. Crystal 3D structures of natural bOBP (A) and monomeric mutant form GCC-bOBP (B). The individual subunits in the bOBP are in gray and orange. RESULTS AND DISCUSSION Structural features of the monomeric and dimeric bOBPs Figure 1 compares structural features of the natural bOBP and its monomeric mutant GCC-bOBP. Structures shown in Figs. 1A and 1B indicate that the domain swapping has been reverted in monomeric GCC-bOBP. Furthermore, Fig. 1C represents the results of the multiple structural alignment of the one of the monomers of the natural dimeric bOBP (blue structure), monomeric mutant GCC-bOBP (red structure), and naturally monomeric pOBP (green structure) and clearly shows that the monomeric GCC-bOBP has the overall fold almost identical to that of the pOBP. Next, we analyzed how introduced mutations affected the structural flexibility of bOBP utilizing the power of the FlexPred tool that rapidly predicts absolute per-residue fluctuations from a three- dimensional structure of a query protein (Jamroz, Kolinski & Kihara, 2012). Results of this analysis are shown in Fig. 1D which clearly indicates that the chains of the bOBP dimer, chains of the naturally monomeric pOBP and the monomeric mutant GCC-bOBP are all characterized by very similar structural flexibility. Since the FlexPred tool provides real-value fluctuations of globular proteins based on their static structures by considering Cα atoms contact number and known B-factor values, the fact that these three proteins, bOBP (PDB ID: 1OBP), GCC-bOBP (PDB ID: 2HLV), and pOBP (PDB ID: 1A3Y), are predicted to have similar structural flexibility indicates that mutations introduced to generate the monomeric GCC-bOBP or present in the naturally monomeric pOBP do not dramatically affect Cα atoms contact number and B-factors of the resulting structures. Note that these observations are in a good agreement with the overall very high structural similarity of these three proteins, the observation supported by the fact that the multiple structural alignment of these three proteins over 136 residues was characterized by the RMSD of 0.89 Å (see Fig. 1C). We also compared these results of structure-based flexibility of the bOBP with the propensity of this protein for intrinsic disorder evaluated by PONDR R⃝VSL2B, which is one of the more accurate stand-alone disorder predictors (Fan & Kurgan, 2014; Peng et al., 2005; Peng & Kurgan, 2012). Results of this analysis are also shown in Fig. 1D which illustrates that there is generally a very good agreement Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 5/23 Figure 1 Analysis of the 3D structure of bOBP. Crystal 3D structures of natural bOBP (A) and monomeric mutant form GCC-bOBP (B). Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 RESULTS AND DISCUSSION The individual subunits in the bOBP are in gray and orange. In the GCC-bOBP short α-helical segment that followed by the 9th β-strand and the disordered C-terminal region of the protein are drawn in orange. The tryptophan residues are indicated in red and blue in the different subunits of bOBP and in blue in GCC-bOBP. The Gly 121+ residue which donates the increased mobility of the loop connecting α-helix and 8th β-strand of the β-barrel and promotes the formation of a monomeric fold of the mutant protein bOBP-Gly121+ is in green. Two cysteines residues Cys 64 and Cys 156 in GCC-bOBP, which are believed to stabilize monomeric structure due to the disulfide bond formation are in yellow. The drawing of bOBP and GCC-bOBP was generated based on the 1OBP (Tegoni et al., 1996) and 2HLV files (Ramoni et al., 2008) from PDB (Dutta et al., 2009) using the graphic software VMD (Hsin et al., 2008) and Raster3D (Merritt & Bacon, 1977). Plot (C) represents the results of the multiple structural alignment of bOBP (PDB ID: 1OBP, blue structure), GCC-bOBP (PDB ID: 2HLV, red structure), and naturally monomeric pOBP (PDB ID: 1A3Y, green structure) using the MultiProt algorithm (http://bioinfo3d.cs.tau.ac.il/MultiProt/) (Shatsky, Nussinov & Wolfson, 2004). The drawing was generated using the graphic software VMD (Hsin et al., 2008). Plot (D) compares flexibility profiles obtained from crystal structures of bOBP (PDB ID: 1OBP, black and red lines for the chains A and B), naturally monomeric pOBP (PDB ID: 1A3Y, green and yellow lines for the chains A and B) and monomeric mutant GCC-bOBP (PDB ID: 2HLV, blue line) with the intrinsic disorder propensity of the bOBP (UniProt ID: P07435, pink dashed line). Flexibility profiles were obtained using the FlexPred software available at http://kiharalab.org/flexPred/ (Jamroz, Kolinski & Kihara, 2012), whereas intrinsic disorder propensity was evaluated using the PONDR R⃝VSL2 algorithm (Peng et al., 2005). Figure 1 Analysis of the 3D structure of bOBP. Crystal 3D structures of natural bOBP (A) and monomeric mutant form GCC-bOBP (B). The individual subunits in the bOBP are in gray and orange. In the GCC-bOBP short α-helical segment that followed by the 9th β-strand and the disordered C-terminal region of the protein are drawn in orange. The tryptophan residues are indicated in red and blue in the different subunits of bOBP and in blue in GCC-bOBP. RESULTS AND DISCUSSION The Gly 121+ residue which donates the increased mobility of the loop connecting α-helix and 8th β-strand of the β-barrel and promotes the formation of a monomeric fold of the mutant protein bOBP-Gly121+ is in green. Two cysteines residues Cys 64 and Cys 156 in GCC-bOBP, which are believed to stabilize monomeric structure due to the disulfide b d f ti i ll Th d i f bOBP d GCC bOBP t d b d th 1OBP Figure 1 Analysis of the 3D structure of bOBP. Crystal 3D structures of natural bOBP (A) and Figure 1 Analysis of the 3D structure of bOBP. Crystal 3D structures of natural bOBP (A) and monomeric mutant form GCC-bOBP (B). The individual subunits in the bOBP are in gray and orange. In the GCC-bOBP short α-helical segment that followed by the 9th β-strand and the disordered C-terminal region of the protein are drawn in orange. The tryptophan residues are indicated in red and blue in the different subunits of bOBP and in blue in GCC-bOBP. The Gly 121+ residue which donates the increased mobility of the loop connecting α-helix and 8th β-strand of the β-barrel and promotes the formation of a monomeric fold of the mutant protein bOBP-Gly121+ is in green. Two cysteines residues Cys 64 and Cys 156 in GCC-bOBP, which are believed to stabilize monomeric structure due to the disulfide bond formation are in yellow. The drawing of bOBP and GCC-bOBP was generated based on the 1OBP (Tegoni et al., 1996) and 2HLV files (Ramoni et al., 2008) from PDB (Dutta et al., 2009) using the graphic software VMD (Hsin et al., 2008) and Raster3D (Merritt & Bacon, 1977). Plot (C) represents the results of the multiple structural alignment of bOBP (PDB ID: 1OBP, blue structure), GCC-bOBP (PDB ID: 2HLV, red structure), and naturally monomeric pOBP (PDB ID: 1A3Y, green structure) using the MultiProt algorithm (http://bioinfo3d.cs.tau.ac.il/MultiProt/) (Shatsky, Nussinov & Wolfson, 2004). The drawing was generated using the graphic software VMD (Hsin et al., 2008). Plot (D) compares flexibility profiles obtained from crystal structures of bOBP (PDB ID: 1OBP, black and red lines for the chains A and B), naturally monomeric pOBP (PDB ID: 1A3Y, green and yellow lines for the chains A and B) and monomeric mutant GCC-bOBP (PDB ID: 2HLV, blue line) with the intrinsic disorder propensity of the bOBP (UniProt ID: P07435, pink dashed line). Equilibrium unfolding of the recombinant bOBP in the presence of natural ligand Equilibrium unfolding of the recombinant bOBP in the presence of natural ligand Equilibrium unfolding of the recombinant bOBP in the presence of natural ligand Previously we have shown that the recombinant bOBP, unlike the natural bOBP extracted from the tissues, represents a mixture of monomeric and dimeric forms suggesting that this protein exists in a stable native-like state with a reduced dimerization capability (Stepanenko et al., 2014c). It has been suggested that the recombinant form of bOBP is characterized by the disturbed package of the α-helical region and some β-strands, which prevents the formation of a native domain-swapped dimer (Stepanenko et al., 2014c). It is likely that the dimer formation via the domain exchange mechanism is a rather complex process that requires specific organization of the secondary and tertiary structure within the monomers. Curiously, recombinant bOBP can form the compact dimeric state under the mild denaturing conditions, namely, in the presence of 1.5 M guanidine hydrochlo- ride (GdnHCl) (Stepanenko et al., 2014c). This process requires bOBP restructuring and is accompanied by the formation of a stable, more compact, intermediate state that is maximally populated at 0.5 M GdnHCl. Noticeably, at GdnHCl concentrations lower than 1.6 M we observed moderate changes in bOBP intrinsic fluorescence and far-UV CD spectra. These changes are not attributed to the bOBP unfolding process but are determined by some local structural changes in the protein globule. The dimeric bOBP in the presence of 1.5 M GdnHCl is characterized by a highly ordered secondary structure and a highly rigid microenvironment around the tryptophan residues. In the absence of GdnHCl, the recombinant bOBP is in a stable state with features similar to the native dimeric bOBP. Still, recombinant bOBP in the absence of GdnHCl is characterized by a less ordered secondary structure compared to the wild-type bOBP crystallographic data and a more rigid microenvironment of tryptophan residues, which is characterized by a decreased capacity of the recombinant bOBP for dimerization in aqueous solutions. This stable state of the recombinant bOBP state was designated as a ‘‘trapped’’ conformation with incorrect packing of α-helices and β-sheet within the protein globule, which may interfere with the formation of the bOBP native state. The reasons for accumulation of this ‘‘trapped’’ state may lie in a relatively complex domain-swapping dimerization mechanism which is also required for the monomers to be correctly folded. Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 RESULTS AND DISCUSSION In the GCC-bOBP short α-helical segment that followed by the 9th β-strand and the disordered C-terminal region of the protein are drawn in orange. The tryptophan residues are indicated in red and blue in the different subunits of bOBP and in blue in GCC-bOBP. The Gly 121+ residue which donates the increased mobility of the loop connecting α-helix and 8th β-strand of the β-barrel and promotes the formation of a monomeric fold of the mutant protein bOBP-Gly121+ is in green. Two cysteines residues Cys 64 and Cys 156 in GCC-bOBP, which are believed to stabilize monomeric structure due to the disulfide bond formation are in yellow. The drawing of bOBP and GCC-bOBP was generated based on the 1OBP (Tegoni et al., 1996) and 2HLV files (Ramoni et al., 2008) from PDB (Dutta et al., 2009) using the graphic software VMD (Hsin et al., 2008) and Raster3D (Merritt & Bacon, 1977). Plot (C) represents the results of the multiple structural alignment of bOBP (PDB ID: 1OBP, blue structure), GCC-bOBP (PDB ID: 2HLV, red structure), and naturally monomeric pOBP (PDB ID: 1A3Y, green structure) using the MultiProt algorithm (http://bioinfo3d.cs.tau.ac.il/MultiProt/) (Shatsky, Nussinov & Wolfson, 2004). The drawing was generated using the graphic software VMD (Hsin et al., 2008). Plot (D) compares flexibility profiles obtained from crystal structures of bOBP (PDB ID: 1OBP, black and red lines for the chains A and B), naturally monomeric pOBP (PDB ID: 1A3Y, green and yellow lines for the chains A and B) and monomeric mutant GCC-bOBP (PDB ID: 2HLV, blue line) with the intrinsic disorder propensity of the bOBP (UniProt ID: P07435, pink dashed line). Flexibility profiles were obtained using the FlexPred software available at http://kiharalab.org/flexPred/ (Jamroz, Kolinski & Kihara, 2012), whereas intrinsic disorder propensity was evaluated using the PONDR R⃝VSL2 algorithm (Peng et al., 2005). 6/23 between the structural flexibility calculated from the protein crystal structure and the propensity of a protein for intrinsic disorder. Equilibrium unfolding of the recombinant bOBP in the presence of natural ligand On the other hand, the intermediate state of bOBP structure which is accumulated at 0.5 M GdnHCl is characterized by the reorganized bOBP structure that has fewer elements of ordered secondary structure, compared with the recombinant bOBP structure both in an aqueous solution and in the solution containing 1.5 M GdnHCl. The increase of the GdnHCl concentration above 1.5 M induces cooperative unfolding of the recombinant bOBP, which is completed by ∼3 M GdnHCl and is indicated by the simultaneous changes of all structural parameters of bOBP analyzed in this study (Stepanenko et al., 2014c). The half- transition point of this unfolding process at >2 M GdnHCl indicates high conformational stability of the recombinant bOBP (Stepanenko et al., 2014c), which is comparable with the stabilities of the native (isolated from tissue) bOBP (Mazzini et al., 2002) and pOBP Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 7/23 Table 1 Hydrodynamic dimensions of recombinant bOBPwt and its mutant forms in the absence and in the presence of natural ligand OCT in different structural states. GdnHCl, M First peak, kDa Second peak, kDa bOBPwt 0.0 43.9 23.8 0.5 34.0 19.3 1.5 43.6 bOBPwt/OCT 0.0 39.6 21.5 0.55 27.2 17.0 1.7 39.6 bOBP/Gly121+ 0.0 23.6 0.25 17.8 1.5–1.9 24.8–28.5 bOBP/Gly121+/OCT 0.0 21.5 0.24–0.5 15.5–16.2 1.7–2.0 24.7–28.5 GCC-bOBP 0.0 23.6 0.25 17.8 1.1–1.82 22.5–25.9 GCC-bOBP/OCT 0.0 22.5 0.27 16.9 1.5–2.0 22.5–27.2 Table 1 Hydrodynamic dimensions of recombinant bOBPwt and its mutant forms in the absence and in the presence of natural ligand OCT in different structural states. Table 1 Hydrodynamic dimensions of recombinant bOBPwt and its mutant forms in the absence and in the presence of natural ligand OCT in different structural states. (Staiano et al., 2007; Stepanenko et al., 2008) and is inherent to β-rich proteins (Stepa- nenko et al., 2012; Stepanenko et al., 2013; Stepanenko et al., 2014b). We have also es- tablished that the unfolding of recombinant protein is a completely reversible process, whereas the preceding process of the dimerization of recombinant bOBP is the irre- versible event (Stepanenko et al., 2014c). To understand how interaction of the recombinant bOBP with its natural ligand, 1-Octen-3-ol (OCT) affects this protein, we investigated structural properties and conformational stability of the recombinant bOBP in the presence and absence of OCT. Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 Equilibrium unfolding of the recombinant bOBP in the presence of natural ligand The formation of the protein-ligand complex does not affect the oligomeric status of the protein, since according to the gel filtration analysis the protein in the bOBP/OCT complex continue to exist as a mixture of monomeric and dimeric molecules. However, both monomeric and dimeric forms of the protein become more compact as a result of the OCT binding (Table 1, Fig. 2). The GdnHCl-induced unfolding of the bOBP/OCT complex is a rather slow process, since the equilibrium unfolding curves are achieved after incubation of the bOBP/OCT in the presence of different concentrations of the denaturing agent for more than 24 h. However, the established equilibrium was not affected by further incubation for up to 5 days (Fig. 3). Figure 3 shows that the complexity of the bOBP/OCT unfolding is clearly illustrated by the complex shapes of the equilibrium dependencies of various characteristics of this complex on GdnHCl concentration. This suggests the accumulation of several interme- diate states, which are similar to partially folded species found during the equilibrium Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 8/23 Figure 2 The changes of hydrodynamic dimensions of recombinant bOBP (A and B) and its com- plex with ligand bOBP/OCT (C and D) in different structural states. The elution profiles for bOBP and bOBP/OCT were recorded during the protein denaturation (A and C) and renaturation from unfolded states (B and D) induced by GdnHCl. The elution profiles for bOBP were measured after pre-incubation of the protein and the solution of GdnHCl in desired concentration for 24 h (A and B), while in the case of bOBP/OCT the incubation time was extended to 72–84 h for denaturation (C) and 6 days for renaturation (D). The figures on the curves are the GdnHCl concentrations. Figure 2 The changes of hydrodynamic dimensions of recombinant bOBP (A and B) and its com- plex with ligand bOBP/OCT (C and D) in different structural states. The elution profiles for bOBP and bOBP/OCT were recorded during the protein denaturation (A and C) and renaturation from unfolded states (B and D) induced by GdnHCl. The elution profiles for bOBP were measured after pre-incubation of the protein and the solution of GdnHCl in desired concentration for 24 h (A and B), while in the case of bOBP/OCT the incubation time was extended to 72–84 h for denaturation (C) and 6 days for renaturation (D). The figures on the curves are the GdnHCl concentrations. Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 Equilibrium unfolding of the recombinant bOBP in the presence of natural ligand The measurements were preceded by incubating the protein in a solution with the appropriate GdnHCl concentration at 4 ◦C for 24 (red circles) in the case of bOBP. The open sym- bols indicate unfolding, whereas the closed symbols represent refolding. While studying the folding of bOBP/OCT (squares), the solution of complex of the protein with its ligand were incubated in a solution with the appropriate GdnHCl concentration at 4 ◦C for less than 24 h (open brown squares), up to 120 h (open pink squares) at the protein denaturation, and 1 h (closed brown squares) and 72 h–30 days (closed pink squares) at the protein renaturation. This hydrophobic fluorescent probe is frequently used for the analysis of the presence of solvent-exposed hydrophobic patches in a protein (Stryer, 1965) and for the detection of accumulation of partially folded intermediates during equilibrium and kinetic protein unfolding-refolding processes due the ability of ANS to bind to such solvent-exposed hydrophobic patches (which are commonly found in partially folded proteins) and due to the fact that this interaction can be easily detected by the significant increase in the ANS fluorescence intensity and a characteristic blue-shift of its fluorescence maximum (Semisotnov et al., 1991). The shape of the unfolding curve monitored by the GdnHCl- induced changes in the fluorescence intensity of ANS added to the bOBP/OCT was remarkably different from the unfolding curve measured for the recombinant bOBP alone. We observe a smooth continuous decrease in the ANS fluorescence intensity at moderate GdnHCl concentrations, with the ANS fluorescence intensity reaching zero at the denaturant concentrations leading to the formation of the compact dimeric form of bOBP (Fig. 3). This hydrophobic fluorescent probe is frequently used for the analysis of the presence of solvent-exposed hydrophobic patches in a protein (Stryer, 1965) and for the detection of accumulation of partially folded intermediates during equilibrium and kinetic protein unfolding-refolding processes due the ability of ANS to bind to such solvent-exposed hydrophobic patches (which are commonly found in partially folded proteins) and due to the fact that this interaction can be easily detected by the significant increase in the ANS fluorescence intensity and a characteristic blue-shift of its fluorescence maximum (Semisotnov et al., 1991). The shape of the unfolding curve monitored by the GdnHCl- induced changes in the fluorescence intensity of ANS added to the bOBP/OCT was remarkably different from the unfolding curve measured for the recombinant bOBP alone. Equilibrium unfolding of the recombinant bOBP in the presence of natural ligand unfolding of the recombinant bOBP in the absence of OCT. However, compared to the bOBP alone, in the case of the unfolding of bOBP/OCT complex, the accumulation of these intermediate states takes place at higher denaturant concentrations. In fact, a more compact intermediate state of the bOBP/OCT complex is formed in the concentration range of 0.26–1.0 M GdnHCl, whereas the transition of the bOBP/OCT complex to the dimeric state occurs only at 2.0 M GdnHCl (Fig. 2). Increasing the GdnHCl concentration over 2.0 M leads to the cooperative unfolding of the bOBP/OCT complex. In comparison with the unfolding of the recombinant bOBP alone, the unfolding of the bOBP/OCT complex occurs in a narrow concentration range, and higher denaturant concentrations are required for complete unfolding of this complex. All these data indicate that forma- tion of the bOBP/OCT complex leads to the substantial stabilization of the protein, but does not affect its unfolding mechanism. To better understand the GdnHCl-induced unfolding of the bOBP/OCT complex, we analyzed the dependence of the 8-anilinonaphthalene-1-sulfonic acid (ANS, also called 1-anilino-8-naphthalenesulfonate), fluorescence on the denaturant concentration. Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 9/23 9/23 Figure 3 bOBP and bOBP/OCT conformational changes induced by GdnHCl. (A): changes in fluores- cence intensity at 320 nm, λex = 297 nm; (B): changes in parameter (A), λex = 297 nm; (C): changes in fluorescence anisotropy at the emission wavelength 365 nm, lex = 297 nm; (D): changes in the ellipticity at 222 nm; (E): changes in the ANS fluorescence intensity at λex = 365 nm, λem = 480 nm. The statistical errors for fluorescence measurements were assessed and were shown to fall (continued on next page...) Figure 3 bOBP and bOBP/OCT conformational changes induced by GdnHCl. (A): changes in fluores- cence intensity at 320 nm, λex = 297 nm; (B): changes in parameter (A), λex = 297 nm; (C): changes in fluorescence anisotropy at the emission wavelength 365 nm, lex = 297 nm; (D): changes in the ellipticity at 222 nm; (E): changes in the ANS fluorescence intensity at λex = 365 nm, λem = 480 nm. The statistical errors for fluorescence measurements were assessed and were shown to fall (continued on next page...) Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 Figure 3 (...continued) within the range of 0.2–1%. Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 Equilibrium unfolding of the recombinant bOBP in the presence of natural ligand In fact, during the refolding process, equilibrium values of the analyzed structural characteristics of the bOBP/OCT complex are reached after the incubation of this complex in the presence of the desired GdnHCl concentration for 72 h. No subsequent changes were detected when protein was incubated for 30 days. This analysis revealed the presence of noticeable hysteresis between the curves describing the equilibrium unfolding and refolding of the bOBP/OCT complex in a wide range of the GdnHCl concentrations. In fact, the equilibrium unfolding and refolding curves coincide only in the vicinity of 2.0 M GdnHCl, where, according to the gel-filtration analysis, the native dimeric state of the bOBP/OCT complex is formed, whereas within the region corresponding to the transition from the native dimeric form to the completely unfolded state of the bOBP/OCT complex, equilibrium curves describing unfolding and refolding of this complex do not coincide. The equilibrium refolding curve describing transition from the unfolded to the compact dimeric state of the bOBP/OCT complex is shifted toward the lower GdnHCl concentrations in comparison with the equilibrium unfolding curve (Fig. 3). However, in comparison with the unfolding of the bOBP alone, this equilibrium refolding curve of the bOBP/OCT complex is still shifted toward higher GdnHCl concentrations. These data suggest that at the same denaturant concentrations, the fractions of native bOBP formed during the refolding from the completely unfolded state are significantly lower than the fraction of native protein remaining within the region of the bOBP/OCT unfolding. However, once formed, the native protein gains the ability to bind ligand. This hypothesis is supported by the results of the gel-filtration analysis (Fig. 2). For example, the elution profiles registered during the unfolding and refolding of the bOBP/OCT complex at 2.5 M GdnHCl show that under these conditions, more native protein is present during the bOBP/OCT unfolding, whereas unfolded species prevail during the refolding of this complex. Therefore, the effective rates of the formation of various bOBP conformers are significantly different during the unfolding and refolding processes and noticeably depend on the denaturant concentration. It is likely that the same reasons define the irreversibility of the unfolding of the bOBP/OCT complex at low GdnHCl concentrations. Under these conditions, the rate of the formation of the monomeric bOBP/OCT complex is significantly higher than the rate of the dimeric bOBP/OCT formation. Equilibrium unfolding of the recombinant bOBP in the presence of natural ligand We observe a smooth continuous decrease in the ANS fluorescence intensity at moderate GdnHCl concentrations, with the ANS fluorescence intensity reaching zero at the denaturant concentrations leading to the formation of the compact dimeric form of bOBP (Fig. 3). These observations suggest that ANS interacts with bOBP at sites close to and/or overlapping with the ligand binding sites. Therefore, the formation of the bOBP/OCT complex prevents ANS binding. Earlier analysis of the dimeric bOBP structure revealed the presence of an additional ligand binding site at the interface between the monomeric subunits (Bianchet et al., 1996; Ikematsu, Takaoka & Yasuda, 2005; Pevsner et al., 1985). However, this inter-subunit binding site was shown to be noticeably weaker than the major ligand binding site located within the β-barrel (Bianchet et al., 1996; Ikematsu, Takaoka & Yasuda, 2005; Pevsner et al., 1985). Our data agree with the presence of an additional ligand binding site in a protein. At the formation of the dimeric bOBP/OCT complex with the native-like compactness at 2.0 M GdnHCl, this additional site is occupied by the ligand, also preventing its interaction with ANS. Moderate ANS fluorescence is detected in solutions containing less than 2 M GdnHCl; i.e., under conditions where the bOBP/OCT complex exists as a mixture of monomeric and dimeric molecules, which are different from the native dimeric form of the bOBP. Under these conditions, ANS fluorescence intensity in the presence of the bOBP/OCT complex is noticeably lower than the ANS fluorescence recorded for the bOBP alone. These observations suggest that under these conditions the additional ligand binding site of the dimeric bOBP/OCT complex is occupied by ANS, whereas the inner cavity of the barrel is engaged in ligand binding. It is likely that the inability of the natural ligand to interact with the additional weak ligand binding site located between the monomeric Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 11/23 subunits can be due to the structural difference of this site in the native dimeric bOBP and in a protein in the original native-like state or an intermediate compact state. Analysis of the bOBP/OCT refolding from the completely unfolded state revealed that the dependencies of various structural characteristics of the bOBP/OCT on GdnHCl concentrations depend on the incubation time of this complex in the presence of the denaturant (see Fig. 3). Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 Equilibrium unfolding of the recombinant bOBP in the presence of natural ligand As a result, refolding of the bOBP/OCT complex at the low GdnHCl concentrations results in the preferential formation of monomeric bOBP/OCT species, whereas under the identical conditions, the unfolding reaction mixture contains roughly equimolar quantities of the bOBP/OCT monomers and dimers (Figs. 2 and 3). Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 12/23 Figure 4 The changes of hydrodynamic dimensions of recombinant bOBP-Gly121+ (A and B) and its complex with ligand bOBP-Gly121+/OCT (C and D) in different structural states. The elution pro- files for bOBP-Gly121+ and bOBP-Gly121+/OCT were recorded during the protein denaturation (A and C) and renaturation from unfolded states (B and D) induced by GdnHCl. The elution profiles for bOBP-Gly121+ were measured after pre-incubation of the protein and the solution of GdnHCl in desired concentration for 24 h (A and B), while in the case of bOBP-Gly121+/OCT the incubation time was ex- tended to 72–84 h for denaturation (C) and 6 days for renaturation (D). The figures on the curves are the GdnHCl concentrations. Figure 4 The changes of hydrodynamic dimensions of recombinant bOBP-Gly121+ (A and B) and its complex with ligand bOBP-Gly121+/OCT (C and D) in different structural states. The elution pro- files for bOBP-Gly121+ and bOBP-Gly121+/OCT were recorded during the protein denaturation (A and C) and renaturation from unfolded states (B and D) induced by GdnHCl. The elution profiles for bOBP-Gly121+ were measured after pre-incubation of the protein and the solution of GdnHCl in desired concentration for 24 h (A and B), while in the case of bOBP-Gly121+/OCT the incubation time was ex- tended to 72–84 h for denaturation (C) and 6 days for renaturation (D). The figures on the curves are the GdnHCl concentrations. Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 Equilibrium unfolding of the monomeric bOBP-Gly121+ While studying the folding of bOBP-Gly121+/OCT (squares), the solution of complex of the protein with its ligand were incubated in a solution with the appropriate GdnHCl concentration at 4 ◦C for less than 24 h (open dark yellow squares), up to 72 h (open light green squares) at the protein denaturation, and 1 h (closed dark yellow squares) and 72 h (closed light green squares) at the protein renaturation. The formation of the bOBP-Gly121+/OCT complex results in a noticeable stabilization of this protein. This is evidenced by the increase in the cooperativity of the unfolding transition, which is also shifted toward higher GdnHCl concentrations. However, the formation of a complex between the bOBP-Gly121+ and OCT does not affect the unfolding mechanism of this protein. Equilibrium unfolding of the monomeric bOBP-Gly121+ Already at relatively low GdnHCl concentrations, the monomeric bOBP-Gly121+ is converted to the compact partially folded state with structural characteristics resembling those of the partially folded species accumulated during the equilibrium unfolding of the recombinant bOBP (see Figs. 4, 5 and Table 1). This compact intermediate is able to bind ANS and exists in a wide range of the GdnHCl concentrations (up to about 1.3 M GdnHCl). Subsequent increase in the denaturant concentration promotes transition to a more loose form, which, at the further increase of the GdnHCl concentration, is converted to the completely unfolded state. This GdnHCl-induced unfolding of the bOBP-Gly121+ is a completely reversible process as evidenced by the coincidence of the equilibrium characteristics of the protein measured at the processes of the bOBP- Gly121+ unfolding and refolding. Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 13/23 Figure 5 bOBP-Gly121+ and bOBP-Gly121+/OCT conformational changes induced by GdnHC changes in fluorescence intensity at 320 nm, λex = 297 nm; (B): changes in parameter (A), λex = 2 (C): changes in fluorescence anisotropy at the emission wavelength 365 nm, lex = 297 nm; (D): cha the ellipticity at 222 nm; (E): changes in the ANS fluorescence intensity at λex = 365 nm, λem = 48 The measurements were preceded by incubating the protein in a solution (continued on next page Figure 5 bOBP-Gly121+ and bOBP-Gly121+/OCT conformational changes induced by GdnHCl. (A): changes in fluorescence intensity at 320 nm, λex = 297 nm; (B): changes in parameter (A), λex = 297 nm; (C): changes in fluorescence anisotropy at the emission wavelength 365 nm, lex = 297 nm; (D): changes in the ellipticity at 222 nm; (E): changes in the ANS fluorescence intensity at λex = 365 nm, λem = 480 nm. The measurements were preceded by incubating the protein in a solution (continued on next page...) Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 Figure 5 (...continued) with the appropriate GdnHCl concentration at 4 ◦C for 24 (dark green circles) in the case of bOBP- Gly121+. The open symbols indicate unfolding, whereas the closed symbols represent refolding. Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 GdnHCl-induced unfolding of the monomeric GCC-bOBP Analysis of the peculiarities of the equilibrium unfolding and refolding processes monitored by the GdnHCl-induced changes in various structural characteristics of the monomeric GCC-bOBP suggests that the unfolding of this protein is a completely reversible process accompanied by the formation of partially folded intermediates similar to those observed during the equilibrium unfolding of the recombinant bOBP and its monomeric bOBP-Gly121+ form (see Figs. 6, 7 and Table 1). However, although qualitatively unfolded processes of these three proteins are similar, there are some noticeable differences. For example, in comparison with the recombinant bOBP and bOBP-Gly121+ unfolding, a compact intermediate with high ANS affinity is formed at higher denaturant concentrations during the GCC-bOBP unfolding (at 1.0 M GdnHCl). This illustrates higher conformational stability of the disulfide-stabilized GCC-bOBP compared to the recombinant bOBP and its monomeric form bOBP-Gly121+. p y GCC-bOBP is further stabilized due to the GCC-bOBP/OCT complex formation. Refolding curves detected by changes in different structural characteristics of this complex and registered after the incubation of the corresponding solutions for one hour coincide with the transition curves describing the equilibrium unfolding of GCC-bOBP, and subsequent incubation of these same solutions for 72 h leads to the detectable shift of the transition curves. As a result, equilibrium unfolding and refolding transitions of the GCC-bOBP/OCT complex coincide suggesting that the unfolding of this protein is a completely reversible process. However, GCC-bOBP becomes able to bind ligand only after the formation of correct native structure stabilized by the disulfide bond. Earlier similar effects were described for other ligand-binding protein, such as the D-glucose/D- galactose-binding protein (GGBP) from E. coli (Stepanenko et al., 2011a; Stepanenko et al., 2009; Stepanenko et al., 2011b). In fact, our analysis of the peculiarities of the GGBP unfolding revealed that ligand binding might constitute a rate-limiting stage of the protein unfolding-refolding process. This phenomenon can be understood considering the fact that the formation of the protein-ligand complex depends on the appearance of the matching configurations between the ligand and the active site of a fully formed native protein (Stepanenko et al., 2011a; Stepanenko et al., 2009; Stepanenko et al., 2011b). Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 15/23 Figure 6 The changes of hydrodynamic dimensions of recombinant GCC-bOBP (A and B) and its complex with ligand GCC-bOBP/OCT (C and D) in different structural states. Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 GdnHCl-induced unfolding of the monomeric GCC-bOBP The elution profiles for GCC-bOBP and GCC-bOBP/OCT were recorded during the protein denaturation (A and C) and renat- uration from unfolded states (B and D) induced by GdnHCl. The elution profiles for GCC-bOBP were measured after pre-incubation of the protein and the solution of GdnHCl in desired concentration for 24 h (A and B), while in the case of GCC-bOBP/OCT the incubation time was extended to 72–84 h for de- naturation (C) and 6 days for renaturation (D). The figures on the curves are the GdnHCl concentrations. Figure 6 The changes of hydrodynamic dimensions of recombinant GCC-bOBP (A and B) and its complex with ligand GCC-bOBP/OCT (C and D) in different structural states. The elution profiles for GCC-bOBP and GCC-bOBP/OCT were recorded during the protein denaturation (A and C) and renat- uration from unfolded states (B and D) induced by GdnHCl. The elution profiles for GCC-bOBP were measured after pre-incubation of the protein and the solution of GdnHCl in desired concentration for 24 h (A and B), while in the case of GCC-bOBP/OCT the incubation time was extended to 72–84 h for de- naturation (C) and 6 days for renaturation (D). The figures on the curves are the GdnHCl concentrations. Our current analysis revealed that the unfolding of the monomeric complexes bOBP- Gly121+/OCT and GCC-bOBP/OCT is not accompanied by the ANS fluorescence enhancement in the whole range of GdnHCl concentrations (see Figs. 5 and 7). These observations support the hypothesis on the existence of the additional ligand-binding site in the dimeric bOBP. Table 2 shows that the equilibrium unfolding transition recorded for the recombinant bOBP coincides with that of its monomeric bOBP-Gly121+ form. On the other hand, unfolding transition of the monomeric GCC-bOBP stabilized by the engineered disulfide bond is noticeably shifted to higher denaturant concentrations. Curiously, the unfolding of complexes of all proteins analyzed in this study with natural ligand OCT happens at the same denaturant concentrations. Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 16/23 Figure 7 GCC-bOBP and GCC-bOBP/OCT conformational changes induced by GdnHCl. Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 GdnHCl-induced unfolding of the monomeric GCC-bOBP (A) Changes in fluorescence intensity at 320 nm, λex = 297 nm; (B) changes in parameter (A), λex = 297 nm; (C) changes in fluorescence anisotropy at the emission wavelength 365 nm, lex = 297 nm; (D) changes in the ellipticity at 222 nm; (E): changes in the ANS fluorescence intensity at (continued on next page...) Figure 7 GCC-bOBP and GCC-bOBP/OCT conformational changes induced by GdnHCl. (A) Changes in fluorescence intensity at 320 nm, λex = 297 nm; (B) changes in parameter (A), λex = 297 nm; (C) changes in fluorescence anisotropy at the emission wavelength 365 nm, lex = 297 nm; (D) changes in the ellipticity at 222 nm; (E): changes in the ANS fluorescence intensity at (continued on next page...) Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 Figure 7 (...continued) λex = 365 nm, λem = 480 nm. The measurements were preceded by incubating the protein in a solution with the appropriate GdnHCl concentration at 4 ◦C for 24 (blue circles) in the case of GCC-bOBP. The open symbols indicate unfolding, whereas the closed symbols represent refolding. While studying the fold- ing of GCC-bOBP/OCT (squares), the solution of complex of the protein with its ligand were incubated in a solution with the appropriate GdnHCl concentration at 4 ◦C for less than 24 h (open dark blue squares), up to 72 h (open light blue squares) at the protein denaturation, and 1 h (closed dark blue squares) and 72 h (closed light blue squares) at the protein renaturation. Table 2 Thermodynamic parameters of GdnHCl-induced denaturation of bOBP, its mutant variants and their complexes with octen-3-ol (OCT). Protein m (kJ mol−1 M−1) Cm (M)a 1G0 N−U b (kJ mol−1) bOBP 3.7±0.2 2.1±0.1 7.7±0.6 bOBP/OCT 4.1±0.2 2.7±0.1 11.0±0.5 bOBP/Gly121+ 2.8±0.2 2.0±0.1 5.5±0.4 bOBP/Gly121+/OCT 5.3±0.2 2.6±0.1 14.0±0.5 GCC-bOBP 2.5±0.3 2.3±0.1 5.8±0.7 GCC-bOBP/OCT 4.5±0.3 2.7±0.1 12.0±0.9 Notes. aCm is the denaturant concentration at midpoint of conformational transition. bThe fluorescence signals of the folded and unfolded states were approximated by linear dependences as function of denaturant concentration (Nolting, 1999). Table 2 Thermodynamic parameters of GdnHCl-induced denaturation of bOBP, its mutant variants and their complexes with octen-3-ol (OCT). Notes. aCm is the denaturant concentration at midpoint of conformational transition. bThe fluorescence signals of the folded and unfolded states were approximated by linear dependences as function of denaturant concentration (Nolting, 1999). Stepanenko et al. (2016), PeerJ, DOI 10.7717/peerj.1574 Competing Interests Irina M Kuznetsova, Vladimir N Uversky and Konstantin K Turoverov are Academic Editors for PeerJ. Data Availability The following information was supplied regarding data availability: All the data obtained during this study are reported in the manuscript in the form of figures and tables. Author Contributions • Olga V. Stepanenko and Vladimir N. Uversky conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. • Denis O. Roginskii and Olesya V. Stepanenko performed the experiments, analyzed the data, prepared figures and/or tables, reviewed drafts of the paper. • Irina M. Kuznetsova conceived and designed the experiments, analyzed the data, prepared figures and/or tables, reviewed drafts of the paper. • Konstantin K. Turoverov conceived and designed the experiments, analyzed the data, reviewed drafts of the paper. GdnHCl-induced unfolding of the monomeric GCC-bOBP It is important to note here that the quantitative characterization of the affinity of the bOBP and its mutants to the natural OCT ligand is beyond the scope of this study. In fact, the binding constant of the protein was determined in previous studies (Bianchet et al., 1996; Ikematsu, Takaoka & Yasuda, 2005; Pevsner et al., 1985). According to published data, binding constant of a natural OCT ligand to the triple mutant GCC-bOBP is in the micromolar range, which is similar to the affinity of the wild type protein (Ramoni et al., 2008). Amino acid substitutions introduced in mutant proteins, namely the Gly 121+ insertion and the W64C and H155C replacements, do not affect the ligand-binding site of the protein. Data reported in the first paper of this series (Stepanenko et al., 2016) revealed that these substitutions do not have significant influence on the protein tertiary and secondary structure. All these data indicate that the mutant forms of the bOBP protein might retain affinity inherent to the wild-type protein. Our data suggest that protein dimerization via the domain-swapping mechanism does not contribute much to the increase in the conformational stability of a protein (at least in the case of the analyzed in this study bOBP), despite the fact that the increased conformational stability was proposed as one of the factors determining the use of this mechanism for dimer and higher oligomer formation (Bennett, Schlunegger & Eisenberg, 1995; Liu & Eisenberg, 2002). In contrast, introduction of a disulfide bond to the structure of a monomeric protein shows significant stabilizing effects. Our data also show that the formation of a protein-ligand complex leads to the significant stabilization of different variants of bOBP and eliminates the original difference in conformational stability caused by their structural differences. Stepanenko et al. 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https://openalex.org/W4362466853	https://www.researchsquare.com/article/rs-1909719/latest.pdf	English	null	Trajectory Optimization of High-Speed Robotic Positioning with Suppressed Motion Jerk via Improved Chicken Swarm Algorithm	Applied sciences	2,023	cc-by	7,898	Trajectory optimization of high-speed robotic positioning with suppressed motion jerk via improved chicken swarm algorithm Yankun Li Qilu University of Technology Yuyang Lu Jiangnan University Dongya Li Jiangnan University Rui Li Jiangnan University Chonghai Xu Qilu University of Technology Xiaozhi Gao University of Eastern Finland Yu Liu (  yuliu@jiangnan.edu.c Jiangnan University https://orc Abstract To address the problems of premature convergence and low solution accuracy of the basic swarm optimization algorithm an improved chicken swarm algorithm based on parallel strategy and dynamic constraints (PDCSO) is proposed for the rooster update method with a parallel strategy while introducing X-best guidance and Levy flight step; dynamic constraints are given for the rooster followed by the hen, and the optimal rooster position is introduced, which improves the convergence accuracy while avoiding falling into local optimum. The simulation experiments on 18 classical test functions show that the PDCSO algorithm outperforms other comparative algorithms in terms of convergence speed, solution accuracy and solution stability. In addition, PDCSO is applied to robotic arm trajectory optimization by fitting the joint trajectory with 5 times B-sample curve, and after algorithm optimization and simulation verification in ADAMS, it is proved that PDCSO can effectively reduce the running time and motion shock of robotic arm and improve the execution efficiency of robotic arm. Keywords: Improved chicken swarm optimization, Levy flight, Robotic arm, Trajectory optimization Yankun Li1,¶ , Yuyang Lu2,¶ , Dongya Li 2 , Rui Li 3, Chonghai Xu 1 , Xiaozhi Gao 4,* an Yu Liu 2,* ∗ Corresponding authors. E-mail addresses: yuliu@jiangnan.edu.cn (Yu Liu); xiao-zhi.gao@uef.fi (Xiaozhi Gao) ∗ Corresponding authors. E-mail addresses: yuliu@jiangnan.edu.cn (Yu Liu); xiao-zhi.gao@uef.fi (Xiaozhi Gao) ¶ These authors contribute to this work equally. ¶ These authors contribute to this work equally. Research Article Keywords: Improved chicken swarm optimization, Levy §ight, Robotic arm, Trajectory optimization Posted Date: February 9th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-1909719/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 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Trajectory optimization of high-speed robotic positioning with suppressed motion jerk via improved chicken swarm algorithm Trajectory optimization of high-speed robotic positioning with suppressed motion jerk via improved chicken swarm algorithm Yankun Li1,¶ , Yuyang Lu2,¶ , Dongya Li 2 , Rui Li 3, Chonghai Xu 1 , Xiaozhi Gao 4,* and Yu Liu 2,* 1School of Mechanical & Automotive Engineering, Qilu University of Technology, Jinan, China； 2School of Mechanical Engineering, Jiangnan University, Wuxi, China 3School of Design, Jiangnan University, Wuxi, China 4Faculty of Science and Forestry, School of Computing, University of Eastern Finland, Joensuu, Kuopio ∗ Corresponding authors. E-mail addresses: yuliu@jiangnan.edu.cn (Yu Liu); xiao-zhi.gao@uef.fi (Xiaozhi Gao) ¶ These authors contribute to this work equally. Yankun Li1,¶ , Yuyang Lu2,¶ , Dongya Li 2 , Rui Li 3, Chonghai Xu 1 , Xiaozhi Gao 4,* and Yu Liu 2,* 1School of Mechanical & Automotive Engineering, Qilu University of Technology, Jinan, China； 2School of Mechanical Engineering, Jiangnan University, Wuxi, China 3School of Design, Jiangnan University, Wuxi, China 4Faculty of Science and Forestry, School of Computing, University of Eastern Finland, Joensuu, Kuopio 1 Introduction 通信作者刘禹yuliu@jiangnan edu cn Chicken Swarm Optimization (CSO), proposed by Meng X et al[1], is a novel swarm intelligence optimization algorithm. The problem was optimized by simulating the intelligent behavior of the swarm composed of roosters, hens, and chicks when foraging for food, which has the characteristics of simple structure and easy implementation. The chicken swarm optimization algorithm has the advantages of clear structure, easy understanding, and robust searching ability. And it further has better convergence than the particle swarm algorithm, differential evolution algorithm, and artificial bee swarm algorithm [1]. It has been applied for task scheduling, route optimization, etc.[2], and has broad research prospects. The chicken swarm algorithm suffers from the problem of falling into local optimality, which leads to premature maturity of the algorithm, and low solution accuracy. In response to the issues of the CSO algorithm, researchers have proposed improvement methods. Irsalinda N et al. [3] proposed a multi-stage CSO algorithm according to the search characteristics of different chickens. In the first stage the algorithm focuses on the hen update method to strengthen the global exploration ability; in the second stage, the algorithm focuses on the chick and rooster update to reflect the local exploitation advantage. Zhang M[4] proposed an improved CSA based on forward and backward learning rooster particles, which learn positively from the optimal particles in each iteration. So that the algorithm quickly enters the most promising region to find food; while learning backward from the worst particles to jump out of the local optimum when the algorithm is stuck in the local optimum solution. Zhang K et al. [5] proposed improving X-best bootstrap individuals and a dynamic rank update mechanism. Not only the best individuals were introduced to accelerate the convergence in the individual update phase, but also the influence of ordinary individuals on the best individuals was properly balanced. And the contribution of the population rank update mechanism to the algorithm convergence was enhanced through dynamic optimization of the rank update parameters. Gu Y et al. [6] removed the chicks, simplified the algorithm, proposed an improved update for both males and females based on inverted s-shaped inertia rights, and added an adaptive update strategy to the update process for females. Liang X et al. [7] introduced an improved search strategy with Levy flight characteristics into the location update equation of hens, which helps to increase the algorithm's perturbation and the population's diversity. 1 Introduction Second, a nonlinear decreasing strategy was added to the chicken's position update equation to improve the chicken's self-learning ability. Trajectory planning is the basis of robot motion control, related to the stability of robot motion, work efficiency and energy consumption, etc.[8-11], Robot trajectory planning is to find the trajectory of the robot's hand end movement to meet the process requirements and robot performance, linking the robot in the process of movement in space and time [12]. Optimized trajectory planning can complete the process requirements, reduce the quick jerk, and improve the construction quality of motion. For example, Peng X et al. [13] used a non-dominated neighborhood immune genetic algorithm to optimize the multi-objective trajectory function of the robot. They obtained the optimal position, velocity, acceleration, and acceleration planning curves for each joint of the robot. Huang J et al. [14] used a 5th order B-spline to interpolate the trajectory into the joint space. They then used an elite non-dominated ranking genetic algorithm (NSGA-II) to optimize the two objectives, i.e., motion time and average acceleration for the entire trajectory. Zhang Y et al. [15] realized the time-jerk optimal trajectory planning of the excavator by using the SQP algorithm with the constraints of joint angular velocity, angular acceleration, and angular acceleration by interpolating in the joint space with three order B-splines. Du Y et al. [16]proposed a method based on segmented polynomial interpolation function and local chaotic particle swarm optimization (LCPSO) algorithm to achieve robotic arm time optimality. Different trajectory planning methods can achieve different planning effects, and among many planning algorithms B-spline curve[17] has outstanding advantages: the trajectory is smooth and can guarantee velocity, acceleration, and acceleration continuity simultaneously; however, it brings substantial computational effort compared to other methods. At the same time, a single optimal trajectory planning cannot meet the comprehensive requirements in modern industrial applications. The integrated consideration of two or more optimality is more suitable for trajectory planning in practical applications[18]. Complex planning methods and multi-objective optimization lead to the increased computational effort and consideration of the complex constraints brought about by the actual working conditions, all placing higher demands on the solution accuracy and the solution speed of the optimization algorithm. 1 Introduction In this paper, the current shortcomings of the CSO algorithm are improved by proposing a parallel strategy for the rooster update method while introducing X-best guidance and Levy flight step; dynamic constraints are proposed for the rooster followed by the hen, and the optimal rooster position following is introduced to improve the convergence accuracy while avoiding falling into local optimum. It is also applied to the problem of robotic arm trajectory optimization, which uses a five order B-spline function to fit the trajectory of the shock continuum from the objectives of time reduction and shock limitation. The simulation results for the above algorithm prove that the proposed optimization method is effective in improving the convergence speed and convergence accuracy of the algorithm, and it is verified in ADAMS that the improved algorithm can effectively reduce the running time and motion shock of the robotic arm, improve the execution efficiency of the robotic arm, and lay the foundation for completing the motion control of the robotic arm. 2 Improved chicken swarm algorithm A stochastic optimization algorithm that simulates the behavioral habits of chickens and the hierarchy within the swarm is called Chicken Swarm Optimization (CSO). The basic idea of the CSO algorithm is to classify chickens into roosters, hens, and chicks according to their fitness values. By assuming a series of rules, different types of chickens follow different movement rules and compete with each other to find food. The CSO algorithm is a global optimization algorithm. g The chicken swarm algorithm follows the following rules： In a chicken swarm, there are several groups. Each group consists of a dominant rooster, several hens d i d i h f h hi k ( h d hi k ) l f h b d In a chicken swarm, there are several groups. Each group consists of a dominant rooster, several hens and chicks. Adaptation determines the status of the chickens (roosters, hens and chicks). Several of the best-adapted chickens will be treated as roosters, with each rooster being the lead rooster in a group. A few of the least well-adapted chickens will be designated as chicks. The others should be hens. The hens randomly choose which group to live in. The mother- In a chicken swarm, there are several groups. Each group consist Adaptation determines the status of the chickens (roosters, hens and chicks). Several of the best-adapted chickens will be treated as roosters, with each rooster being the lead rooster in a group. A few of the least well-adapted chickens will be designated as chicks. The others should be hens. The hens randomly choose which group to live in. The mother- child relationship between the hen and the chick is also established randomly. p y The hierarchical order, dominance and mother-child relationships within th The hierarchical order, dominance and mother-child relationships within the group remain unchanged. These states are renewed every 𝐺 generations. Each group is renewed differently, with the hens in each group following the roosters in that grou food, or randomly stealing food from other groups; the chicks in each group follow the mother hen to fo Each group is renewed differently, with the hens in each group following the roosters in that group to forage for food, or randomly stealing food from other groups; the chicks in each group follow the mother hen to forage for food. 2 Improved chicken swarm algorithm 𝑥𝑖 𝑗(𝑡+ 1) = 𝑥𝑖 𝑗+ 𝐹𝐿⋅(𝑥𝑚 𝑗(𝑡) −𝑥𝑖 𝑗(𝑡)) (6) 𝑥𝑖 𝑗(𝑡+ 1) = 𝑥𝑖 𝑗+ 𝐹𝐿⋅(𝑥𝑚 𝑗(𝑡) −𝑥𝑖 𝑗(𝑡)) (6) where the subscript 𝑚 denotes the mother hen of chick 𝑖 ; 𝐹𝐿 is the coefficient of the chick following the mother hen, which takes the value within[0,2]. 2 Improved chicken swarm algorithm The individual's position corresponds to the optimization problem's solution, and the algorithm eliminates the lagging individuals of the previous generation with the new generation of outstanding individuals at each iteration. food, or randomly stealing food from other groups; the chicks in each group follow the mother hen to forage for food. The individual's position corresponds to the optimization problem's solution, and the algorithm eliminates the lagging individuals of the previous generation with the new generation of outstanding individuals at each iteration. The individual's position corresponds to the optimization problem's solution, and the algorithm lagging individuals of the previous generation with the new generation of outstanding individuals at eac The total number of clusters in the population is 𝑁𝑝𝑜𝑝, the number of roosters is 𝑁𝑟,the number of hens is 𝑁ℎ, the number of chicks is 𝑁ℎ,and the number of mother hens is 𝑁𝑚. The mother hen is chosen randomly among the hens and has chicks that follow her. Roosters dominate the entire foraging process and can forage in a much larger space. The rooster p equation is as follows: 𝑥𝑖 𝑗(𝑡+ 1) = 𝑥𝑖 𝑗(𝑡)(1 + 𝑅𝑎𝑛𝑑𝑛(0, 𝜎2)) (1) 𝜎2 = { 1 𝑓𝑖< 𝑓𝑖 𝑒𝑥𝑝( 𝑓𝑘−𝑓𝑖 \|𝑓𝑖\|+𝜀) else (2) (1) (2) 𝑓𝑖 Where 𝑅𝑎𝑛𝑑𝑛(0, 𝜎2) is the Gaussian distribution with mean0 and variance 𝜎2 ; 𝜀 is the small constant introduced to prevent the denominator from being; 𝑘 is another individual in the rooster population different from the current 𝑖individual, and,𝑘∈[1, 𝑁𝑟], 𝑘≠𝑖, 𝑓𝑘 is its fitness. Hen location updated： p 𝑥𝑖 𝑗(𝑡+ 1) = 𝑥𝑖 𝑗(𝑡) + 𝑆1 ⋅𝑅𝑎𝑛𝑑⋅(𝑥𝑟1 𝑗(𝑡) −𝑥𝑖 𝑗(𝑡)) + 𝑆2 ⋅𝑅𝑎𝑛𝑑⋅(𝑥𝑟2 𝑗(𝑡) −𝑥𝑖 𝑗(𝑡)) (3) 𝑆1 = 𝑒𝑥𝑝( 𝑓𝑖−𝑓𝑟1 \|𝑓𝑖\|+𝜀) (4) 𝑆2 = 𝑒𝑥𝑝( 𝑓𝑟2 −𝑓𝑖) (5) (3) (3) (4) (5) (3) (4) (5) dual (4) (5) (5) (5) Where 𝑅𝑎𝑛𝑑 is the random number within [0,1];𝑟1 is the rooster corresponding to hen 𝑖,𝑟2 is different from 𝑟1 randomly selected in the rooster population and the hen population. Where 𝑅𝑎𝑛𝑑 is the random number within [0,1];𝑟1 is the rooster corresponding to hen 𝑖,𝑟2 is an individual different from 𝑟1 randomly selected in the rooster population and the hen population. Chick position update Chick position update. 2.1.1 Parallel policy-based X-best bootstrap and Levy flight cock update mechanism 2.1.1 Parallel policy-based X-best bootstrap and Levy flight cock update mechanism In order to overcome the poor solution accuracy and prematureness when X-best is introduced this p 2.1.1 Parallel policy-based X-best bootstrap and Levy flight cock update mechanism In order to overcome the poor solution accuracy and prematureness when X-best is introduced, this paper propos a rooster update mechanism based on a parallel strategy for X-best guidance and Levy flight. In order to overcome the poor solution accuracy and prematureness when X-best is introduced, this pap rooster update mechanism based on a parallel strategy for X-best guidance and Levy flight. First, the global most individual𝑥𝑔𝑏𝑒𝑠𝑡bootstrap is introduced in the rooster update equation and in order to avoid the over-dependence on𝑥𝑔𝑏𝑒𝑠𝑡that causes it to fall into the local optimum, the adjustment coefficient 𝜔 is introduced before the𝑥𝑔𝑏𝑒𝑠𝑡term, and the improved rooster update equation is as follows. 𝑥𝑖 𝑗(𝑡+ 1) = 𝑥𝑖 𝑗(𝑡) + 𝑅𝑎𝑛𝑑𝑛(0, 𝜎2) ⋅(𝜔⋅𝑥𝑔𝑏𝑒𝑠𝑡 𝑗 (𝑡) −𝑥𝑖 𝑗(𝑡)) (7) 𝑥𝑖 𝑗(𝑡+ 1) = 𝑥𝑖 𝑗(𝑡) + 𝑅𝑎𝑛𝑑𝑛(0, 𝜎2) ⋅(𝜔⋅𝑥𝑔𝑏𝑒𝑠𝑡 𝑗 (𝑡) −𝑥𝑖 𝑗(𝑡)) (7) 𝑗 (7) Where 𝑥𝑔𝑏𝑒𝑠𝑡 𝑗 (𝑡) is the position of the optimal individual in the 𝑡th iteration, and 𝜔 is the regulat Where 𝑥𝑔𝑏𝑒𝑠𝑡 𝑗 (𝑡) is the position of the optimal individual in the 𝑡th iteration, and 𝜔 is the regulation coefficient. The modulation coefficient𝜔 is designed as three segments, which are unreferenced, progressively referenced, and fully referenced. The equation for the regulation factor 𝜔 is as follows: The modulation coefficient𝜔 is designed as three segments, which are unreferenced, progressively referenced, and fully referenced. The equation for the regulation factor 𝜔 is as follows: g { 𝜔= 0 𝑡< 𝑇/3 𝜔= 1 2 − 𝑐𝑜𝑠((𝑡−𝑇/3)⋅𝑝𝑖/(𝑇/3)) 2 𝑇/3 < 𝑡< 2𝑇/3 𝜔= 1 𝑒𝑙𝑠𝑒 (8) q g { 𝜔= 0 𝑡< 𝑇/3 𝜔= 1 2 − 𝑐𝑜𝑠((𝑡−𝑇/3)⋅𝑝𝑖/(𝑇/3)) 2 𝑇/3 < 𝑡< 2𝑇/3 𝜔= 1 𝑒𝑙𝑠𝑒 (8) (8) Where 𝑇 is the maximum number of iterations, and 𝑡 is the 𝑡th iteration. mum number of iterations, and 𝑡 is the 𝑡th iteration. Where 𝑇 is the maximum number of iterations, and 𝑡 is the 𝑡th iteration. Unreferenced stage 𝜔= 0, equation (7) is the same as the original algorithm equation(1) after simplification, At this point the cock position update is not affected by 𝑥𝑔𝑏𝑒𝑠𝑡. Progressive reference Stage, 𝜔 according to equation (8) non-linear increasing, when 𝑥𝑔𝑏𝑒𝑠𝑡 gr venes to update the position of the cockerel. 2.1 Improved CSO The rooster occupies the dominant position in the whole population, which guides the hens and chicks toward the optimal solution, and the update equation in the standard CSO algorithm is more conducive to maintaining the diversity of the population[1],but leads to the problems of low solution accuracy and weak local solution capability in the chicken swarm algorithm. A reasonable X-best bootstrap mechanism has the advantage of improving the solution accuracy, but its unreasonable use will make the population individuals overly dependent on X-best individuals, which in turn leads to the reduction of population diversity and increases the possibility of falling into local optimal solutions. To address this problem, many scholars have introduced the optimal global position into the rooster position update [4,5,19]. 2.1.1 Parallel policy-based X-best bootstrap and Levy flight cock update mechanism Full reference stage 𝜔= 1,at which point 𝑥𝑔𝑏𝑒𝑠𝑡 fully intervenes to update the position of the rooster. To visualize the three stages of the modulation coefficient 𝜔, whose image is shown in Fig. 1, the maximum number of iterations 𝑇= 1000. And it can be seen that the three stages of unreferenced, asymptotically referenced and fully referenced are clearly distinguished following the above requirements. and fully referenced are clearly distinguished following the above requirements. Fig. 1𝝎 modulation factor curve Fig. 1𝝎 modulation factor curve (A; Unreferenced stage; B: Progressive reference stage; C: Full reference stage.) (A; Unreferenced stage; B: Progressive reference stage; C: Full reference stage.) Levy flight is a random wandering model according to the Levy distribution proposed by the French mathematician Levy [20]. The Levy flight process simulates smaller steps in most cases. Occasionally, larger steps are introduced into the original rooster update equation to increase the randomness of roosters further and increase the diversity of the population. y p p Introducing Levy's cock position update equation 𝑗 𝑗 cock position update equation 𝑥𝑖 𝑗(𝑡+ 1) = 𝑥𝑖 𝑗(𝑡) ⋅(1 + 𝑅𝑎𝑛𝑑𝑛(0, 𝜎2)) ⊗𝐿𝑒𝑣𝑦(𝜆) (9) Introducing Levy s cock position update equation 𝑥𝑖 𝑗(𝑡+ 1) = 𝑥𝑖 𝑗(𝑡) ⋅(1 + 𝑅𝑎𝑛𝑑𝑛(0, 𝜎2)) ⊗𝐿𝑒𝑣𝑦(𝜆) (9) Where 𝐿𝑒𝑣𝑦(𝜆) is a Levy distribution obeying parameter 𝜆, 𝜆=1.5 𝐿𝑒𝑣𝑦(𝜆) = 𝜇 \|𝜈\| 1 𝜆 (10) Where 𝜇、𝜐 is the definition of the positive-terrestrial distribution {𝜇∼𝑁(0, 𝜎𝜇 2) 𝜈∼𝑁(0, 𝜎𝜈 2) (11) in the equation: 𝜎 { 𝛤（1+𝛽）⋅𝑠𝑖𝑛(𝜋⋅𝛽 2 )} 1 𝛽 p p q 𝑥𝑖 𝑗(𝑡+ 1) = 𝑥𝑖 𝑗(𝑡) ⋅(1 + 𝑅𝑎𝑛𝑑𝑛(0, 𝜎2)) ⊗𝐿𝑒𝑣𝑦(𝜆) (9 (9) 𝑥𝑖(𝑡+ 1) = 𝑥𝑖(𝑡) ⋅(1 + 𝑅𝑎𝑛𝑑𝑛(0, 𝜎)) ⊗𝐿𝑒𝑣𝑦(𝜆) (9) Where 𝐿𝑒𝑣𝑦(𝜆) is a Levy distribution obeying parameter 𝜆, 𝜆=1.5 𝐿𝑒𝑣𝑦(𝜆) = 𝜇 \|𝜈\| 1 𝜆 (10) (10) \|𝜈\| Where 𝜇、𝜐 is the definition of the positive-terrestrial distribution 𝑁(0 2) (11) in the equation: { 𝜎𝜇= { 𝛤（1+𝛽）⋅𝑠𝑖𝑛(𝜋⋅𝛽 2 ) 𝛤[（1+𝛽） 2 ]⋅2 𝛽−1 2 ⋅𝛽 } 1 𝛽 𝜎𝜈=1 (12) (12) As for the rooster update equation (7) and equation (9) a parallel update strategy is used, where both equations exist simultaneously but the vast majority of roosters update their positions according to equation (7) a so as to improve the solution accuracy of the algorithm. 2.1.1 Parallel policy-based X-best bootstrap and Levy flight cock update mechanism A small number of roosters are updated according to equation (9), to ensure the diversity of solutions and to improve the possibility of jumping out of the local optimal solution. Moreover, each rooster is not updated in a fixed way, and is selected among the two with a certain probability. In this paper, the probability of equation (7) is 30% and the probability of equation (9) is 70%. { 𝑥𝑖 𝑗(𝑡+ 1) = 𝑥𝑖 𝑗(𝑡) + 𝑅𝑎𝑛𝑑𝑛(0, 𝜎2) ⋅(𝜔⋅𝑥𝑔𝑏𝑒𝑠𝑡 𝑗 (𝑡) −𝑥𝑖 𝑗(𝑡)) 30% 𝑥𝑖 𝑗(𝑡+ 1) = 𝑥𝑖 𝑗(𝑡) ⋅(1 + 𝑅𝑎𝑛𝑑𝑛(0, 𝜎2)) ⊗𝐿𝑒𝑣𝑦(𝜆) 70% (13) (13) 2.1.2 Dynamic constrained hen-following goal mechanism The number of hens is the largest in the whole swarm. Led by the rooster, the hens perform local search near the rooster, so the merit of the rooster determines the merit of the hen's search space. In the original algorithm, the rooster followed by the hen is randomly selected from the whole rooster swarm, and although this approach can improve the diversity of solutions, it limits the solution accuracy. In this regard, a dynamic constraint is proposed for the roosters, which is converted from randomly selecting all roosters to high-quality roosters, while an inferior rooster is added to the high-quality roosters to ensure the diversity of solutions. As the number of followed roosters gradually decreases, the number of follower hens per rooster gradually increases, which in turn improves the search accuracy under the region around the current rooster. Fig. 2 shows the idea of the constraint strategy for hen-following roosters. Fig. 2 Being followed by cock-binding strategies Fig. 2 Being followed by cock-binding strategies The constraint mechanism for hens to follow the males should have the following characteristics： The total number of roosters is constant, and the number of roosters followed by hens in the process of following all roosters to following quality roosters is decreasing, and the number of roosters followed should be a decreasing function. In the early iteration, the number of followed roosters should be quickly reduced to determine the approximate range of the optimal solution quickly. In the late iteration, keep constant and reduce variation to achieve exact search and improve the accuracy of the solution. There is always the worst rooster among good roosters to improve the diversity of solutions and avoid falling into local optima. p Based on the above analysis, the hen follows the rooster constraint equation as follows: 1 𝑅𝑁𝑢𝑚= {𝑓𝑙𝑜𝑜𝑟（ 𝑟𝑁𝑢𝑚 10 × 10 1 1+2×𝑡/𝑇）+ 1 𝐴 𝑏𝑎𝑑 𝑐𝑜𝑐𝑘 (14) (14) Where 𝑅𝑁𝑢𝑚 is the total number of roosters, 𝑟𝑁𝑢𝑚 is the bound roosters. The change in the number of constrained roosters curve is shown in Fig. 3, where the maximum number of iterations𝑇= 1000, the total number of roosters𝑟𝑁𝑢𝑚= 20, It can be seen that the number of constrained roosters decreases abruptly in the first 500 iterations and changes relatively little in the last 500 iterations. The above requirements are met. Fig. 3 Constraint rooster number curve Fig. 2.1.2 Dynamic constrained hen-following goal mechanism 3 Constraint rooster number curve g By introducing the best quality rooster into the hen's position update, the hen is attracted by the best value rooster while following the target rooster of the group in which it is located. So that it does not just search randomly around the target rooster of the group, giving it a certain target, which can improve the convergence accuracy to some extent. The idea of the hen's update strategy with the introduction of the best quality rooster is shown in Fig.4. Fig. 4 Double cock-following strategy Fig. 4 Double cock-following strategy The equation is as follows： 𝑥𝑖 𝑗(𝑡+ 1) = 𝑥𝑖 𝑗(𝑡) + 𝑆1 ⋅𝑅𝑎𝑛𝑑⋅(𝑥𝑟1 𝑗(𝑡) −𝑥𝑖 𝑗(𝑡)) + 𝑆2 ⋅𝑅𝑎𝑛𝑑⋅(𝑥𝑟2 𝑗(𝑡) −𝑥𝑖 𝑗(𝑡)) + 𝑆3 ⋅𝑅𝑎𝑛𝑑⋅(𝑥𝑟3 𝑗(𝑡) −𝑥𝑖 𝑗(𝑡)) (15) 𝑆3 = 𝑒𝑥𝑝( 𝑓𝑟3 −𝑓𝑖) (16) Where 𝑟3 is the optimal value cock g g gy The equation is as follows： 𝑥𝑖 𝑗(𝑡+ 1) = 𝑥𝑖 𝑗(𝑡) + 𝑆1 ⋅𝑅𝑎𝑛𝑑⋅(𝑥𝑟1 𝑗(𝑡) −𝑥𝑖 𝑗(𝑡)) + 𝑆2 ⋅𝑅𝑎𝑛𝑑⋅(𝑥𝑟2 𝑗(𝑡) −𝑥𝑖 𝑗(𝑡)) + 𝑆3 ⋅𝑅𝑎𝑛𝑑⋅(𝑥𝑟3 𝑗(𝑡) −𝑥𝑖 𝑗(𝑡)) (15) 𝑆3 = 𝑒𝑥𝑝( 𝑓𝑟3 −𝑓𝑖) (16) Where 𝑟3 is the optimal value cock (15) (16) (16) Where 𝑟3 is the optimal value cock Table 1 PDCSO Pseudocode Pseudocode of the PDCSO algorithm. Initialize a population of N chickens and define the related parameters; 2.2 Implementation steps of the algorithm p p g The CSO is PDCSO after introducing the parallel policy based on X-best bootstrap and Levy flight cock update mechanism with dynamic constraint hen following target mechanism at the same time, the flowchart is as Fig. 5 and its pseudo code is as Table 1. Fig. 5 The flowchart of PDCSO Fig. 5 The flowchart of PDCSO 2.3 Improved algorithm performance testing To verify the effectiveness of the proposed improvement mechanism with PDCSO, the improved algorithm was tested with 18 test functions. The simulation environment was MATLAB, and the simulation computer was configured with Inter® Core™ i7-6700HQ CPU@2.60Hz 2.60Hz,16GB running memory. The information about the test function is shown in Table 3, which contains the test function's search range, test dimension, and theoretical optimal value. The adjustable dimensionality of the function can be used to test the performance of the algorithm in high dimensionality. And the fixed dimensionality of the test function 𝑓1~𝑓12 function with adjustable dimensionality can be used to test the performance of the algorithm in high-dimensional cases, and 𝑓13~𝑓18is a fixed-dimensional test function. The main algorithms involved in the simulation comparison are CSO, PDCSO, ICSO[7]and ASCSO-S[6]. The parameter settings of each algorithm are shown in Table 2 , the maximum number of iterations of each algorithm is 1000, and the test results after 30 independent runs are shown in Table 4. Table 4. Initialize a population of N chickens and define the related parameters; Evaluate the𝑁chickens’ fitness values,𝑡= 0; Evaluate the𝑁chickens’ fitness values,𝑡= 0; While (𝑡< 𝑀𝑎𝑥_𝐺𝑒𝑛𝑒𝑟𝑎𝑡𝑖𝑜𝑛) While (𝑡< 𝑀𝑎𝑥_𝐺𝑒𝑛𝑒𝑟𝑎𝑡𝑖𝑜𝑛) If (𝑚𝑜𝑑( 𝑡, 𝐺) == 1) Rank the chickens’ fitness values and establish a hierarchal order in the swarm; Divide the swarm into different groups, and determine the relationship between the chicks and mother hens in a group; End Select Rooster update method using equation (13) 2.3 Improved algorithm performance testing Table 2 Test function parameters Algorithm Parameter Settings CSO 𝑁𝑝𝑜𝑝= 100，𝑁𝑟= 𝑁𝑐= 0.2𝑁𝑝𝑜𝑝，𝑁ℎ= 0.6𝑁𝑝𝑜𝑝，𝑁𝑚= 0.1𝑁ℎ，𝐺= 10，0.4 ≤𝐹𝐿≤1 PDCSO 𝑁𝑝𝑜𝑝= 100、𝑁𝑟= 𝑁𝑐= 0.2𝑁𝑝𝑜𝑝、𝑁ℎ= 0.6𝑁𝑝𝑜𝑝、𝑁𝑚= 0.1𝑁ℎ、𝐺= 10、0.4 ≤𝐹𝐿≤1 ICSO 𝑁𝑝𝑜𝑝= 100、𝑁𝑟= 𝑁𝑐= 0.2𝑁𝑝𝑜𝑝、𝑁ℎ= 0.6𝑁𝑝𝑜𝑝、𝑁𝑚= 0.1𝑁ℎ、𝐺= 100、𝜔max = 0.9、𝜔min = 0.4、𝑐= 10 ASCSO-S 𝑁𝑝𝑜𝑝= 100、𝑁𝑟= 𝑁𝑐= 0.2𝑁𝑝𝑜𝑝、𝑁ℎ= 0.6𝑁𝑝𝑜𝑝、𝑁𝑚= 0.1𝑁ℎ、𝐺= 100、𝜔max = 0.7、𝜔min = 0.1、𝑎= 6、𝑏 = 0.7、𝐾= 10 Table 2 Test function parameters Algorithm Parameter Settings CSO 𝑁𝑝𝑜𝑝= 100，𝑁𝑟= 𝑁𝑐= 0.2𝑁𝑝𝑜𝑝，𝑁ℎ= 0.6𝑁𝑝𝑜𝑝，𝑁𝑚= 0.1𝑁ℎ，𝐺= 10，0.4 ≤𝐹𝐿≤1 PDCSO 𝑁𝑝𝑜𝑝= 100、𝑁𝑟= 𝑁𝑐= 0.2𝑁𝑝𝑜𝑝、𝑁ℎ= 0.6𝑁𝑝𝑜𝑝、𝑁𝑚= 0.1𝑁ℎ、𝐺= 10、0.4 ≤𝐹𝐿≤1 ICSO 𝑁𝑝𝑜𝑝= 100、𝑁𝑟= 𝑁𝑐= 0.2𝑁𝑝𝑜𝑝、𝑁ℎ= 0.6𝑁𝑝𝑜𝑝、𝑁𝑚= 0.1𝑁ℎ、𝐺= 100、𝜔max = 0.9、𝜔min = 0.4、𝑐= 10 ASCSO-S 𝑁𝑝𝑜𝑝= 100、𝑁𝑟= 𝑁𝑐= 0.2𝑁𝑝𝑜𝑝、𝑁ℎ= 0.6𝑁𝑝𝑜𝑝、𝑁𝑚= 0.1𝑁ℎ、𝐺= 100、𝜔max = 0.7、𝜔min = 0.1、𝑎= 6、𝑏 = 0.7、𝐾= 10 Table 3 Test functions and parameters Function Range Dimension Theoretically Optimal 𝑓1(𝑥) = ∑𝑥𝑖 2 𝐷 𝑖=1 [-100,100] D 0 𝑓2(𝑥) = ∑\|𝑥𝑖\| 𝐷 𝑖=1 + ∏\|𝑥𝑖\| 𝐷 𝑖=1 [-10,10] D 0 𝑓3(𝑥) = ∑(∑𝑥𝑗 𝑖 𝑗=1 𝐷 𝑖=1 )2 [-100，100] D 0 𝑓4(𝑥) = 𝑚𝑎𝑥{\|𝑥𝑖\|, 1 ≤𝑖≤𝐷} [-100，100] D 0 𝑓5(𝑥) = ∑(\|𝑥𝑖+ 0.5\|)2 𝐷 𝑖=1 [-100，100] D 0 𝑓6(𝑥) = ∑𝑖𝑥𝑖 4 + 𝑟𝑎𝑛𝑑𝑜𝑚0,1) 𝐷 𝑖=1 [-1.28，1.28] D 0 𝑓7(𝑥) = ∑[𝑥𝑖 2 −10 𝑐𝑜𝑠( 2𝜋𝑥𝑖)] 𝐷 𝑖=1 + 10𝐷 [-5.12，5.12] D 0 𝑓8(𝑥) = −20 𝑒𝑥𝑝( −0.2√1 30 ∑𝑥𝑖 2 𝐷 𝑖=1 ) −𝑒𝑥𝑝1 30 ∑𝑐𝑜𝑠2 𝜋𝑥𝑖 30 𝑖=1 ) + 20 + 𝑐 [-32，32] D 0 𝑓9(𝑥) = 1 4000 ∑𝑥𝑖 2 𝐷 𝑖=1 −∏𝑐𝑜𝑠( 𝑥𝑖 √𝑖 ) 𝐷 𝑖=1 + 1 [-600,600] D 0 𝑓10(𝑥) = 𝜋 30 {10 𝑠𝑖𝑛2( 𝜋𝑦1) + ∑(𝑦𝑖−1)2 𝐷−1 𝑖=1 ⋅[1 + 10 𝑠𝑖𝑛2( 𝜋𝑦𝑖+ 1)] + (𝑦𝑛−1)2} + ∑𝑢(𝑥𝑖, 10,100,4) 𝐷 𝑖=1 [-50,50] D 0 𝑓11(𝑥) = ∑ [(𝑥4𝑖−3 + 10𝑥4𝑖−2)2 + 5(𝑥4𝑖−1 −𝑥4𝑖)2 𝐷/4 𝑖=1 + (𝑥4𝑖−2 −2𝑥4𝑖−1)4 + 10(𝑥4𝑖−3 −𝑥4𝑖)4] [-4,5] D 0 𝑓12(𝑥) = ∑ 𝑥𝑖 2 + (∑ 0.5𝑖𝑥𝑖 𝐷 𝑖=1 )2 + (∑ 0.5𝑖𝑥𝑖 𝐷 𝑖=1 )4 𝐷 𝑖=1 [-5,10] D 0 Table 3 Test functions and parameters 𝑓13 = ∑[𝑎𝑖−𝑥1(𝑏𝑖 2 + 𝑏𝑖𝑥2) 𝑏𝑖 2 + 𝑏𝑖𝑥3 + 𝑥4 ] 11 𝑖=1 2 𝑎𝑖= [0.1957,0.1947,0.1735,0.16,0.0844,0.0627, 0.0456,0.0342,0.0323,0.0235,0.0246]; 𝑏𝑖 −1 = [0.25,0.5,1,2,4,6,8,10,12,14,16] [-5,5] 4 0.0003075 𝑓14(𝑥) = (𝑥2 −5.1 4𝜋2 𝑥1 2 + 5 𝜋𝑥1 −6)2 + 10(1 −1 8𝜋) 𝑐𝑜𝑠𝑥1 + 10 [(-5,0),(10,15)] 2 0.398 𝑓15(𝑥) = [1 + (𝑥1 + 𝑥2 + 1)2(19 −14𝑥1 + 3𝑥1 2 −14𝑥2+6𝑥1𝑥2 + 3𝑥2 2)] × [30 + (2𝑥1 −3𝑥2)2× (18 −32𝑥1 + 12𝑥1 2 + 48𝑥2 −36𝑥1𝑥2 + 27𝑥2 2)] [-2,2] 2 3 𝑓16(𝑥) = (𝑥1 + 2𝑥2 −7)2 + (2𝑥1 + 𝑥2 −5)2 [-10,10] 2 0 𝑓17(𝑥) = −∑𝑠𝑖𝑛( 𝑥𝑖) 𝑠𝑖𝑛2𝑚( 𝑖𝑥𝑖 2 𝜋) 10 𝑖=1 [0, ] 10 -9.66015 𝑓18(𝑥) = −(𝑥2 + 47) 𝑠𝑖𝑛( √\|𝑥2 + 𝑥1 2 + 47\|) −𝑥1 𝑠𝑖𝑛( √\|𝑥1 −(𝑥2 + 47)\|) [-512,512] 2 -959.6407 𝑖 𝑖=1 𝑎𝑖= [0.1957,0.1947,0.1735,0.16,0.0844,0.0627, 0.0456,0.0342,0.0323,0.0235,0.0246]; Table 4 Test results of different improved algorithms Function CSO PDCSO ICSO ASCSO-S 𝐷= 10 𝐷= 50 𝐷= 10 𝐷= 50 𝐷= 10 𝐷= 50 𝐷= 10 𝐷= 50 F1 Ave 0 0 2E-131 1.04E-31 2.1E-142 1.86E-45 0 0 Std 0 0 9.5E-131 4.53E-31 8.6E-142 7.68E-45 0 0 Best 0 0 6.2E-136 3.79E-50 7.5E-151 7.47E-50 0 0 Worst 0 0 5.2E-130 2.47E-30 4.6E-141 4.22E-44 0 0 F2 Ave 0 0 7.57E-78 4.88E-35 2.41E-91 4.89E-38 0 0 Std 0 0 3.23E-77 1.72E-34 1.27E-90 8.78E-38 0 0 Best 0 0 2.58E-80 6.16E-40 4.03E-96 1.96E-40 0 0 Worst 0 0 1.78E-76 9.37E-34 6.94E-90 4.05E-37 0 0 F3 Ave 0 0 1.59E-46 149.2781 4.75E-70 19.57705 0 0 Std 0 0 8.66E-46 457.8035 1.38E-69 98.51109 0 0 Best 0 0 1.27E-71 1.29E-06 7.13E-81 1.93E-08 0 0 Worst 0 0 4.74E-45 2038.464 6.39E-69 539.5373 0 0 F4 Ave 0 0 4.14E-47 18.97023 1.21E-59 17.23904 0 0 Std 0 0 2.21E-46 6.744785 2.52E-59 6.530568 0 0 Best 0 0 2.03E-52 1.201056 2.89E-68 0.125546 0 0 Worst 0 0 1.21E-45 28.29731 8.94E-59 25.15158 0 0 F5 Ave 1.13E-26 0.073115 0.043912 4.416301 0.019847 5.616788 0.966116 10.02114 Std 2.58E-26 0.09572 0.031135 0.376249 0.048784 0.338539 0.152327 0.330825 Best 9.21E-31 0.00795 0.001415 3.844698 0.000216 5.041785 0.655284 8.975014 Worst 1.18E-25 0.282306 0.124232 5.418371 0.270026 6.263295 1.259867 10.49705 F6 Ave 5.64E-05 6.44E-05 0.000227 0.001258 0.000344 0.001001 7.63E-05 0.000118 Std 5.37E-05 6.66E-05 0.000133 0.000405 0.000222 0.000536 7.68E-05 0.000112 Best 9.17E-07 2.49E-06 3.51E-05 0.000596 1.9E-05 0.000415 2.75E-06 4.85E-06 Worst 0.000262 0.000263 0.000475 0.002215 0.000832 0.003155 0.000332 0.00047 F7 Ave 0 0 0 0 0 0 0 0 Std 0 0 0 0 0 0 0 0 Best 0 0 0 0 0 0 0 0 Worst 0 0 0 0 0 0 0 0 F8 Ave 8.88E-16 8.88E-16 1.6E-15 1.14E-14 8.88E-16 4.20E-15 8.88E-16 8.88E-16 Std 0 0 1.45E-15 2.90E-14 0 9.01E-16 0 0 Best 8.88E-16 8.88E-16 8.88E-16 4.44E-15 8.88E-16 8.88E-16 8.88E-16 8.88E-16 Worst 8.88E-16 8.88E-16 4.44E-15 1.64E-13 8.88E-16 4.44E-15 8.88E-16 8.88E-16 F9 Ave 0 0 0 0 0 0 0 0 Std 0 0 0 0 0 0 0 0 Best 0 0 0 0 0 0 0 0 Worst 0 0 0 0 0 0 0 0 F10 Ave 1.84E-27 0.000681 0.002161 0.216774 0.001866 0.329059 0.317632 0.855516 Std 5.35E-27 0.001182 0.001818 0.04494 0.002993 0.035752 0.096942 0.040541 Best 4.74E-32 5.48E-05 1.29E-05 0.15463 7.2E-07 0.233194 0.107997 0.743935 Worst 2.69E-26 0.004427 0.007248 0.344528 0.012607 0.389021 0.49548 0.924134 F11 Ave 0 0 1.07E-06 6.97E-07 3.05E-09 1.82E-30 0 0 Std 0 0 5.85E-06 1.67E-06 1.66E-08 7.41E-30 0 0 Best 0 0 3.5E-42 9.37E-36 3.18E-71 1.54E-48 0 0 Worst 0 0 3.2E-05 5.83E-06 9.12E-08 3.99E-29 0 0 F12 Ave 0 0 1.02E-57 3.239553 4.47E-65 3.550345 0 0 Std 0 0 2.49E-57 2.054349 2.43E-64 1.95467 0 0 Best 0 0 2.78E-62 0.525498 3.44E-78 1.092479 0 0 Worst 0 0 9.85E-57 8.636726 1.33E-63 9.225863 0 0 F13 Ave 0.000338 0.00062 0.000593 0.001151 Std 0.000167 0.000187 0.000205 0.000363 Best 0.000307 0.000308 0.000307 0.000521 Worst 0.001223 0.001223 0.001223 0.002194 F14 Ave 0.397887 0.397887 0.397887 0.414706 Std 0 2.52E-09 1E-10 0.01935 Best 0.397887 0.397887 0.397887 0.398084 Table 4 Test results of different improved algorithms Worst 0.397887 0.397887 0.397887 0.47301 F15 Ave 3 3 3 3.229174 Std 1.32E-15 1.34E-15 8.29E-08 0.332011 Best 3 3 3 3.001369 Worst 3 3 3 4.782669 F16 Ave 0 0 0 0.02634 Std 0 0 0 0.025041 Best 0 0 0 0.001075 Worst 0 0 0 0.122481 F17 Ave -9.3287 -9.22455 -9.16256 -3.67902 Std 0.174558 0.279614 0.256986 0.42561 Best -9.65524 -9.61852 -9.60838 -4.55678 Worst -9.00491 -8.2349 -8.58308 -2.91759 F18 Ave -959.641 -959.641 -959.641 -909.584 Std 5.78E-13 5.78E-13 1.63E-06 45.37918 Best -959.641 -959.641 -959.641 -959.641 Worst -959.641 -959.641 -959.641 -820.583 The results in Table 4 show that the performance of the PDCSO algorithm is much better than that of the other algorithms in terms of optimization accuracy, convergence speed, and stability. 2.3 Improved algorithm performance testing Compared with ASCSO-S, PDCSO has the same search accuracy, and it can be found by y y that the parallel strategy of PDCSO can avoid the situation of falling into local optimum due to over-reliance on X-best bootstrap as in ASCSO-S, and has the ability to jump out of local optimum in some function tests. In summary, PDCSO outperforms CSO, ICSO and ASCSO-S, which reflects the superiority of PDCSO. 3.1 Description of Optimal Time Shock Planning Problem By discretizing the motion trajectory to be executed by the robot arm in Cartesian space, a sequence of spatial positional matrices𝑇𝑖can be obtained, and the spatial positional sequence can be converted into a sequence of robot arm joint positions𝑝𝑖= [𝑝1,𝑖 𝑝2,𝑖 ⋯ 𝑝𝑁,𝑖], 𝑖= 0,1,2, ⋯, 𝑛 by inverse kinematics,,𝑁 denotes the number of joints of the robot arm, and,𝑖 denotes the time node 𝑡𝑖. The joint position-time-node sequence is j p q 𝑄= [(𝑝, 𝑡𝑖)\|𝑖= 0,1, ⋯, 𝑛] (17) j p q 𝑄= [(𝑝, 𝑡𝑖)\|𝑖= 0,1, ⋯, 𝑛] i d b i l i h b j i (17) The joint trajectory profile can be obtained by interpolating the above joint position-time node sequence with polynomial or spline functions. The former improves the execution efficiency of the arm, and the latter ensures that shocks are minimized during the execution of the motion trajectory, optimizing two conflicting kinematic performance metrics. The less the accumulation of joint shocks and the less the accumulation of drive torque fluctuations, the smoother the arm motion. In this regard, by constructing trajectory profiles for each joint of the robot arm using a 5 order B-spline function and using both joint motion time and shocks as optimization metrics, the optimal time-shock planning problem for the robot arm can be generalized as a multi-objective optimization problem as follows: 𝑓1(𝑥) = ∑△𝑡𝑖 𝑖−1 𝑛=0 = 𝑇 𝑓2(𝑥) = ∑√1 𝑇∫(𝐽(𝑡))2𝑑𝑡 𝑇 0 𝑀 𝑚=0 (18) (18) (18) Consider the motion constraint of the robot arm and define the following constraint: \|𝑉( )\| ≤𝑉 { \|𝑉(𝑡)\| ≤𝑉𝑚𝑎𝑥 \|𝐴(𝑡)\| ≤𝐴𝑚𝑎𝑥 \|𝐽(𝑡)\| ≤𝐽𝑚𝑎𝑥{ { \|𝑉(𝑡)\| ≤𝑉𝑚𝑎𝑥 \|𝐴(𝑡)\| ≤𝐴𝑚𝑎𝑥 \|𝐽(𝑡)\| ≤𝐽𝑚𝑎𝑥{ (19) (19) The weight method is also used to convert the multi-objective optimization problem into a single-objective optimization problem, and the penalty function method is used to convert the constrained problem into an unconstrained problem. 𝐹(𝑥) = 𝑘1𝑓1(𝑥) + 𝑘2𝑓2(𝑥) + ∑ 𝜉𝑛𝑓𝑝𝑒𝑛𝑎𝑙𝑡𝑦 (𝑛) 𝑛𝑢𝑚−𝑁 𝑛=1 𝑓𝑝𝑒𝑛𝑎𝑙𝑡𝑦 (𝑛) = {1, 𝑚𝑎𝑥( \|𝑁(𝑡)\|) > 𝑁𝑚𝑎𝑥 0, 𝑒𝑙𝑠𝑒 (20) (20) To satisfy the jerk continuous trajectory, each joint trajectory curve is at least third-order geometrically continuous, and since the 𝑘th order B-spline curve has the property of 𝐶𝑘+1 continuity, it is required that 𝑘≥4 . Therefore, the interpolation curve is constructed using the 5 order B-spline function. (a)F1 (b)F5 (c)F7 (d)F10 (e)F13 (f)F17 Fig. 3.1 Description of Optimal Time Shock Planning Problem 6 Partial function convergence curve 3.2 B-spline interpolation trajectory construction The 𝑘 order B-spline curve is defined as： 𝑃(𝑢) = ∑ 𝑃𝑖𝐵𝑖,𝑘(𝑢) 𝑛 𝑖=0 (21) Where 𝑃𝑖 is the vertex of the characteristic polygon; 𝐵𝑖,𝑘 is called the 𝑘th order (𝑘−1th order) basis function and 𝑢∈[𝑢𝑖,𝑢𝑖+1] ∈[𝑢𝑘,𝑢𝑘+𝑛] is the normalized time vector(0 ≤𝑢≤1). To satisfy the jerk continuous trajectory, each joint trajectory curve is at least third-order geometrically continuous, and since the 𝑘th order B-spline curve has the property of 𝐶𝑘+1 continuity, it is required that 𝑘≥4 . Therefore, the interpolation curve is constructed using the 5 order B-spline function. (b)F5 (b)F5 (a)F1 (c)F7 (d)F10 Fig. 6 Partial function convergence curve Fig. 6 Partial function convergence curve 3.2 B-spline interpolation trajectory construction The 𝑘 order B-spline curve is defined as： 𝑃(𝑢) = ∑ 𝑃𝑖𝐵𝑖,𝑘(𝑢) 𝑛 𝑖=0 (21) Where 𝑃𝑖 is the vertex of the characteristic polygon; 𝐵𝑖,𝑘 is called the 𝑘th order (𝑘−1th order) basis function and 𝑢∈[𝑢𝑖,𝑢𝑖+1] ∈[𝑢𝑘,𝑢𝑘+𝑛] is the normalized time vector(0 ≤𝑢≤1). 3.2 B-spline interpolation trajectory construction The 𝑘 order B-spline curve is defined as： (21) ( ) ∑ 𝑖 𝑖,𝑘( ) 𝑖=0 ( ) Where 𝑃𝑖 is the vertex of the characteristic polygon; 𝐵𝑖,𝑘 is called the 𝑘th order (𝑘−1th order) basis function and 𝑢∈[𝑢𝑖,𝑢𝑖+1] ∈[𝑢𝑘,𝑢𝑘+𝑛] is the normalized time vector(0 ≤𝑢≤1). ( ) 𝑖 𝑖,𝑘( ) 𝑖0 ( ) Where 𝑃𝑖 is the vertex of the characteristic polygon; 𝐵𝑖,𝑘 is called the 𝑘th order (𝑘−1th order) basis function and 𝑢∈[𝑢𝑖,𝑢𝑖+1] ∈[𝑢𝑘,𝑢𝑘+𝑛] is the normalized time vector(0 ≤𝑢≤1). The expression of the basis function𝐵𝑖,𝑘(𝑢) is derived from the deBoor-Cox recurrence equation. 𝐵𝑖,𝑘(𝑢) = 𝑢−𝑢𝑖 𝑢𝑖+𝑘−1 −𝑢𝑖 ⋅𝐵𝑖,𝑘−1(𝑢) + 𝑢𝑖+𝑘−𝑢 𝑢𝑖+𝑘−𝑢𝑖+1 ⋅𝐵𝑖+1,𝑘−1(𝑢) 1 The expression of the basis function𝐵𝑖,𝑘(𝑢) is derived from the deBoor-Cox recurrence equation. 𝑢−𝑢𝑖 𝑢𝑖+𝑘−𝑢 𝐵𝑖,𝑘(𝑢) = 𝑢−𝑢𝑖 𝑢𝑖+𝑘−1 −𝑢𝑖 ⋅𝐵𝑖,𝑘−1(𝑢) + 𝑢𝑖+𝑘−𝑢 𝑢𝑖+𝑘−𝑢𝑖+1 ⋅𝐵𝑖+1,𝑘−1(𝑢) 1 𝑢< 𝑢< 𝑢 ; 𝐵𝑖,𝑘(𝑢) = 𝑢 𝑢𝑖 𝑢𝑖+𝑘−1 −𝑢𝑖 ⋅𝐵𝑖,𝑘−1(𝑢) + 𝑢𝑖+𝑘 𝑢 𝑢𝑖+𝑘−𝑢𝑖+1 ⋅𝐵𝑖+1,𝑘−1(𝑢) 𝐵𝑖,1(𝑢) = {1 𝑢𝑖< 𝑢< 𝑢𝑖+1; 0 𝑂𝑡ℎ𝑒𝑟𝑤𝑖𝑠𝑒; (22) 0 ( ) Where 𝑘 is the number of B-sample functions,𝑖 is the sequence number of B-sample functions, and specifies 0 0 = 0. ( ) Where 𝑘 is the number of B-sample functions,𝑖 is the sequence number of B-sample functions, and specifies 0 0 = 0. Where 𝑘 is the number of B-sample functions,𝑖 is the sequence number of B-sample functions, and spec 0. 3.1 Description of Optimal Time Shock Planning Problem In order to make each joint trajectory pass through 𝑛+ 1 position nodes in𝑄 the control points of the B-sample trajectory equation need to be inverted, and the node values of the B-sample curve located in the node interval[𝑢𝑘,𝑢𝑘+𝑛] are brought into the equation(21) in turn to obtain 𝑛+ 1 which meets the constraint conditions equation. 𝑃( ) ∑ 𝑃𝐵 ( ) 𝑖+𝑘 𝑃(𝑖) 𝑖 1 2 (23) q 𝑃(𝑢𝑖+𝑘) = ∑ 𝑃𝑗𝐵𝑗,𝑘(𝑢) 𝑖+𝑘 𝑗=𝑖 = 𝑃(𝑖), 𝑖= 1,2, ⋯, 𝑛 (23) (23) 𝑗 𝑗 𝑗 Therefore it is also necessary to add 𝑘−1 conditions, this paper takes𝑘= 5,and obtain the remai conditions by configuring the boundary conditions. it is also necessary to add 𝑘−1 conditions, this paper takes𝑘= 5,and obtain the remaining boundary onfiguring the boundary conditions. {𝑃′(𝑢0) = 𝑣0, 𝑃′(𝑢𝑒) = 𝑣𝑒 𝑃″(𝑢0) = 𝑎0, 𝑃″(𝑢𝑒) = 𝑎𝑒 (24) (24) Where𝑣0, 𝑣𝑒, 𝑎0, 𝑎𝑒 is the joint beginning and end velocity and beginning and end acceleration, respectiv [ ] The trajectory curve of joint 𝑗on 𝑡∈[𝑡0,𝑡𝑛] is derived from the solved control vertex and time node vec Table 6 Sequence of the position of each joint of the robot arm 4 The simulation test Using the KUKA kr10-900 arm as the simulation object, the arm is modeled D-H with parameters shown in Table 5 and the sequence of joints shown in Using the KUKA kr10-900 arm as the simulation object, the arm is modeled D-H with parameters shown in Table 5 and the sequence of joints shown in Table 6. Five times B-sample is used as the interpolation curve, and the start-stop speed of all joints is set, and the acceleration is 0. Standard CSO and PDCSO perform the trajectory optimization simulation, and the parameters of both CSO and PDCSO algorithms are 𝑁𝑝𝑜𝑝= 100、𝑁𝑟= 𝑁𝑐= 0.2𝑁𝑝𝑜𝑝、𝑁ℎ= 0.6𝑁𝑝𝑜𝑝、𝑁𝑚= 0.1𝑁ℎ、𝐺= 10、0.4 ≤𝐹𝐿≤1. The iterative results in Fig. 7 are obtained, and the position curves of each joint are shown in Fig. Fig. 7 Comparison of iterative convergence Fig. 7 Comparison of iterative convergence Table 5 Robotic arm D-H parameter table 𝑖 𝜃𝑖(∘) 𝑎𝑖(𝑚𝑚) 𝑑𝑖(𝑚𝑚) 𝛼𝑖(∘) 1 -170∘< 𝜃1 < 170∘ 400 25 −𝑝𝑖/2 2 -190∘< 𝜃2 < 45∘ 0 455 0 3 -210∘< 𝜃3 < 68∘ 0 25 −𝑝𝑖/2 4 -180∘< 𝜃4 < 180∘ 420 0 𝑝𝑖/2 5 -120∘< 𝜃5 < 120∘ 0 0 −𝑝𝑖/2 6 -350∘< 𝜃6 < 350∘ 90 0 0 Table 6 Sequence of the position of each joint of the robot arm Nodes Joint position /(∘) Joint 1 Joint 2 Joint 3 Joint 4 Joint 5 Joint 6 1 0 0 0 0 0 0 2 0 -10 -5 10 20 0 3 0 -20 10 20 20 0 4 0 -50 20 30 10 0 5 0 -30 60 60 30 0 6 0 -10 40 50 20 0 7 0 0 10 30 30 0 8 0 10 0 10 10 0 9 0 20 10 0 5 0 10 0 0 0 0 0 0 The kinematics simulation analysis of the robot is run on the platform ADAMS. The driving of each joint is carried out in this dynamic simulation, and the torque change curve of each joint is obtained. In this paper, there is no position change between joint 1 and joint 6, so there are only the other 4 joints, and the torque change comparison curve is shown in Fig. 9. As seen from Fig. 7, DRCSO has higher search accuracy than CSO and can jump out of the optimal local solution. It can be seen from the joint moment curve in Fig. 5 Conclusion To address the shortcomings of the basic swarm optimization algorithm, we propose a rooster update mechanism based on X-best guidance and Levy flight with the parallel strategy and a dynamically constrained hen-following target mechanism, which improve the accuracy of the algorithm and have the ability to avoid the local optimum. The optimization simulations of 18 test functions show that the proposed PDCSO algorithm has a significant improvement in terms of the search accuracy, convergence speed, and stability of the algorithm compared with CSO and other improved algorithms. p g Finally, PDCSO is applied to robotic arm trajectory optimization. The experimental results through ADAMS simulation show that the PDCSO algorithm. can effectively reduce the running time and motion shock of the robotic arm and improve the execution efficiency of the robotic arm. Data Availability Statements The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Compliance with ethical standards Conflict of interest: The authors declare that there is no conflict of interest regarding the publication of this paper. Ethical approval: This article does not contain any studies with animals performed by any of the authors. Acknowledgements We acknowledge the financial support from National Natural Science Foundation of China (Grant No. 51875253), and Key Research and Development Plan Program of Jiangsu (Grant No. SBE2022020081). 4 The simulation test 9 that the joint motion curve of the manipulator obtained by dynamic simulation is smooth and continuous without sudden change. By comparing the changes of joint torque before and after optimization, the required moving time is shortened from the original 44.5s to 35s, and the peak torques of the optimized joints 2, 3, and 5 are improved. The overall torque variation range of the joint is much smaller than that of other joints, and does not affect the overall robotic arm. The cumulative effect of the trajectory obtained by the DRCSO optimization method on execution time and impact satisfies various kinematic constraints. The accumulation of movement shock is reduced, and the joint movement is more stable. Fig. 8 Six- joint angle curve (a) Joint 2 (b) Joint 3 Fig. 8 Six- joint angle curve (b) Joint 3 (c) Joint 4 (d) Joint 5 Fig. 9 Comparison of joint torque curves 5 Conclusion To address the shortcomings of the basic swarm optimization algorithm, we propose a rooster update mechanism based on X-best guidance and Levy flight with the parallel strategy and a dynamically constrained hen-following target mechanism, which improve the accuracy of the algorithm and have the ability to avoid the local optimum. The optimization simulations of 18 test functions show that the proposed PDCSO algorithm has a significant improvement in terms of the search accuracy, convergence speed, and stability of the algorithm compared with CSO and other improved algorithms. Finally, PDCSO is applied to robotic arm trajectory optimization. The experimental results through ADAMS simulation show that the PDCSO algorithm. can effectively reduce the running time and motion shock of the robotic arm and improve the execution efficiency of the robotic arm (c) Joint 4 (d) Joint 5 Fig. 9 Comparison of joint torque curves (d) Joint 5 Fig. 9 Comparison of joint torque curves [5] Zhang KW, Zhao XL, He L, Li ZZ (2021) A chicken swarm algorithm with improved x-best guided individuals and dynamic rank update mechanism, Journal of Beijing University of Aeronautics and Astronautics 47:15 6 References [1] Meng XB, Liu Y, Gao XZ, Zhang HZ (2014), “A NEW BIO-INSPIRED ALGORITHM: CHICKEN SWARM OPTIMIZATION”, Advances in Swarm Intelligence, PT1, Lecture Notes in Computer Science,1-15. https://doi.org/10.1007/978-3-319-11857-4_10 [2] Deb S, Gao XZ, Tammi K, Kalita K, Mahanta P (2020) Recent Studies on Chicken Swarm Optimization algorithm: a review (2014–2018). 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https://openalex.org/W4300096648	https://jurnal.unipasby.ac.id/index.php/adi_raga/article/download/4588/3443	Indonesian	null	Pengembangan Model Pembelajaran Fair Play dalam Perkuliahan Permainan Bola Tangan Terhadap Pembentukan Sikap dan Prilaku Mahasiswa S1 Pendidikan Jasmani	Jurnal Ilmiah Adiraga/Jurnal ilmiah adiraga	2,021	cc-by-sa	4,381	Jurnal Ilmiah ADIRAGA Vol. 7 No. 2, November 2021, pp. 92-107 Jurnal Ilmiah ADIRAGA Vol. 7 No. 2, November 2021, pp. 92-107 Jurnal Ilmiah ADIRAGA Vol. 7 No. 2, November 2021, pp. 92-107 Develoment of a fair play learning model in handball game lectures on the behavior formation of S1 Physical Education Studens Ujang Rohman1 Abd Cholid2 Sumardi3 dst Ujang Rohman1, Abd. Cholid2, Sumardi3, dst. 1,2,3Pendidikan Jasmanii/Sekolah Pascasarjana, Universitas PGRI Adi Buana Surabaya, Jln. Dukuh Menanggal XII No. 4, Surabaya, Jawa Timur 60234, Indonesia Abstrak Penelitian ini bertujuan mengembangkan suatu model Pembelajaran Fair Play (PFP) yang terintegrasi dalam perkuliahan Permainan Bola Tangan dan tertuang dalam bentuk model sebagai pedoman dalam mengiplementasikan proses perkuliahan bagi mahasiswa S1 Pendidikan Jasmani. Jenis penelitian ini adalah penelitian pengembangan atau research and development (R&D), menggunakan metode dengan pendekatan mixed methods. Objek penelitian adalah mahasiswa S1 Program Studi Pendidikan Jasmani, FPP Universitas PGRI Adi Buana Surabaya. Rancangan penelitian model desain eksplanatoris sekuensial (sequential exploratory design) dan pengumpulan data menggunakan teknik survey). Hasil analisis kuantitatif menunjukkan bahwa setelah dilakukan pemberian perlakuan model pembelajaran fair play (PFP) sebanyak 16 kali pertemuan diperoleh nilai pre test dan post test melalui uji Independent sample t test nilai out put fair play sikap dan perilaku lebih kecil dari Sig.(p-value) dimana α < 0,05 artinya model PFP berpengaruh terhadap perubahan sikap dan perilaku. Sedangkan hasil analisis kualitatif menggunakan model interactive menunjukkkan bahwa model PFP memberikan perubahan dan dapat mengembangkan sikap dan perilaku. Temuan penelitian ini menunjukkan bahwa penelitian model pembelajaran fair play dapat membentuk dan mengembangankan sikap serta menanamkan perilaku mahasiswa Pendidikan Jasmani dalam pembelajaran permainan bola tangan Kata kunci: model pembelajaran, fair play, bola tangan Kata kunci: model pembelajaran, fair play, bola tangan Abstract This study aims to develop a Fair Play Learning (PFP) model that is integrated in the Handball Game lecture and is contained in the form of a model as a guide in implementing the lecture process for S1 Physical Education students. This type of research is research and development (R&D), using a mixed methods approach. The object of the research is the undergraduate student of the Physical Education Study Program, FPP, PGRI Adi Buana University, Surabaya. The research design is a sequential explanatory design model (sequential exploratory design) and data collection using survey techniques). The results of quantitative analysis show that after giving the fair play learning model (PFP) treatment for 16 meetings, the pre-test and post-test values obtained through the Independent sample t-test, the out put value of fair play attitudes and behavior is smaller than Sig. (p-value ) where < 0.05 means that the PFP model has an effect on changes in attitudes and behavior. While the results of the interactive model analysis show that the PFP model provides change and can develop attitudes and behavior. The findings of this study indicate that the fair play learning model research can shape and develop attitudes and instill the behavior of Physical Education students in learning handball games. Keywords: learning model, fair play, handball indicate that the fair play learning model research can shape and develop attitudes and instill the behavior of Physical Education students in learning handball games. Keywords: learning model, fair play, handball PENDAHULUAN Proses pendidikan di perguruan tinggi merupakan aktivitas untuk menghasilkan perubahan individu baik secara fisik, mental, dan emosional. Oleh karena itu proses pendidikan dalam pembelajaran didesain untuk mengorientasi aktivitas mahasiswa dengan tujuan memperoleh hasil belajar berupa perpaduan antara aspek kognitif, afektif dan psikomotor secara proposional sesuai bidang keilmuannya. Selain itu aspek penting dalam proses pendidikan adalah pembentukan karakter yang merupakan pengembangan atas kemampuan diri (self esteem) dalam membentuk berkepribadian luhur sesuai dengan nilai-nilai budaya dan memotivasi keberhasilan seseorang sesuai keilmuannya. Berpijak pada upaya tersebut, untuk meningkatkan kualitas pembelajaran yaitu dengan memberikan sumbangan nyata bagi usaha pengembangan sumber daya manusia (SDM) tidak terbatas pada lingkup di kampus melainkan juga disiapkan untuk memberikan kontribusi pada lembaga pendidikan tinggi melalui pengembangan bentuk pembelajaran yang berorientasi pada kebutuhan pasar (market oriented). Berdasarkan hal tersebut maka struktur kurikulum harus berorientasi pada kebutuhan pasar dengan meningkatkan kualitas pembelajaran dan kompetensi lulusan. Prinsip-prinsip dasar kurikulum berorientasi pada tujuan pendidikan nasional dan berpedoman pada kesinambungan antara teori dan praktek yang dirancang sesuai dengan visi, misi, dan tujuan dari lembaga pendidikan. Salah satu lembaga pendidikan tersebut diantaranya Universitas PGRI Adi Buana Surabaya yang memiliki 7 fakultas dan 21 program studi. Salah satunya adalah program studi pendidikan jasmani. Kurikulum program studi pendidikan jasmani didasarkan pada SK Menteri Pendidikan Nasional Republik Indonesia Nomor 232/U/2000 tentang penyusunan kurikulum pendidikan tinggi dan penilaian hasil belajar mahasiswa. Prinsip-prinsip dasar kurikulum berorientasi pada tujuan 7 (2) November 2021 \| 92-107 94 Jurnal Ilmiah ADIRAGA Vol. 7 No. 2, November 2021, pp. 92-107 Jurnal Ilmiah ADIRAGA Vol. 7 No. 2, November 2021, pp. 92-107 Jurnal Ilmiah ADIRAGA Vol. 7 No. 2, November 2021, pp. 92-107 pendidikan nasional dan berpedoman pada kesinambungan antara teori dan praktek dirancang sesuai dengan visi, misi, dan tujuan program studi yang tertuang di dalam buku Pedoman Akademik Universitas PGRI Adi Buana Surabaya. Deskriptif mata kuliah program studi pendidikan jasmani di susun sefleksibel mungkin, mengikuti perkembangan teknologi khususnya yang berbasis pada Information and Communication Technology (ICT) yang pada pelaksanaan kurikulum memegang peranan yang sangat penting dalam pelaksanaan pembelajaran. Perkembangan teknologi ini memungkinkan mengadakan perbaikan dan evaluasi dari RPS dan SAP yang dibahas dalam rapat internal fakultas dan program studi bersama dosen yang terkait. Salah satu mata kuliah yang wajib di program dan ditempuh mahasiswa berdasarkan kurikulum pendidikan jasmani adalah mata kuliah Permainan Bola tangan. PENDAHULUAN Dalam mata kuliah permainan bola tangan ada unsur-unsur yang harus dikuasai oleh mahasiswa yaitu kemampuan teknik (lempar, tangkap, menggiring dan menembak) dan memiliki karakter yang baik. Oleh karena itu, untuk mencapai hasil maksimal khususnya dalam bermain bola tangan, maka dalam proses pembelajaran permainan bola tangan disamping memerlukan kemampuan penguasaan teknik juga dibutuhkan pembentukan karakter selama perkuliahan. Salah satu karakter yang mendasari permainan bola tangan adalah nilai-nilai fair play (Kasih, 2009). Fair play dimaknai sebagai semangat atlet sejati atau kesatria (the finest sport maship). Seorang atlet bertindak secarai fair play apabila melakukan suatu perbuatan yang tunduk dan mematuhi peraturan yang berlaku secara tertulis. Menurut Suharjana (2011) karakter yang dapat dikembangkan antara lain dalam bentuk perilaku sportifitas, menghargai, pengendalian diri, kemauan dan tanggung jawab. Fair play yang merupakan sebagai konsep moral berisi penghargaan terhadap lawan dan sesama teman yang bertumpu pada standar moral yang dihayati dan dilaksanakan oleh setiap orang (Aji Setiawan, 2016., Butler, 2000). Oleh karena itu nilai-nilai fair play yang dilakukan para mahasiswa selama proses pembelajaran permainan bola tangan harus ditanamkan dan dijalankan oleh para mahasiswa sebagai bagian dari pembentukan karakter mahasiswa dalam mewujudkan keberhasilan proses pembelajaran nilai-nilai fair play tersebut. Hasil pengamatan (observasi) dilapangan selama proses pembelajaran permainan bola tangan terhadap mahasiswa yang melaksanakan praktek permainan bola tangan, kejadian yang sering terjadi masih rendahnya karakter mahasiswa secara sikap dan perilaku. Upaya yang dilakukan untuk mengatasi permasalahan tersebut yaitu dengan memberikan model pembelajaran yang didalamnya terintegrasi nilai-nilai fair play selama proses pembelajaran berlangsung. Permasalahan mendasar dalam penelitian ini masih rendahnya karakter para mahasiswa yang ditunjukkan oleh perilaku yang bertentantangan dengan nilai-nilai fair play selama proses pembelajaran. Pengamatan yang dilakukan selama proses pembelajaran permainan bola tangan menunjukkan bahwa dalam proses pembelajaran permainan bola tangan, dosen memiliki peran sentral dalam upaya menanamkan nilai-nilai fair play pada mahasiswa. Oleh karena itu dibutuhkan suatu model pembelajaran fair play yang terintegrasi dalam pembelajaran permainan bola tangan sehingga dapat dijadikan acuan oleh para dosen dalam mengajarkan nilai-nilai fair play. Model pembelajaran adalah perencanaan dan penerapan untuk mencapai tujuan pembelajaran dalam keanekaragaman isi (Raharja, 2019). Berdasarkan permasalahan tersebut menarik peneliti untuk dianalisis dan diteliti yaitu mengenai model pembelajaran permainan bola tangan dengan menerapkan Model Pembelajaran Fair Play (PFP). Model PFP memberikan pengalaman pada mahasiswa dalam suasana belajar dan bermain yang edukatif dan afektif dalam bentuk pengembangan sikap dan perilaku mahasiswa selama pembelajaran. PENDAHULUAN Sehubungan dengan hal tersebut, peneliti mengangkat permasalahan ini dalam bentuk penelitian yang berjudul “Pengembangan model pembelajaran fair play (PFP) dalam perkuliahan permainan bola tangan terhadap pembentukan sikap dan perilaku mahasiswa S1 pendidikan jasmani. Hasil temuan penelitian ini diharapkan dapat direkomendasikan sebagai salah satu model pembelajaran yang dapat membentuk nilai-nilai fair play dalam merubah dan mengembangkan sikap serta menanamkan perilaku bagi para mahasiswa. 7 (2) November 2021 \| 92-107 96 Jurnal Ilmiah ADIRAGA Vol. 7 No. 2, November 2021, pp. 92-107 METODE Penelitian ini adalah jenis penelitian pengembangan atau research and development (R&D) dengan menggunakan pendekatan mixed methods yang difokuskan pada pengembangan suatu produk model pembelajaran fair play (PFP) yang diintegrasikan dalam perkuliahan permainan bola tangan terhadap perubahan sikap dan perilaku mahasiswa S1 Pendidikan Jasmani FPP Universitas PGRI Adi Buana Surabaya. Model disain penelitian pengembangan (R&D) adalah desain eksplanatoris sekuensial (sequential exploratory design) dengan langkah-langkah dan prosedur meliputi; (1) pengembangan model dan (2) menguji kefektifan model sesuai tujuan yang ingin dicapai. Desain eksplanatoris sekuensial (sequential exploratory design) dijabarkan pada bagan berikut: TAHAPAN PENYUSUNAN MODEL Eksplorasi (Investigasi Penyusunan Model) Identifikasi (Komponen Materi Model) Konten (Kurikulum Metode dan Produk) TAHAPAN PENGEMBANGAN MODEL Data Kuantitatif Analisis Data Kuantitatif Simpulan Angket Uji-t Interpretasi Model Observasi Wawancara Model Interaktif Rekomendasi Data Kualitatif Analisis Data Kualitatif Gambar 1. Bagan Model Rancangan Penelitian Desain Eksplanatoris Sekuensial (Sugiono, 2017) TAHAPAN PENYUSUNAN MODEL Eksplorasi (Investigasi Penyusunan Model) Konten (Kurikulum Metode dan Produk) Identifikasi (Komponen Materi Model) Eksplorasi (Investigasi nyusunan Model) Eksplorasi (Investigasi TAHAPAN PENGEMBANGAN MODEL Data Kuantitatif Analisis Data Kuantitatif Simpulan Angket Uji-t Interpretasi Model Observasi Wawancara Model Interaktif Rekomendasi Data Kualitatif Analisis Data Kualitatif Gambar 1. Bagan Model Rancangan Penelitian Desain Eksplanatoris Sekuensial (Sugiono, 2017) TAHAPAN PENGEMBANGAN MODEL Data Kuantitatif Analisis Data Kuantitatif Simpulan Angket Interpretasi Model Observasi Wawancara Model Interaktif Rekomendasi Gambar 1. Bagan Model Rancangan Penelitian Desain Eksplanatoris Sekuensial (Sugiono, 2017) Populasi yang dijadikan objek dalam penelitian pengembangan ini adalah mahasiswa Program Studi S1 Pendidikan Jasmani FPP Universitas PGRI Ad Buana Surabaya yang memprogram mata kuliah permainan bola tangan semester genap tahun akademik 2020-2021 dan sampel penelitian sebanyak 15 mahasiswa yang diambil secara acak (random sampling) dari kelas A, B dan C. Penggolongan subjek penelitian yang dijadikan sampel berdasarkan tahapan-tahapan penelitian pengembangan seperti dijabarkan pada bagan disain penelitian tersebut di atas. Model pembelajaran fair play (PFP) yang terintegrasi dalam perkuliahan Permainan Bola Tangan dilakukan secara konprehensip yang berkenaan dengan seluruh aspek pembelajaran permainan bola tangan baik secara tatap muka di kelas maupun praktek di lapangan. Prosedur pengumpulan data menggunakan instrumen penelitian seperti dalam bentuk (1) angket atau kuesioner (questionnaires). (2) observasi (observations) dan (3) wawancara (interviews) yang sudah divalidasi ahli (expert) dan bertujuan mengungkap perubahan sikap dan perilaku mahasiswa melalui nilai-nilai fair play. Subyek penelitian yang terkait dengan nilai-nilai fair play disajikan pada tabel 1 berikut: Tabel 1. Komponen dan Sub-komponen Pembelajaran Fair Play Tabel 1. Jurnal Ilmiah ADIRAGA Vol. 7 No. 2, November 2021, pp. 92-107 pendapat dan persepsi seseorang atau kelompok orang tentang fenomena atau gejala sosial yang terjadi, hal ini secara spesifik telah ditetapkan oleh peneliti, yang selanjutnya disebut variabel penelitian. Analisis hasil skala pengukuran jawaban angket menggunakan rancangan One Group Pretest - Posttest Design yang dianalisis menggunakan Uji-t (t- test) dalam bentuk Independent sample test yaitu untuk menguji pengaruh satu variabel bebas (variable independent) terhadap satu atau lebih variabel terikat (variable dependent) 2. Analisis deskriptif kualitatif Analisis data yang didapat dari hasil observasi dan wawancara (interviews) bertujuan untuk melengkapi dan menguatkan data yang didapat dari angket melalui sumber data yang berbeda. Analisis deskriptif kualitatif dengan cara mencermati dan memilah data yang dapat dianalisis (screening dan coding data) serta menginterpretasikan dan memaknai hasil analisis untuk menentukan hasilnya. Untuk pengumpulan data kualitatif hasil wawancara jenis in depth interview menggunakan model interaktif (interactive model) yang dikembangkan oleh Miles dan Huberman (Sugiyono, 2017) dengan merujuk pada pedoman wawancara yang dikembangkan Moleong (Moleong, 2010). Model inetraktif menggunakan matriks yang dimulai dari paparan data, kemudian dikelompok (collection data) kemudian data dikumpulkan dan selanjutnya di display sesuai dengan bentuk datanya, setelah itu mereduksi data (reduction of data) agar data-data yang didapat dianalisis dan dikaji untuk diambil sari pati dari setiap data yang ada dan dilanjutkan dilakukan verfikasi dengan data lainnya agar ditemukan satu bentuk kesimpulan. METODE Komponen dan Sub-komponen Pembelajaran Fair Play Komponen Sub Komponen Indikator Pembelajaran Fair Play (PFP) Sikap 1. Mengembangkan disiplin 2. Mengembangkan sportifitas 3. Mengembangkan saling meng hargai Perilaku 1. Menanamkan tanggungjawab 2. Menanamkan kejujuran 3. Menanamkan rasa hormat Teknik analisis data yang digunakan melalui tahapan sebagai berikut: Analisis data kuantitatif melalui tahap tabulasi data (coding sheet) dan pengolahan analisis data. Kode data dalam proses analisis ini melalui penilaian dari tiap-tiap butir pernyataan melalui pengukuran skala Likers. Menurut Iskandar (2013), skala Likers digunakan untuk mengukur sikap, Analisis data kuantitatif melalui tahap tabulasi data (coding sheet) dan pengolahan analisis data. Kode data dalam proses analisis ini melalui penilaian dari tiap-tiap butir pernyataan melalui pengukuran skala Likers. Menurut Iskandar (2013), skala Likers digunakan untuk mengukur sikap, 7 (2) November 2021 \| 92-107 98 Jurnal Ilmiah ADIRAGA Vol. 7 No. 2, November 2021, pp. 92-107 A. Analisis Data Kuantitatif Mengacu pada prosedur pengumpulan data, maka sebelum dilakukan uji analisis data menggunakan uji-t (independent sample t test) melalui aplikasi SPSS versi 21.0, maka dilakukan terlebih dahulu uji normalitas dan homogenitas data dengan asumsi data berdistribusi normal dan homogen. Data hasil uji normalitas dan homogenitas dapat di lihat pada tabel 2 dan 3 berikut. Tabel 2. Hasil Uji Normalitas Data Komponen Pembelajaran Fair Play (PFP) Indikator Kolmogorof- Smirnov Test Sig. (p- value) Keterangan Sikap Disiplin 4.26 8.0 Normal Sportifitas 3.11 8.0 Normal Meng Hargai 2.78 8.0 Normal Perilaku Tanggungjawab 4.16 8.0 Normal Kejujuran 3.92 8.0 Normal Rasa Hormat 2.24 8.0 Normal Tabel 2. Hasil Uji Normalitas Data Tabel 2. Hasil Uji Normalitas Data Data pada tabel 2 menunjukkan hasil uji normalitas menggunakan analisis Kolmogorov Smirnov Test, dengan tarap signifikansi (α) sebesar 0.05 dan df = 1 diperoleh nilai setiap indikator komponen fair play kurang dari 0.05 [< Sig.(p-value)] artinya data setiap indikator fair play berdistribusi normal. Sedangkan hasil uji homogenitas data dengan tarap signifikansi 0.05 atau [Sig.(p-value)] dengan df. pembilang dan penyebut (15;15) dapat dilihat pada tabel 3 berikut Tabel 3. Hasil Uji Homogenitas Data Komponen Pembelajaran Fair Play (PFP) Indikator F FSig. (p- Value) Keterangan Sikap Disiplin 0.89 2.39 Homogen Sportifitas 0.76 2.39 Homogen Meng hargai 0.92 2.39 Homogen Perilaku Tanggungjawab 0,96 2.39 Homogen Kejujuran 0.83 2.39 Homogen Rasa Hormat 0.77 2.39 Homogen Tabel 3. Hasil Uji Homogenitas Data Data pada tabel 3 menunjukkan hasil uji homogenitas, dimana diperoleh nilai setiap indikator komponen fair play Fhitung lebih kecil dari Ftabel [< Sig.(p-value)] atau Fhitung < Ftabel artinya data setiap indikator fair play adalah homogen. Setelah data tes dinyatakan normal dan homogen, tahap 100 7 (2) November 2021 \| 92-107 Jurnal Ilmiah ADIRAGA Vol. 7 No. 2, November 2021, pp. 92-107 Jurnal Ilmiah ADIRAGA selanjutnya adalah dilakukan untuk menguji pengaruh perbedaan satu variabel bebas (variable independent) terhadap satu atau lebih variabel terikat (variable dependent). Hasil analisis uji-t dapat dilihat pada tabel 4 berikut. Tabel 4. Hasil Uji Independent Sample T-test Komponen Pembelajaran Fair Play (PFP) Indikator uji-t t Sig. (p-value) Keterangan Sikap Disiplin -2.75 0.05 Signifikan Sportifitas -1.98 0.05 Signifikan Mengh argai -2.01 0.05 Signifikan Perilaku Tanggungjawab -3.82 0.05 Signifikan Kejujuran -2.21 0.05 Signifikan Rasa Hormat -1.89 0.05 Signifikan Tabel 4. Hasil Uji Independent Sample T-test Tabel 4. B. Analisis Data Kualitatif . Analisis Data Kualitatif Setelah mengumpulkan data komponen nilai-nilai fair play dalam bentuk sikap dan perilaku melalui observasi dan wawancara, selanjutnya direduksi untuk memperoleh respon mahasiswa dengan berbagai jawaban yang cederung mendukung dan mengikuti apa yang disampaikan dosen. Setelah mengumpulkan data komponen sikap yang meliputi disiplin, sportifitas dan saling menghargai melalui observasi dan wawancara pada mahasiswa, selanjutnya data tersebut direduksi, maka diperoleh hasil bahwa mahasiswa cenderung mempunyai tingkat disiplin, sportifitas dan saling menghargai yang bisa diterima. Hal tersebut menunjukkan bahwa selama proses perlakuan model PFP cenderung adanya perubahan dalam memahami dan mengembangkan sikap mahasiswa yang diperlihatkan dalam bentuk disiplin, sportif dan saling menghargai selama proses pembelajaran. Sedangkan hasil pengumpulan data perilaku dalam bentuk tanggungjawab, kejujuran, rasa hormat melalui observasi dan wawancara serta selanjutnya data direduksi, maka diperoleh hasil bahwa mahasiswa cenderung memiliki rasa tanggungjawab, jujur dan hormat yang bisa diterima. Hal tersebut menunjukkan bahwa selama proses perlakuan model PFP menyebabkan adanya perubahan dalam memahami dan menanamkan rasa tanggungjawab, jujur serta rasa hormat selama proses pembelajaran. Adanya perubahan dalam mengembangkan sikap dan menanamkan perilaku mahasiswa tidak terlepas dari penerapan PFP, karena penerapan fair play sebagai nilai dalam dalam aktivitas olahraga seseorang dihadapkan dengan struktur sosial yang dapat diterima dalam kehidupan sesungguhkan (Pradipta, 2015). Setelah mengumpulkan data komponen nilai-nilai fair play dalam bentuk sikap dan perilaku melalui observasi dan wawancara, selanjutnya direduksi untuk memperoleh respon mahasiswa dengan berbagai jawaban yang cederung mendukung dan mengikuti apa yang disampaikan dosen. Setelah mengumpulkan data komponen sikap yang meliputi disiplin, sportifitas dan saling menghargai melalui observasi dan wawancara pada mahasiswa, selanjutnya data tersebut direduksi, maka diperoleh hasil bahwa mahasiswa cenderung mempunyai tingkat disiplin, sportifitas dan saling menghargai yang bisa diterima. Hal tersebut menunjukkan bahwa selama proses perlakuan model PFP cenderung adanya perubahan dalam memahami dan mengembangkan sikap mahasiswa yang diperlihatkan dalam bentuk disiplin, sportif dan saling menghargai selama proses pembelajaran. Sedangkan hasil pengumpulan data perilaku dalam bentuk tanggungjawab, kejujuran, rasa hormat melalui observasi dan wawancara serta selanjutnya data direduksi, maka diperoleh hasil bahwa mahasiswa cenderung memiliki rasa tanggungjawab, jujur dan hormat yang bisa diterima. Hal tersebut menunjukkan bahwa selama proses perlakuan model PFP menyebabkan adanya perubahan dalam memahami dan menanamkan rasa tanggungjawab, jujur serta rasa hormat selama proses pembelajaran. Adanya perubahan dalam mengembangkan sikap dan menanamkan perilaku mahasiswa tidak terlepas dari penerapan PFP, karena penerapan fair play sebagai nilai dalam dalam aktivitas olahraga seseorang dihadapkan dengan struktur sosial yang dapat diterima dalam kehidupan sesungguhkan (Pradipta, 2015). A. Analisis Data Kuantitatif Hasil Uji Independent Sample T-test Data pada tabel 4 menunjukkan adanya perbedaan yang signifikan antara out put indikator komponen fair play antara sebelum diberi perlakukan model PFP (pre-test) dengan sesudah diberi perlakuan (post-test) model PFP dalam permainan bola tangan pada mahasiswa Program Studi Pendidikan Jasmani. Adanya perbedaan tersebut menunjukkan adanya perubahan dalam pengenalan, pemahaman dan praktik-praktik nilai-nilai Fair Play dalam bentuk sikap dan perilaku para mahasiswa pendidikan jasmani. Hal ini dibuktikan dengan nilai uji-t pada komponen fair play sikap yang terdiri dari ujit-t indikator disiplin sebesar -2.75 [< Sig.(p-value)], indikator sportifitas sebesar -1.98 [< Sig.(p-value)] dan indikator menghargai sebesar -2.01[< Sig.(p-value)]. Sedangkan pada komponen fair play perilaku yang terdiri dari uji-t indikator tangggungjawab sebesar -3.82 [< Sig.(p- value)], indikator kejujuran sebesar -2.21 [< Sig.(p-value)] dan indikator rasa hormat sebesar -1.89 [< Sig.(p-value)]. Adanya nilai uji-t negatif pada seluruh indikator komponen fair play sikap dan perilaku hal ini artinya bahwa model PFP menunjukkan adanya perubahan pengembangan out put indikator komponen fair play yang berarti (signifikan) sesudah diberi perlakuan model PFP. B. Analisis Data Kualitatif PEMBAHASAN Berdasarkan hasil analisis data kuantitatif dan kualitatif, maka pembelajaran permainan bola tangan dengan menggunakan model pembelajaran fair play (PFP) dapat memberikan perubahan pada pengembangan sikap dan penanaman perilaku mahasiswa program studi Pendidikan jasmani, Fakultas Pedagogi dan Psikologi (FPP) Universitas PGRI Adi Buana Surabaya. Fakta tersebut sebagai gambaran yang 7 (2) November 2021 \| 92-107 102 Jurnal Ilmiah ADIRAGA Vol. 7 No. 2, November 2021, pp. 92-107 Jurnal Ilmiah ADIRAGA memperlihatkan betapa pentingnya pembelajaran yang efektif dalam mengembangkan dan menanamkan fair play berupa sikap dan perilaku pada proses perkuliahan permainan bola tangan para mahasiswa. Oleh karena itu dapat dijelaskan bahwa model PFP dirumuskan dan disusun melalui proses interaksi antara kebiasaan mahasiswa sebagai individu dalam mengorganisasikan pengalamannya ke dalam pola pengalaman lingkungan melalui peran dosen (pendidik) saat memberikan informasi mengenai realitas sosial dalam proses pembelajaran permainan bola tangan yang merupakan proses reorganisasi dan transformasi struktur dasar pengembangan sikap dan perilaku mahasiswa sebagai individu. Berdasarkan nilai-nilai fair play yang ada pada model PFP, maka tujuan utama dari model PFP ini yaitu pengembangkan sikap dan penanaman perilaku pada mahasiswa yang terbentuk melalui sikap dan perilaku dosen sebagai role model dalam proses pembelajaran permainan bola tangan yang menghasilkan perubahan sikap dan perilaku. Oleh karena itu dosen dituntut harus memiliki kemampuan atau strategi pengembangkan sikap dalam bentuk disiplin, sportifitas, saling menghargai dan menanamkan perilaku dalam bentuk tanggungjawab, kejujuran, rasa hormat. Adanya perubahan pengembangan disiplin, sportif dan saling menghargai dalam pembelajaran permainan bola tangan ternyata sebagai akibat dari proses perlakuan model PFP yang didalamnya terkandung nilai- nilai luhur dan merupakan nilai-nilai yang ada pada kehidupan sehari-hari sesuai dalam kehidupan nyata. Perubahan sikap disiplin yang terjadi pada mahasiswa Prodi Pendidikan Jasmani disebabkan adanya kebutuhan yang sangat mendasar yang memberikan kekuatan dalam mengendalikan hidup dan meningkatkan standar kehidupan dalam mentaati dan mematuhi aturan yang ada. Sedangkan perubahan sikap sportif yang terjadi pada mahasiswa Prodi Pendidikan Jasmani disebabkan adanya kesadaran yang selalu melekat bahwa dalam setiap aktivitas jasmani selalu menjungjung tinggi rasa persaudaraan di antara individu, dimana komitmen terhadap suatu aktivitas olahraga dalam pembelajaran permainan bola tangan standar etika lebih diutamakan. Karena menurut Herdiyana & Prakoso (2016), pembentukan karakter melalui pendidikan jasmani sangat efektif untuk meningkatkan jiwa sportif. Seseorang yang sportif maka akan mendapatkan kepercayaan penuh ketika melakukan suatu hal. Selanjutnya perubahan sikap rasa hormat (menghormati) yang terjadi pada mahasiswa Prodi Pendidikan Jasmani disebabkan adanya perasaan yang lebih dekat, saling memahami, menghargai dan sifat tauladan terhadap sesama teman. KESIMPULAN Berdasar pada hasil analisis dan pembahasan tersebut di atas, dapat disimpulkan bahwa pembelajaran permainan bola tangan dengan menggunakan model Pembelajaran Fair Play (PFP) dapat membentuk dan mengembangkan sikap sportif, disiplin dan menghargai. Selain itu model PFP dapat membentuk dan menanamkan perilaku tanggungjawab, jujur, rasa hormat pada mahasiswa Program Studi Pendidikan Jasmani yang memprogram mata kuliah permainan bola tangan. Usaha pengembangan perubahan sikap dan penanaman perilaku tersebut diperoleh melalui pembelajaran model (PFP) yang menampilkan dan memperbaiki karakter mahasiswa melalui nilai-nilai fair play yang diterapkan sehingga model PFP dapat membudayakan nilai- nilai fair play secara sistematis dan merupakan wahana yang ampuh sebagai pondasi dalam pembentukan karakter mahasiswa selama proses pembelajaran permainan bola tangan. PEMBAHASAN Perubahan yang terjadi dalam menanamkan perilaku yang berbentuk tanggungjawab, kejujuran, rasa hormat saat proses pembelajaran permainan bola tangan sebagai akibat dari proses perlakuan model PFP. Perubahan yang terjadi dalam menanamkan tanggungjawab diperlihatkan melalui perilaku mahasiswa dalam melaksanakan tugas dan kewajibannya, sebagaimana dilakukan terhadap dirinya sendiri, masyarakat dan lingkungannya. Sedangkan perubahan yang terjadi dalam menanamkan kejujuran didasarkan pada upaya menjadikan diri mahasiswa sebagai orang yang selalu dipercaya. Hal tersebut diperlihatkan dalam hal perkataan, tindakan dan kinerja terhadap diri sendiri maupun pihak lain. Selanjutnya perubahan yang terjadi dalam menanamkan rasa hormat diperlihatkan dalam bentuk saling menghargai akan ketentuan dan peraturan yang ditetapkan selama proses pembelajaran permainan bola tangan dan saling menghargai kepada sesama teman baik sebagai lawan maupun kawan selama proses pembelajaran permainan bola tangan. Dari sejumlah nilai-nilai fair play dalam bentuk sikap dan perilaku, hasilnya ternyata telah diterapkan oleh mahasiswa program studi Pendidikan jasmani, karena sikap dan perilaku ini merupakan faktor yang dipengaruhi oleh suasana hati, emosi dan kepribadian, dimana menurut Suprapti (2010), sikap positif seseorang akan menyebabkan perilaku yang prositif terhadap suatu objek. Mengacu pada pembahasan dan konsep tersebut di atas, maka temuan hasil penelitian ini menunjukkan adanya perubahan yang berarti (signifikan) dalam pengembangan model PFP terhadap sikap dan perilaku mahasiswa Program Studi Pendidikan Jasmani, Fakultas Pedagogi dan Psikologi, Universitas PGRI Adi Buana Surabaya yang terintegrasi dalam pembelajaran permainan bola tangan. 7 (2) November 2021 \| 92-107 104 Jurnal Ilmiah ADIRAGA Vol. 7 No. 2, November 2021, pp. 92-107 REFERENSI Abdurrochim, Muhamad. & Rachman, Hari Amirullah. (2016). Pengembangan Model Permainan Bolatangan Untuk Anak Usia Sekolah Dasar Kelas Atas. Jurnal Keolahragaan. 4 (1) 60-73 (2016). Abdullah, 2017., Pendekatan dan Model Pembelajaran yang Mengaktifkan Siswa. Jurnal Edureligia 1(1) 45-62 Aji Setiawan, Danang., (2016). Fair Play Dalam Olahraga. Jurnal Jendela 1(1) 1-13 Butler. (2000). Fair play; respect for all. Journal of Physical Education Recreation and Dance. 71 (2) 4-32 Departemen Pendidikan Nasional. (2008). 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International Journal of Science and Research (IJSR). 3 (2) 256-257 Manchado, C., Tortosa-Martínez, J., Vila, H., Ferragut, C., & Platen, P. (2013). Performance factors in women’s team handball: Physical and Physiological Aspects- A Review. Journal of Strength and Conditioning Research. 27 (6) 1708-1719. ( ) Maksum, Ali. (2009). Konstruksi Nilai Melalui Olahraga, Cakrawala Pendidikan, Jurnal Ilmiah Pendidikan. XXVIII (1) 25-34 Moleong, J.L, (2010), Metodologi Penelitian Kualitatif, Bandung, Edisi Revisi, Remaja RosdaKarya. Pradipta, Galih Dwi, (2015). Sportifitas Dalam Olahraga Sebagai Bagian Pembentukan Generasi Muda dan Nasionalisme. Jurnal Civic 5 (1) 713-724 Raharja, Didik S.P. (2019), Model Pendidikan Olahraga Dalam Meningkatkan Sikap Sportivitas. Jurnal of Sport. 3 (1) 29-36 Rusman. (2013). Model-model Pembelajaran: Mengembangkan Profesionalisme Guru. Jakarta: Rajawali Pers. Siahaan, Jonni. (2017). 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REFERENSI https://doi.org/10.21831/pep.v21i1.15784 7 (2) November 2021 \| 92-107 106 Jurnal Ilmiah ADIRAGA Vol. 7 No. 2, November 2021, pp. 92-107 Jurnal Ilmiah ADIRAGA Vol. 7 No. 2, November 2021, pp. 92-107 Jurnal Ilmiah ADIRAGA Vol. 7 No. 2, November 2021, pp. 92-107 Kilding E, Andrew., Tunstall, Helen., and Kuzmic, Dejan. (2008). Suitability of FIFA’s “The 11” Training Programme for Young Football Players Impact on Physical Performance, Journal of Sport Sceince and Medicine, 7 (11) 320-326 Kisaalita, Nkaku R. and Robinson, Michael E. (2014). Attitudes and Motivations of Competitive Cyclists Regarding Use of Banned and Legal Performance Enhancers, Journal of Sport Sceince and Medicine, 13 (21) 44-49 Kisaalita, Nkaku R. and Robinson, Michael E. (2014). Attitudes and Motivations of Competitive Cyclists Regarding Use of Banned and Legal Performance Enhancers, Journal of Sport Sceince and Medicine, 13 (21) 44-49
https://openalex.org/W3200197358	https://www.preprints.org/manuscript/202107.0362/v2/download	English	null	Efficacy and Toxicity of VarroMed® Used for Controlling Varroa destructor Infestation in Different Seasons and Geographical Areas	Applied sciences	2,021	cc-by	8,548	Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 doi:10.20944/preprints202107.0362.v2 doi:10.20944/preprints202107.0362.v2 Article Abstract VarroMed® is a soft acaricide registered for honey bees on the European Union market since 2017 for Varroa control. Researchers involved were partners of different countries of the Varroa control task force of COLOSS Association. Our goal was to eval- uate performances (acaricide efficacy and toxic effects on honey bees) of VarroMed® in different climatic conditions. Our results in the tested apiaries showed an efficacy rang- ing from 71.2 to 89.3 % in summer/autumn, and from 71.8 to 95.6 % in winter. No toxic effects on bees were observed, except in one apiary, where severe cold climatic conditions played a crucial role. The treatment could be efficiently applied in broodright as well as in broodless colonies. Integrated pest management (IPM) recommendations for bee- keepers are provided in order to apply the best Varroa control protocol. Keywords: VarroMed®; Varroa destructor; winter treatment; summer-autumn treatment; queen caging; brood interruption Efficacy and toxicity of VarroMed® used for controlling Varroa destructor infestation in different seasons and geographical areas Maja Ivana Smodiš Škerl 1, Jorge Rivera-Gomis 2, Ivana Tlak Gajger 3, Jernej Bubnič 1, Gabriela Talakić 3, Giovanni Formato 2, Alessandra Baggio 4, Franco Mutinelli 4, Wim Tollenaers 5, Dries Laget 6, Valeria Malagnini 7, Livia Zanotelli 7 and Marco Pietropaoli 2 1 Agricultural institute of Slovenia, Ljubljana, Slovenia; maja.smodis.skerl@kis.si, jernej.bubnic@kis.si 2 Istituto Zooprofilattico Sperimentale del Lazio e della Toscana “M. Aleandri”, Rome, Italy; giovanni.formato@izslt.it, marco.pietropaoli@izslt.it, jorgeriveragomis@gmail.com 3 Faculty of Veterinary Medicine University of Zagreb, Zagreb, Croatia; ivana.tlak@vef.hr, galincic10@gmail.com 4 Instituto Zooprofilattico Sperimentale delle venezie, Legnaro (PD), Italy; fmutinelli@izsvenezie.it , baggio.ale@libero.it gg 5 De Lieteberg, Zutendaal, Belgium; wim@lieteberg.be 6 Ghent University, Honeybee Vallex, Gent, Belgium; dries.laget@ugent.be 6 Ghent University, Honeybee Vallex, Gent, Belgium; drie 7 Fondazione Edmund Mach, Centro Trasferimento Technologico, S. Michele all’Adige (TN), Italy; l i l i i f h i li i lli f h i 7 Fondazione Edmund Mach, Centro Trasferimento Technologico, S. Michele all’Adige (TN), valeria.malagnini@fmach.it , livia.zanotelli@fmach.it valeria.malagnini@fmach.it , livia.zanotelli@fmach.it * Correspondence: maja.smodis.skerl@kis.si ; Tel.: (+38612805150) Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 doi:10.20944/preprints202107.0362.v2 2 of 15 From previous studies it is known that soft acaricides [7] have one or more compo- nents as an active ingredient: organic acids like oxalic [9-13], formic [14-16], lactic acid [17], other acids [18], thymol [19-22] and other essential oils [23,24]. Recently, different formulations of natural ingredients were evaluated, like a formulation, composed of ox- alic acid, thymol and oregano oil [25] and some compared or combined with brood in- terruption technique [26,8]. Since 2017, VarroMed®, an organic acid-based VMP against Varroa, was registered for its use on the honey bees in EU. g y In the context of the Varroa control task force of the COLOSS association [27], we decided to test the performances of VarroMed® concerning efficacy against Varroa mite and toxicity for honey bees in different European climatic conditions in agreement with EMA guidelines [28], following the instructions given by the producer. The aim of our study was to test the application of VarroMed® in different condi- tions and to give detailed and reliable information to European beekeepers in the framework of a Varroa management strategy. Table 1. Number of hives used in each trial and location. 1. Introduction The mite Varroa destructor [1] is one of the main threats to the European honeybee Apis mellifera and thus for the beekeeping sector [2]. The number of suitable veterinary medicine products (VMPs) for the treatment is limited [3] due to the need of low honey bee toxicity [4], the risk of residues in honey bee products [5,6] and the V. destructor mite capability to develop resistance to the frequently used active substances [7]. In this con- text, the availability of new VMPs in the market, as well as upgraded treatment measures or tools (i.e. brood interruption techniques) can be valuable help for beekeepers [4,8]. 2. Materials and Methods The active ingredients of VarroMed® (BeeVital GmbH, Handelstrasse 65162 Pber- trum am See, Austria) are formic acid (5 mg/ml) and oxalic acid dihydrate (44 mg/ml). Other ingredients in the product include caramel colour (E150d), sucrose syrup, propolis tincture (20 %), citric acid monohydrate, lemon (Citrus limon) essential oil, star anise (Il- licium verum) essential oil and pure water. There is no specific range of environmental temperature or humidity for the use of VarroMed® indicated by the producer. p y y p We evaluated the performances of VarroMed® in Italy (temperate continen- tal/Mediterranean climate), Slovenia and Croatia (moderately warm and rainy conti- nental climate), and Belgium (maritime temperate climate) in 2018. The protocol was structured in accordance with the European Medicines Agency (EMA) guidelines on VMPs for controlling V. destructor parasitosis in bees [28]. It was designed by the group of participants of the Varroa Control Task Force (COLOSS association). A summary scheme of the activities and their timing is presented in Figures 1-3. Honey bee colonies were set up in 10-frame Dadant-Blatt (DB), Langstroth Root (LR) or AŽ (Alberti-Žnideršič) hives, and placed in the same apiary each. We established two protocols: a summer/autumn protocol and winter protocol. The trials were carried out in absence of honey-flow and honey supers. Two ex- perimental groups homogenous in strength and Varroa infestation levels were organized for each trial. In both protocols, the colonies were treated with VarroMed® (treated group), while the other group was left untreated (control). The main differences between the two protocols were the absence of brood during the winter protocol versus its pres- ence during the summer/autumn protocol, and the duration of the treatment due to the number of VarroMed® applications. According to label instructions, the treatments in autumn should be performed according to decreasing colony population, 3 to 5 times, 6 days apart. In winter it should be applied once only, at the start of the broodless period and in hives with Varroa infestation. The summer/autumn protocol was evaluated in Italy, Slovenia, Croatia and Bel- gium, once per country, and the winter protocol was only carried out by three institu- tions in Italy and one in Croatia. The protocol followed by the participants and the number of honey bee colonies used for each trial are summarized in Table 1. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 doi:10.20944/preprints202107.0362.v2 3 of 15 Location Protocol Apiary code Number of treated colo- nies/control colonies Treatment period Zutendaal, Belgium Summer/autumn (x5)1 BELGIUM 10/10 23.9.-3.11.2018 Rome, Italy Summer/autumn (x5) ITALY_IZSLT 10/10 6.8.-20.9.2018 Brdo pri Kranju, Slo- venia Summer/autumn (x5) SLOVENIA 8/73 13.9.-15.10.2018 Karlovac, Croatia Summer/autumn (x3)2 CROATIA 10/10 20.7.-2.8.2018 Levico Terme, Italy Winter ITALY_FEM 10/10 22.11.-6.12.2018 Rome, Italy Winter ITALY_IZSLT 10/10 Bassano del Grappa, Italy Winter ITALY_IZSVE 10/10 17.11.-1.12.2018 Karlovac, Croatia Winter CROATIA 10/10 29.1.-20.2.2018 1 Five applications of VarroMed®. 2 Three applications of VarroMed®. 3 Number of colonies and hives was limited due to apiary size. In order to evaluate the variation of the colony strength due to the VarroMed® treatment, the number of adult bees and brood coverage was estimated [29] immediately before the beginning and after the end of the treatment period. According to manufacturers’ indications, VarroMed® was warmed at 25-35° C, shook before its use and administered between the hive frames fully occupied by bees. The dose was adjusted to the colony size according to dosage instructions of the pro- ducer. On the day of the first VarroMed® treatment (day 0 of the protocol – see Figures 1-3), the presence of the queen was checked, and colony strength was assessed in both groups. In order to calculate the acaricidal efficacy, mites fallen during the trial were counted every 2 to 3 days by inserting sticky boards. For the summer/autumn protocol, boards were checked until 17 days after the beginning of the last VarroMed® treatment considering the number of days of efficacy on dispersal mites (6 days as described in the leaflet) and for more 11 days to evaluate the eventual efficacy of the treatment on mites inside the capped brood cells susceptible to Varroa (Figures 1-3). doi:10.20944/preprints202107.0362.v2 4 of 15 Figure 1. Summer/autumn protocol for the treated group (5 repeated treatments) and control group. The following two treatments are additionally applied, if more than 150 mites are detected on the sticky boards within 6 days after the third treatment. Figure 1. Summer/autumn protocol for the treated group (5 repeated treatments) and control group. The following two treatments are additionally applied, if more than 150 mites are detected on the sticky boards within 6 days after the third treatment. doi:10.20944/preprints202107.0362.v2 5 of 15 Figure 2. Summer/autumn protocol for the treated group (3 repeated treatments) and control group. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 Only three treatments are needed, if less than 150 mites are detected on the sticky boards. Figure 2. Summer/autumn protocol for the treated group (3 repeated treatments) and control group. Only three treatments are needed, if less than 150 mites are detected on the sticky boards. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 doi:10.20944/preprints202107.0362.v2 7 of 15 During the experiments, mean environmental temperatures were recorded by weather stations near the apiaries. Statistical analysis was performed using XLSTATTM software [32]. It was verified, if differences in acaricide efficacy were statistically significant using Mann-Whitney Test [33]. The amount of adult honey bees and brood coverage between groups (treated and control) was evaluated using Kruskal-Wallis Test [34]. For the latter, multiple pairwise comparison with Dunn’s Test [35] was applied with Bonferroni correction. 3.1. Summer/autumn trial Acaricide efficacy of VarroMed® recorded during the summer/autumn trial ranged from 71.2 %, when applied three times (Croatia), to a maximum of 89.4 % (Belgium) when applied five times (Table 2; Figure 4). All treated groups efficacies differed from control groups (Kruskal-Wallis test: H(7)=59.14, p< 0.0001; Dunn’s tests: p < 0.05). There were no statistically significant differences recorded between the locations, and the number of applied treatments (3 or 5) was not related to the natural mite fall rates before treatment. Table 2. Acaricide efficacy recorded during the summer/autumn trial in the apiaries and relative mite fall during the same period in control group (%). Statistics VARROMED X5 (BELGIUM) VARROMED X5 (ITALY_IZSLT) VARROMED X5 (SLOVENIA) VARROMED X3 (CROATIA) CONTROL (BELGIUM) CONTROL (ITALY_IZSLT) CONTROL (SLOVENIA) CONTROL (CROATIA) Minimum 81.5 73.9 83.4 13.3 6.8 28.2 21.4 0.4 Maximum 100 99.2 93.8 80.7 32.8 75.3 69.2 1.5 1st Quartile 83.6 82.9 84.9 76.1 9.8 32.1 28.8 0.5 Median 89.2 89.7 88.6 77.3 12.9 39.8 46.7 0.6 3rd Quartile 94.1 98.2 91.3 78.9 13.5 53.7 48.7 0.7 Mean 89.4 88.2 88.3 71.2 15.3 44.6 42.7 0.7 Variance (n-1) 0.5 0.9 0.2 4.2 0.9 2.7 3.2 0 Standard deviation (n-1) 7.2 9.3 4.0 20.4 9.7 16.3 17.9 0.4 Figure 4. Box plot of acaricide efficacies recorded during the summer/autumn trial and relative mite fall during the same period in control group (%). The horizontal line in boxes shows median. The box shows 1st and 3rd quartile. Whiskers extend to 1.5 x interquartile range (IQR). Diamonds mark the outliers outside the 1.5 x IQR. Addition- ally, the pluses show mean values. y recorded during the summer/autumn trial in the apiaries and relative mite fall during the same %) Table 2. Acaricide efficacy recorded during the summer/autumn trial in the apiaries and relative mite fall during the same period in control group (%). Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 doi:10.20944/preprints202107.0362.v2 6 of 15 Figure 3. Winter protocol for the treated and control group in broodless colonies. Figure 3. Winter protocol for the treated and control group in broodless colonies. For the winter protocol, boards were checked until 14 days after the VarroMed® treatment. A follow-up treatment consisting in the application of synthetic miticides with dif- ferent mechanism of action based on amitraz and tau-fluvalinate (in combination or in double dose), in order to avoid low efficacy due to possible mite resistance to one of the active substances, was applied to verify the residual number of mites. We used VPMs registered for honey bees with active substances not present in VarroMed®. In the summer/autumn protocol all queens were caged in VAR-CONTROL cages (Api-Mo.Bru, Campodoro, Padova, Italy – http://www.apimobru.com/en/ppe/ppe.htm) for 24 days. The follow-up treatment was applied during the caging period and for more 18 days. The residual number of mites was counted on sticky boards during the whole timeframe [30,25] In brood absence (winter treatment) residual mites were counted on the sticky boards for 14 days after the follow-up treatment [30,25]. The untreated colonies of all control groups were checked for the natural mite fall and received the same follow-up treatment. The percentage of acaricidal efficacy (AE) in each hive was evaluated using the formula: AE=(VT/VT+follow-up)*100, where VT is the total number of mites killed by the Var- roMed® treatment, not considering the mites fallen during the queen caging period, and VT+follow-up represents the total number of mites killed by the tested treatment and the fol- low-up treatments [31]. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 Statistics VARROMED X5 (BELGIUM) VARROMED X5 (ITALY_IZSLT) VARROMED X5 (SLOVENIA) VARROMED X3 (CROATIA) CONTROL (BELGIUM) CONTROL (ITALY_IZSLT) CONTROL (SLOVENIA) CONTROL (CROATIA) Minimum 7.5 11.5 15.3 69.7 92.4 56.4 10.9 53.3 Maximum 122.2 219.4 63.2 94.6 242.7 448.1 109.2 111.7 1st Quartile 23.1 55.2 18.5 76.5 152.3 61.9 23.7 61.2 Median 44.0 90.1 20.4 80.2 180.0 117.5 28.8 70.8 3rd Quartile 63.7 121.2 38.4 85.2 208.2 126.4 .48.5 78.1 Mean 49.6 94.2 30.4 80.9 179.7 137.8 42.5 72.1 Variance (n-1) 15.3 37.0 3.5 0.5 25.1 147.1 12.7 2.9 Standard devi- ation (n-1) 39.1 60.8 18.8 7.3 50.1 121.3 35.6 16.9 Figure 5. Box plot of adult bee coverage in all groups recorded during the summer/autumn trial compared to the beginning of the treatment (%). The horizontal line in boxes shows median. The box shows 1st and 3rd quartile. Whiskers extend to 1.5 x interquartile range (IQR). Diamonds mark the outliers outside the 1.5 x IQR. Addi- tionally, the pluses show mean values. Mean brood area compared to the control group decreased after VarroMed treat- ment in Italy and Slovenia (-71.1 % and -3.0 % respectively) and increased in Croatia lt honey bee coverage in all groups recorded during the summer/autumn trial compared to the be- (%). ginning of the treatment (%). Statistics VARROMED X5 (BELGIUM) VARROMED X5 (ITALY_IZSLT) VARROMED X5 (SLOVENIA) VARROMED X3 (CROATIA) CONTROL (BELGIUM) CONTROL (ITALY_IZSLT) CONTROL (SLOVENIA) CONTROL (CROATIA) Minimum 7.5 11.5 15.3 69.7 92.4 56.4 10.9 53.3 Maximum 122.2 219.4 63.2 94.6 242.7 448.1 109.2 111.7 1st Quartile 23.1 55.2 18.5 76.5 152.3 61.9 23.7 61.2 Median 44.0 90.1 20.4 80.2 180.0 117.5 28.8 70.8 3rd Quartile 63.7 121.2 38.4 85.2 208.2 126.4 .48.5 78.1 Mean 49.6 94.2 30.4 80.9 179.7 137.8 42.5 72.1 Variance (n-1) 15.3 37.0 3.5 0.5 25.1 147.1 12.7 2.9 Standard devi- ation (n-1) 39.1 60.8 18.8 7.3 50.1 121.3 35.6 16.9 Figure 5. Box plot of adult bee coverage in all groups recorded during the summer/autumn trial compared to the beginning of the treatment (%). The horizontal line in boxes shows median. The box shows 1st and 3rd quartile. Whiskers extend to 1.5 x interquartile range (IQR). Diamonds mark the outliers outside the 1.5 x IQR. Addi- tionally, the pluses show mean values. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 doi:10.20944/preprints202107.0362.v2 8 of 15 The amount of adult bees was reduced in treated groups, compared to the control ones, in Belgium, Italy and Slovenia, ranging from -130.1 % to -12.1 %, and increased in Croatia +8.8 % (Table 3 and Figure 5). These differences were statistically significant only in one apiary (Belgium) (Dunn’s tests p value: 0.0001) where colonies were smaller and three of them died after the treatment. Table 3. Variation of adult honey bee coverage in all groups recorded during the summer/autumn trial compared to the be- ginning of the treatment (%). Statistics VARROMED X5 (BELGIUM) VARROMED X5 (ITALY_IZSLT) VARROMED X5 (SLOVENIA) VARROMED X3 (CROATIA) CONTROL (BELGIUM) CONTROL (ITALY_IZSLT) CONTROL (SLOVENIA) CONTROL (CROATIA) Minimum 7.5 11.5 15.3 69.7 92.4 56.4 10.9 53.3 Maximum 122.2 219.4 63.2 94.6 242.7 448.1 109.2 111.7 1st Quartile 23.1 55.2 18.5 76.5 152.3 61.9 23.7 61.2 Median 44.0 90.1 20.4 80.2 180.0 117.5 28.8 70.8 3rd Quartile 63.7 121.2 38.4 85.2 208.2 126.4 .48.5 78.1 Mean 49.6 94.2 30.4 80.9 179.7 137.8 42.5 72.1 Variance (n-1) 15.3 37.0 3.5 0.5 25.1 147.1 12.7 2.9 Standard devi- ation (n-1) 39.1 60.8 18.8 7.3 50.1 121.3 35.6 16.9 Figure 5. Box plot of adult bee coverage in all groups recorded during the summer/autumn trial compared to the beginning of the treatment (%). The horizontal line in boxes shows median. The box shows 1st and 3rd quartile. Whiskers extend to 1.5 x interquartile range (IQR). Diamonds mark the outliers outside the 1.5 x IQR. Addi- tionally, the pluses show mean values. Mean brood area compared to the control group decreased after VarroMed treat- ment in Italy and Slovenia (-71 1 % and -3 0 % respectively) and increased in Croatia Table 3. Variation of adult honey bee coverage in all groups recorded during the summer/autumn trial compared to the be- ginning of the treatment (%). Table 3. Variation of adult honey bee coverage in all groups recorded during the summer/autumn trial compared to the be- ginning of the treatment (%). 3.1. Summer/autumn trial Statistics VARROMED X5 (BELGIUM) VARROMED X5 (ITALY_IZSLT) VARROMED X5 (SLOVENIA) VARROMED X3 (CROATIA) CONTROL (BELGIUM) CONTROL (ITALY_IZSLT) CONTROL (SLOVENIA) CONTROL (CROATIA) Minimum 81.5 73.9 83.4 13.3 6.8 28.2 21.4 0.4 Maximum 100 99.2 93.8 80.7 32.8 75.3 69.2 1.5 1st Quartile 83.6 82.9 84.9 76.1 9.8 32.1 28.8 0.5 Median 89.2 89.7 88.6 77.3 12.9 39.8 46.7 0.6 3rd Quartile 94.1 98.2 91.3 78.9 13.5 53.7 48.7 0.7 Mean 89.4 88.2 88.3 71.2 15.3 44.6 42.7 0.7 Variance (n-1) 0.5 0.9 0.2 4.2 0.9 2.7 3.2 0 Standard deviation (n-1) 7.2 9.3 4.0 20.4 9.7 16.3 17.9 0.4 Figure 4. Box plot of acaricide efficacies recorded during the summer/autumn trial and relative mite fall during the same period in control group (%). The horizontal line in boxes shows median. The box shows 1st and 3rd quartile. Whiskers extend to 1.5 x interquartile range (IQR). Diamonds mark the outliers outside the 1.5 x IQR. Addition- ally, the pluses show mean values. cide efficacy recorded during the summer/autumn trial in the apiaries and relative mite fall during the sam rol group (%). Figure 4. Box plot of acaricide efficacies recorded during the summer/autumn trial and relative mite fall during the same period in control group (%). The horizontal line in boxes shows median. The box shows 1st and 3rd quartile. Whiskers extend to 1.5 x interquartile range (IQR). Diamonds mark the outliers outside the 1.5 x IQR. Addition- ally, the pluses show mean values. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 Variation of brood area in all groups recorded during the summer/autumn trial compared to the begin- ning of the treatment (%). . Variation of brood area in all groups recorded during the summer/autumn trial compared to the begin- the treatment (%). Median 75.9 8.6 50.4 115.6 12.1 26.5 3rd Quartile 117.6 16.2 62.8 173.3 24.7 28.8 Mean 81.1 10.7 51.1 152.3 13.7 26.4 Variance (n-1) 33.7 0.8 1.9 154.5 1.7 0.3 Standard deviation (n-1) 58.0 8.9 13.8 124.3 13.0 5.5 Figure 6. Box plot of brood area in all groups recorded during the summer/autumn trial compared to the begin- ning of the treatment (%). The horizontal line in boxes shows median. The box shows 1st and 3rd quartile. Figure 6. Box plot of brood area in all groups recorded during the summer/autumn trial compared to the begin- ning of the treatment (%). The horizontal line in boxes shows median. The box shows 1st and 3rd quartile. Whiskers extend to 1.5 x interquartile range (IQR). Diamonds mark the outliers outside the 1.5 x IQR. Addi- tionally, the pluses show mean values. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 Mean brood area compared to the control group decreased after VarroMed treat- ment in Italy and Slovenia (-71.1 % and -3.0 % respectively) and increased in Croatia (+24.7 %) but all variations were not statistically significant (Table 4; Figure 6). Brood area was not recorded in Belgium as bees started clustering and weather conditions were prohibitive to check brood coverage properly. doi:10.20944/preprints202107.0362.v2 9 of 15 Table 4. Variation of brood area in all groups recorded during the summer/autumn trial compared to the begin- ning of the treatment (%). Statistics VARROMED X5 (ITALY_IZSLT) VARROMED X5 (SLOVENIA) VARROMED X3 (CROATIA) CONTROL (ITALY_IZSLT) CONTROL (SLOVENIA) CONTROL (CROATIA) Minimum 1.5 1.3 34.0 42.6 0 19.0 Maximum 170.5 23.8 71.4 450.0 29.1 36.7 1st Quartile 40.1 4.5 39.5 76.8 3.0 23.3 Median 75.9 8.6 50.4 115.6 12.1 26.5 3rd Quartile 117.6 16.2 62.8 173.3 24.7 28.8 Mean 81.1 10.7 51.1 152.3 13.7 26.4 Variance (n-1) 33.7 0.8 1.9 154.5 1.7 0.3 Standard deviation (n-1) 58.0 8.9 13.8 124.3 13.0 5.5 Figure 6. Box plot of brood area in all groups recorded during the summer/autumn trial compared to the begin- ning of the treatment (%). The horizontal line in boxes shows median. The box shows 1st and 3rd quartile. Whiskers extend to 1.5 x interquartile range (IQR). Diamonds mark the outliers outside the 1.5 x IQR. Addi- tionally, the pluses show mean values. 3 2 Winter trial Table 4. Variation of brood area in all groups recorded during the summer/autumn trial compared to the begin- ning of the treatment (%). Statistics VARROMED X5 (ITALY_IZSLT) VARROMED X5 (SLOVENIA) VARROMED X3 (CROATIA) CONTROL (ITALY_IZSLT) CONTROL (SLOVENIA) CONTROL (CROATIA) Minimum 1.5 1.3 34.0 42.6 0 19.0 Maximum 170.5 23.8 71.4 450.0 29.1 36.7 1st Quartile 40.1 4.5 39.5 76.8 3.0 23.3 Median 75.9 8.6 50.4 115.6 12.1 26.5 3rd Quartile 117.6 16.2 62.8 173.3 24.7 28.8 Mean 81.1 10.7 51.1 152.3 13.7 26.4 Variance (n-1) 33.7 0.8 1.9 154.5 1.7 0.3 Standard deviation (n-1) 58.0 8.9 13.8 124.3 13.0 5.5 Figure 6. Box plot of brood area in all groups recorded during the summer/autumn trial compared to the begin- ning of the treatment (%). The horizontal line in boxes shows median. The box shows 1st and 3rd quartile. Whiskers extend to 1.5 x interquartile range (IQR). Diamonds mark the outliers outside the 1.5 x IQR. Addi- tionally, the pluses show mean values. 3.2. Winter trial Table 4. 3.2. Winter trial In colonies where VarroMed was applied during wintertime, in the absence of brood, the recorded acaracide efficacy ranges from 71.8 % to 95.6 % (Table 5; Figure 7). Efficacies in all treated groups differed when compared to control groups (Kruskal-Wallis test: H(7)=62.07, p<0.0001; Dunn’s tests: p<0.05). In colonies where VarroMed was applied during wintertime, in the absence of brood, the recorded acaracide efficacy ranges from 71.8 % to 95.6 % (Table 5; Figure 7). Efficacies in all treated groups differed when compared to control groups (Kruskal-Wallis test: H(7)=62.07, p<0.0001; Dunn’s tests: p<0.05). Table 5. Acaricide efficacy recorded during the summer/autumn trial in the apiaries and relative mite fall during the same period in control group (%). Table 5. Acaricide efficacy recorded during the summer/autumn trial in the apiaries and relative mite fall during the same period in control group (%). Statistics VARROMED (ITALY_FEM) VARROMED (ITALY_IZSLT) VARROMED (ITALY_IZSVE) VARROMED (CROATIA) CONTROL (ITALY_FEM) CONTROL (ITALY_IZSLT) CONTROL (ITALY_IZSVE) CONTROL (CROATIA) Minimum 73.1 87.8 64.8 42.9 1.1 0.6 1.7 0 Maximum 100 98.9 98.7 87.3 19.6 23.8 33.3 35.7 1st Quartile 79.3 93.8 84.6 64.6 5.4 2.8 2.4 3.4 Median 94.6 97.0 93.7 74.1 6.6 6.5 14.4 16.0 3rd Quartile 99.8 97.6 96.0 82.3 10.2 11.8 23.3 20.3 Mean 89.9 95.6 89.7 71.8 8.3 8.6 15.0 14.8 Variance (n-1) 1.2 0.1 1.1 1.9 0.3 0.5 1.6 1.5 Standard deviation (n-1) 11.1 3.5 10.5 13.7 5.3 7.3 12.7 12.0 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 doi:10.20944/preprints202107.0362.v2 10 of 15 Figure 7. Box plot of acaricide efficacies recorded during the winter trial and relative mitefall during the same period in control group (%). The horizontal line in boxes shows median. The box shows 1st and 3rd quartile. Whiskers extend to 1.5 x interquartile range (IQR). Additionally, the pluses show mean values. Figure 7. Box plot of acaricide efficacies recorded during the winter trial and relative mitefall during the same period in control group (%). The horizontal line in boxes shows median. The box shows 1st and 3rd quartile. Whiskers extend to 1.5 x interquartile range (IQR). Additionally, the pluses show mean values. Small variations of coverage of adult honey bees were recorded but no statistically significant differences were observed (Table 6; Figure 8). No queen mortality and no clinical signs of other economically important diseases were observed. Small variations of coverage of adult honey bees were recorded but no statistically significant differences were observed (Table 6; Figure 8). No queen mortality and no clinical signs of other economically important diseases were observed. Table 6. Variation of adult honey bee coverage in all groups recorded during the winter trial compared to the beginning of th treatment (%). honey bee coverage in all groups recorded during the winter trial compared to the beginning of the Table 6. Variation of adult honey bee coverage in all groups recorded during the winter trial compared to the beginning of th treatment (%). Statistics VARROMED (ITALY_FEM) VARROMED (ITALY_IZSLT) VARROMED (ITALY_IZSVE) VARROMED (CROATIA) CONTROL (ITALY_FEM) CONTROL (ITALY_IZSLT) CONTROL (ITALY_IZSVE) CONTROL (CROATIA) Minimum 70.6 62.4 84.6 81.1 71.6 80.6 91.3 85.7 Maximum 199.6 95.4 120.7 100 187.6 93.8 112.5 100 1st Quartile 98.7 88.9 97.3 85.7 81.9 83.3 95.7 99.0 Median 122.9 92.2 101.3 88.3 93.4 90.3 98.2 100 3rd Quartile 135.6 94.2 104.2 99.5 124.1 90.9 105.9 100 Mean 120.2 88.1 101.9 91.1 106.6 88.3 100.3 98.0 Variance (n-1) 13.1 1.1 1.0 0.6 13.4 0.2 0.5 0.2 Standard deviation (n-1) 36.2 10.4 10.2 7.6 36.6 4.8 7.0 4.5 doi:10.20944/preprints202107.0362.v2 11 of 15 11 of 15 Figure 8. Boxplot of adult honey bee coverage in all groups recorded during the winter trial and compared to the beginning of the treatment (%). The horizontal line in boxes shows median. The box shows 1st and 3rd quartile. Whiskers extend to 1 5 x interquartile range (IQR) Additionally the pluses show mean values Figure 8. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 Boxplot of adult honey bee coverage in all groups recorded during the winter trial and compared to the beginning of the treatment (%). The horizontal line in boxes shows median. The box shows 1st and 3rd quartile. Whiskers extend to 1.5 x interquartile range (IQR). Additionally, the pluses show mean values. Figure 8. Boxplot of adult honey bee coverage in all groups recorded during the winter trial and compared to the beginning of the treatment (%). The horizontal line in boxes shows median. The box shows 1st and 3rd quartile. Whiskers extend to 1.5 x interquartile range (IQR). Additionally, the pluses show mean values. During the summer/autumn trials, the mean environmental temperatures recorded during VarroMed treatments were as follows: 30.2±2.5° C in Croatia; 23.6±1.3° C in Italy; 15.6±4.2° C in Slovenia; 12.4±2.7° C in Belgium. During winter trials we recorded the following mean environmental temperatures: 7.4±3.4° C in Italy_IZSVE; 6.4±3.2° C in Italy_IZSLT; 4.2±4.6° C in Croatia, and 3.3±2.6° C in Italy_FEM. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 doi:10.20944/preprints202107.0362.v2 12 of 15 12 of 15 [42], the efficacy of winter treatment with VarroMed® was 88 %. The level of efficacy of VarroMed® treatment in summer/autumn and winter was found sufficient, which can be supported by the average number of mite fall after queen caging and follow-up treat- ment. Another critical aspect to the use of mixture of organic acids (oxalic and formic acid) and additional substances against Varroa mites that should be considered is a possible toxicity to honey bees and colonies. The use of oxalic acid by trickling, evaporation and spraying, honey bee tolerability and efficacy has been reviewed by Rademacher & Hartz [12]. In general, the application of oxalic acid in a formulation with sugar syrup increases the efficacy against Varroa mites. However, there are some negative effects of oxalic acid on queen health [44,45], and on worker bees’ digestive system [45]. Formic acid operates through inhibition of mitochondrial energetic metabolism of Varroa mites binding the cytochrome C oxidase enzyme [46], as well as through a sig- nificant neuroexcitation process [47]. It also affects honey bee colony as it reduces lon- gevity of worker bees [48] and affects brood survival [48]. There is a report on increased number of dead bees in front of the hive, queen rejection and decrease of honey yield during treatment [50]. Considering the above-mentioned toxicity aspects, we evaluated the effects on the strength of the colonies after VarroMed® treatments. We found that the reduction in number of honey bees and brood in treated hives was very low or insignificant in all countries and in all application’s seasons except in Belgium, where less honey bees were observed in treated colonies and some of them even died. This finding could be ex- plained by the lack of an ideal combination of temperature (below 15° C), treatment (several applications) and colony condition (decreasing colony population) at the time of field trial. It is important to highlight that our data showed that there is no need to treat in absence of brood (e.g. applying the queen caging) as a high acaricide efficacy was ob- served in both cases (with or without brood). The treatments were repeated several times and the duration of the treatment covered the period longer than worker developmental stage from egg to emerging. 4. Discussion Varroa mite is a major cause of overwintering honey bee (A. mellifera) colony losses across the globe [36]. In recent years, several surveys were conducted to analyze bee- keepers’ treatment practices and overwintering [36-40]. One of the findings of the above-mentioned studies was that the worst scenario concerning overwintering ability was a combination of weakness of honey bee populations, low food reserves and high Varroa infestation levels [37]. In our study, we administered VarroMed® in several apiaries with different climatic conditions. The average temperatures near the experimental apiaries in Croatia, Italy, Slovenia and Belgium show a versatile range (30.2 – 12.4° C) only in summer/autumn treatment period in 2018. Our results show that the efficacy of VarroMed® treatment was above 70 % and not highly variable, regardless of the presence of honey bee brood and environmental temperature, despite some relevant differences between minimum and maximum efficacy values in some sites as shown in Tables 2 and 5. High efficacies of other oxalic acid-based treatments (application by trickling or sublimation) were re- ported by Büchler et al. [8]. Colonies were treated in a broodless period in summer time (using brood interruption techniques, like queen caging or brood removal) and the effi- cacy ranged from 48 to 89 % mite removal. The protocols of the treatments against V. destructor may require quite a long time (over 40 days), to reach a high acaricide efficacy, especially in broodright colonies with high levels of infestation [31]. The field trials were carried out in medium infested colo- nies and the treatment was therefore appropriate. In the experimental apiaries, the effi- cacy ranged from 71.2 to 89.3 % in summer/autumn treatment. Tlak Gajger & Sušec [41] reported even higher acaricide efficacy (91.5 %) in summer, after three consecutive ap- plications of the oxalic acid based complementary feed HiveClean® (former product, like VarroMed®). In winter treatments reported in this paper, the overall acaricide efficacies ranged from 71.8 to 95.6 %. According to the report of the European Medicines Agency References Available online: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A32010R0037 (accessed on 19 June 2021) stances and their classification regarding maximum residue limits in foodstuffs of animal origin. OJ L 15, 1 72, 2010. Available online: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A32010R0037 (accessed on 19 June 2021) a e a ei a i i a io ega i g a i u e i ue i i i oo u o a i a o igi OJ 5, , 0 0 A ai a e online: https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A32010R0037 (accessed on 19 June 2021) 7. Rosenkranz, P.; Aumeier, P.; Ziegelmann, B. Biology and control of Varroa destructor. J Invert Pathol 2010, 103, S96-S119. https://doi.org/10.1016/j.jip.2009.07.016 7. Rosenkranz, P.; Aumeier, P.; Ziegelmann, B. Biology and control of Varroa destructor. J Invert Pathol 2010, 103, S96-S119. https://doi.org/10.1016/j.jip.2009.07.016 8. Büchler, R.; Uzunov, A.; Kovačić, M.; Prešern, J.; Pietropaoli, M.; Hatjina, F.; Pavlov, B.; Charistos, L.; Formato, G.; Galarza, E.; Gerula, D.; Gregorc, A.; Malagnini, V.; Meixner, M.; Nedić, N.; Puškadija, Z.; Rivera-Gomis, J.; Rogelj Jenko, M.; Smodiš Škerl, M. I.; Vallon, J.; Vojt, D.; Wilde, J.; Nanetti, A. Summer brood interruption as integrated management strategy for effective Varroa control in Europe. J Apicult Res 2020, 59, 764-773. https://doi.org/10.1080/00218839.2020.1793278 8. Büchler, R.; Uzunov, A.; Kovačić, M.; Prešern, J.; Pietropaoli, M.; Hatjina, F.; Pavlov, B.; Charistos, L.; Formato, G.; Galarza, E.; Gerula, D.; Gregorc, A.; Malagnini, V.; Meixner, M.; Nedić, N.; Puškadija, Z.; Rivera-Gomis, J.; Rogelj Jenko, M.; Smodiš Škerl, M. I.; Vallon, J.; Vojt, D.; Wilde, J.; Nanetti, A. Summer brood interruption as integrated management strategy for effective Varroa control in Europe. J Apicult Res 2020, 59, 764-773. https://doi.org/10.1080/00218839.2020.1793278 p p p g 9. Radetzki, T.; Reiter, M.; Negelein, B.; Fischermuhle, H. Oxalsaure zur Varroabekampfung [Oxalic acid for Varroa control]. Schweizerische Bienen-Zeitung 1994, 117, 263-267. p p p g 9. Radetzki, T.; Reiter, M.; Negelein, B.; Fischermuhle, H. Oxalsaure zur Varroabekampfung [Oxalic acid for Varroa control]. Schweizerische Bienen-Zeitung 1994, 117, 263-267. 10. Imdorf, A.; Charriere, J.-E.; Bachofen, B. Wann ist die Oxalsaure als Varroazid geeignet [When is oxalic acid suitable as var- roacide?]. Schweizerische Bienen-Zeitung 1995, 7, 389-391. 11. Mutinelli, F.; Baggio, A.; Capolongo, F.; Piro, R.; Prandin, L.; Biasion, L. A scientific note on oxalic acid by topical application for the control of varroosis. Apidologie 1997, 28, 461-462. https://doi.org/10.1051/apido:19970612 12. Nanetti, A. Oxalic acid for mite control - Results and review. In Coordination in Europe of research on integrated varroa mites in honey bee colonies. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 doi:10.20944/preprints202107.0362.v2 13 of 15 13 of 15 Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the man- uscript, or in the decision to publish the results. References Commission of the European Communities 1999, 9-15. Available online: http://varroa.fr/wp-content/uploads/2017/08/Fries-I.-et-al-1999.-Coordination-in-europe-of-integrated-control-of-varroa-mites- in-honey-bees-colonies.pdf (accessed on 19 June 2021) 13. Rademacher, E.; Harz, M. Oxalic acid for the control of varroosis in honey bee colonies – a review. Apidologie 2006, 37, 98-120. https://doi.org/10.1051/apido:2005063 p g p 14. Imdorf, A.; Gerig, L. Lutte intégrée contre varroa: acide formique. J Suisse d’Apiculture 1988, 85, 311-32 14. Imdorf, A.; Gerig, L. Lutte intégrée contre varroa: acide formique. J Suisse d’Apiculture 1988, 85, 311-320. 15. Bolli, H.K. ; Bogdanov, S. ; Imdorf, A. ; Fluri, P. Zur Wirkungsweise von Ameisensäure bei Varroa jacobsoni Oud und der Ho- nigbiene (Apis mellifera L). [Title in English: Action of formic acid on Varroa jacobsoni Oud and the honeybee (Apis mellifera L). Apidologie 1993, 24, 51-57. https://doi.org/10.1051/apido:19930106 15. Bolli, H.K. ; Bogdanov, S. ; Imdorf, A. ; Fluri, P. Zur Wirkungsweise von Ameisensäure bei Varroa jacobsoni Oud und der Ho- nigbiene (Apis mellifera L). [Title in English: Action of formic acid on Varroa jacobsoni Oud and the honeybee (Apis mellifera L). Apidologie 1993, 24, 51-57. https://doi.org/10.1051/apido:19930106 p g p g p 16. Pietropaoli, M.; Formato, G. Liquid formic acid 60% to control varroa mites (Varroa destructor) in honey bee colonies (Apis mellifera): protocol evaluation. J Apicult Res 2018, 57, 300-307. https://doi.org/10.1080/00218839.2017.1376767 p g p 16. Pietropaoli, M.; Formato, G. Liquid formic acid 60% to control varroa mites (Varroa destructor) in honey bee colonies (Apis mellifera): protocol evaluation. J Apicult Res 2018, 57, 300-307. https://doi.org/10.1080/00218839.2017.1376767 f p p p g 17. Girisgin, A.O.; Aydin, L. Efficacies of formic, oxalic and lactic acids against Varroa destructor in nat (Apis mellifera L.) colonies in Tureky. Kafkas Univ Vet Fak Derg 2010, 16, 941-945. 18. Rademacher, E., Harz, M., Schneider, S. (2015). The development of HopGuard® as a winter treatment against Varroa destructor in colonies of Apis mellifera. Apidologie; 46(6), 748-759. https://doi.org/10.1007/s13592-015-0363-0 18. Rademacher, E., Harz, M., Schneider, S. (2015). The development of HopGuard® as a winter treatment against Varroa destructor in colonies of Apis mellifera. Apidologie; 46(6), 748-759. https://doi.org/10.1007/s13592-015-0363-0 p f p g ( ) p g 19. Imdorf, A. ; Kilchenmann, V. ; Maquelin, C. ; Bogdanov, S. Optimierung der Anwendung von ’Apilife VAR’ zur Bekämpfung von Varroa jacobsoni Oud. in Bienenvölkern, Apidologie 1994, 25, 49-60. https://doi.org/10.1051/apido:19940106 p f p g ( ) p g 19. Imdorf, A. ; Kilchenmann, V. ; Maquelin, C. ; Bogdanov, S. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 However, we want to underline the repeated applications of the product, that demand extended efforts of the beekeeper in terms of time and costs and result in higher toxicity to adult honey bees, especially in case of environmental temperatures lower than 15° C (mean autumn temperatures in Belgium were 12.4±2.7° C). Finally, it is noteworthy that the producer of VarroMed® leaves the decision to apply an extra-treatment or not to the beekeepers, based on the Varroa infestation levels ob- tained after the previous treatment [51]. This approach to the treatment against Varroa mites based on an integrated pest management (IPM) includes the implementation of good beekeeping practices [52] and beekeepers’ education to a sustainable and successful beekeeping. Author Contributions: Conceptualization, all authors; methodology, all authors; investigation, all authors; writing—original draft preparation, J.R.-G., M.I.S.Š., M.P. and I.T.G.; writing—review and editing, all authors.; visualization, M.P.; funding acquisition, all authors. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Slovenian Research Agency, research program P4-0133 ‘Trajnostno kmetijstvo’. This work was supported by the Project BPractices (ERA-NET SusAn) co-financed by the European Union's Horizon 2020 research and Innovation Program, and core financing of Slovenian Research Agency (grants P4-0133 for M.I.S.Š.). Acknowledgments: We thank COLOSS (Honey bee research association) for support in the meet- ings and successful collaboration of partners. References 1. Anderson, D.L.; Trueman, J.W.H. Varroa jacobsoni (Acari: Varroidae) is more than one species. Exp appl acarol 2000, 24, 165-189. https://doi.org/10.1023/A:1006456720416 1. Anderson, D.L.; Trueman, J.W.H. Varroa jacobsoni (Acari: Varroidae) is more than one species. Exp appl acarol 2000, 24, 165-189. https://doi.org/10.1023/A:1006456720416 1. Anderson, D.L.; Trueman, J.W.H. Varroa jacobsoni (Acari: Varroidae) is more than one species. Exp appl acarol 2000, 24, 165-189. https://doi.org/10.1023/A:1006456720416 p g 2. Noël, A. ; Le Conte, Y. ; Mondet, F. 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Beyer, M.; Junk, J.; Eickermann, M.; Clermont, A.; Kraus, F.; Georges, C.; Reichart, A.; Hoffmann, L. Winter honey bee colony 35. Dunn, O.J. Multiple comparisons among means. J Am Stat Assoc 1961, 56, 52-64. https://doi.org/10.1080/01621459.1961.10482090 36. Beyer, M.; Junk, J.; Eickermann, M.; Clermont, A.; Kraus, F.; Georges, C.; Reichart, A.; Hoffmann, L. Winter honey bee colony losses, Varroa destructor control strategies, and the role of weather conditions: Results from a survey among beekeepers. Res Vet Sci 2018, 118, 52-60. https://doi.org/10.1016/j.rvsc.2018.01.012 p g j 37. Guzmán-Novoa, E.; Eccles, L.; Calvete, Y.; Mcgowan, J.; Kelly, P.G.; Correa-Benítez, A. Varroa destructor is the main culprit for the death and reduced populations of overwintered honey bee (Apis mellifera) colonies in Ontario, Canada. Apidologie 2010, 41, 443–450. https://doi.org/10.1051/apido/2009076 38. Haber, A.I.; Steinhauer, N.A.; vanEngelsdorp D. Use of chemical and nonchemical methods for the control of Varroa destructor (Acari: Varroidae) and associated winter colony losses in U.S. beekeeping operations. J Econ Entomol 2019, 112, 1509-1525. https://doi.org/doi: 10.1093/jee/toz088 p g j 39. Oberreiter, H.; Brodschneider, R. Austrian COLOSS Survey of honey bee colony winter losses 2018/19 and Analysis of Hive Management Practices. Diversity 2020, 12, 99. https://doi.org/doi:10.3390/d12030099 p g j 39. Oberreiter, H.; Brodschneider, R. Austrian COLOSS Survey of honey bee colony winter losses 2018/19 and Analysis of Hive Management Practices. Diversity 2020, 12, 99. https://doi.org/doi:10.3390/d12030099 g y p g 40. Vercelli, M.; Croce, L.; Mancuso, T. An economic approach to assess the annual stock in beekeeping farms: The honey bee colony inventory tool. Sustainability 2020, 12, 9258. https://doi.org/10.3390/su12219258 40. Vercelli, M.; Croce, L.; Mancuso, T. An economic approach to assess the annual stock in beekeeping farms: The honey bee colony inventory tool. Sustainability 2020, 12, 9258. https://doi.org/10.3390/su12219258 Vercelli, M.; Croce, L.; Mancuso, T. An economic approach to assess the annual stock in beekeeping colony inventory tool. Sustainability 2020, 12, 9258. https://doi.org/10.3390/su12219258 y y y p g 41. Tlak Gajger, I.; Sušec, P. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 doi:10.20944/preprints202107 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 doi:10.20944/preprints202107.0362.v2 14 of 15 14 of 15 23. Damiani, N.; Gende, L. B.; Bailac, P.; Marcangeli, J.A.; Eguaras, M. J. Acaricidal and insecticidal activity of essential oils on Varroa destructor (Acari: Varroidae) and Apis mellifera (Hymenoptera: Apidae). Parasitol Res 2009, 106, 145-152. https://doi.org/10.1007/s00436-009-1639-y p g y 24. Ghasemi, V.; Moharramipour, S.; Tahmasbi, G. Biological activity of some plant essential oils against Varroa destructor (Acari: Varroidae), an ectoparasitic mite of Apis mellifera (Hymenoptera: Apidae). Exp App Acarol 2011, 55, 147-154. https://doi.org/10.1007/s10493-011-9457-1 p g 25. Sabahi, Q.; Morfin, N.; Emsen, B.; Gashout, H.A.; Kelly, P.G.; Otto, S.; Rod Merrill, A.; Guzman-Novoa, E. Evaluation of dry and wet formulations of oxalic acid, thymol, and oregano oil for Varroa mite (Acari: Varroidae) control in honey bee (Hyme- noptera: Apidae) Colonies. J Econ Entomol, 2020, 113, 2588-2594. https://doi.org/10.1093/jee/toaa218 p p p g j 26. Giacomelli, A.; Pietropaoli, M.; Carvelli, A.; Iacoponi, F.; Formato, G. Combination of thymol treatment (Apiguard®) and cag- ing the queen technique to fight Varroa destructor. Apidologie 2016, 47, 606-616. https://doi.org/10.1007/s13592-015-0408-4 27. COLOSS. Available online: https://coloss.org/varroa-control/ p g 28. EMA, 2008. European Medicines Agency, Committee for Medicinal Products for Veterinary use. Guidelines on veterinary medicinal products controlling Varroa destructor parasitosis in bees. EMA/CVMP/459883/2008. https://www.ema.europa.eu/en/veterinary-medicinal-products-controlling-varroa-destructor-parasitosis-bees (accessed on 19 June 2021) 29. Delaplane, K.S.; van der Steen, J.; Guzman-Novoa, E. Standard methods for estimating strength parameters of Apis mellifera colonies. J Apicult Res 2013, 52, 1-12. https://doi.org/10.3896/IBRA.1.52.1.03 30. Pietropaoli, M.; Giacomelli, A.; Macrì, S.; Volterrani, A.; Pizzariello M.; Formato, G. Considerazioni sui risultati nel Centro Italia dell'impiego di acido formico in gel (MAQS™), nella lotta alla varroa, in condizioni di presenza di covata e di melario. Apimondia Italia 2012, 1/2, 20-23. p 31. Dietemann, V.; Nazzi, F.; Martin, S.J.; Anderson, D.L.; Locke, B.; Delaplane, K.S.; Wauquiez, Q.; Tannahill, C.; Frey, E.; Ziegelmann, B.; Rosenkranz, P.; Ellis, J.D. Standard methods for varroa research. J Apicul Res 2013, 52, 1-54. https://doi.org/10.3896/IBRA.1.52.1.09 p g 32. Addinsoft, S.A.R.L. 2010. XLSTAT-software user’s manual, version 10. Paris: Addinsoft. p g 32. Addinsoft, S.A.R.L. 2010. XLSTAT-software user’s manual, version 10. Paris: Addinsoft. p g 32. Addinsoft, S.A.R.L. 2010. XLSTAT-soft Whitney, D.R. On a test of whether one of two random variables is stochastically larger than the other. A ey, D.R. On a test of whether one of two random variables is stochastically larger than the other. References Optimierung der Anwendung von ’Apilife VAR’ zur Bekämpfung von Varroa jacobsoni Oud. in Bienenvölkern, Apidologie 1994, 25, 49-60. https://doi.org/10.1051/apido:19940106 20. Imdorf, A.; Bogdanov, S.; Ibáñez Ochoa, R.; Calderone, N.W. Use of essential oils for the control of Varroa jacobsoni Oud. in honey bee colonies. Apidologie 1999, 30, 209-228. https://doi.org/10.1051/apido:19990210 20. Imdorf, A.; Bogdanov, S.; Ibáñez Ochoa, R.; Calderone, N.W. Use of essential oils for th honey bee colonies. Apidologie 1999, 30, 209-228. https://doi.org/10.1051/apido:19990210 21. Floris, I.; Satta, A.; Cabras, P.; Garau, V.L.; Angioni, A. Comparison between two thymol formulations in the control of Varroa destructor: effectiveness, persistence, and residues. J Econ Entomol 2004, 97, 187-191. https://doi.org/10.1603/0022-0493-97.2.187 21. Floris, I.; Satta, A.; Cabras, P.; Garau, V.L.; Angioni, A. Comparison between two thymol formulations in the control of Varroa destructor: effectiveness, persistence, and residues. J Econ Entomol 2004, 97, 187-191. https://doi.org/10.1603/0022-0493-97.2.187 22. Marinelli, E.; De Santis, L.; De Pace, F.M.; Dell’Aira, E.; Saccares, S.; Nisi, S.; Formato, G. Impiego del timolo e dell’acido for- mico per il controllo della varroatosi nel Lazio [Use of thymol and formic acid to control varroatosis in Latium region]. Apitalia 2007, 1, 1-4. 22. Marinelli, E.; De Santis, L.; De Pace, F.M.; Dell’Aira, E.; Saccares, S.; Nisi, S.; Formato, G. Impiego del timolo e dell’acido for- mico per il controllo della varroatosi nel Lazio [Use of thymol and formic acid to control varroatosis in Latium region]. Apitalia 2007, 1, 1-4. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 doi:10.20944/preprints202107 Efficacy of varroacidal food additive appliance during summer treatment of honeybee colonies (Apis mellifera). Veterinarski arhiv 2019, 89, 87-96. https://doi.org/10.24099/vet.arhiv.0441 y y y p g 41. Tlak Gajger, I.; Sušec, P. Efficacy of varroacidal food additive appliance during summer treatment of honeybee colonies (Apis mellifera). Veterinarski arhiv 2019, 89, 87-96. https://doi.org/10.24099/vet.arhiv.0441 f ) p g 42. EMA, 2017. European Medicines Agency, EPAR summary for the public. Available online: https://www.ema.europa.eu/ (accessed on 19 June 2021). f p g 42. EMA, 2017. European Medicines Agency, EPAR summary for the public. Available online: https://www.ema.europa.eu/ (accessed on 19 June 2021). 43. Higes, M.; Meana, A.; Suárez, M.; Llorente, J. Negative long-term effects on bee colonies treated with o jacobsoni Oud. Apidologie 1999, 30, 289-292. https://doi.org/10.1051/apido:19990404 43. Higes, M.; Meana, A.; Suárez, M.; Llorente, J. Negative long-term effects on bee colonies treated w jacobsoni Oud. Apidologie 1999, 30, 289-292. https://doi.org/10.1051/apido:19990404 44. Martin-Hernandez, R.; Higes, M.; Perez, J.L.; Nozal, M.J.; Gomez, L.; Meana, A. Short term negative effect of oxalic acid in Apis mellifera iberiensis. Span J Agric Res 2007, 5, 474-480. https://doi.org/10.5424/sjar/2007054-270 44. Martin-Hernandez, R.; Higes, M.; Perez, J.L.; Nozal, M.J.; Gomez, L.; Meana, A. Short term negative effect of oxalic acid in Apis mellifera iberiensis. Span J Agric Res 2007, 5, 474-480. https://doi.org/10.5424/sjar/2007054-270 f p J g p g j 45. Gregorc, A. & Škerl, M.I. Toxicological and immunohistochemical testing of honeybees after oxalic acid and rotenone treat- ments. Apidologie 2007, 38(3), 296-305. https://doi.org/10.1051/apido:2007014 f p g p g j 45. Gregorc, A. & Škerl, M.I. Toxicological and immunohistochemical testing of honeybees after oxalic acid and rotenone treat- ments. Apidologie 2007, 38(3), 296-305. https://doi.org/10.1051/apido:2007014 f p g p g j 45. Gregorc, A. & Škerl, M.I. Toxicological and immunohistochemical testing of ments. Apidologie 2007, 38(3), 296-305. https://doi.org/10.1051/apido:2007014 f p g p g j 45. Gregorc, A. & Škerl, M.I. Toxicological and immunohistochemical testing o ments. Apidologie 2007, 38(3), 296-305. https://doi.org/10.1051/apido:2007014 p g p g p 46. Keyhani, J.; Keyhani, E. EPR study of the effect of formate on cytochrome c oxidase (1980). Biochem Biophys Res Commun 1980, 92, 327-333. https://doi.org/10.1016/0006-291X(80)91556-9 46. Keyhani, J.; Keyhani, E. EPR study of the effect of formate on cytochrome c oxidase (1980). Biochem Biophys Res Commun 1980, 92, 327-333. https://doi.org/10.1016/0006-291X(80)91556-9 47. Song, C.; Scharf, M.E. Formic acid: A neurologically active, hydrolyzed metabolite of insecticidal formate esters. Pestic Biochem Physiol 2008, 92, 77-82. https://doi:10.1016/j.pestbp.2008.06.005 47. Song, C.; Scharf, M.E. pp p g pp 52. Rivera-Gomis, J.; Bubnič, J.; Ribarits, A.; Moosbeckhofer, R.; Alber, O.; Kozmus, P.; Jannoni-Sebastianini, R.; Haefeker, W.; Köglberger, H.; Smodiš Škerl, M.I.; Tiozzo, B.; Pietropaoli, M.; Lubroth, J.; Raizman, E.; Lietaer, C.; Zilli, R.; Eggenhoeffner, R.; Higes, M.; Muz, M.N.; D'Ascenzi, C.; Riviere, M.P.; Gregorc, A.; Cazier, J.; Hassler, E; Wilkes, J.; Formato, G. Good farming practices in apiculture. Rev Sci Tech 2019, 38(3), 879-890. https://doi.org/10.20506/rst.38.3.3032 49. Fries, I. Treatment of sealed honey bee brood with formic acid for control of Varroa jacobsoni. Am Bee J 1991, 131, 313-314. 51. Pietropaoli, M.; Tlak Gajger, I.; Costa, C.; Gerula, D.; Wilde, J.; Adjlane, N.; Aldea-Sánchez, P.; Smodiš Škerl, M.; Bubnič, J.; Formato, G. Evaluation of Two Commonly Used Field Tests to Assess Varroa destructor Infestation on Honey Bee (Apis mel- lifera) Colonies. Appl Sci 2021, 11(10), 4458. https://doi.org/10.3390/app11104458 y j J 50. Johnson, R.M. ; Ellis, M.D. ; Mullin, C.A.; Frazier, M. Pesticides and honey bee toxicity USA. Apid https://doi.org/10.1051/apido/2010018 49. Fries, I. Treatment of sealed honey bee brood with formic acid for control of Varroa jacobsoni. A s, M.D. ; Mullin, C.A.; Frazier, M. Pesticides and honey bee toxicity USA. Apidologie 2010, 41, 312-331 051/apido/2010018 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 30 August 2021 doi:10.20944/preprints202107 Formic acid: A neurologically active, hy Physiol 2008, 92, 77-82. https://doi:10.1016/j.pestbp.2008.06.005 47. Song, C.; Scharf, M.E. Formic acid: A neurologically active, hydrolyzed metabolite of insecticidal formate esters. Pestic Biochem Physiol 2008, 92, 77-82. https://doi:10.1016/j.pestbp.2008.06.005 y p j p p 48. Underwood, R.M. ; Currie, R. The effects of temperature and dose of formic acid on treatment efficacy against Varroa destructor (Acari: Varroidae), a parasite of Apis mellifera (Hymenoptera: Apidae). Exp App Acarol 2003, 29, 303-313. https://doi.org/10.1023/A:1025892906393 y p j p p 48. Underwood, R.M. ; Currie, R. The effects of temperature and dose of formic acid on treatment efficacy against Varroa destructor (Acari: Varroidae), a parasite of Apis mellifera (Hymenoptera: Apidae). 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https://openalex.org/W2919331777	https://www.nature.com/articles/s41598-019-40386-z.pdf	English	null	Pathophysiological background and prognostic implication of systolic aortic root motion in non-ischemic dilated cardiomyopathy	Scientific reports	2,019	cc-by	9,519	Pathophysiological background and prognostic implication of systolic aortic root motion in non- ischemic dilated cardiomyopathy 2- and 3-dimensional sys- tems have improved its diagnostic potential continuously6,7 but they still face limitations especially when deal- ing with poor acoustic windows. In such cases M-mode echocardiography is a helpful alternative. Due to its high temporal resolution movement of echogenic structures can easily be visualized even when image quality is reduced8. The first description of moving ultrasound signals using M-mode echocardiography dates back to the early fifties when Edler assumed these patterns to originate from the anterior left atrial wall9. By contrast enhanced echocardiography using saline injection in the supravalvular position Gramiak et al. confirmed that undulating parallel signals medial to the mitral valve actually arise distal from the aortic valve and thus represent a portion of the aorta. Furthermore, they could demonstrate, that the pattern of motion obtained from the aortic root equals 1Department of Internal Medicine III, Cardiology, Angiology and Pneumology, University of Heidelberg, Heidelberg, Germany. 2Institute of Medical Biometry and Informatics, University of Heidelberg, Heidelberg, Germany. Correspondence and requests for materials should be addressed to M.A. (email: matthias.aurich@med.uni- heidelberg.de) Recordings of aortic root movement represent one of the first accomplishments of ultrasound in medicine and mark the beginning of functional cardiac imaging. However, the underlying mechanism is not completely understood. Since the aortic root is directly connected to the cardiac skeleton we hypothesize, that the amplitude of systolic aortic root motion (SARM) may be mainly caused by displacement of the cardiac base towards the apex and might therefore be used as measure of left ventricular longitudinal function (LV-LF). One hundred and eighty patients with dilated cardiomyopathy and 180 healthy controls were prospectively included into this study. SARM was lower in patients compared to controls (9 ± 3 mm vs. 12 ± 2 mm, p < 0.001) and lowest in patients with cardiovascular events (9 ± 3 mm vs. 7 ± 3 mm, p < 0.001). During a median follow-up time of 38 months, the combined end-point of cardiovascular death or hospitalization for heart failure was reached by 25 patients (13.9%). Reduced SARM had significant prognostic impact on outcome (hazard ratio 0.74, 95% confidence interval 0.63–0.88, p < 0.001) and remained an independent predictor in the multivariate analysis. Compared to parameters with potential influence on its mechanism, SARM correlated best (r = 0.75, p < 0.001) with global longitudinal strain (GLS). www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 21 November 2018 Accepted: 13 February 2019 Published: xx xx xxxx Pathophysiological background and prognostic implication of systolic aortic root motion in non- ischemic dilated cardiomyopathy Received: 21 November 2018 Accepted: 13 February 2019 Published: xx xx xxxx Matthias Aurich 1, Matthias Niemers1, Patrick Fuchs1, Sebastian Greiner1, Matthias Müller- Hennessen1, Lorenz Uhlmann2, Evangelos Giannitsis1, Philipp Ehlermann1, Benjamin Meder1, Hugo A. Katus1 & Derliz Mereles 1 1 Scientific Reports \| (2019) 9:3866 \| https://doi.org/10.1038/s41598-019-40386-z Hugo A. Katus & Derliz Mereles Recordings of aortic root movement represent one of the first accomplishments of ultrasound in medicine and mark the beginning of functional cardiac imaging. However, the underlying mechanism is not completely understood. Since the aortic root is directly connected to the cardiac skeleton we hypothesize, that the amplitude of systolic aortic root motion (SARM) may be mainly caused by displacement of the cardiac base towards the apex and might therefore be used as measure of left ventricular longitudinal function (LV-LF). One hundred and eighty patients with dilated cardiomyopathy and 180 healthy controls were prospectively included into this study. SARM was lower in patients compared to controls (9 ± 3 mm vs. 12 ± 2 mm, p < 0.001) and lowest in patients with cardiovascular events (9 ± 3 mm vs. 7 ± 3 mm, p < 0.001). During a median follow-up time of 38 months, the combined end-point of cardiovascular death or hospitalization for heart failure was reached by 25 patients (13.9%). Reduced SARM had significant prognostic impact on outcome (hazard ratio 0.74, 95% confidence interval 0.63–0.88, p < 0.001) and remained an independent predictor in the multivariate analysis. Compared to parameters with potential influence on its mechanism, SARM correlated best (r = 0.75, p < 0.001) with global longitudinal strain (GLS). SARM may therefore represent an alternative echocardiographic parameter for the assessment of LV-LF, particularly when GLS is not feasible or apical views are not available. Left ventricular (LV) contraction is determined by a complex arrangement of muscle fiber layers and comprises longitudinal shortening and axial twist. Impairment of the longitudinal component is often the first sign of LV dysfunction even when ejection fraction (EF) is still normal1,2. Beyond that diagnostic significance, LV longitu- dinal function (LF) has additive prognostic value when EF is already reduced3. Therefore, techniques that enable assessment of LV-LF are highly relevant and should nowadays complement every cardiac imaging report4,5. Echocardiography is by far the most widely used imaging modality in cardiology. Pathophysiological background and prognostic implication of systolic aortic root motion in non- ischemic dilated cardiomyopathy SARM may therefore represent an alternative echocardiographic parameter for the assessment of LV-LF, particularly when GLS is not feasible or apical views are not available. Left ventricular (LV) contraction is determined by a complex arrangement of muscle fiber layers and comprises longitudinal shortening and axial twist. Impairment of the longitudinal component is often the first sign of LV dysfunction even when ejection fraction (EF) is still normal1,2. Beyond that diagnostic significance, LV longitu- dinal function (LF) has additive prognostic value when EF is already reduced3. Therefore, techniques that enable assessment of LV-LF are highly relevant and should nowadays complement every cardiac imaging report4,5. Echocardiography is by far the most widely used imaging modality in cardiology. 2- and 3-dimensional sys- tems have improved its diagnostic potential continuously6,7 but they still face limitations especially when deal- ing with poor acoustic windows. In such cases M-mode echocardiography is a helpful alternative. Due to its high temporal resolution movement of echogenic structures can easily be visualized even when image quality is reduced8.hi The first description of moving ultrasound signals using M-mode echocardiography dates back to the early fifties when Edler assumed these patterns to originate from the anterior left atrial wall9. By contrast enhanced echocardiography using saline injection in the supravalvular position Gramiak et al. confirmed that undulating parallel signals medial to the mitral valve actually arise distal from the aortic valve and thus represent a portion of the aorta. Furthermore, they could demonstrate, that the pattern of motion obtained from the aortic root equals 1Department of Internal Medicine III, Cardiology, Angiology and Pneumology, University of Heidelberg, Heidelberg, Germany. 2Institute of Medical Biometry and Informatics, University of Heidelberg, Heidelberg, Germany. Correspondence and requests for materials should be addressed to M.A. (email: matthias.aurich@med.uni- heidelberg.de) Scientific Reports \| (2019) 9:3866 \| https://doi.org/10.1038/s41598-019-40386-z 1 www.nature.com/scientificreports/ Figure 1. Parasternal echocardiographic B-mode image at the level of the valvular plane. The M-mode beam (light green) is directed through the center of the aortic root (Ao). LA, left atrium; PA, pulmonary artery; PV, pulmonary valve; RA, right atrium; RVOT, right ventricular outflow tract; TV, tricuspid valve Figure 1. Parasternal echocardiographic B-mode image at the level of the valvular plane. The M-mode beam (light green) is directed through the center of the aortic root (Ao). LA, left atrium; PA, pulmonary artery; PV, pulmonary valve; RA, right atrium; RVOT, right ventricular outflow tract; TV, tricuspid valve Figure 2. that of earlier M-mode recordings from the mitral ring10,11. Aortic root motion has subsequently been investi- gated as a surrogate parameter of left ventricular systolic and diastolic function. As part of the cardiac skeleton the aortic annulus and the attached aortic root follow the valvular plane dis- placement during the heart cycle. Therefore, we hypothesize, that the amplitude of systolic aortic root motion (SARM) obtained by M-Mode echocardiography may be used as a measure of global LV-LF (Figs 1 and 2). that of earlier M-mode recordings from the mitral ring10,11. Aortic root motion has subsequently been investi- gated as a surrogate parameter of left ventricular systolic and diastolic function. As part of the cardiac skeleton the aortic annulus and the attached aortic root follow the valvular plane dis- placement during the heart cycle. Therefore, we hypothesize, that the amplitude of systolic aortic root motion (SARM) obtained by M-Mode echocardiography may be used as a measure of global LV-LF (Figs 1 and 2). www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports ficreports/ Parameter Patients (n = 180) Controls (n = 180) p-value Baseline Male gender, n (%) 139 (77) 139 (77) 1 Age, years 56 (48;65) 58 (50;67) 0.191 BSA, m2 2.0 ± 0.2 1.9 ± 0.2 <0.001 BMI, kg/m2 27 (24;30) 25 (23;27) <0.001 Heart rate, min−1 73 ± 18 62 ± 9 <0.001 BP systolic, mmHg 121 ± 18 136 ± 15 <0.001 BP diastolic, mmHg 75 ± 11 86 ± 9 <0.001 MAP, mmHg 91 ± 13 102 ± 10 <0.001 Clinical chemistry NT-proBNP, ng/L 489 (108;1,339) 55(30;97) <0.001 hs-TNT, pg/mL 11 (6;24) 5 (4;7) <0.001 Echocardiography IVS, mm 9 (8;10) 10 (9;11) 0.011 PW, mm 8 (7;9) 8 (7;9) 0.656 EDD, mm 57 ± 9 48 ± 4 <0.001 ESD, mm 45 (38;54) 34 (31;37) <0.001 LV mass/BSA, g/m2 187 ± 64 140 ± 30 <0.001 EDV, mL 152 (117;209) 116 (93;137) <0.001 ESV, mL 93 (67;149) 48 (38;57) <0.001 EF, % 38 (26;44) 58 (56;61) <0.001 MAPSE, mm 12 (8;15) 16 (14;17) <0.001 MASV, cm/s 6 (5;9) 10 (8;11) <0.001 GLS, % −12.7 ± 4.8 −19.5 ± 1.7 <0.001 SARM, mm 9 ± 3 12 ± 2 <0.001 LA-Volume/BSA, mL/m2 37 (28;48) 26 (22;31) <0.001 LA-VC, % 46 (29;54) 58 (52;64) <0.001 E/A 1.0 (0.8;1.3) 1.1 (0.8;1.3) 0.577 E/e’ 7 (5;9) 6 (5;7) <0.001 E-DT, ms 197 (157;253) 209 (183;244) 0.239 SPVF/DPVF 1.1 ± 0.5 1.4 ± 0.4 0.005 Table 1. Characteristics of patients and healthy controls. BMI, body mass index; BP, blood pressure; BSA, body surface area; DPVF, diastolic pulmonary venous flow; E/A, ratio of mitral inflow velocity (E) to atrial contraction velocity (A); E/e’, ratio of mitral inflow velocity (E) to tissue Doppler mitral annular velocity (e’); E-DT, E-wave deceleration time; EDD, end-diastolic diameter; EDV, end-diastolic volume; EF, ejection fraction; ESD, end-systolic diameter; ESV, end-systolic volume; GLS, global longitudinal strain; IVS, interventricular septum; hs-TNT, high sensitive Troponin T; LA-VC, left atrial volume change; MAP, mean arterial pressure; MAPSE, mitral annular plane systolic excursion; MASV, mitral annular systolic velocity; NT-proBNP, N-terminal pro Brain natriuretic peptide; PW, posterior wall; SARM, systolic aortic root motion; SPVF, systolic pulmonary venous flow. www.nature.com/scientificreports/ BMI, body mass index; BP, blood pressure; BSA, body surface area; DPVF, diastolic pulmonary venous flow; E/A, ratio of mitral inflow velocity (E) to atrial contraction velocity (A); E/e’, ratio of mitral inflow velocity (E) to tissue Doppler mitral annular velocity (e’); E-DT, E-wave deceleration time; EDD, end-diastolic diameter; EDV, end-diastolic volume; EF, ejection fraction; ESD, end-systolic diameter; ESV, end-systolic volume; GLS, global longitudinal strain; IVS, interventricular septum; hs-TNT, high sensitive Troponin T; LA-VC, left atrial volume change; MAP, mean arterial pressure; MAPSE, mitral annular plane systolic excursion; MASV, mitral annular systolic velocity; NT-proBNP, N-terminal pro Brain natriuretic peptide; PW, posterior wall; SARM, systolic aortic root motion; SPVF, systolic pulmonary venous flow. Table 1. Characteristics of patients and healthy controls. BMI, body mass index; BP, blood pressure; BSA, body surface area; DPVF, diastolic pulmonary venous flow; E/A, ratio of mitral inflow velocity (E) to atrial contraction velocity (A); E/e’, ratio of mitral inflow velocity (E) to tissue Doppler mitral annular velocity (e’); E-DT, E-wave deceleration time; EDD, end-diastolic diameter; EDV, end-diastolic volume; EF, ejection fraction; ESD, end-systolic diameter; ESV, end-systolic volume; GLS, global longitudinal strain; IVS, interventricular septum; hs-TNT, high sensitive Troponin T; LA-VC, left atrial volume change; MAP, mean arterial pressure; MAPSE, mitral annular plane systolic excursion; MASV, mitral annular systolic velocity; NT-proBNP, N-terminal pro Brain natriuretic peptide; PW, posterior wall; SARM, systolic aortic root motion; SPVF, systolic pulmonary venous flow. Pathophysiological background and prognostic implication of systolic aortic root motion in non- ischemic dilated cardiomyopathy Left: Schematic representation of one cardiac cycle recorded by M-mode echocardiography at the level of the aortic root (Ao). Right: Two examples of SARM measurement in a healthy individual (B) and a patient with markedly depressed left ventricular longitudinal function (C). AW, anterior wall; LA, left atrium; PW, posterior wall; RVOT, right ventricular outflow tract Figure 2. Left: Schematic representation of one cardiac cycle recorded by M-mode echocardiography at the level of the aortic root (Ao). Right: Two examples of SARM measurement in a healthy individual (B) and a patient with markedly depressed left ventricular longitudinal function (C). AW, anterior wall; LA, left atrium; PW, posterior wall; RVOT, right ventricular outflow tract that of earlier M-mode recordings from the mitral ring10,11. Aortic root motion has subsequently been investi- gated as a surrogate parameter of left ventricular systolic and diastolic function. As part of the cardiac skeleton the aortic annulus and the attached aortic root follow the valvular plane dis- placement during the heart cycle. Therefore, we hypothesize, that the amplitude of systolic aortic root motion (SARM) obtained by M-Mode echocardiography may be used as a measure of global LV-LF (Figs 1 and 2). that of earlier M-mode recordings from the mitral ring10,11. Aortic root motion has subsequently been investi- gated as a surrogate parameter of left ventricular systolic and diastolic function. As part of the cardiac skeleton the aortic annulus and the attached aortic root follow the valvular plane dis- placement during the heart cycle. Therefore, we hypothesize, that the amplitude of systolic aortic root motion (SARM) obtained by M-Mode echocardiography may be used as a measure of global LV-LF (Figs 1 and 2). gt y As part of the cardiac skeleton the aortic annulus and the attached aortic root follow the valvular plane dis lacement during the heart cycle. Therefore, we hypothesize, that the amplitude of systolic aortic root motion SARM) obtained by M-Mode echocardiography may be used as a measure of global LV-LF (Figs 1 and 2). Scientific Reports \| (2019) 9:3866 \| https://doi.org/10.1038/s41598-019-40386-z 2 www.nature.com/scientificreports/ Parameter Patients (n = 180) Controls (n = 180) p-value Baseline Male gender, n (%) 139 (77) 139 (77) 1 Age, years 56 (48;65) 58 (50;67) 0.191 BSA, m2 2.0 ± 0.2 1.9 ± 0.2 <0.001 BMI, kg/m2 27 (24;30) 25 (23;27) <0.001 Heart rate, min−1 73 ± 18 62 ± 9 <0.001 BP systolic, mmHg 121 ± 18 136 ± 15 <0.001 BP diastolic, mmHg 75 ± 11 86 ± 9 <0.001 MAP, mmHg 91 ± 13 102 ± 10 <0.001 Clinical chemistry NT-proBNP, ng/L 489 (108;1,339) 55(30;97) <0.001 hs-TNT, pg/mL 11 (6;24) 5 (4;7) <0.001 Echocardiography IVS, mm 9 (8;10) 10 (9;11) 0.011 PW, mm 8 (7;9) 8 (7;9) 0.656 EDD, mm 57 ± 9 48 ± 4 <0.001 ESD, mm 45 (38;54) 34 (31;37) <0.001 LV mass/BSA, g/m2 187 ± 64 140 ± 30 <0.001 EDV, mL 152 (117;209) 116 (93;137) <0.001 ESV, mL 93 (67;149) 48 (38;57) <0.001 EF, % 38 (26;44) 58 (56;61) <0.001 MAPSE, mm 12 (8;15) 16 (14;17) <0.001 MASV, cm/s 6 (5;9) 10 (8;11) <0.001 GLS, % −12.7 ± 4.8 −19.5 ± 1.7 <0.001 SARM, mm 9 ± 3 12 ± 2 <0.001 LA-Volume/BSA, mL/m2 37 (28;48) 26 (22;31) <0.001 LA-VC, % 46 (29;54) 58 (52;64) <0.001 E/A 1.0 (0.8;1.3) 1.1 (0.8;1.3) 0.577 E/e’ 7 (5;9) 6 (5;7) <0.001 E-DT, ms 197 (157;253) 209 (183;244) 0.239 SPVF/DPVF 1.1 ± 0.5 1.4 ± 0.4 0.005 Table 1. Characteristics of patients and healthy controls. BMI, body mass index; BP, blood pressure; BSA, body surface area; DPVF, diastolic pulmonary venous flow; E/A, ratio of mitral inflow velocity (E) to atrial contraction velocity (A); E/e’, ratio of mitral inflow velocity (E) to tissue Doppler mitral annular velocity (e’); E-DT, E-wave deceleration time; EDD, end-diastolic diameter; EDV, end-diastolic volume; EF, ejection fraction; ESD, end-systolic diameter; ESV, end-systolic volume; GLS, global longitudinal strain; IVS, interventricular septum; hs-TNT, high sensitive Troponin T; LA-VC, left atrial volume change; MAP, mean arterial pressure; MAPSE, mitral annular plane systolic excursion; MASV, mitral annular systolic velocity; NT-proBNP, N-terminal pro Brain natriuretic peptide; PW, posterior wall; SARM, systolic aortic root motion; SPVF, systolic pulmonary venous flow. Table 1. Characteristics of patients and healthy controls. www.nature.com/scientificreports/ Characteristics of patients stratified according to an event or no event. BMI, body mass index; BP, blood pressure; BSA, body surface area; DPVF, diastolic pulmonary venous flow; E/A, ratio of mitral inflow velocity (E) to atrial contraction velocity (A); E/e’, ratio of mitral inflow velocity (E) to tissue Doppler mitral annular velocity (e’); E-DT, E-wave deceleration time; EDD, end-diastolic diameter; EDV, end-diastolic volume; EF, ejection fraction; ESD, end-systolic diameter; ESV, end-systolic volume; GLS, global longitudinal strain; IVS, interventricular septum; hs-TNT, high sensitive Troponin T; LA-VC, left atrial volume change; LVEDP, left ventricular end-diastolic pressure; MAP, mean atrial pressure; MAPSE, mitral annular plane systolic excursion; MASV, mitral annular systolic velocity; NT-proBNP, N-terminal pro Brain natriuretic peptide; PW, posterior wall; SPVF, systolic pulmonary venous flow. Table 2. Characteristics of patients stratified according to an event or no event. BMI, body mass index; BP, blood pressure; BSA, body surface area; DPVF, diastolic pulmonary venous flow; E/A, ratio of mitral inflow velocity (E) to atrial contraction velocity (A); E/e’, ratio of mitral inflow velocity (E) to tissue Doppler mitral annular velocity (e’); E-DT, E-wave deceleration time; EDD, end-diastolic diameter; EDV, end-diastolic volume; EF, ejection fraction; ESD, end-systolic diameter; ESV, end-systolic volume; GLS, global longitudinal strain; IVS, interventricular septum; hs-TNT, high sensitive Troponin T; LA-VC, left atrial volume change; LVEDP, left ventricular end-diastolic pressure; MAP, mean atrial pressure; MAPSE, mitral annular plane systolic excursion; MASV, mitral annular systolic velocity; NT-proBNP, N-terminal pro Brain natriuretic peptide; PW, posterior wall; SPVF, systolic pulmonary venous flow. Table 2. Characteristics of patients stratified according to an event or no event. BMI, body mass index; BP, blood pressure; BSA, body surface area; DPVF, diastolic pulmonary venous flow; E/A, ratio of mitral inflow velocity (E) to atrial contraction velocity (A); E/e’, ratio of mitral inflow velocity (E) to tissue Doppler mitral annular velocity (e’); E-DT, E-wave deceleration time; EDD, end-diastolic diameter; EDV, end-diastolic volume; EF, ejection fraction; ESD, end-systolic diameter; ESV, end-systolic volume; GLS, global longitudinal strain; IVS, interventricular septum; hs-TNT, high sensitive Troponin T; LA-VC, left atrial volume change; LVEDP, left ventricular end-diastolic pressure; MAP, mean atrial pressure; MAPSE, mitral annular plane systolic excursion; MASV, mitral annular systolic velocity; NT-proBNP, N-terminal pro Brain natriuretic peptide; PW, posterior wall; SPVF, systolic pulmonary venous flow. Youden’s index and yielded a cutoff value of 11 mm (area under the curve [AUC] = 0.85, 95% confidence interval [CI] 0.82–0.89). Results Ch Characteristics of the study population. One hundred and eighty patients with dilated cardiomyopathy were matched by age and gender with 180 healthy control subjects. Median follow-up time was 1,150 days (38 months), 4 patients were lost to follow-up. Clinical, laboratory, and echocardiographic parameters are summa- rized in Table 1 and Table 2. Male subjects predominated in this study (n = 278 [77%]). Determining factors of SARM. A potential association between different hemodynamic as well as func- tional cardiac parameters and SARM was tested by linear regression analysis. Results of correlation between SARM and global longitudinal strain (GLS), EF, LV stroke volume (SV), left atrial volume change (LA-VC) and mean arterial pressure (MAP) are presented in Table 3, Fig. 3 and Supplementary Fig. S1. Best correlations were found for SARM and GLS (r = 0.75 and 0.78, respectively, Fig. 3A,B) as well as SARM and EF (r = 0.74, Supplementary Fig. S1A). Weaker associations were found to SV and LA-VC (r = 0.57 and 0.61, respectively, Supplementary Fig. S1B,C) and no correlation to MAP (r = 0.21, Supplementary Fig. S1D). SARM in patients and healthy controls. Total excursion of SARM was lower in the patient cohort com- pared with healthy individuals (9 ± 3 mm vs. 12 ± 2 mm, p < 0.001) and was even stronger diminished in patients with compared to patients without an event (7 ± 3 mm vs. 9 ± 3 mm, p < 0.001). The ability of SARM to differen- tiate patients with DCM from healthy subjects was tested by Receiver operating characteristic (ROC) analysis and Scientific Reports \| (2019) 9:3866 \| https://doi.org/10.1038/s41598-019-40386-z 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Parameter No event (n = 155) Event (n = 25) p-value Baseline Male gender, n (%) 121 (78) 18 (72) 0.502 Age, years 55 ± 14 56 ± 15 0.940 BSA, m2 2.0 ± 0.2 1.9 ± 0.3 0.077 BMI, kg/m2 27 (24;30) 25 (27;29) 0.095 Heart rate, min−1 71 ± 19 79 ± 16 0.023 BP systolic, mmHg 122 ± 19 120 ± 15 0.748 BP diastolic, mmHg 75 ± 11 76 ± 10 0.758 MAP, mmHg 91 ± 13 91 ± 10 0.975 NYHA > II, n 19 (13) 7 (29) 0.040 Clinical chemistry NT-proBNP, ng/L 432 (89;1,164) 1,293 (662;3,934) <0.001 hs-TNT, pg/mL 10 (6;22) 14 (8;48) 0.020 Comorbidities Hypertension, n (%) 80 (52) 15 (63) 0.351 Dyslipidemia, n (%) 41 (27) 8 (33) 0.506 Diabetes, n (%) 27 (18) 8 (33) 0.072 Renal dysfunction, n (%) 83 (58) 12 (52) 0.623 Heart catheterization LVEDP, mmHg 16 (12;24) 23 (13;31) 0.020 Echocardiography IVS, mm 9 ± 2 9 ± 2 0.971 PW, mm 8 (7;9) 7 (6;9) 0.365 EDD, mm 56 ± 8 60 ± 9 0.035 ESD, mm 45 ± 11 53 ± 9 0.004 LV mass/BSA, g/m2 88 (73;105) 101 (90;113) 0.012 EDV, mL 145 (116;203) 199 (153;267) 0.007 ESV, mL 88 (63;135) 147 (91;204) <0.001 EF, % 39 (27;45) 24 (16;36) <0.001 MAPSE, mm 12 (9;15) 8 (7;11) <0.001 MASV, cm/s 7 (5;10) 5 (4;7) <0.001 GLS, % −13.2 ± 4.7 −9.4 ± 3.8 <0.001 SARM, mm 9 ± 3 7 ± 3 <0.001 LA-Volume/BSA, mL/m2 35 (27;48) 42 (35;48) 0.014 LA-VC, % 48 (32;55) 32 (21;45) 0.014 E/A 1.0 (0.8;1.2) 1.1 (0.8;2.4) 0.421 E/e’ 7 (5;9) 10 (8;11) <0.001 E-DT, ms 217 ± 71 169 ± 53 0.002 SPVF/DPVF 1.2 ± 0.5 1.0 ± 0.5 0.060 T bl 2 Ch i i f i ifi d di BMI b BSA, m2 2.0 ± 0.2 1.9 ± 0.3 0.077 BMI, kg/m2 27 (24;30) 25 (27;29) 0.095 Heart rate, min−1 71 ± 19 79 ± 16 0.023 BP systolic, mmHg 122 ± 19 120 ± 15 0.748 BP diastolic, mmHg 75 ± 11 76 ± 10 0.758 MAP, mmHg 91 ± 13 91 ± 10 0.975 NYHA > II, n 19 (13) 7 (29) 0.040 Clinical chemistry NT-proBNP, ng/L 432 (89;1,164) 1,293 (662;3,934) <0.001 hs-TNT, pg/mL 10 (6;22) 14 (8;48) 0.020 Comorbidities Hypertension, n (%) 80 (52) 15 (63) 0.351 Dyslipidemia, n (%) 41 (27) 8 (33) 0.506 Diabetes, n (%) 27 (18) 8 (33) 0.072 Renal dysfunction, n (%) 83 (58) 12 (52) 0.623 Heart catheterization LVEDP, mmHg 16 (12;24) 23 (13;31) 0.020 Echocardiography IVS, mm 9 ± 2 9 ± 2 0.971 PW, mm 8 (7;9) 7 (6;9) 0.365 EDD, mm 56 ± 8 60 ± 9 0.035 ESD, mm 45 ± 11 53 ± 9 0.004 LV mass/BSA, g/m2 88 (73;105) 101 (90;113) 0.012 EDV, mL 145 (116;203) 199 (153;267) 0.007 ESV, mL 88 (63;135) 147 (91;204) <0.001 EF, % 39 (27;45) 24 (16;36) <0.001 MAPSE, mm 12 (9;15) 8 (7;11) <0.001 MASV, cm/s 7 (5;10) 5 (4;7) <0.001 GLS, % −13.2 ± 4.7 −9.4 ± 3.8 <0.001 SARM, mm 9 ± 3 7 ± 3 <0.001 LA-Volume/BSA, mL/m2 35 (27;48) 42 (35;48) 0.014 LA-VC, % 48 (32;55) 32 (21;45) 0.014 E/A 1.0 (0.8;1.2) 1.1 (0.8;2.4) 0.421 E/e’ 7 (5;9) 10 (8;11) <0.001 E-DT, ms 217 ± 71 169 ± 53 0.002 SPVF/DPVF 1.2 ± 0.5 1.0 ± 0.5 0.060 Table 2. www.nature.com/scientificreports/ The combined end point of cardiovascular death or hospitalization for heart failure was observed in 25 patients, including 22 patients with acute heart failure with need for hospital admission and 3 cases of cardio- vascular death. An optimal cutoff value for SARM to discriminate patients at risk for a cardiac event was found to be <7 mm calculated by ROC analysis and Youden’s index (AUC = 0.72, 95% CI 0.61–0.83). The frequency of cardiac events over time is displayed by Kaplan-Meier curves (Fig. 4), which were compared using the Log-rank test. Results of univariate Cox regression analysis are shown in Table 4. N-terminal pro–brain natriuretic peptide (NT-proBNP), New York Heart Association (NYHA) functional classes III and IV and Diabetes as clinical as well as LV end-diastolic pressure assessed by left heart catheterization as invasively determined parameter were Scientific Reports \| (2019) 9:3866 \| https://doi.org/10.1038/s41598-019-40386-z 4 www.nature.com/scientificreports/ Parameter Equation r SEE p-value SARM - GLS f(x) = −0.5x + 3.3 0.75 1.93 <0.001 SARM/BSA - GLS f(x) = −0.3x + 1.4 0.78 0.99 <0.001 SARM - EF f(x) = 0.2x + 3.5 0.74 2.01 <0.001 SARM - SV f(x) = 0.1x + 5.0 0.57 2.44 <0.001 SARM - LA-VC f(x) = 0.1x + 4.3 0.61 2.38 <0.001 SARM - MAP f(x) = 0.1x + 6.0 0.21 2.81 <0.001 Table 3. Linear regression analysis. BSA, body surface area; EF, ejection fraction; GLS, global longitudinal strain; LA-VC, left atrial volume change; MAP, mean arterial pressure; SARM, systolic aortic root motion; SEE, standard error of estimate; SV, stroke volume. Parameter Equation r SEE p-value SARM - GLS f(x) = −0.5x + 3.3 0.75 1.93 <0.001 SARM/BSA - GLS f(x) = −0.3x + 1.4 0.78 0.99 <0.001 SARM - EF f(x) = 0.2x + 3.5 0.74 2.01 <0.001 SARM - SV f(x) = 0.1x + 5.0 0.57 2.44 <0.001 SARM - LA-VC f(x) = 0.1x + 4.3 0.61 2.38 <0.001 SARM - MAP f(x) = 0.1x + 6.0 0.21 2.81 <0.001 Table 3. Linear regression analysis. BSA, body surface area; EF, ejection fraction; GLS, global longitudinal strain; LA-VC, left atrial volume change; MAP, mean arterial pressure; SARM, systolic aortic root motion; SEE, standard error of estimate; SV, stroke volume. Table 3. Linear regression analysis. www.nature.com/scientificreports/ BMI, body mass index; BP, blood pressure; BSA, body surface area; DPVF, diastolic pulmonary venous flow; E/A, ratio of mitral inflow velocity (E) to atrial contraction velocity (A); E/e’, ratio of mitral inflow velocity (E) to tissue Doppler mitral annular velocity (e’); E-DT, E-wave deceleration time; EDD, end-diastolic diameter; EDV, end-diastolic volume; EF, ejection fraction; ESD, end- systolic diameter; ESV, end-systolic volume; GLS, global longitudinal strain; IVS, interventricular septum; LA- VC, left atrial volume change; LVEDP, left ventricular end-diastolic pressure; hs-TnT, high sensitive Troponin T; MAP, mean arterial pressure; MAPSE, mitral annular plane systolic excursion; MASV, mitral annular systolic velocity; NTproBNP, N-terminal pro Brain natriuretic peptide; NYHA, New York Heart Association Functional Classification; PW, posterior wall; SARM, systolic aortic root motion; SPVF, systolic pulmonary venous flow. Table 4. Univariate Cox regression analysis. BMI, body mass index; BP, blood pressure; BSA, body surface area; DPVF, diastolic pulmonary venous flow; E/A, ratio of mitral inflow velocity (E) to atrial contraction velocity (A); E/e’, ratio of mitral inflow velocity (E) to tissue Doppler mitral annular velocity (e’); E-DT, E-wave deceleration time; EDD, end-diastolic diameter; EDV, end-diastolic volume; EF, ejection fraction; ESD, end- systolic diameter; ESV, end-systolic volume; GLS, global longitudinal strain; IVS, interventricular septum; LA- VC, left atrial volume change; LVEDP, left ventricular end-diastolic pressure; hs-TnT, high sensitive Troponin T; MAP, mean arterial pressure; MAPSE, mitral annular plane systolic excursion; MASV, mitral annular systolic velocity; NTproBNP, N-terminal pro Brain natriuretic peptide; NYHA, New York Heart Association Functional Classification; PW, posterior wall; SARM, systolic aortic root motion; SPVF, systolic pulmonary venous flow. GLS, MAPSE and MASV (model 2, Table 5) and the second including EF and GLS as parameters of systolic LV function (model 3, Table 5). In the clinical model SARM remained independently predictive regarding cardiac death and hospitalization, whereas none of the longitudinal or systolic function parameters in both echocardio- graphic models was independently predictive.fi GLS, MAPSE and MASV (model 2, Table 5) and the second including EF and GLS as parameters of systolic LV function (model 3, Table 5). In the clinical model SARM remained independently predictive regarding cardiac death and hospitalization, whereas none of the longitudinal or systolic function parameters in both echocardio- graphic models was independently predictive.fi g p p y p Reproducibility analysis revealed coefficients of variation of 5.8 for intra- and 7.6 for interobserver variability. Discussion www.nature.com/scientificreports/ BSA, body surface area; EF, ejection fraction; GLS, global longitudinal strain; LA-VC, left atrial volume change; MAP, mean arterial pressure; SARM, systolic aortic root motion; SEE, standard error of estimate; SV, stroke volume. Figure 3. Correlations between systolic aortic root motion (SARM) and global longitudinal Strain (A) and SARM adjusted for body surface area (BAS) and GLS (B). SEE, standard error of estimate. Figure 3. Correlations between systolic aortic root motion (SARM) and global longitudinal Strain (A) and SARM adjusted for body surface area (BAS) and GLS (B). SEE, standard error of estimate. Figure 4. Kaplan-Meier curve displaying the frequency of cardiac events over time for a given cutoff value of systolic aortic root motion (SARM). Figure 4. Kaplan-Meier curve displaying the frequency of cardiac events over time for a given cutoff value of systolic aortic root motion (SARM). associated with the occurrence of adverse events (p < 0.05 each). Among echocardiographic parameters EF and SARM had the highest impact on patient outcomes (p < 0.001 each). Based on the univariate Cox regression SARM was entered into a clinical Model including NT-proBNP, NYHA functional class III and IV and Diabetes (model 1, Table 5) and 2 echocardiographic models, the first consisting of LV longitudinal function parameters Scientific Reports \| (2019) 9:3866 \| https://doi.org/10.1038/s41598-019-40386-z 5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Parameter HR CI p-value Baseline Male gender 1.234 0.509–2.990 0.641 Age, years 0.999 0.971–1.028 0.948 BSA, m2 0.270 0.047–1.561 0.143 BMI, kg m2 0.912 0.821–1.013 0.085 Heart rate, min−1 1.015 0.996–1.034 0.133 BP systolic, mmHg 0.993 0.970–1.017 0.561 BP diastolic, mmHg 1.009 0.971–1.050 0.638 MAP, mmHg 1.000 0.966–1.035 0.996 NYHA > II 2.694 1.092–6.644 0.031 Clinical chemistry NT-proBNP, ng/L 1.162 1.072–1.260 <0.001 hs-TNT, pg/mL 1.000 0.998–1.002 0.877 Comorbidities Hypertension 1.426 0.617–3.299 0.407 Dyslipidemia 1.433 0.607–3.384 0.411 Diabetes 2.405 1.018–5.683 0.045 Renal dysfunction 0.806 0.355–1.827 0.605 Heart catheterization LVEDP, mmHg 1.066 1.028–1.107 0.001 Echocardiography IVS, mm 1.042 0.813–1.336 0.745 PW, mm 1.003 0.759–1.325 0.984 EDD, mm 1.042 0.997–1.089 0.070 ESD, mm 1.047 1.009–1.086 0.016 LV mass/BSA, g/m2 1.015 1.003–1.027 0.017 EDV, mL 1.005 1.001–1.009 0.008 ESV, mL 1.006 1.002–1.011 0.003 LA-Volume/BSA, mL/m2 1.032 1.009–1.056 0.007 E/A 1.762 1.026–3.025 0.040 E/e’ 1.147 1.054–1.248 0.002 E-DT, ms 0.988 0.981–0.996 0.002 SPVF/DPVF 0.293 0.086–0.996 0.049 EF, % 0.935 0.903–0.968 <0.001 MAPSE, mm 0.813 0.721–0.917 0.001 MASV, cm/s 0.707 0.576–0.867 0.001 GLS, % 1.191 1.079–1.315 0.001 SARM, mm 0.741 0.627–0.877 <0.001 LA-VC, % 0.967 0.942–0.992 0.009 Parameter HR CI p-value Baseline Male gender 1.234 0.509–2.990 0.641 Age, years 0.999 0.971–1.028 0.948 BSA, m2 0.270 0.047–1.561 0.143 BMI, kg m2 0.912 0.821–1.013 0.085 Heart rate, min−1 1.015 0.996–1.034 0.133 BP systolic, mmHg 0.993 0.970–1.017 0.561 BP diastolic, mmHg 1.009 0.971–1.050 0.638 MAP, mmHg 1.000 0.966–1.035 0.996 NYHA > II 2.694 1.092–6.644 0.031 Clinical chemistry NT-proBNP, ng/L 1.162 1.072–1.260 <0.001 hs-TNT, pg/mL 1.000 0.998–1.002 0.877 Comorbidities Hypertension 1.426 0.617–3.299 0.407 Dyslipidemia 1.433 0.607–3.384 0.411 Diabetes 2.405 1.018–5.683 0.045 Renal dysfunction 0.806 0.355–1.827 0.605 Heart catheterization LVEDP, mmHg 1.066 1.028–1.107 0.001 Echocardiography IVS, mm 1.042 0.813–1.336 0.745 PW, mm 1.003 0.759–1.325 0.984 EDD, mm 1.042 0.997–1.089 0.070 ESD, mm 1.047 1.009–1.086 0.016 LV mass/BSA, g/m2 1.015 1.003–1.027 0.017 EDV, mL 1.005 1.001–1.009 0.008 ESV, mL 1.006 1.002–1.011 0.003 LA-Volume/BSA, mL/m2 1.032 1.009–1.056 0.007 E/A 1.762 1.026–3.025 0.040 E/e’ 1.147 1.054–1.248 0.002 E-DT, ms 0.988 0.981–0.996 0.002 SPVF/DPVF 0.293 0.086–0.996 0.049 EF, % 0.935 0.903–0.968 <0.001 MAPSE, mm 0.813 0.721–0.917 0.001 MASV, cm/s 0.707 0.576–0.867 0.001 GLS, % 1.191 1.079–1.315 0.001 SARM, mm 0.741 0.627–0.877 <0.001 LA-VC, % 0.967 0.942–0.992 0.009 Table 4. Univariate Cox regression analysis. Discussion With regard to the anatomical axes most former studies published on aortic root motion relied on M-mode echocardiography and described the aortic walls as pair of parallel linear signals moving anterior in systole and posterior in diastole12–21. This assumption is insufficient though and might be due to the fact that M-mode echo- cardiography is an unidimensional technique and therefore obtains signals only in one direction. However, the echo-probe is not only directed posteriorly but the imaging plane additionally has to be angulated medial and cephalic to display SARM10 which already indicates that a pure forward-backward motion may not completely be true. Two-dimensional B-Mode echocardiography can already display motion in 2 directions simultaneously but the restriction to specific cardiac ultrasound windows still hinders an exact alignment of SARM to the anatomic body planes. Using cardiac magnetic resonance (CMR) imaging, however, the direction of SARM was exemplary analyzed in one of the authors (MA) applying strictly orientated cine slices in the coronal and sagittal plane of the thorax. In the coronal plane the aortic root shows a downward and lateral-left displacement (Fig. 5, row 1; Supplementary Video 1), in the sagittal plane it moves downward and anterior (Fig. 5, row 2; Supplementary Video 2). Thus, the resulting vector of SARM consists of 3 components: downward, anterior and lateral which equal the motion direction of the cardiac base towards the apex during systole. q p g y Regarding to the main axis of the heart, SARM is best visualized using cine slices of the 3 chamber view (Fig. 5, row 3; Supplementary Video 3). Here it becomes obvious, that throughout the heart cycle the aortic root paral- lels the motion of the mitral annulus in the longitudinal axis of the left ventricle which was already assumed by Tandon et al.22. Determining factors of aortic root motion. Different attempts have been made to identify potential influence factors on SARM. Pratt et al. correlated the amplitude of the posterior aortic wall motion with cardiac flow parameters and found the strongest relationship with LV stroke volume (r = 0.77). Thus, they concluded that SARM is a response to the action of the whole LV12. Keltai et al. and Burggraf et al. Discussion In the present study we investigated basic properties, influencing factors as well as the diagnostic and prognostic value of systolic aortic root motion (SARM) assessed by M-mode echocardiography. 6 Scientific Reports \| (2019) 9:3866 \| https://doi.org/10.1038/s41598-019-40386-z www.nature.com/scientificreports/ Parameter in the model HR CI p value Model 1 NT-proBNP, ng/L 1.035 0.918–1.167 0.578 NYHA > II 1.601 0.559–4.587 0.381 Diabetes 1.822 0.733–4.531 0.197 SARM, mm 0.809 0.663–0.987 0.037 Model 2 GLS, % 1.091 0.905–1.315 0.363 MAPSE, mm 1.069 0.832–1.374 0.603 MASV, cm/s 0.818 0.613–1.091 0.171 SARM, mm 0.831 0.628–1.100 0.196 Model 3 EF, % 0.926 0.855–1.002 0.057 GLS, % 0.919 0.720–1.173 0.499 SARM, mm 0.839 0.637–1.105 0.212 Table 5. Multivariate Cox regression analysis. GLS, global longitudinal strain; EF, ejection fraction; MAPSE, mitral annular plane systolic excursion; MASV, mitral annular systolic velocity; NTproBNP, N-terminal pro Brain natriuretic peptide; NYHA, New York Heart Association Functional Classification; SARM, systolic aortic root motion. Parameter in the model HR CI p value Model 1 NT-proBNP, ng/L 1.035 0.918–1.167 0.578 NYHA > II 1.601 0.559–4.587 0.381 Diabetes 1.822 0.733–4.531 0.197 SARM, mm 0.809 0.663–0.987 0.037 Model 2 GLS, % 1.091 0.905–1.315 0.363 MAPSE, mm 1.069 0.832–1.374 0.603 MASV, cm/s 0.818 0.613–1.091 0.171 SARM, mm 0.831 0.628–1.100 0.196 Model 3 EF, % 0.926 0.855–1.002 0.057 GLS, % 0.919 0.720–1.173 0.499 SARM, mm 0.839 0.637–1.105 0.212 Table 5. Multivariate Cox regression analysis. GLS, global longitudinal strain; EF, ejection fraction; MAPSE, mitral annular plane systolic excursion; MASV, mitral annular systolic velocity; NTproBNP, N-terminal pro Brain natriuretic peptide; NYHA, New York Heart Association Functional Classification; SARM, systolic aortic root motion. Table 5. Multivariate Cox regression analysis. GLS, global longitudinal strain; EF, ejection fraction; MAPSE, mitral annular plane systolic excursion; MASV, mitral annular systolic velocity; NTproBNP, N-terminal pro Brain natriuretic peptide; NYHA, New York Heart Association Functional Classification; SARM, systolic aortic root motion. Table 5. Multivariate Cox regression analysis. GLS, global longitudinal strain; EF, ejection fraction; MAPSE, mitral annular plane systolic excursion; MASV, mitral annular systolic velocity; NTproBNP, N-terminal pro Brain natriuretic peptide; NYHA, New York Heart Association Functional Classification; SARM, systolic aortic root motion. Direction of systolic aortic root motion. SARM can be described in two different ways: on the one hand in relation to the cardinal axes and planes of the body and on the other hand in relation to the main axis of the heart within the thorax. Discussion confirmed some of the earlier observations, even though the correlation of SARM with LV stroke volume was weaker in their stud- ies21,23 (r = 0.72 and r = 0.59, respectively) as it was in ours (r = 0.57). Slightly conflicting results were reported by Rosenblatt et al. who found only a poor correlation24 (r < 0.5). While an exercise induced rise in blood pressure (BP) did not affect SARM in the study by Pratt et al., pharmacological lowering of BP resulted in an increase in amplitude12. Furthermore, the onset of SARM coincides with the rise of blood pressure and flow velocity in the ascending aorta and thus reflects the hemodynamic changes caused by LV contraction15. Nevertheless, no con- clusive connection between mean arterial pressure and the amplitude of SARM could be established in our study (r = 0.21).f At the same time Strunk et al. and Biamino et al. offered an alternative explanation and hypothesized that pos- terior aortic wall motion is largely determined by left atrial (LA) volume change13,17. This idea has subsequently been further investigated in patients with valvular heart disease14. By calculating peak relative volume change between atrial diastole and systole our results reveal a moderate relationship with total excursion of the aortic root (r = 0.61). In our opinion this relationship seems logical because both, the aortic root and the LA, share a same anatomical wall. Nevertheless, this connectivity does not explain the simultaneous motion of the aorta’s root ante- rior wall whose motion parallels that of the posterior wall and has an even higher amplitude12. Except for active contraction in sinus rhythm, the atrial volume change occurs passively as a result of valvular plane displacement caused by ventricular contraction. Thus, we believe that SARM is related to but not exclusively caused by left atrial volume change and that SARM has to be attributed mainly to LV systolic function. Scientific Reports \| (2019) 9:3866 \| https://doi.org/10.1038/s41598-019-40386-z 7 www.nature.com/scientificreports/ Figure 5. Cardiac magnetic resonance images to illustrate the position of the aortic root at different time-points during the heart cycle. Rows display the aortic root in the coronal (1st row), sagittal (2nd row) and an angulated plane of the left ventricular 3 chamber view (3rd row). Columns represent end-diastole (1st column), end-systole (2nd row) and a superimposed image of the 1st and 2nd column. Discussion The location of the aortic root is marked blue in end-diastole and yellow in end-systole. Red dots demonstrate the displacement of the mitral anulus from end- diastole to end-systole. Figure 5. Cardiac magnetic resonance images to illustrate the position of the aortic root at different time-points during the heart cycle. Rows display the aortic root in the coronal (1st row), sagittal (2nd row) and an angulated plane of the left ventricular 3 chamber view (3rd row). Columns represent end-diastole (1st column), end-systole (2nd row) and a superimposed image of the 1st and 2nd column. The location of the aortic root is marked blue in end-diastole and yellow in end-systole. Red dots demonstrate the displacement of the mitral anulus from end- diastole to end-systole. This could be confirmed in our study by correlating SARM to LV ejection fraction, the parameter most fre- quently used to grade systolic performance (r = 0.74). A comparable result was found between SARM and LV longitudinal deformation determined by global longitudinal strain (r = 0.75). This association was even higher when SARM was normalized to body surface area (r = 0.78). This could be confirmed in our study by correlating SARM to LV ejection fraction, the parameter most fre- quently used to grade systolic performance (r = 0.74). A comparable result was found between SARM and LV longitudinal deformation determined by global longitudinal strain (r = 0.75). This association was even higher when SARM was normalized to body surface area (r = 0.78). Aortic root motion in cardiac disease. SARM can be altered by different pathologic conditions. Keltai et al. found a reduced SARM after myocardial infarction and significant differences according to Killip classification with highest amplitude for Killip class I and lowest for Killip class IV21. In a study investigating various cardiac disorders Hall et al. described significant elevated SARM in patients with severe mitral regurgitation and atrial septal defect16. Similar results for mitral regurgitation were reported by Akgün et al.14 while SARM was dimin- ished in mitral stenosis. A flattened profil of SARM was also observed by Chandraratna et al.15 in patients with hypertrophic obstructive cardiomyopathy and by Ochs et al.25 in patients with cardiac involvement of light chain Amyloidosis.h y The present study investigated SARM in a prospectively recruited cohort of DCM patients and an age and gender matched control group. Methods S d Study population. The present study is part of the project “New echocardiographic parameters for assess- ment of longitudinal left ventricular function” (ClinicalTrials.gov Identifier: NCT01275963). A corresponding recruitment strategy, inclusion and exclusion criteria as well as the follow-up process have therefore already been described in detail previously27. Initial analysis for the current study is based on data derived from a recent trial and included the complete cohort of 202 patients with dilated cardiomyopathy who were recruited between January 2009 and December 2015 in our cardiology department and the same number of age and gender matched control subjects. Digitally stored echocardiographic examinations were screened for availability of SARM measurements and all suitable datasets have subsequently been re-matched for age and gender. The final study population consisted of 180 patients and 180 healthy controls.ht This study was carried out after approval by the ethics committee of the University of Heidelberg in concord- ance with the Declaration of Helsinki. Written informed consent was provided by all participants of this study. Echocardiography. Echocardiography was conducted with a commercially available ultrasound machine (Vivid E9 BT 11; GE Vingmed Ultrasound, Horten, Norway) using a 1.5- to 4.6-MHz phased-array probe (M5S-D). The sampling rate was adjusted to 55 to 60 frames/sec for B-mode, M-mode was recorded at 1.000 to 2.000 frames/sec. Images from 3 consecutive heart cycles were acquired and stored digitally in RAW-DICOM for- mat for later offline analysis. Measurements were performed according to the recommendation of the American Society of Echocardiography and the European Association of Cardiovascular Imaging28 using commercially available software (EchoPAC version 110.1.1 BT 11; GE Vingmed Ultrasound). Global longitudinal strain (GLS) was determined using the embedded Automated Function Imaging tool.tt g g g SARM was displayed starting from a left parasternal B-mode short axis view of the left ventricle with the echo probe directing posteriorly. The imaging plane was then tilted medial and angulated cephalic until the opening and closing of the aortic valve was visible. Finally, the M-mode beam was placed through the center of the aortic roots cross section (Figs 1 and 2). Statistical analysis. Statistical data analysis was performed using SPSS version 24 (IBM Corporation, Armonk, New York). Continuous variables are presented as mean ± SD or as median with interquartile range as appropriate. Discussion Like in former studies, systolic heart failure lead to a significantly reduced ampli- tude of total aortic root excursion compared to healthy control subjects (9 ± 3 mm vs. 12 ± 2 mm, p < 0.001). Furthermore, our results indicate that SARM is primarily driven by LV systolic function, in particular by longitu- dinal deformation. It is well established, that an impaired LV longitudinal function is associated with a poor prog- nosis in cardiac disease3,26 which could be confirmed by our present data. When adjusted for clinical parameters, Scientific Reports \| (2019) 9:3866 \| https://doi.org/10.1038/s41598-019-40386-z 8 www.nature.com/scientificreports/ diminished SARM is an independent predictor of cardiac death and hospitalization due to cardiac decompen- sation in DCM patients while a value below 7 mm was associated with worse outcome. Among multivariate models including only echocardiographic parameters, however, neither SARM nor any other measured value of longitudinal or systolic LV function could be detected as independently predictive. Although both EF and GLS are approved indicators of cardiac outcome in patients with heart failure, the contradicting information gained through the several multivariate models in our analyses might be caused by the low number of adverse events during follow-up. Furthermore, cases in which it is impossible to calculate EF and/or GLS are rare, especially in times of echocardiography contrast agents. This might limit the use of SARM as a general index of systolic LV function. Limitations First, this was a monocentric study conducted with a specific echocardiographic machine for image acquisition and its proprietary software package was used for analysis. Second, due to the divergent acoustic window, the dis- placement axis of SARM may not be completely concordant with the global longitudinal motion of the LV. Thus, compared to speckle tracking echocardiography SARM serves only as a surrogate parameter of global longitudi- nal LV deformation and is not suitable to determine regional abnormalities. Third, discrepancy in information obtained by multivariate models in our analyses might be caused by the low number of adverse events during follow-up. Scientific Reports \| (2019) 9:3866 \| https://doi.org/10.1038/s41598-019-40386-z Conclusion Due to its high echogenicy, SARM can easily be visualized by M-mode echocardiography. Contrary to many pre- vious reports we demonstrated that SARM is not exclusively directed anterior but shows an additional downward and lateral-left displacement similar to the systolic movement of the cardiac base towards the apex.ii t Abnormal SARM is a frequent finding in cardiac disease. Alterations are, however, not specific to a particular pathology but can generally be regarded as a prognostically unfavorable sign in patients with systolic heart failure. Our data suggest that SARM is closely related to global longitudinal strain and thus might represent an alter- native measure of LV longitudinal function especially when strain assessment is not feasible or available. References Aortic root motion: a marker of left ventricular diastolic dysfunction using M-mode echocardiography? An observational study. Conn. Med. 75(10), 591–598 (2011). g y y 3. Burggraf, G. W., Mathew, M. T. & Parker, J. O. Aortic root motion determined by ultrasound: Relation to cardiac performance in man. Cathet. Cardiovasc. Diagn. 4, 29–41 (1978). g , ( ) 24. Rosenblatt, A. & Clark, R. Echocardiographic Assessment of the Level of Cardiac Compensation in Valvular Heart Disease. 509–519 (1976). g ( ) 4. Rosenblatt, A. & Clark, R. Echocardiographic Assessment of the Level of Cardiac Compensation in Valvular Heart Disease. 509–519 (1976). ( ) 5. Ochs, M. M. et al. Anterior Aortic Plane Systolic Excursion: A Novel Indicator of Transplant-Free Survival in Systemic Light-Chain Amyloidosis. J. Am. Soc. Echocardiogr. 29, 1188–1196 (2016). y J g , ( ) 26. Kalam, K., Otahal, P. & Marwick, T. H. Prognostic implications of global LV dysfunction: a systematic review and meta-analysis of global longitudinal strain and ejection fraction. Heart 100, 1673–1680 (2014). y g ( ) 26. Kalam, K., Otahal, P. & Marwick, T. H. Prognostic implications of global LV dysfunction: a systematic review and meta-analysis of global longitudinal strain and ejection fraction. Heart 100, 1673–1680 (2014). global longitudinal strain and ejection fraction. Heart 100, 1673– g g j ( ) 27. Aurich, M. et al. Unidimensional Longitudinal Strain: A Simple Approach for the Assessment of Longitudinal Myoca Deformation by Echocardiography. J. Am. Soc. Echocardiogr. 1–10, https://doi.org/10.1016/j.echo.2017.12.010 (2018).i 28. Lang, R. M. et al. Recommendations for cardiac chamber quantification by echocardiography in adults: An update from the Ame society of echocardiography and the European association of cardiovascular imaging. J. Am. Soc. Echocardiogr. 28, 1–39 (2015).i y g p y p g g g 9. Hicks, K. A. et al. 2014 ACC/AHA Key Data Elements and Definitions for Cardiovascular Endpoint Events in Clinical Trials Circulation 132 (2015). Acknowledgementsi We acknowledge financial support by Deutsche Forschungsgemeinschaft within the funding programme Open Access Publishing, by the Baden-Württemberg Ministry of Science, Research and the Arts and by Ruprecht-Karls- Universität Heidelberg. Methods S d Values were compared with Student’s t test for normally distributed data, otherwise the Wilcoxon signed rank test for paired observations (patients vs control subjects) or the Mann-Whitney-Wilcoxon test for unpaired observations (patients with or without events) was used. Categorical values are expressed as number (percentage) and were compared using χ2 or McNemar depending on data distribution. All analyses were of explorative nature. A p-value of < 0.05 was denoted statistically significant.l i Association between SARM and potential influence factors was explored by Pearson’s correlation and linear regression analysis. The ability of SARM to discriminate patients with DCM from healthy individuals as well as to discriminate patients with from those without a cardiac event was tested using receiver operating characteristic analyses. Cutoff values were calculated using Youden’s index. f To evaluate the prognostic value of SARM a combined end-point consisting of cardiovascular death29 and hos- pitalization for heart failure was defined. The occurrence of events over time is displayed by Kaplan-Meier curves. Groups were compared using the Log-rank test. Univariate Cox regression was used to calculate hazard ratios of Scientific Reports \| (2019) 9:3866 \| https://doi.org/10.1038/s41598-019-40386-z 9 9 www.nature.com/scientificreports/ clinical and echocardiographic variables. Parameters with significant impact on patient outcome were put into different multivariate models to identify independently prognostic factors. Hazard ratios with 95% confidence intervals and p-values are provided. According to the number of events in this study and with the intention not to overfit the analysis, multivariate models were restricted to a maximum of 4 variables. clinical and echocardiographic variables. Parameters with significant impact on patient outcome were put into different multivariate models to identify independently prognostic factors. Hazard ratios with 95% confidence intervals and p-values are provided. According to the number of events in this study and with the intention not to overfit the analysis, multivariate models were restricted to a maximum of 4 variables. i y Reproducibility of SARM measurements was tested by reanalyzing 20 randomly chosen patients and control subjects in a blinded fashion by the same and by a second experienced investigator. Intra- and interobserver var- iability results are expressed as coefficients of variation.h y pfi The datasets generated and analyzed during the current study are available from the corresponding author on easonable request. References References 1. Russo, C. et al. Prevalence and prognostic value of subclinical left ventricular systolic dysfunction by global longitudinal strain in a community-based cohort. Eur. J. Heart Fail. 16, 1301–1309 (2014). 1. Russo, C. et al. Prevalence and prognostic value of subclinical left ventricular systolic dysfunction by global longitudinal strain in a community-based cohort. Eur. J. Heart Fail. 16, 1301–1309 (2014).h y ( ) 2. Thavendiranathan, P. et al. Use of myocardial strain imaging by echocardiography for the early detection of cardiotoxicity in patients during and after cancer chemotherapy: A systematic review. J. Am. Coll. Cardiol. 63, 2751–2768 (2014). during and after cancer chemotherapy: A systematic review. J. Am. Coll. 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Author Contributions M.A. and D.M. conception and design of the study. M.A., S.G. and M.M.H. data acquisition. M.N. and P.F. data analysis. L.U. biostatistical analysis. P.E. and B.M. patient recruitment. H.A.K. and E.G. critical review of the manuscript. Additional Information Additional Information Supplementary information ac Additional Information Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-40386-z. Scientific Reports \| (2019) 9:3866 \| https://doi.org/10.1038/s41598-019-40386-z 10 www.nature.com/scientificreports/ 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. 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https://openalex.org/W3101508532	https://link.springer.com/content/pdf/10.1007/JHEP05(2019)106.pdf	English	null	A supersymmetric color superconductor from holography	The Journal of high energy physics/The journal of high energy physics	2,019	cc-by	6,406	Published for SISSA by Springer Received: March 14, 2019 Accepted: May 15, 2019 Published: May 20, 2019 Received: March 14, 2019 Accepted: May 15, 2019 Published: May 20, 2019 Received: March 14, 2019 Accepted: May 15, 2019 Published: May 20, 2019 A supersymmetric color superconductor from holography JHEP05(2019)106 Open Access, c⃝The Authors. Article funded by SCOAP3. Published for SISSA by Springer Contents 1 Introduction 2 Model 3 Higgs branch 4 Solution 5 Physical interpretation 6 Symmetry breaking 7 Spectrum 8 Discussion Contents 1 Introduction 1 2 Model 2 3 Higgs branch 3 4 Solution 4 5 Physical interpretation 5 6 Symmetry breaking 6 7 Spectrum 7 8 Discussion 7 Contents 1 Introduction 2 Model 3 Higgs branch 4 Solution 5 Physical interpretation 6 Symmetry breaking 7 Spectrum 8 Discussion 1 2 3 4 5 6 7 7 JHEP05(2019)106 Ant´on F. Faedo,a David Mateos,a,b Christiana Pantelidouc and Javier Tarr´ıod Ant´on F. Faedo,a David Mateos,a,b Christiana Pantelidouc and Javier Tarr´ıod Ant´on F. Faedo,a David Mateos,a,b Christiana Pantelidouc and Javier Tarr´ıod aDepartament de F´ısica Qu´antica i Astrof´ısica and Institut de Ci`encies del Cosmos (ICC), Universitat de Barcelona, Mart´ı i Franqu`es 1, ES-08028, Barcelona, Spain bInstituci´o Catalana de Recerca i Estudis Avan¸cats (ICREA), Passeig Llu´ıs Companys 23, ES-08010, Barcelona, Spain cCentre for Particle Theory and Department of Mathematical Sciences, Durham University, Durham, DH1 3LE, U.K. dPhysique Th´eorique et Math´ematique, Universit´e Libre de Bruxelles (ULB), and International Solvay Institutes, Campus de la Plaine CP 231, B-1050, Brussels, Belgium E-mail: afaedo@ffn.ub.es, dmateos@icrea.cat, christiana.pantelidou@durham.ac.uk, tarrio@gmail.com Mart´ı i Franqu`es 1, ES-08028, Barcelona, Spain cCentre for Particle Theory and Department of Mathematical Sciences, Durham University, Durham, DH1 3LE, U.K. dPhysique Th´eorique et Math´ematique, Universit´e Libre de Bruxelles (ULB), and International Solvay Institutes, Campus de la Plaine CP 231, B-1050, Brussels, Belgium E-mail: afaedo@ffn.ub.es, dmateos@icrea.cat, christiana.pantelidou@durham.ac.uk, tarrio@gmail.com Abstract: We use holography to study d = 4, N = 4, SU(Nc) super Yang-Mills cou- pled to Nf ≪Nc quark ﬂavors. We place the theory at ﬁnite isospin density nI by turning on an isospin chemical potential µI = Mq, with Mq the quark mass. We also turn on two R-symmetry charge densities n1 = n2. We show that the ground state is a supersymmetric, superﬂuid, color superconductor, namely a ﬁnite-density state that preserves a fraction of supersymmetry in which part of the global symmetries and part of the gauge symmetries are spontaneously broken. The holographic description consists of Nf D7-brane probes in AdS5 × S5. The symmetry breaking is due to the dissolution of some D3-branes inside the D7-branes triggered by the electric ﬁeld associated to the isospin charge. The mass- less spectrum contains Goldstone bosons and their fermionic superpartners. The massive spectrum contains long-lived, mesonic quasi-particles if nI ≪µ3 I , and no quasi-particles otherwise. We discuss the possibility that, despite the presence of mass scales and charge densities in the theory, conformal and relativistic invariance arise as emergent symmetries in the infrared. Keywords: AdS-CFT Correspondence, Gauge-gravity correspondence ArXiv ePrint: 1807.09712 Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. https://doi.org/10.1007/JHEP05(2019)106 Contents 1 Introduction 1 2 Model 2 3 Higgs branch 3 4 Solution 4 5 Physical interpretation 5 6 Symmetry breaking 6 7 Spectrum 7 8 Discussion 7 1 Introduction Quantum Chromodynamics (QCD) at non-zero baryon density nb is notoriously diﬃcult to analyze. Because of asymptotic freedom, the preferred phase at asymptotically high density can be shown to be a color-ﬂavor locked (CFL) conﬁguration [1, 2] (for a review see [3]). The ground state in this regime is a color superconductor, namely a ﬁnite-density state in which the color symmetry is Higgsed. Following a common abuse of language, we will refer to this as the spontaneous breaking of the color symmetry. In addition, the CFL ground state is also a superﬂuid, since the baryon number symmetry is spontaneously broken too. In the regime of high but ﬁnite density, such as at the core of neutron stars, no ﬁrst-principle calculations are possible. The only non-perturbative tool, namely lattice QCD, is of limited applicability due to the so-called sign problem [4]. This situation provides one motivation to study the physics of QCD as some other conserved charge is taken to be large, for example the isospin charge. In this case the sign problem is absent and the theory can be simulated on the lattice (see e.g. [5]). Analytical methods can also be used [6, 7]. The emergent picture is that the ground state is a superﬂuid with superﬂuidity driven by a pion condensate at low density and by a quark-antiquark condensate at high density. No color superconductivity was found in these analysis. In this paper we give a step towards the holographic description of color supercon- ducting phases. In this context the goal is not to do precision physics but to perform ﬁrst-principle calculations that may lead to interesting insights [8]. In the case of QCD at non-zero temperature, the insights obtained through this program range from static properties to far-from-equilibrium dynamics of strongly coupled plasmas (see e.g. [9] and references therein). – 1 – We will investigate a simple yet extremely rich holographic model which exhibits both color superconductivity and superﬂuidity when a certain combination of conserved charges is taken to be large. Our model diﬀers from QCD in many respects, including the fact that it is supersymmetric, that it exhibits no chiral symmetry and hence no pions, and that it possesses an R-symmetry that is absent in QCD. Therefore we do not claim that our results have any direct implications for real-world QCD. However, we believe that they are interest- ing for three reasons. 1 Introduction First, they show that color superconductivity does appear in hologra- phy when some conserved charges are large (in this case a combination of isospin charge and R-charge). Second, we expect that a similar holographic mechanism will give rise to color superconductivity in the presence of baryon density [10]. Third, to the best of our knowl- edge our model is the ﬁrst example of a supersymmetric color superconductor. Color su- perconductivity in supersymmetric theories has been previously considered in e.g. [11–13], but in these cases all the supersymmetries are broken by the ground state. In contrast, in our model the ground state leaves some supersymmetry unbroken. We expect that this property will facilitate a precise comparison between the strong-coupling limit described by holography and the weak-coupling regime accessible via perturbative ﬁeld theory methods. JHEP05(2019)106 Color superconductivity in the holographic context has been previously explored. Refs. [14, 15] considered baryon density instead of isospin density, ref. [16] studied a bottom- up model instead of a top-down model, and refs. [17, 18] mimicked the breaking of the color symmetry as the breaking of a global symmetry. 2 Model Type IIB string theory on the near-horizon geometry of Nc D3-branes and Nf D7-branes is dual to d = 4, N = 4, SU(Nc) super Yang-Mills theory coupled to Nf hypermultiplets in the fundamental representation. The presence of the hypermultiplets breaks supersymmetry to N = 2, so we will refer to this theory simply as “the N = 2 gauge theory”. Although the hypermultiplets contain both bosons and fermions, we will loosely refer to them as “ﬂavors” or “quarks”. In the regime Nf ≪Nc the D7-branes can be treated as probes [19] in the AdS5 × S5 geometry ds2 = H−1 2 −dt2 + d⃗x2 + H 1 2 dy2 i + dz2 α , (2.1) (2.1) where t, ⃗x are the four gauge theory directions parallel to the D3-branes, yi with i = 1, . . . , 4 are the coordinates along the D7-branes orthogonal to the D3-branes, and zα with α = 1, 2 are the coordinates orthogonal to both sets of branes. We will often write the metric in the yi directions in spherical coordinates as dy2 i = dr2 + r2 ω2 1 + ω2 2 + ω2 3 , (2.2) (2.2) where ωn are the left-invariant forms on S3. H is the usual harmonic function in the six-dimensional space transverse to the D3-branes: where ωn are the left-invariant forms on S3. H is the usual harmonic function in the six-dimensional space transverse to the D3-branes: H = L4 r2 + z2 1 + z2 2 2 , (2.3) (2.3) with L the radius of AdS5 and S5. with L the radius of AdS5 and S5. – 2 – – 2 – The dynamics of the Nf D7-branes may be described by the non-Abelian action of [20]. At the lowest order in the string tension this reduces to a super-Yang-Mills-Higgs (SYMH) action together with extra couplings to background ﬂuxes coming from the Wess-Zumino term. As emphasised by the author himself, the action in [20] is known to be incomplete, but it seems to capture the exact physics for supersymmetric conﬁgurations [21, 22]. In fact, supersymmetric solutions of the SYMH action often become solutions of the full action. This is also the case here [23], and therefore we will eﬀectively work with the SYMH action. 2 Model For simplicity we will focus on the case Nf = 2 and we will refer to the two ﬂavors as u and d quarks. In the background (2.1) the SYMH action takes the form In the background (2.1) the SYMH action takes the form JHEP05(2019)106 S TD7 = − Z 1 2Tr F ∧∗F + H 1 2 δαβDZα ∧∗DZβ − Z 1 2H−1dt ∧d3x ∧Tr (F ∧F) . (2.4) (2.4) Throughout this paper we set 2πℓ2 s = 1, so all quantities are eﬀectively dimensionless. The last term in (2.4) comes from the coupling to the RR ﬁve-form that supports the geometry (2.1). F is the U(2)f non-Abelian ﬁeld strength on the world volume of the D7- branes and D is the gauge covariant derivative. Zα are non-Abelian scalars (Higgs ﬁelds) in the adjoint of U(2)f, which parametrize the (in general non-commuting) positions of the D7-branes in the zα-plane. The Hodge dual ∗is taken with respect to the eight-dimensional induced metric on the branes, g. The solutions that we will consider will all lie within the SU(2)f subgroup of U(2)f = SU(2)f × U(1)b and will be translationally-invariant along the ⃗x-directions. Therefore we will be eﬀectively studying SU(2)f conﬁgurations in the ﬁve dimensions {t, ⃗y}. The U(1)b charge can be thought of as the baryon number and will play no role here. 1There is also a Coulomb branch parametrized by the VEVs of the adjoint scalars of the theory. 3 Higgs branch The N = 2 gauge theory possesses a continuous moduli space of vacua parametrized by the vacuum expectation values (VEVs) of (s)quark bilinear operators. Supersymmetry guarantees that all the ground states in this so-called Higgs branch are exactly degenerate.1 Holographic studies of the Higgs branch include [24–27]. In these papers no baryon or isospin density was considered. At a generic vacuum on the Higgs branch part of the SU(Nc) gauge symmetry is spontaneously broken (some global symmetries are broken too, see section 6). At weak coupling in the gauge theory the breaking can be seen by analysing the part of the SU(Nc) symmetry that is broken by a speciﬁc set of VEVs in a ﬁxed gauge (see e.g. [24]). At strong coupling the breaking is due to the separation of some D3-branes from the others, which gives a mass to the strings stretching between them. Since these strings are dual to the gluons in the gauge theory this signals the breaking of the SU(Nc) gauge group. The – 3 – separated D3-branes dissolve inside the D7-branes and appear as instantons of the SYMH theory on the D7-branes.2 The breaking can also be seen explicitly by considering the backreaction of the D7- branes-plus-instanton on the spacetime ﬁelds. This results in a position-dependent RR ﬁve-form ﬂux [31] and therefore in a scale-dependent eﬀective rank of the gauge group. The size of the instanton Λ is dual to the scale of gauge symmetry breaking and, in the absence of charge densities, it is arbitrary. A key point of our paper is that, in the presence of isospin and R-charge densities, the scale of gauge symmetry breaking is ﬁxed by the charges. In other words, in the absence of charge densities one may place the theory at an arbitrary point on the Higgs branch and thus break the gauge symmetry “by hand”. In contrast, in the presence of charges the system is driven to a speciﬁc point on the Higgs branch ﬁxed by the charges and the gauge symmetry is broken dynamically. JHEP05(2019)106 4 Solution We consider a direct importation of the dyonic instanton solution of [32] A = at(r) dt ⊗σ3 + a(r) δmn ωn ⊗σn , (4.1a) Z1 = Z = φ(r) σ3 , Z2 = 0 , (4.1b) (4.1a) (4.1b) (4.1b) where σn are the Pauli matrices. We split the ﬁeld strength into purely electric and purely magnetic parts: F = dt ∧E + Fmag. If the following ﬁrst-order BPS conditions are satisﬁed (4.2) Fmag = −⋆Fmag , E = −DZ , (4.2) where the Hodge dual ⋆is taken with respect to the ﬂat metric along the y-directions, then the conﬁguration (4.1) preserves N = 1 supersymmetry and it solves the second-order equations of motion of SYMH theory in ﬂat space [32]. The ﬁrst equation is the usual (anti)selfduality condition associated to instantonic conﬁgurations, whereas the second one relates the electric ﬁeld and the scalar and, via Gauss’ law, implies that D ⋆DZ = 0. As expected from supersymmetry, this conﬁguration also solves the equations of motion of the full action for a pair of D-branes [20] in ﬂat space [33]. Crucially, the same is true for a pair of D7-branes placed in the background (2.1) [23]. The solution of the BPS equations of interest to us is The solution of the BPS equations of interest to us is a(r) = Λ2 r2 + Λ2 , at(r) = φ(r) = Mqr2 r2 + Λ2 , (4.3) (4.3) where Λ and Mq are integration constants. The solution is completely regular everywhere. The asymptotic form where Λ and Mq are integration constants. The solution is completely regular everywhere. The asymptotic form 2 at(r) = φ(r) ≃Mq −MqΛ2 r2 + · · · (4.4) (4.4) will be useful below. will be useful below. 2This is a particular example of the more general phenomenon of “Branes within branes” [28, 29] whereby a low-dimensional brane dissolves inside a high-dimensional brane. From the viewpoint of the eﬀective ﬁeld theory on the high-dimensional brane, the low-dimensional brane appears as a ﬂux of the worldvolume gauge ﬁeld. A pedagogical discussion can be found in [30]. In the context of holography only the case of an instanton ﬂux on the D7-branes leads to color superconductivity because this is the case where the dissolved objects are precisely the D3-branes associated to the color symmetry of the dual gauge theory. – 4 – -6 -4 -2 2 4 6 -1.0 -0.5 0.5 1.0 ±φ/Mq r Figure 1. Brane proﬁles for Λ = 1 (dashed, red curve) and Λ = 1/10 (solid, blue curve). JHEP05(2019)106 Figure 1. Brane proﬁles for Λ = 1 (dashed, red curve) and Λ = 1/10 (solid, blue curve). Choosing the scalar to point in the σ3 direction explicitly breaks the SU(2)f gauge symmetry down to U(1)f. This allows us to deﬁne the electric charge of the instanton by projecting onto the unbroken U(1)f as q = lim r→∞ 1 Mq Z S3 r3Tr (ZEr) = 2π2Λ2Mq , (4.5) (4.5) where the integral is taken on a three-sphere of radius r in the metric (2.2). Despite the explicit breaking of SU(2)f to an Abelian subgroup, the instanton is prevented from collapse by the non-zero angular momentum produced by the crossed electric and magnetic non- Abelian ﬁelds [34]. The Poynting momentum density is aligned with the ω3 left-invariant form in (2.2), which results in a self-dual angular momentum with equal skew-eigenvalues in the yi-directions n1 = n2 ∝q. 5 Physical interpretation The fact that Z(r) is proportional to σ3, which is diagonal with entries ±1, means that the branes bend in opposite directions along the z1-axis with otherwise identical proﬁles, as shown in ﬁgure 1. The asymptotic behaviour (4.4) has two immediate consequences. First, the constant Mq corresponds to the quark mass [19]. To be precise, the quark mass is a complex number and we see that the masses of the u and d quarks are equal in magnitude but have opposite phases. The same is true for the corresponding quark condensates ⟨¯uu⟩= −⟨¯dd⟩∝−MqΛ2 . (5.1) (5.1) Note that this corresponds to a vanishing chiral condensate Note that this corresponds to a vanishing chiral condensate ⟨¯ΨΨ⟩= ⟨¯uu + ¯dd⟩= 0 (5.2) (5.2) but to a nonzero condensate of the form ⟨¯Ψσ3Ψ⟩= ⟨¯uu −¯dd⟩∝−MqΛ2 . (5.3) (5.3) Second, the isospin chemical potential and the charge densities are given by [35, 36] µI = Mq , nI = nu = −nd ∝−MqΛ2 . (5.4) (5.4) – 5 – From the viewpoint of the dual gauge theory, the angular momentum corresponds to equal R-charge densities [37–39] From the viewpoint of the dual gauge theory, the angular momentum corresponds to equal R-charge densities [37–39] (5.5) nR ≡n1 = n2 (5.5) along two of the three U(1) factors in the Cartan subalgebra of the SO(6) R-symmetry of N = 4 SYM. Note that all the charge densities are comparable since along two of the three U(1) factors in the Cartan subalgebra of the SO(6) R-symmetry of N = 4 SYM. Note that all the charge densities are comparable since nI ∼nR ∼q . (5.6) (5.6) Moreover, the size of the instanton is not a free parameter but is ﬁxed as Moreover, the size of the instanton is not a free parameter but is ﬁxed as Λ2 ∝nR µI . (5.7) Λ2 ∝nR µI . JHEP05(2019)106 (5.7) In summary, the solution (4.3) represents a state in the N = 2 gauge theory with non-zero isospin and R-charge densities related to one another and to the quark mass through (5.4), (5.5) and (5.6). The N = 2 gauge theory has been previously studied both at non-zero isospin density [35, 40, 41] and at non-zero baryon density [14, 36, 42–44]. In these papers no supersymmetric ground state was identiﬁed. 6 Symmetry breaking Four-dimensional N = 4 SYM is invariant under conformal transformations and possesses an SO(6) R-symmetry. Coupling the theory to massive quarks has several eﬀects. First, it breaks conformal invariance. Second, it decreases the supersymmetry to N = 2. Third, it reduces the SO(6) symmetry to SO(4) = SU(2)L×SU(2)R, where SU(2)R is the R-symmetry of the N = 2 algebra and SU(2)L is a global symmetry that does not act on the N = 2 supercharges. For massless quarks there would be an extra U(1)R factor. The addition of Nf = 2 quark ﬂavors of equal mass would introduce a new SU(2)f × U(1)b global symmetry that would rotate the quarks into one another. If the quark masses diﬀer even just in phase, as in our case, then this symmetry is explicitly broken to U(1)f × U(1)b. On the gravity side these breakings can be understood geometrically. The original SO(6) symmetries are the isometries of the S5. The addition of the D7-branes selects a four-plane in the R6 space transverse to the D3-branes and thus reduces the symmetry to SO(4). The SU(2)L factor leaves each ωn form in (2.2) invariant, whereas the SU(2)R rotates them into one another. For massless quarks the branes lie at the origin of the z12-plane and an additional U(1)R corresponding to rotations in this plane is preserved. Instead, a non-zero quark mass breaks the U(1)R explicitly. The SU(2)f × U(1)b global symmetry of the gauge theory becomes the non-Abelian gauge symmetry on the pair of D7-branes, which is broken explicitly to U(1)f × U(1)b by the fact that the branes bend in opposite direction in the z1-axis. In summary, N = 4 SYM coupled to massive quarks of unequal masses is invariant under an SU(Nc) gauge symmetry and an SU(2)L × SU(2)R × U(1)f × U(1)b global sym- metry. Placing the theory at non-zero density by turning on nI and nR results in the spontaneous breaking of some of these symmetries. This can be easily seen on the grav- ity side. First, the presence of an instanton indicates that some D3-branes have dissolved – 6 – inside the D7-branes. As explained in section 3 this implies that the gauge group is sponta- neously broken, with the scale of the breaking given by (5.7). 6 Symmetry breaking Second, the self-dual angular momentum generated by the simultaneous presence of the isospin electric ﬁeld and the instanton spontaneously breaks part of the global symmetries as SU(2)R × U(1)f →U(1)D, where the group on the right-hand side is the diagonal U(1) in the Cartan subalgebra of the left-hand side. This breaking can be seen geometrically as follows. The fact that the Poynting vector distinguishes between ω3 and ω1,2 breaks SU(2)R to the U(1)R subgroup that rotates ω1,2 into one another. The fact that only simultaneous rotations of ωn and σn leave the second term in (4.1a) invariant then breaks U(1)R × U(1)f to the diagonal U(1)D. This is similar to the breaking of SU(2)R × SU(2)f to SU(2)D that takes place on the Higgs branch in the absence of the electric ﬁeld (see e.g. [24]). JHEP05(2019)106 7 Spectrum Since several global symmetries are broken spontaneously, we expect the spectrum to con- tain massless bosons. Given that the ground state preserves N = 1 supersymmetry, we ex- pect them to be accompanied by the corresponding massless, fermionic superpartners. The fact that the ground state breaks Lorentz invariance means that the number of these modes need not coincide with the number of broken generators [45, 46]. Holographic examples of this phenomenon include [47–49]. We will report on the massless modes elsewhere [23]. [ ] [ ] The qualitative properties of the spectrum of massive modes depend on the value of the ratio ϵ = Λ2/M 2 q ∝nI/µ3 I . For small ϵ the spectrum contains long-lived, mesonic quasi-particles; otherwise no massive quasi-particles are present. This is illustrated in ﬁgure 2, where we show the spectral function at zero spatial momentum for the gauge theory operator dual to the σ3 component of the scalar ﬁeld Z2, calculated via standard methods [50] from ﬂuctuations governed by the action (2.4). For small ϵ we see high and narrow peaks associated to long-lived excitations, which on the gravity side correspond to quasi-normal modes (QNM) of the Z2 ﬁeld on the D7-branes with very small imaginary parts compared to their real parts. The position of these peaks agrees almost exactly with the masses of mesons on a single D7-brane with the same value of Mq [51]. For ϵ = 1 no such peaks are present. This feature is straightforward to understand on the gravity side. As seen in ﬁgure 1, for small ϵ the branes’ proﬁles approach those of [51] everywhere except on a very thin throat that connects the branes to the Poincar´e horizon at the origin of AdS. The imaginary part of the QNMs measures the absorption probability by the horizon. Since this vanishes as the throat closes oﬀ, in this limit the imaginary parts of the QNMs frequencies vanish and the real parts converge to the real frequencies of [51]. 8 Discussion We have considered d = 4, N = 4, SU(Nc) super Yang-Mills coupled to Nf ≪Nc quarks, which breaks the supersymmetry to N = 2. We have placed the theory at non-zero isospin and R-charge densities. We have shown that, if the conditions (5.4) and (5.5) are obeyed, then the ground state of the system is a supersymmetric, superﬂuid, color superconductor. – 7 – 0 5 10 15 20 25 30 20 40 60 80 100 120 140 χ L4ω2/M 2 q Figure 2. Spectral function described in the text (with arbitrary normalization) for ϵ = 1 (dashed, red curve) and ϵ = 1/100 (solid, blue curve). 0 5 10 15 20 25 30 20 40 60 80 100 120 140 χ L4ω2/M 2 q JHEP05(2019)106 Figure 2. Spectral function described in the text (with arbitrary normalization) for ϵ = 1 (dashed, red curve) and ϵ = 1/100 (solid, blue curve). The solution that we considered is a unit-charge instanton centered at r = 0. In this case the scale of gauge symmetry breaking is ﬁxed by the charge densities. This is an essential diﬀerence with the physics of the Higgs branch3 in which this scale is arbitrary. The solution that we considered is a unit-charge instanton centered at r = 0. In this case the scale of gauge symmetry breaking is ﬁxed by the charge densities. This is an essential diﬀerence with the physics of the Higgs branch3 in which this scale is arbitrary. Solutions for the case Nf > 2 with any instanton number are straightforward to con- struct because multidyonic instanton solutions for higher-rank gauge groups are known [34]. For these conﬁgurations the possible symmetry-breaking patterns are richer [23]. The superconducting property in our system is fundamentally diﬀerent from that in what are usually referred to as “holographic superconductors”, in which the broken sym- metry is actually a global symmetry [53–55]. In contrast, in our case the broken color symmetry is a strongly coupled, non-Abelian gauge symmetry. In our probe approxima- tion this breaking is encoded in the dissolution of some D3-brane charge on the D7-branes in the form of an instanton. The fact that the instanton size is completely ﬁxed by the asymptotic charges proves that the Fermi seasickness [56] that causes the dissolution of the D3-branes in the ﬁrst place need not result in a runaway potential. 3And in a certain approximation in the presence of a baryon density [52]. Acknowledgments We thank Stefano Carignano, Roberto Emparan, Bartomeu Fiol, Eduardo Fraga, Jaume Garriga, Prem Kumar, and very specially Carlos Hoyos, for discussions. We are grate- ful to Jorge Casalderrey-Solana for a critical reading of the manuscript. AF and DM are supported by grants FPA2016-76005-C2-1-P, FPA2016-76005-C2-2-P, 2014-SGR-104, 2014-SGR-1474, SGR-2017-754 and MDM-2014-0369. CP is supported by the STFC Con- solidated Grant ST/P000371/1. JT is supported by the Advanced ARC project “Holog- raphy, Gauge Theories and Quantum Gravity” and by the Belgian Fonds National de la Recherche Scientiﬁque FNRS (convention IISN 4.4503.15). Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. 8 Discussion Were we to include the backreaction of the D7-branes-plus-instanton on spacetime then the color breaking would be visible in the running of the color gauge group, as in [31]. In this backreacted scenario one would also be able to work in the grand-canonical ensemble for the R-charges. Instead, we are limited to the canonical ensemble because the gauge ﬁelds whose asymptotic values would allow us to deﬁne the R-charge chemical potentials are oﬀ-diagonal components of the ten-dimensional metric, whose dynamics is frozen in our probe approximation. To the best of our knowledge our solution is the ﬁrst example of a supersymmetric color superconductor. The BPS equations (4.2) are crucial to prove that the solution of the SYMH equations is also a solution of the full non-Abelian action of [20]. It would be interesting to investigate whether the full open string equations of motion are also satisﬁed, as in [57]. Supersymmetry requires the isospin to be critical, µI = Mq, and the R-charges to be equal, n1 = n2. It would be interesting to relax these conditions both on the gravity side and on the ﬁeld theory side. In the case of near-critical values it may be possible to compare a weak-coupling ﬁeld-theory analysis along the lines of [58, 59] with the strong-coupling holographic result. – 8 – We have referred to our system as a superﬂuid because it breaks some global sym- metries spontaneously. However, this does not directly imply that the system is able to support a superﬂow. 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https://openalex.org/W2290355460	https://www.frontiersin.org/articles/10.3389/fnins.2016.00051/pdf	English	null	Neuroendocrinal, Neurodevelopmental, and Embryotoxic Effects of Recombinant Tissue Plasminogen Activator Treatment for Pregnant Women with Acute Ischemic Stroke	Frontiers in neuroscience	2,016	cc-by	5,206	MINI REVIEW published: 25 February 2016 doi: 10.3389/fnins.2016.00051 Keywords: rTPA, alteplase, brain development, toxicity, haemorrhagic, intravenous, teratogenic, uterine Anna Steinberg 1, 2* and Tiago P. Moreira 1, 2 1 Department of Neurology, Karolinska University Hospital, Stockholm, Sweden, 2 Stroke Research Unit, Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden Edited by: Edited by: Fumihiko Maekawa, National Institute for Environmental Studies, Japan Edited by: Fumihiko Maekawa, National Institute for Environmental Studies, Japan 1 Department of Neurology, Karolinska University Hospital, Stockholm, Sweden, 2 Stroke Research Unit, Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden Thrombolysis with recombinant tissue plasminogen activator (rTPA) was the ﬁrst evidence-based treatment approved for acute stroke. Ischemic stroke is relatively uncommon in fertile women but treatment is often delayed or not given. In randomized trials, pregnancy has been an exclusion criterion for thrombolysis. Physiologic TPA has been shown to have neuroendocrine effects namely in vasopressin secretion. Important TPA effects in brain function and development include neurite outgrowth, migration of cerebellar granular neurons and promotion of long-term potentiation, among others. Until now, no neuroendocrine side-effects have been reported in pregnant women treated with rTPA. The effects of rTPA exposure in the fetus following intravenous thrombolysis in pregnant women are still poorly understood. This depends on low case frequency, short-duration of exposure and the fact that rTPA molecule is too large to pass the placenta. rTPA has a short half-life of 4–5 min, with only 10% of its concentration remaining in circulation after 20 min, which may explain its safety at therapeutically doses. Ischemic stroke during pregnancy occurs most often in the third trimester. Complication rates of rTPA in pregnant women treated for thromboembolic conditions and ischemic stroke were found to be similar when compared to non-pregnant women (7–9% mortality). In embryos of animal models so far, no indications of a teratogenic or mutagenic potential were found. Pregnancy is still considered a relative contraindication when treating acute ischemic stroke with rTPA, however, treatment risk must be balanced against the potential of maternal disability and/or death. Reviewed by: Tin-TIn Win-Shwe, National Institute for Environmental Studies, Japan Masaki Kakeyama, Waseda University, Japan Specialty section: This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience NEUROENDOCRINE EFFECTS OF TISSUE PLASMINOGEN ACTIVATOR were able to show a speciﬁc localization of TPA at neurosecretory granules containing vasopressin, indicating that TPA is co-released along with the exocytosis of vasopressin and might be an early regulator of vasopressin release (Imamura et al., 2010). TPA has been implicated in neurite outgrowth of neuronal cultures (Pittman et al., 1989), neuronal regeneration, migration of cerebellar granule neurons (Seeds et al., 1995), and prohormone synthesis (Sappino et al., 1993). TPA is capable of potentiating N-methyl-D-aspartate (NMDA) receptor activation by cleaving the NMDA receptor 1 (NMDAR1) subunit (Nicole et al., 2001). The signiﬁcance TPA-induced cleavage of NMDAR1, as well as cleavage of pro-BDNF by plasmin is particularly relevant for learning and memory. On the one hand, TPA knockout (KO) mice show reduced maintenance of the long- term potential in the hippocampal CA1 area and exhibit less open-ﬁeld exploration and poor performance in a context- conditioning task (Calabresi et al., 2000). On the other hand, mice overexpressing TPA show an enhancement of the long- term potential in the hippocampus with improved performance in spatial navigation learning tasks (Baranes et al., 1998; Madani et al., 1999). Long-term depression is absent in the striatum of TPA KO mice and has been coupled with decreased rearing activity and object exploration, as well as with poorer performance in a two-way active avoidance task (Calabresi et al., 2000). In 3 month-old Fischer rats, increased TPA mRNA expression is detected in Purkinje cerebellar neurons following 1 h of complex motor task learning in rats (Seeds et al., 1995). Ocular dominance plasticity in the visual cortex was also shown to be related to TPA and plasmin activities (Müller and Griesinger, 1998; Mataga et al., 2004). The authors have also suggested that a cascade of plasmin generated by TPA may selectively mediate cortical plasticity, perhaps via structural remodeling of axons (Müller and Griesinger, 1998; Mataga et al., 2004). Evidence for a function of TPA and the brain-speciﬁc protease inhibitor neuroserpin in regulating axonal TPA-cleaved plasmin regulates proteolysis of among other, laminin, collagen IV, proteoglycans, pro-brain-derived neurotrophic factor (pro-BDNF), and protease activated receptor-1 (Dityatev and Schachner, 2003; Pang et al., 2004). TPA has been shown to directly interact with low-density lipoprotein receptor-related protein (LRP) leading to phosphorylation of mitogen-activated protein kinase (MAPK) 1 and extracellular signal-regulated kinases-1 and 2 (Zhuo et al., 2000; Hu et al., 2006). NEUROENDOCRINE EFFECTS OF TISSUE PLASMINOGEN ACTIVATOR In the brain, wide expression of neuronal TPA and the plasmin inhibitor neuroserpin are found in the developing and adult nervous system and have been shown to play a role in neuronal plasticity (Lee et al., 2015). TPA expression predominate in the lobar hemispheres, thalamus, medulla oblongata, and mesencephalon whereas neuroserpin, although also overlapping with TPA in the lobar hemispheres and mesencephalon, is more abundant in the spinal cord, substantia nigra and Purkinje cells (Teesalu et al., 2004). Moreover, TPA mRNA expression is seen in ventricular ependymal cells and meningeal blood vessel cells (Hashimoto et al., 1998). Tissue plasminogen activator (TPA) is a serine protease that converts plasminogen into the ﬁbrinolytic enzyme plasmin thus promoting ﬁbrin dissolution in blood clots (Carmeliet et al., 1994). Endothelial cells are the major source of circulating TPA, which is released upon stimulation by factor X-a, bradykinin, ﬁbrin, platelet activating factor, and thrombin (Booyse et al., 1986; Emeis, 1992). Other triggers of TPA released into the bloodstream include among others, anxiety, exercise, surgery, and electroconvulsive therapy, however, these conditions are also coupled to catecholamine release, which may thus be the shared trigger mechanism for both direct TPA release and TPA release from endothelial cells in these conditions. In support of this notion, TPA was demonstrated to be co- expressed and traﬃcked simultaneously with noradrenaline in the chromaﬃn cells of the adrenal glands (Parmer et al., 1993). Chromogranin A is one soluble protein that is co-released with TPA and catecholamines. It works as prohormone which, when cleaved into active peptides, inhibits the further release of catecholamines (Parmer et al., 1993). Experiments with knockout mice lacking TPA provided further evidence for a role of TPA in behavioral stress responses and catecholamine release. These mice exhibit deﬁcient stress-induced anxiety behavior (Pawlak et al., 2003, 2005) and show anxiety-like behavior after intracerebroventricular injection of corticotrophin releasing factor (Matys et al., 2004). In particular, a role of TPA in the neurohypophysis has been proposed for the osmotic regulation of body ﬂuids. The antidiuretic hormone vasopressin (arginine-vasopressin or AVP) is synthesized in the magnocellular neurons of the hypothalamic supra-optic nucleus and paraventricular nucleus, and packed into neurosecretory granules, which are transported through their axons over to the neurohypophysary terminals (Miyata and Hatton, 2002). TPA immunoreactivity was observed at neurosecretory granules of vasopressin-positive magnocellular terminals and that of plasminogen was seen at astrocytes. With electron microscopy, Imamura et al. Citation: Citation: Steinberg A and Moreira TP (2016) Neuroendocrinal, Neurodevelopmental, and Embryotoxic Effects of Recombinant Tissue Plasminogen Activator Treatment for Pregnant Women with Acute Ischemic Stroke. Front. Neurosci. 10:51. doi: 10.3389/fnins.2016.00051 February 2016 \| Volume 10 \| Article 51 Frontiers in Neuroscience \| www.frontiersin.org TPA Exposure in Pregnant Women with Stroke Steinberg and Moreira Frontiers in Neuroscience \| www.frontiersin.org NEUROENDOCRINE EFFECTS OF TISSUE PLASMINOGEN ACTIVATOR So far, only one mother treated with rTPA for acute stroke suﬀered a signiﬁcant uterine bleeding complication (Demchuk, 2013), however, caution about bias publication should be taken into account when reviewing case reports. Intravenously administered rTPA has a high aﬃnity for ﬁbrin strands and a short half-life of 4–5 min via liver Finally, ischemic damage is suggested to lead to excess endogenous TPA activity in the brain and contribute to neurodegeneration via extracellular matrix degradation, microglia activation, and blood brain barrier leakage (Lee et al., 2015). Neuroserpin-knockout mice have worse ischemic damage and neurological outcomes than controls, with the eﬀects attributed to TPA-mediated activation of microglia (Gelderblom et al., 2013). Experimental intravenous (exogenous) TPA administration was shown to increase cerebrovascular permeability and decrease cerebrovascular resistance (Tsirka et al., 1995; Yepes et al., 2003; Nassar et al., 2004). NEUROENDOCRINE EFFECTS OF TISSUE PLASMINOGEN ACTIVATOR In turn, serpins (including the CNS variant neuroserpin) and plasminogen activator inhibitor PAI-1 and -2 are the main inhibitors of the serine protease family including TPA, urokinase-type plasminogen activator (uPA), plasmin, and thrombin (Yepes and Lawrence, 2004). Endogenous TPA is widely distributed in the neuroendocrine system. In the neuroendocrine cells of the hypophysis, the magnocellular neurons of the hypothalamic supra-optic nucleus, the chromaﬃn cells of the adrenal medulla, thyroid and parathyroid glands, endogenous TPA ﬁrst enters the endoplasmic reticulum where it binds to a signal peptide and is transported through the Golgi complex. It is then released either via the regulated secretory pathway (vesicular) or the constitutive secretory pathway (direct release) (Kelly, 1985). Although there is one regulated secretory pathway, TPA gets rapidly released from storage vesicles originated from the Golgi complex and appears to be mediated by calcium ion inﬂux (Gualandris et al., 1996). A similar mechanism of TPA co-release with parathyroid hormone has been described in parathyroid cells (Bansal and MacGregor, 1992). February 2016 \| Volume 10 \| Article 51 Frontiers in Neuroscience \| www.frontiersin.org 2 TPA Exposure in Pregnant Women with Stroke Steinberg and Moreira growth has come from studies of cultured cells (for a recent review see Lee et al., 2015). Hashimoto and colleagues found evidence supporting TPA involvement in long-lasting cortical plasticity following psychotomimetic administration in the rat by observing increased mRNA expression in prefrontal cortex neurons projecting to the medial striatum (Hashimoto et al., 1998). rTPA being able to cross the human placenta. In 2006, Leonhardt et al. had reviewed 18 cases of pregnant women treated with rTPA for other thromboembolic conditions, mainly pulmonary embolism, deep vein thrombosis and thrombosed cardiac valve prosthesis and 10 cases of pregnant women treated with rTPA for acute stroke, including an own stroke case (Leonhardt et al., 2006). Good maternal neurological outcome was reported for all but two mothers who died (one with stroke, the other with mitral valve thrombosis) and one who developed cerebral infarction. Ineﬀective thrombolysis or partial arterial recanalization was reported in four mothers. Twenty children were born with good outcome, however, there were two spontaneous abortions, three pregnancy interruptions owing to maternal cause and one infant died at 2 weeks’ post-partum. NEUROENDOCRINE EFFECTS OF TISSUE PLASMINOGEN ACTIVATOR Thus, there was a similar rate of complications in pregnant women compared to non- pregnant women, with mortality at about 7% for the mother and about 23% for the child (half of the child losses occurred in three stroke cases; the other half in two pulmonary embolisms and one valve thrombosis). Possible explanations for child loss not addressed by this review may include the severity of the underlying maternal medical condition rather than a direct eﬀect of rTPA treatment alone. Interruptions of pregnancy may also have been carried following medical decision. Later in 2006, Wiese et al. reported use of intravenous rTPA thrombolysis in a pregnant woman with acute cardioembolic stroke. The patient improved clinically, did not develop complications after receiving rTPA, and at 37 weeks’ gestation, delivered a healthy infant (Wiese et al., 2006). Yamaguchi et al. reported a 36 year-old woman, who was 18 weeks pregnant and developed a sudden onset of motor aphasia and hemiparesis on the right side. The NIH stroke scale was 6, and the brain MRI indicated occlusion of the left middle cerebral artery branches. She was treated with intravenous rTPA with subsequent recanalization of the occluded left middle cerebral artery branches. The symptoms disappeared within a few hours after treatment. She delivered a healthy infant without any apparent complications (Yamaguchi et al., 2010). There are further cases of successful use of rTPA in pregnant women with acute stroke, the majority in the third trimester of pregnancy (Dapprich and Boessenecker, 2002; Elford et al., 2002; Johnson et al., 2005; Murugappan et al., 2006). In 2012, Li et al. reported one own stroke case and reviewed 10 previously published stroke cases. They reported good to complete recovery in 10 mothers and one death during endovascular treatment, resulting in the delivery of eight healthy infants, two medical terminations of pregnancy, and one fetus death (Li et al., 2012). In 2013 and 2014, two additional stroke cases with good outcome for the mothers and the fetuses were reported by Tassi and Ritter, respectively (Tassi et al., 2013; Mantoan Ritter et al., 2014). The most recent case of successful rTPA treatment in a pregnant woman at 39 weeks of gestation with normal delivery was reported in 2015 (Ritchie et al., 2015). Frontiers in Neuroscience \| www.frontiersin.org TREATMENT WITH RECOMBINANT TPA IN PREGNANT WOMEN About 85% of all strokes are ischemic and the remaining are hemorrhagic. Spontaneous reperfusion may occur through endogenous release of plasminogen activator, which stimulates plasmin formation from plasminogen. For larger occlusions this release seems insuﬃcient to induce reperfusion in time to avoid a cerebral lesion. Administration of alteplase, a recombinant tissue plasminogen activator (rTPA) as an injectable drug, which is commonly used to treat myocardial infarction, stroke and thrombosis, is thus one method to enhance this endogenous procedure (for a recent review see Prabhakaran et al., 2015). Acute ischemic stroke in pregnant women occurs most commonly in the third trimester and is potentiated by an increased pro-coagulant state during pregnancy, higher risk for cervical and intracranial artery dissection peri-partum, as well as by persistent foramen ovale and other underlying cardiac conditions. The thrombolytic eﬀect of rTPA varies among species. Humans are proposed to have a more sensitive ﬁbrinolytic system to the eﬀects of rTPA (Korninger and Collen, 1981). Thus, in humans the eﬀective and safe dose for acute stroke treatment is 0.9 mg/kg. In rats, a dose of 1.8 mg/kg up to 10 mg/kg induced recanalization of carotid artery occlusion in 17–71%, whereas in humans this is only achieved in 10–30% of cases. The 1.8 mg/kg dose in the rat is proposed to be equivalent to the human dose of 0.9 mg/kg in terms of eﬃcacy (Tomkins et al., 2015). In rabbits, a dose of 5 mg/kg—but not of 3 or 10 mg/kg—is capable of dissolving an intracerebral clot embolized from the carotid artery (Bednar et al., 1993). Until now, randomized controlled trials have excluded pregnant women and patients with increased hemorrhage risk from participation in studies regarding thrombolysis treatment. In animals rTPA does not cross the placenta and there has been no evidence of teratogenicity (Chan et al., 2000; Leonhardt et al., 2006; De Keyser et al., 2007). To date there are no reports on February 2016 \| Volume 10 \| Article 51 Frontiers in Neuroscience \| www.frontiersin.org 3 TPA Exposure in Pregnant Women with Stroke Steinberg and Moreira contraindication. The risk during pregnancy must be balanced against the potential of a disabled outcome without treatment (Demchuk, 2013). TREATMENT WITH RECOMBINANT TPA IN PREGNANT WOMEN To explore the safety of thrombolysis in pregnant women with acute stroke, and indeed within the whole group of fertile women, the Safe Implementation of Treatments in Stroke International Stroke Thrombolysis Register (SITS- ISTR), a prospective, international, observational registry for medical centers documenting stroke treatments (Wahlgren et al., 2007) has been expanded to include speciﬁc questions for women in the age group 13–50. The aim is to systematically collect data, to contribute to knowledge about treatment safety for these women, and to explore whether treatment in pregnant women, or indeed all women in fertile age is safe and not delayed. We estimate that a number of women will be treated despite pregnancy, partly because the condition was not considered when treatment was initiated, or because the potential beneﬁt was judged higher than the risk. The overall aim of the study, Safe Implementation of Treatments in Stroke-Fertile Women Stroke Thrombolysis Study (SITS-FW), is to determine if pregnancy and even menstruation constitutes any safety issue when treated with thrombolysis, or if these patients can be given the same opportunity for treatment as other patients. in circulation after 20 min, which may explain its safety at therapeutic doses. In menstruating women, Wein et al. described ﬁve subjects in the active arm of the National Institute of Neurological Disorders and Stroke (NINDS) intravenous thrombolysis trial, who were coded as actively menstruating. One subject who had a 1-year history of dysfunctional uterine bleeding required urgent uterine artery ligation. The authors also reported a case of a woman requiring transfusion after intravenous thrombolysis for acute ischaemic stroke (Wein et al., 2002). So far, no neuroendocrine side-eﬀects have been reported in pregnant women treated with alteplase. ONGOING STUDIES Uncertainty whether fertile women with potential or known pregnancy should be treated may delay or halt thrombolysis and worsen stroke outcome. Maternal hemorrhagic complications have been reported in 8% with systemic thrombolysis across the spectrum of clinical thromboembolic indications (Cronin et al., 2008). More speciﬁcally, mortality in 172 pregnant women treated with a potent thrombolytic agent, streptokinase, was reported at 1.2% (Turrentine et al., 1995), which is far lower than the 9.5% mortality owed to stroke alone in pregnant women (Ritchie et al., 2015). Thus, considering this limited risk, pregnancy should not be considered an absolute EFFECTS OF EXPOSURE TO rTPA IN BRAIN DEVELOPMENT As stated above, intravenous rTPA is too large a molecule (7200 KDa) to be able to pass the placental blood barrier. From clinical reports of IVT-treated pregnant women with stroke, there have been no signs of brain development issues on the surviving fetuses. The European Medicines Agency license for alteplase includes information on embryotoxicity (in the form of embryolethality and growth retardation) in pregnant rabbits given 3 mg/kg alteplase, which is over 3 times the therapeutical dose (0.9 mg/kg). However, no teratogenic eﬀects were observed in animals treated with i.v. therapeutical doses and no eﬀects on peri- or post-natal development or fertility were observed in rats treated with doses up to 10 mg/kg (Kojima et al., 1988) In subchronic toxicity studies in rats and marmosets no unexpected adverse eﬀects were observed. No indicative signs of mutagenesis were found (preclinical safety data included in the European license documentation) (EMEA, 2002). AUTHOR CONTRIBUTIONS AS, Planned litterature review, wrote ﬁrst draft with references. TM, Planned litterature review, wrote abstract, edited ﬁrst draft, and wrote ﬁnal version. CONCLUSIONS Pregnancy is still considered a relative contraindication for intravenous thrombolysis with rTPA for acute ischemic stroke within 4.5 h of symptom onset. However, the present and previous reviews indicate a similar maternal safety proﬁle compared with non-pregnant women. This should be further analyzed in future prospective studies. It is reasonable to weigh in the beneﬁt of rTPA vs. the risk for the fetus in this patient group and oﬀer treatment for moderate to severe disabling stroke, particularly if there is no access to endovascular treatment. With the current ongoing implementation of mechanical thrombectomy for acute ischemic stroke in routine practice (Wahlgren et al., 2016), we expect more pregnant women to beneﬁt from acute reperfusion strategies that may or not include intravenous thrombolysis in addition to mechanical thrombectomy for large vessel occlusions. Frontiers in Neuroscience \| www.frontiersin.org REFERENCES Bednar, M. M., Raymond, S. J., and Gross, C. E. (1993). Tissue plasminogen activator: comparison of dose and route of administration in a rabbit model of thromboembolic stroke. Neurol. Res. 15, 405–408. Bansal, D. D., and MacGregor, R. R. (1992). Calcium-regulated secretion of tissue plasminogen activator and parathyroid hormone from human parathyroid cells. J. Clin. Endocrinol. 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https://openalex.org/W4308267894	https://zenodo.org/records/7293720/files/Clinical%20observation%20of%20gonadal%20dysgenesis%20with%20surgical%20correction.pdf	Ukrainian	null	Clinical observation of gonadal dysgenesis with surgical correction	Reproduktivnaâ èndokrinologiâ	2,022	cc-by	4,104	Р.M. ЮСЕФ ● ●із хромосомними порушеннями; к. мед. н., медичний директор Центру прогресивної медицини «Авіценна Мед», м. Київ ORCID: 0000-0003-0608-0171 ● ●з нормальним чоловічим каріотипом; ● ●з жіночим каріотипом. Розлади статевого розвитку з каріотипом 46ХY поділяють на розлади розвитку тестикул і порушення синтезу та активності андрогенів. О.С. ЗАГОРОДНЯ д. мед. н., доцент кафедри акушерства та гінекології № 1 Національного медичного університету імені О.О. Богомольця, м. Київ ORCID: 0000-0003-0424-8380 О.С. ЗАГОРОДНЯ д. мед. н., доцент кафедри акушерства та гінекології № 1 Національного медичного університету імені О.О. Богомольця, м. Київ ORCID: 0000-0003-0424-8380 Синдром Сваєра – рідкісна форма розла- дів статевого розвитку, в основі якої лежить дисгенезія (порушення розвитку) гонад за нормального чоловічого каріотипу [17]. Наз- ва синдрому походить від імені дослідника, який уперше описав його у 1955 р., – Gerald Swyer [3]. Причина повного припинення розвитку яєчка в особи з каріотипом 46ХY полягає у зміні активності певних зон Y-хро- мосоми, відповідальних за детермінацію статі. Однією з таких мутацій є мутація гена SRY (Sex-determining Region Y) [18]. Розта- шована в короткому плечі Y-хромосоми, ця зона містить ген, який кодує структуру про- теїну – фактора, що визначає розвиток яєчок (testis-determining factor, TDF). Безпосередня реалізація дії цього протеїну полягає у транс формації клітин-попередників на клітини Сертолі, які є структурно-функціональним елементом яєчка, а не гранульозні клітини, які формують яєчник [20]. Клітини Сертолі виділяють антимюллерів гормон, що пригні- чує розвиток похідних мюллерової протоки (матки, маткових труб, піхви). Клітини Лейдіга секретують тестостерон, що сприяє диферен- ціації похідних вольфової протоки (сім’яних Синдромом дисгенезїі гонад типу 2 назва- но мутацію гена DAX-1, зчепленого з Х-хро- мосомою, який визнано головним антияєч- никовим гормоном. Наразі відомо, що цей ген бере участь у регуляції роботи всіх залоз внутрішньої секреції, його мутація, крім пору- шення статевого розвитку, супроводжується і частковою наднирниковою недостатністю. Контакти: Загородня Олександра Сергіївна Перинатальний центр м. Києва 03150, Київ, Предславинська, 9 Тел.: +38 (050) 687-32-68 Email: gyner2007@gmail.com Тип 3 дисгенезії гонад, який виявляють при генотипуванні кожного четвертого пацієнта, – мутація гена, що кодує структуру стероїдного фактора 1. Ген локалізовано на довгому плечі 9-ї хромосоми, а сам протеїн є регулятором функції ендокринних залоз, спермато- та оогенезу. Мутація супроводжується широ- ким спектром розладів – від незначної ано- малії зовнішніх статевих органів (гіпоспадія тощо) до повної дисгенезії гонад із супутніми проявами недостатності наднирників [2, 14]. Описано ще декілька типів дисгенезії гонад із їхньою залежністю від точкових мутацій, про- те їхня частота значно нижча. К ЛІНІЧНЕ СПОС ТЕРЕЖЕННЯ ГОНАДА ЛЬНОГО ДИСГЕНЕЗУ З ОПЕРАТИВНОЮ КОРЕКЦІЄЮ К ЛІ Н ІЧ Н И Й В И П А ДО К DOI: http://dx.doi.org/10.18370/2309-4117.2022.65.118-121 DOI: http://dx.doi.org/10.18370/2309-4117.2022.65.118-121 канальців, сім’явиносної протоки). Тому де- леція короткого плеча Y-хромосоми, що є носієм цього гена, – причина формування аномалії статевого розвитку на тлі нормаль- ного чоловічого генотипу – синдрому Сваєра. Описано випадки мікроделеції, що супрово- джуються формуванням жіночого генотипу з затримкою статевого розвитку та гіперогона- дотропним гіпогонадизмом [22]. В.В. БІЛА к. мед. н., директор КНП «Перинатальний центр м. Києва», м. Київ ORCID: 0000-0002-3139-2313 Н.М. КОЛЕСНИК к. мед. н., лікар – акушер- гінеколог гінекологічного відділення КНП «Перинатальний центр м. Києва», м. Київ ORCID: 0000-0002-3249-2447 В.Г. ЖЕГУЛОВИЧ к. мед. н., доцент кафедри акушерства та гінекології № 1 Національного медичного університету імені О.О. Богомольця, м. Київ ORCID: 0000-0002-8165-5214 DOI: http://dx.doi.org/10.18370/2309-4117.2022.65.118-121 В.Г. ЖЕГУЛОВИЧ У 10–20% усіх випадків дисгенезії гонад при каріотипуванні виявляють сaме мутацію ло- кусу SRY, є дані як про її спонтанність, так і про можливість успадкування. Синдром інверсії статі, що спричинений цією мутацією, назва- но типом 1. Причиною синдрому можуть бути й точкові мутації гена, а також різного роду мутації інших генів, зчеплені як із Х-хромо- сомою, так і з деякими аутосомами. Кількість таких відомостей буде зростати, з огляду на розширення даних про людський геном [7]. Контакти: Загородня Олександра Сергіївна Перинатальний центр м. Києва 03150, Київ, Предславинська, 9 Тел.: +38 (050) 687-32-68 Email: gyner2007@gmail.com № 3(65)/вересень 2022 WWW.REPRODUCT-ENDO.COM / WWW.REPRODUCT-ENDO.COM.UA ISSN 2309-4117 В.В. БІЛА к. мед. н., директор КНП «Перинатальний центр м. Києва», м. Київ ORCID: 0000-0002-3139-2313 Аномалії статевого розвитку посідають тре- тє місце серед усіх вад розвитку. Ця проблема має два паралельних аспекти – від глибокого вивчення механізмів дисгенезу, їх генетич- ного і генних рівнів, до розробки сучасних методів хірургічної, медикаментозної, психо- логічної та соціальної корекції, покликаних забезпечити адаптацію пацієнтів. Н.М. КОЛЕСНИК к. мед. н., лікар – акушер- гінеколог гінекологічного відділення КНП «Перинатальний центр м. Києва», м. Київ ORCID: 0000-0002-3249-2447 i.org/10.18370/2309- Натомість транслокація короткого плеча Y-хромосоми на Х-хромосому призводить до формування синдрому де ля Шапеля – чоло- вічого фенотипу на тлі нормального жіночого генотипу. Стать є багатокомпонентним поняттям, що включає хромосомну, гонадну, гормональну, фенотипову та соціальну складові. Форму- вання статі розпочинається внутрішньоут- робно й завершується в пубертатному періо- ді. Досить часто розлади статевого розвитку поєднуються з аномаліями формування ін- ших органів [15]. Аномалії статевого розвитку поділяють на три групи [2]: ОПИС ВИПАДКУ Д і До уваги читачів опис клінічного випадку дисгенезії гонад у пацієнтки, її лікування та подальше спостереження відбу- вається в Перинатальному центрі м. Києва. Чинники ризику розвитку захворювання включають за- гальні мутагенні ефекти – дія іонізувального опромінення, інтоксикація, вірусна інфекція, незбалансоване та дефіцитне харчування, а також вік батька. Проте в більшості випадків важко окреслити безпосередній чинник, що сприяв розвит- ку аномалії [3]. Пацієнтка Наталія Д., 1993 року народження, звернулася до закладу для розгляду можливості оперативного втручан- ня. З анамнезу відомо, що вперше пацієнтка звернулася по медичну допомогу 2017 р. через відсутність менструацій, огрубіння голосу та ріст волосся на обличчі. Виконано каріо- типування – виявлено каріотип 46ХY. На підставі результатів огляду (неправильна будова зовнішніх статевих органів, але за жіночим типом, гіпертрофований клітор, аплазія піхви), УЗД (виявлено гіпопластичну матку, високо в порожнині таза – гонади), лабораторного обстеження (високий вміст гонадотропних гормонів) встановлено діагноз дисгенезії гонад. При проведенні генетичного дослідження виявлено мутацію MYRF. Частота виявлення синдрому гонадальної дисгенезії ста- новить 1:30 000 – 1:80 000 всіх осіб із каріотипом 46ХY [26]. Описано випадки родинного повторення синдрому [6]. Па- цієнти з синдромом гонадальної дисгенезії при народженні мають фенотипові ознаки жіночої статі – зовнішні статеві органи за жіночим типом. Гонади представлено в них фі- брозними тяжами. Проте вже в підлітковому віці реєструють затримку статевого розвитку – відсутність менархе та вто- ринних статевих ознак (росту молочних залоз, оволосіння лобка тощо). При більш глибокому обстеженні, зокрема при візуалізації органів малого таза, виявляють деривати мюлле- рової протоки – гіпоплазовану матку та іноді маткові труби. При гістологічному дослідженні гонад виявляють оваріопо- дібну строму без фолікулів [2, 5]. При гормональному обсте- женні – гіпергонадотропний гіпогонадизм. 2018 р. пацієнтку прооперовано – видалено фрагменти сім’явивідного протоку зліва. 2019 р. за даними МРТ вияв- лено рудиментарну матку, гіпоплазований канал піхви, над лобковим симфізом – рудиментарні печеристі тіла, у про- світі пахвинного каналу – структура овоїдної форми, за бу- довою – недорозвинена тестикула. 2019 р. виконано орхек- томію. Розпочато замісну гормональну терапію комбінацією естрадіолу валеріат/дидрогестерон. Диференціювати синдром гонадальної дисгенезії необ- хідно з іншими станами, що супроводжуються формуванням жіночого фенотипу на тлі каріотипу 46ХY, як-от: вроджена гіперплазія наднирників із дефіцитом 17α-гідроксилази або 17,20-ліази, вроджений синдром нечутливості до андро- генів [13], дефекти рецептора лютеїнізувального гормону (гіпо- або аплазія клітин Лейдіга), дефіцит антимюллерового гормону або дефект його рецепторів (синдром персистент- ної мюллерової протоки) [2]. Крім того, описані синдромаль- ні форми дисгенезії гонад, коли порушення статевого роз- витку асоційовані з іншими аномаліями розвитку, зокрема Дані зовнішнього огляду 2021 р. Р.M. ЮСЕФ припускають, що, крім участі в процесах мієлінізації, цей білок виконує функцію посередника в дії всіх інших регуля- торів диференціації статі [10]. L. Rossetti et al. довели роль мутації білка в розвитку PAGOD-синдрому – комбінації гіпо- плазії легень і легеневої артерії, агонадизму, омфалоцеле та діафрагмальних дефектів [24]. Описано також вади розвитку ока у вигляді тяжкої гіперопії в комбінації з аномаліями діаф- рагми та серця на тлі одного з варіантів мутації [25]. Оскільки при народженні пацієнти з синдромом гона- дальної дисгенезії мають зовнішні статеві органи дівчинки, їх переважно реєструють як осіб жіночої статі. З настанням пубертату затримка появи вторинних статевих ознак спону- кає до більш детального обстеження, включно з генотипу- ванням, що часто дозволяє виявити чоловічу стать. Питання реєстрації статі розв’язує консиліум лікарів за участі гіне- колога, уролога, ендокринолога, психолога з урахуванням можливостей хірургічної корекції. Причиною наполегливої рекомендації щодо видалення рудиментарних гонад є ви- сока частота злоякісних новоутворень [7, 19, 21]. У деяких випадках саме пухлина малого таза зі стрімкими темпами росту стає причиною глибокого обстеження та виявлення синдрому [23]. Для підтримки гормонального статусу при- значають замісну гормональну терапію залежно від визна- ченої юридичної статі [2]. WWW.REPRODUCT-ENDO.COM.UA / WWW.REPRODUCT-ENDO.COM № 3(65)/вересень 2022 ISSN 2309-4117 Р.M. ЮСЕФ Одним із новітніх патогенетичних чинни- ків формування синдрому дисгенезії гонад є мутація гена MYRF, локалізованого в 11-й 118 № 3(65)/вересень 2022 WWW.REPRODUCT-ENDO.COM / WWW.REPRODUCT-ENDO.COM.UA ISSN 2309-4117 МІЖДИСЦИПЛІНАРНИЙ КОНСИЛІУМ хромосомі. Фактор, що регулює мієлін (MYelin Regulatory Factor, MYRF), називають таємничим мембранним транс портним білком через політропність його функцій [12]. Го- ловна функція білка, який кодує цей ген, – регуляція тран- скрипції білків, що відповідають за повноцінну мієлінізацію. Білок є трансмембранним транскриптором, описано щонай- менше 4 варіанти мутації, які призводять до порушення ди- ференціації нервових клітин в ембріональному періоді, зни- ження пластичності синапсів [8]. До фенотипових проявів цих мутацій належить кардіоурогенітальний синдром, а та- кож енцефалопатія з мієліновою вакуолізацією, порушення формування діафрагми [4, 9]. Перший варіант фенотипової реалізації мутацій полягає в комбінації різних уражень сер- ця та різного ступеня дисгенезії гонад обох статей. Клітини Сертолі та Лейдіга мають походження з клітин целомічного епітелію, у фетальних монадах такі клітини активно секре- тують білок, що кодується геном MYRF [11]. K. Humanaka et al. припускають, що, крім участі в процесах мієлінізації, цей білок виконує функцію посередника в дії всіх інших регуля- торів диференціації статі [10]. L. Rossetti et al. довели роль мутації білка в розвитку PAGOD-синдрому – комбінації гіпо- плазії легень і легеневої артерії, агонадизму, омфалоцеле та діафрагмальних дефектів [24]. Описано також вади розвитку ока у вигляді тяжкої гіперопії в комбінації з аномаліями діаф- рагми та серця на тлі одного з варіантів мутації [25]. синдромом WAGR (нейробластома Вільмса, відсутність райдужної оболонки і когнітивні розлади) [9], синдромом Деніса–Драша (ранній початок нефротичного синдрому, що призводить до ниркової недостатності ще в малюковому пе- ріоді), синдромом Фрез’є (гломерулопатія) [1]. хромосомі. Фактор, що регулює мієлін (MYelin Regulatory Factor, MYRF), називають таємничим мембранним транс портним білком через політропність його функцій [12]. Го- ловна функція білка, який кодує цей ген, – регуляція тран- скрипції білків, що відповідають за повноцінну мієлінізацію. Білок є трансмембранним транскриптором, описано щонай- менше 4 варіанти мутації, які призводять до порушення ди- ференціації нервових клітин в ембріональному періоді, зни- ження пластичності синапсів [8]. До фенотипових проявів цих мутацій належить кардіоурогенітальний синдром, а та- кож енцефалопатія з мієліновою вакуолізацією, порушення формування діафрагми [4, 9]. Перший варіант фенотипової реалізації мутацій полягає в комбінації різних уражень сер- ця та різного ступеня дисгенезії гонад обох статей. Клітини Сертолі та Лейдіга мають походження з клітин целомічного епітелію, у фетальних монадах такі клітини активно секре- тують білок, що кодується геном MYRF [11]. K. Humanaka et al. ОПИС ВИПАДКУ Д і при зверненні до Пери- натального центру м. Києва: конституція астенічна, вторинні статеві ознаки за жіночим типом. Зовнішні статеві органи: клі- тор гіперплазований, вхід до піхви не візуалізується (рис. 1). Виконано оперативне втручання: Виконано оперативне втручання: Виконано оперативне втручання: 1. Цистоскопія – вільно прохідна уретра, вічка сечоводів звичайно розташовані, проведено їх стентування. 2. Лапароскопія – матка в порожнині малого таза відсутня (розташована екстраперитонеально), яєчники відсутні, на рівні пахвинного кільця ліворуч виявлено гонаду й видалено. 119 WWW.REPRODUCT-ENDO.COM.UA / WWW.REPRODUCT-ENDO.COM № 3(65)/вересень 2022 ISSN 2309-4117 МІЖДИСЦИПЛІНАРНИЙ КОНСИЛІУМ Рисунок 2. Інтраопераційно, після видалення клітора та резекції малих статевих губ, сечовий катетер уведено в піхву 3. Трансперитонеальна тунелізація піхви під лапароскопіч- ним і цистоскопічним контролем цілісності прямої кишки та сечового міхура. Розсічення вертикальної перетинки піхви. Видалення клітора, резекція малих статевих губ (рис. 2). Післяопераційний період перебігав без ускладнень. Са- мостійне сечовипускання – за 48 годин після операції. Про- тягом 7 діб післяопераційного періоду повторювали там- понаду піхви. Реабілітація з використанням фалоімітатора впродовж 2 місяців. При огляді за 6 місяців після операції пацієнтка повідомляє про задовільне самопочуття, регулярні місячні (відповідно до циклічного застосування гормонального препарату). При УЗД виявлено збільшення розмірів матки до 50 × 40 × 35 мм, візуалізовано ендометрій (рис. 3). Протягом одного року відбулася соціальна адаптація, у грудні 2021 р. пацієнтка вийшла заміж. 120 № 3(65)/вересень 2022 WWW.REPRODUCT-ENDO.COM / WWW.REPRODUCT-ENDO.COM.UA ISSN 2309-4117 у руд р ц ВИСНОВОК Описане клінічне спостереження доповнює наявні дані про варіанти клінічного перебігу рідкісної аномалії – син- дрому Сваєра на тлі мутації гена MYRF і можливості сучасної комплексної корекції з повною фізичною, психологічною та соціальною адаптацією. Рисунок 1. Стан перед оперативним втручанням, гіпертрофований клітор Рисунок 3. Стан за 6 місяців після операції Рисунок 2. Інтраопераційно, після видалення клітора та резекції малих статевих губ, сечовий катетер уведено в піхву Рисунок 1. Стан перед оперативним втручанням, гіпертрофований клітор Рисунок 2. Інтраопераційно, після видалення клітора та резекції малих статевих губ, сечовий катетер уведено в піхву REPRODUCT-ENDO.COM / WWW.REPRODUCT-ENDO.COM.UA Рисунок 3. Стан за 6 місяців після операції Рисунок 1. Стан перед оперативним втручанням, гіпертрофований клітор Рисунок 1. Стан перед оперативним втручанням, гіпертрофований клітор gy ( ) 8. Fan, C., An, H., Sharif, M., et al. j g 19. Milewicz, T., Mrozińska, S., Szczepański, W., Białas, M. 19. Milewicz, T., Mrozińska, S., Szczepański, W., Białas, M. “Dysgerminoma and gonadoblastoma in the course of Swyer syndrome.” Pol J Pathol 67.4 (2016): 411–4. DOI: 10.5114/pjp.2016.65876 “Functional mechanisms of MYRF DNA-binding domain “Functional mechanisms of MYRF DNA-binding domain g mutations implicated in birth defects.” J Biol Chem 296 (2021) 100612. DOI: 10.1016/j.jbc.2021.100612 mutations implicated in birth defects.” J Biol Chem 296 (2021) 100612. DOI: 10.1016/j.jbc.2021.100612 p 100612. DOI: 10.1016/j.jbc.2021.100612 ВИСНОВОК Описане клінічне спостереження доповнює наявні дані про варіанти клінічного перебігу рідкісної аномалії – син- дрому Сваєра на тлі мутації гена MYRF і можливості сучасної комплексної корекції з повною фізичною, психологічною та соціальною адаптацією. Рисунок 3. Стан за 6 місяців після операції 120 № 3(65)/вересень 2022 WWW.REPRODUCT-ENDO.COM / WWW.REPRODUCT-ENDO.COM.UA ISSN 2309-4117 № 3(65)/вересень 2022 WWW.REPRODUCT-ENDO.COM / WWW.REPRODUCT-ENDO.COM.UA ISSN 2309-4117 КЛІНІЧНЕ СПОСТЕРЕЖЕННЯ ГОНАДАЛЬНОГО ДИСГЕНЕЗУ З ОПЕРАТИВНОЮ КОРЕКЦІЄЮ Клінічний випадок В.В. Біла, к. мед. н., директор КНП «Перинатальний центр м. Києва», м. Київ , д , д р р р ц р , Н.М. Колесник, к. мед. н., лікар – акушер-гінеколог гінекологічного відділення КНП «Перинатальний центр м. Києва», м. Київ Н.М. Колесник, к. мед. н., лікар – акушер-гінеколог гінекологічного відділення КНП «Перинаталь , д , р у р дд р ц р В.Г. Жегулович, к. мед. н., доцент кафедри акушерства та гінекології № 1 НМУ ім. О.О. Богомольця, м. Київ сеф, к. мед. н., медичний директор Центру прогресивної медицини «Авіценна Мед», м. Київ Р.М. Юсеф, к. мед. н., медичний директор Центру прогресивної медицини «Авіценна О.С. Загородня, д. мед. н., доцент кафедри акушерства та гінекології № 1 НМУ ім. О.О. Богомольця, м. Київ Аномалії статевого розвитку, попри незначну поширеність, є актуальною проблемою з огляду на стрімке розширення діагностичних можливостей на генному та генетичному рівнях і зростання уваги до соціальної адаптації таких пацієнток. Статтю присвячено опису клінічного спостереження рідкісної форми аномалій статевого розвитку – синдрому Сваєра, що полягає у недорозвиненні чоловічих гонад в осіб із каріотипом 46ХY. Найбільш поширеною та вивченою причиною дисгенезії чоловічих гонад є мутація гена SRY, який відповідає за розвиток яєчок. Наразі виявлено кілька точкових мутацій генів, що беруть участь у реалізації розвитку чоловічих гонад. Гени розташовано як в аутосомах, так і в обох статевих хромосомах. Один із новітніх патогенетичних механізмів розвитку синдрому – мутація гена MYRF. Головним наслідком цієї мутації є порушення мієлінізації, нещодавно виявлено її роль у порушеннях диференціації статі. Пацієнти з синдромом Сваєра при народженні мають фенотипові ознаки жіночої статі. У підлітковому віці реєструють затримку статевого розвитку, при ультразвуковому дослідженні виявляють гіпоплазовану матку, гонади у вигляді фіброзних тяжів. Наведено клінічне спостереження пацієнтки 27 років, яка звернулася зі скаргами на відсутність менструацій, огрубіння голосу та ріст волосся на обличчі. При огляді виявлено будову зовнішніх статевих органів за жіночим типом, гіпертрофію клітора, аплазію піхви, при каріотипуванні – генотип 46ХY, при генетичному обстеженні – мутацію MYRF. Проведено оперативне втручання, що включало цистоскопію (уретра вільно прохідна, виконано стентування сечоводів), лапароскопію (матка та яєчники в порожнині малого таза не візуалізуються, на рівні пахвинного кільця виявлено гонаду й видалено), трансперитонеальну тунелізацію піхви, розсічення вертикальної перетинки піхви, видалення клітора, резекцію малих статевих губ. У післяопераційному періоді застосовували замісну гормональну терапію, місцеву реабілітацію. За 6 місяців після операції відбулася повна фізіологічна та соціальна адаптація пацієнтки. Ключові слова: синдром Сваєра, дисгенезія гонад, мутація MYRF, аплазія піхви. 11. Huang, H., Teng, P., Du, J., et al. 22. Mutlu, G., Kirmizibekmez, H., Aydin, H., et al. “Pure gonadal dysgenesis (Swyer syndrome) due to microdeletion in the SRY gene: a case report.” J Pediatr Endocrinol Metab 28 (2015): 207–10. g, , g, , , , “Interactive Repression of MYRF Self-Cleavage and Activity in Oligodendrocyte Differentiation by TMEM98 Protein.” Neurosci 14.38 (2018): 9829–39. ( ) 2. Щербак, Ю.О. Клінічна презентація 46ХУ порушення статі – дисгенезія гонад // Український журнал дитячої ендокринології. – 2016. – № 2. – С. 44–49. DOI: 10.1523/JNEUROSCI.0154-18.2018 DOI: 10.1523/JNEUROSCI.0154-18.2018 12. Huang, H., Zhou, F., Zhou, S., Qiu, M. 12. Huang, H., Zhou, F., Zhou, S., Qiu, M. “MYRF: A Mysterious Membrane-Bound Transcription Factor Involved in Myelin Development and Human Diseases.” Neurosci Bull 37.6 (2021): 881–4. DOI: 10.1007/s12264-021-00678-9 1. Майданник, В.Г. 1. Майданник, В.Г. 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Aliyev, N., Aliyev, Z. “Case about Swyer syndrome (complete, or “pure” gonadal dysgenesis).” J Clin Res and Rep 5.5 (2020). DOI: 10.31579/2690-1919/0128 “Clinical presentation of 46XV gonadal dysgenesis disorder.” Ukrainian journal of child endocrinology 2 (2016): 44–9. 13. Meyer, K.F., Freitas Filho, L.G., Silva, K.I., et al. “The XY femaleand SWYER syndrome.” Urology Case Reports 26 (2019): 104–9. DOI: 10.1016/j.eucr.2019.100939 3. Aliyev, N., Aliyev, Z. “Case about Swyer syndrome (complete, or “pure” gonadal dysgenesis).” J Clin Res and Rep 5.5 (2020). DOI: 10.31579/2690-1919/0128 ( ) 10. Hamanaka, K., Takata, A., Uchiyama, Y., et al. fi ( ) 10. Hamanaka, K., Takata, A., Uchiyama, Y., et al. fi 10. Hamanaka, K., Takata, A., Uchiyama, Y., et al. “MYRF haploinsufficiency causes 46,XY and 46,XX disorders of sex development: bioinformatics consideration.” Hum Mol Genet 28.14 (2019): 2319–29. DOI: 10.1093/hmg/ddz066 21. Moreira, A.I., Silva, J.C., Ferreira, M.S., Lanhoso, A. “Bilateral dysgerminoma in a patient with a previous diagnosis of Swyer syndrome.” J Obstet Gynaecol Res 38.2 (2012): 452–4. DOI: 10.1111/j.1447-0756.2011.01689.x 21. Moreira, A.I., Silva, J.C., Ferreira, M.S., Lanhoso, A. “Bilateral dysgerminoma in a patient with a previous diagnosis of Swyer syndrome.” J Obstet Gynaecol Res 38.2 (2012): 452–4. DOI: 10.1111/j.1447-0756.2011.01689.x 21. Moreira, A.I., Silva, J.C., Ferreira, M.S., Lanhoso, A. “Bilateral dysgerminoma in a patient with a previous diagnosis of Swyer syndrome.” J Obstet Gynaecol Res 38.2 (2012): 452–4. DOI: 10.1111/j.1447-0756.2011.01689.x 10. Hamanaka, K., Takata, A., Uchiyama, Y., et al. “MYRF haploinsufficiency causes 46,XY and 46,XX disorders of sex development: bioinformatics consideration.” Hum Mol Genet 28.14 (2019): 2319–29. DOI: 10.1093/hmg/ddz066 CLINICAL OBSERVATION OF GONADAL DYSGENESIS WITH SURGICAL CORRECTION V.V. Bila, PhD, director of the Kyiv City Perinatal Center, Kyiv V.V. Bila, PhD, director of the Kyiv City Perinatal Center, Kyiv y y y . Kolesnyk, PhD, obstetrician-gynecologist, Gynecology Department, Kyiv City Perinatal Center, Kyiv V.H. Zhehulovych, PhD, associate professor, Obstetrics and Gynecology Department No. 1, O.O. Bogomolets National Medical University, Kyiv R.M. Yusef, PhD, medical director, Center for Progressive Medicine “Avicenna Med”, Kyiv OS Zahorodnia MD associate professor Obstetrics and Gynecology Department No 1 R.M. Yusef, PhD, medical director, Center for Progressive Medicine “Avicenna Med”, Kyiv Anomalies of sexual development, despite their low prevalence, are relevant both due to the rapid expansion of diagnostic capabilities at the genetic and genetic levels, and due to increasing attention to the social adaptation of such patients. This article is devoted to the description of clinical observation of a rare form of sexual rosette anomalies – Swyer syndrome, which consists in the underdevelopment male gonads in persons with 46XY karyotype. The most common and studied cause of male gonadal dysgenesis is a SRY gene mutation, which is responsible for testicular development. So far, several point mutations in genes involved in the development of male gonads have been identified. Genes are located in both autosomes and both sex chromosomes. One of the newest pathogenetic mechanisms of this syndrome is a MYRF gene mutation. The main consequence of this mutation is a violation of myelination, its role in disorders of sex differentiation has recently been shown. Patients with Swyer syndrome at birth have phenotypic traits of the female sex. Delayed sexual development is registered in adolescence; ultrasound reveals a hypoplasia of the uterus, gonads in the form of fibrous bands. Clinical observation of a 27-year-old patient with complaints of lack of menstruation, coarsening of the voice and facial hair growth is present. Examination revealed the female type structure of the external genitalia, clitoral hypertrophy, vaginal aplasia, 46XY genotype, MYRF mutation. Surgery was performed. It included cystoscopy (free urethra, ureteral stenting), laparoscopy (uterus and ovaries in the pelvic cavity are not visualized, gonads are found at the level of the inguinal ring and removed), transperitoneal tunneling of the vagina, dissection of the vertical membrane of the vagina, removal of the clitoris, resection of the labia minora. Patient underwent complete physiological and social adaptation 6 months after operation. Keywords: Swyer syndrome, gonadal dysgenesis, MYRF mutation, vaginal aplasia.
https://openalex.org/W4393152973	https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2024.1371748/pdf	English	null	Diagnosing arsenic-mediated biochemical responses in rice cultivars using Raman spectroscopy	Frontiers in plant science	2,024	cc-by	8,483	OPEN ACCESS OPEN ACCESS EDITED BY Shikha Verma, Ben-Gurion University of the Negev, Israel REVIEWED BY Arnab Majumdar, Jadavpur University, India Bo-Fang Yan, Guangdong Academy of Agricultural Sciences (GDAAS), China Fawad Ali, Southern Cross University, Australia CORRESPONDENCE Dmitry Kurouski dkurouski@tamu.edu RECEIVED 16 January 2024 ACCEPTED 05 March 2024 PUBLISHED 25 March 2024 CITATION Jua´ rez ID, Dou T, Biswas S, Septiningsih EM and Kurouski D (2024) Diagnosing arsenic- mediated biochemical responses in rice cultivars using Raman spectroscopy. Front. Plant Sci. 15:1371748. doi: 10.3389/fpls.2024.1371748 OPEN ACCESS EDITED BY Shikha Verma, Ben-Gurion University of the Negev, Israel REVIEWED BY Arnab Majumdar, Jadavpur University, India Bo-Fang Yan, Guangdong Academy of Agricultural Sciences (GDAAS), China Fawad Ali, Southern Cross University, Australia CORRESPONDENCE Dmitry Kurouski dkurouski@tamu.edu RECEIVED 16 January 2024 ACCEPTED 05 March 2024 PUBLISHED 25 March 2024 CITATION Jua´ rez ID, Dou T, Biswas S, Septiningsih EM and Kurouski D (2024) Diagnosing arsenic- mediated biochemical responses in rice cultivars using Raman spectroscopy. Front. Plant Sci. 15:1371748. doi: 10.3389/fpls.2024.1371748 Isaac D. Jua´rez 1,2, Tianyi Dou 1, Sudip Biswas 3, Endang M. Septiningsih 3 and Dmitry Kurouski 1,2* Isaac D. Jua´rez 1,2, Tianyi Dou 1, Sudip Biswas 3, Endang M. Septiningsih 3 and Dmitry Kurouski 1,2* 1Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, United States, 2Interdisciplinary Faculty of Toxicology, Texas A&M University, College Station, TX, United States, 3Department of Soil and Crop Sciences, Texas A&M University, College Station, TX, United States Rice (Oryza sativa) is the primary crop for nearly half of the world’s population. Groundwater in many rice-growing parts of the world often has elevated levels of arsenite and arsenate. At the same time, rice can accumulate up to 20 times more arsenic compared to other staple crops. This places an enormous amount of people at risk of chronic arsenic poisoning. In this study, we investigated whether Raman spectroscopy (RS) could be used to diagnose arsenic toxicity in rice based on biochemical changes that were induced by arsenic accumulation. We modeled arsenite and arsenate stresses in four different rice cultivars grown in hydroponics over a nine-day window. Our results demonstrate that Raman spectra acquired from rice leaves, coupled with partial least squares- discriminant analysis, enabled accurate detection and identiﬁcation of arsenic stress with approximately 89% accuracy. We also performed high-performance liquid chromatography (HPLC)-analysis of rice leaves to identify the key molecular analytes sensed by RS in conﬁrming arsenic poisoning. Oryza sativa, phenylpropanoids, carotenoids, stress pathways, non-invasive analysis, analytical techniques, bioaccumulation TYPE Original Research PUBLISHED 25 March 2024 DOI 10.3389/fpls.2024.1371748 TYPE Original Research PUBLISHED 25 March 2024 DOI 10.3389/fpls.2024.1371748 TYPE Original Research PUBLISHED 25 March 2024 DOI 10.3389/fpls.2024.1371748 Introduction Numerous pieces of evidence show that Raman spectroscopy (RS) can be used for conﬁrmatory diagnosis of biotic and abiotic stresses in plants (Altangerel et al., 2017; Egging et al., 2018). RS is an optical technique based on inelastic scattering caused by light interacting with molecules present in the sample (Orlando et al., 2021). The resulting spectra provide information about the molecular structure and composition of analyzed specimens. RS has shown promise for analyzing plant tissues, capable of diagnosing plant diseases, detecting environmental stress, and label-free phenotyping (Payne and Kurouski, 2021). This technique is primarily based on the relative concentrations of metabolites present within a plant, namely Raman active molecules phytochemicals such as terpenes, phenolics, and alkaloids. For example, a study in 2022 showed that RS could detect Fusarium head blight in wheat kernels due to the spectral changes in bands associated with lignin, carotenoids, pectin, cellulose, protein, and starch (Qiu et al., 2022). It was also recently demonstrated that RS could be used to diagnose nutritional deﬁciencies, salinity stress, and aluminum and iron toxicities with high accuracy in rice crops (Sanchez et al., 2020; Higgins et al., 2022a). Based on these previous ﬁndings, we hypothesize that RS can be used to detect changes caused by arsenic stress in rice. Rice is the primary staple crop for more than 3.5 billion people worldwide, with 90% of it grown in Asia (U.S. Department of Agriculture). With up to 60 million people at risk of arsenic-related health conditions in Southeast Asia alone, ﬁnding effective strategies for mitigating the risk of consuming arsenic-rich rice is key to protecting global human health. Arsenic is considered a non- threshold human carcinogen by the International Agency for Research on Cancer (IARC), meaning that even small doses increase the risk of cancer (Signes-Pastor et al., 2016). Arsenic is associated with various cancers, including bladder, kidney, liver, lung, prostate, and skin cancers. Furthermore, arsenic exerts serious effects on many human biological systems, such as the gastrointestinal, hepatic, renal, cardiovascular, dermal, respiratory, and neurological systems (Prevention CfDCa). In this study, we investigated the extent to which RS could be reliably used for the detection of arsenate and arsenite stresses within different cultivars of rice (Oryza sativa). We acquired Raman spectra from rice crops after their exposure to either arsenate or arsenite. Introduction cause signal interference with each other. Moreover, the complexity of ICP-MS analysis is costly and labor-intensive, requiring experienced laboratories to process samples. AAS has lower cost association per analysis and is also reliable, but equipment is equally expensive (Schneider et al., 2018). IC is generally the most cost- effective method but is inconsistent due to variations among column manufacturers while still requiring the involvement of an experienced laboratory. All these methods provide limitations to the average farmer who may not have the resources available to get crop samples analyzed by far-off laboratories. Lastly, the complicated pretreatment for these methods is destructive to the crop sample. As the human population grows, food demand increases proportionally. In just under 30 years, the need for food is estimated to increase by 30% (Agriculture USDo Food Security). To meet the growing food demands, numerous strategies are used in farming, including genetically modiﬁed plants and timely diagnostics of biotic and abiotic stresses (Jiehua et al., 2019; Rusinque et al., 2021). Among these stressors, arsenic ions, including arsenite (As+3) and arsenate (As+5), are among the greatest threats to human health. Arsenate is more commonly found in groundwater compared to arsenite (Arsenic, metals, ﬁbres, and dusts, 2012), although their ratio is dependent on water conditions, notably pH and the presence of oxygen. In some parts of Southeast Asia, both naturally occurring and anthropogenic arsenites and arsenates contaminate the groundwater at levels toxic to human health (Kim et al., 2011). This same groundwater is used for irrigation of rice paddies, where the anaerobic environment of the rice paddy soil readily renders arsenic ions available (Signes-Pastor et al., 2016). The combination of irrigating rice paddy soils with arsenic-polluted water and rice’s inherent plant physiology results in its accumulation of arsenic at levels 10-20 times higher than other grains (Nunes and Otero, 2017; Liao et al., 2018). In rice, arsenate mimics phosphate and is heavily taken up by phosphate transporters, while arsenite is primarily transported by nodulin 26-like intrinsic proteins (NIPs) which are aquaporin channels. Arsenic accumulation in plant roots results in the deceleration of root growth. Once these toxic ions translocate to the shoot, they begin to inhibit cellular metabolism which ultimately leads to plant death (Finnegan and Chen, 2012; Angulo-Bejarano et al., 2021). OPEN ACCESS We found that RS primarily detected a decrease in the concentration of lutein and an increase in the concentration of vanillic and ferulic acids due to the accumulation of arsenite and arsenate in rice. This showed that these molecules are detectable indicators of biochemical response to arsenic accumulation. Finally, a cross-correlation of RS with HPLC and ICP-MS demonstrated RS’s potential for a label-free, non-invasive, and non- destructive quantiﬁcation of arsenic accumulation in rice. CITATION Jua´ rez ID, Dou T, Biswas S, Septiningsih EM and Kurouski D (2024) Diagnosing arsenic- mediated biochemical responses in rice cultivars using Raman spectroscopy. Front. Plant Sci. 15:1371748. doi: 10.3389/fpls.2024.1371748 COPYRIGHT © 2024 Jua´rez, Dou, Biswas, Septiningsih and Kurouski. 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. 01 01 Frontiers in Plant Science frontiersin.org 10.3389/fpls.2024.1371748 Jua´ rez et al. Frontiers in Plant Science Introduction Using partial least squares discriminant analysis (PLS- DA), we demonstrated that such spectra could be used for the conﬁrmatory diagnosis of both arsenate and arsenite stresses in rice. We also performed ICP-MS to quantify the amount of arsenic accumulated by the rice crops. Finally, we utilized high- performance liquid chromatography (HPLC) analysis to reveal the plant metabolites sensed by RS. We found that RS detected changes in the concentration of lutein, ferulic acid, and vanillic acid due to the bioaccumulation of arsenic in rice. Considering the importance of sustaining rice production and mitigating the risk of consuming arsenic-contaminated rice, several methods have been developed to quantify arsenic levels in crops (Ladha et al., 2021). Inductively coupled plasma-mass spectroscopy (ICP-MS), atomic absorption spectroscopy (AAS), and ion chromatography (IC) are commonly used to detect arsenic in plants (Wang et al., 2022). Although ICP-MS is a robust and reliable method to detect trace levels of elements, it suffers from several limitations (Spanu et al., 2019; Wilschefski and Baxter, 2019). Speciﬁcally, many different elements and compounds can frontiersin.org High-performance liquid chromatography HPLC was performed to quantify the amount of carotenoids and phenylpropanoids in all groups of plants. Carotenoids were extracted by homogenizing 150 mg of rice leaf tissue with a mortar and pestle (Higgins et al., 2022b). 1.5 mL of a 1:2 v/v dichloromethane: chloroform mixture was added to the homogenized plant tissue, and the resultant solution was mixed on a thermomixer for 30 minutes at 4°C and 500 rpm. This agitation allowed for thoroughly mixing and extracting of the carotenoids from the plant tissue. The solution was then phase separated by adding 1 mL of 1 M NaCl before centrifugation for 10 minutes at 5,000 g. The aqueous and organic phases were separated, and 0.75 mL of the dichloromethane: chloroform mixture was added again to the aqueous phase. This portion then underwent a second centrifugation for 10 minutes at 5,000 g. The second organic phase from this portion was added to the original organic phase, and all aqueous phases were discarded. The organic phase was then dried in a Multivapor™vacuum evaporator. Finally, the dried pellet was resuspended in 1 mL of methanol prior to HPLC injection. For phenylpropanoids, extraction began similarly by homogenizing 100 mg of rice leaf tissue with a mortar and pestle. 1.5 mL of methanol was then added to the homogenized plant tissue. The solution was sonicated for 1 hour to aid in efﬁcient extraction. Both the phenylpropanoid and carotenoid samples were ﬁltered with a 0.45 µm ﬁlter before injection. The plants in the experiment were divided into three experimental groupings based on the oxidation state of arsenic administered: arsenite (As3+), arsenate (As5+), and the control grouping. This totaled 12 different groups of rice plants, accounting for the combinations of experimental conditions and cultivars. 50µM NaAsO2 was used for modelling arsenite stress, and 50µM Na2AsO4·7H2O was used for arsenate stress. The arsenic was added to the Yoshida solution for administration. Administration of the arsenic marked stress day 1. The extracts were analyzed by reverse-phase HPLC using a C30, 3 mm column (250 × 4.6 mm) (Thermo Fisher Scientiﬁc Inc, part number 075723). We used a Waters 1525 pump equipped with the Waters 2707 autosampler and the 2489 Waters photodiode array detector. For carotenoid analysis, the mobile phases were (A) methanol:water (95:5, v/v) and (B) MTBE. Experimental design The rice plants were cultivated in a hydroponic system using plastic bins and Styrofoam boards. The Styrofoam boards were 02 frontiersin.org Jua´ rez et al. 10.3389/fpls.2024.1371748 DA models were built for a binary comparison of each experimental group, with 2 to 6 latent variables used for each model. All HPLC and ICP-MS data were then checked for normality using QQ-plots, before performing unpaired two-tailed T-tests to measure signiﬁcance. designed with circular holes for each rice plant, and a plastic mesh was securely attached to the bottom of each board panel. The rice seeds were pre-germinated before being placed into the hydroponics, with one seed per hole. For the performed experiments, 18 plants of each of the following cultivars were grown: IR64-Sub1A, IR154, Ciherang-Sub1A, and Presidio, Supplementary Table S1. These different cultivars were selected primarily to study the plant response to arsenic in a diverse array of rice. Presidio is a Southern U.S. tropical japonica rice cultivar while the rest were indica cultivars. The plants received nutrients via a Yoshida solution mixture consisting of macronutrients (114.30 mg/ L NH4NO3, 50.40 mg/L NaH2PO4·2H2O, 89.30 mg/L K2SO4, 108.25 mg/L CaCl2 and 405 mg/L MgSO4·7H2O), and micronutrients [1.875 mg/L MnCl2·4H2O, 0.093 mg/L (NH4)6Mo7O24·4H2O, 1.09 mg/L H3BO3, 0.038 mg/L CuSO4·5H2O, 9.62 mg/L FeCl3·6H2O, 14.88 mg/L C6H8O7·H2O and 0.043 mg/L ZnSO4·7H2O] (Higgins et al., 2022a). The water and solution for the crops were completely replaced every 3 days, and the hydroponics were maintained at a pH of 5. Experimental growth conditions were controlled in a growth chamber set to a day/night cycle of 12h/12h, humidity to 55%, and day/night temperatures to 29°C/26°C (Higgins et al., 2022a). Rice was grown in such conditions for two weeks before initiation of arsenic stress. High-performance liquid chromatography The method for elution gradient began with 97% A until minute 6, where B increased from 3% to 100% linearly until minute 20. The ﬂow then gradually returned to starting conditions at minute 23. The elution peaks were recorded at 450 nm. For phenylpropanoid analysis, the mobile phases were (A) 0.1% H3PO4 and (B) acetonitrile. The elution gradient method began at 95% A until minute 1. The gradient then decreased linearly to 40% A and 60% B until minute 13. The gradient rapidly dropped to 5% A over the next minute, before returning to starting conditions over the ﬁnal minute. The elution peaks were recorded at 280 nm. All peaks were quantiﬁed for comparison by measuring the peak area under the curve (AUC) using Breeze software. Frontiers in Plant Science Raman spectroscopy and photography For data collection, an Agilent Resolve hand-held Raman spectrophotometer was used to collect spectra from the rice leaves at 830 nm. Acquisition time was 1 second at a laser power of 495 milliwatts. 40 Raman spectra were acquired for each group of plants every other day, stopping at day 9. All spectra were baselined and normalized at the 1440 cm-1 peak. Photographs of the crops were also collected at these time points. There were some visual differences between the arsenic- stressed crops and the control by day 5, noticeably in the number of leaves for each rice plant. Still, in the larger scope of a ﬁeld containing arsenic, these differences were not strong enough to indicate high levels of arsenic stress (Supplementary Figure 1). frontiersin.org Raman spectroscopy The Raman spectra collected from rice leaves contained several peaks that corresponded to different biomolecules, such as carotenoids (1003, 1155, 1185, 1213, 1525 cm-1), phenylpropanoids (1604, 1632 cm-1), cellulose (917, 1048 cm-1), carbohydrates (1155 cm-1), and pectin (747 cm-1) (Sanchez et al., 2020; Higgins et al., 2022a, Higgins et al., 2022b) (Figure 1). The peaks at 1286, 1326, 1386, and 1440 cm-1 could be assigned to CH and CH2 vibrations, which are present in nearly all classes of biological molecules. Therefore, these vibrations cannot be assigned to a speciﬁc class of molecular analytes. Our ﬁndings also showed that arsenic toxicity caused an increase in the concentration of phenylpropanoids in rice (Figure 2). Phenylpropanoids are a large class of biomolecules found in plants. These molecules are the major constituents of lignin, and they protect plants against UV light and pathogens (Deng and Lu, 2017). In the Raman spectra acquired from rice plants exposed to arsenic, we observed an increase in the intensity of the 1604 cm-1 peak compared to the intensity of this band in the spectra acquired from the leaves of healthy plants. These spectroscopic changes point to the production of phenylpropanoids by rice as a response to arsenic stress. In the Raman spectra acquired from rice plants exposed to arsenic, we observed substantial changes in the intensities of the discussed above vibrational bands. We primarily observed a decrease in intensity of carotenoid vibrations and an increase in intensity of phenylpropanoid vibrations. A slight decrease was also noticed in vibrational bands that could be assigned to cellulose. These ﬁndings indicate that arsenic toxicity in rice is linked to a reduction in the concentration of carotenoids (Figure 2). These molecules are crucial in photosynthesis and photoprotection by protecting the plant against reactive oxygen species (ROS) generated by chlorophyll under high light conditions (Khorobrykh et al., 2020; Swapnil et al., 2021). This protective mechanism involves carotenoid degradation, such as the enzymatic oxidation of neoxanthin to abscisic acid. This molecular analyte triggers the plant’s immune response to both biotic and abiotic stresses (Havaux, 2014; Stanley and Yuan, 2019). The predominant plant carotenoid is lutein, which serves a crucial function in the assembly and preservation of photosystems (Lv et al., 2012). Decreased concentrations of lutein are associated with oxidative damage due to light stress inducing more ROS than a plant can process. Inductively coupled plasma mass spectroscopy PLS_toolbox (Eigenvector Research Inc) was used in MATLAB to perform all statistical analyses. Data was downloaded from the instrument as CSV ﬁles then imported into MATLAB. First, ANOVA was performed for all peaks with visual change. Next, PLS- ICP-MS was performed to quantify the amount of arsenic present in each group. To prepare the sample, 200 mg of rice 03 frontiersin.org Jua´ rez et al. 10.3389/fpls.2024.1371748 FIGURE 1 Average Raman spectra collected from each treatment group at experimental day 5. Blue regions correspond with carotenoids, purple region corresponds with phenylpropanoids, and yellow region corresponds with cellulose. Spectrum normalized at 1440 peak indicated by the asterisk (). stem tissue was dried and then predigested in 10 mL of HNO3 in 50mL centrifuge tubes overnight. The next day, the tubes were placed in a water bath at 100°C for 3 hours to complete digestion. Beforehand, a hole was made in the cap of each tube to allow for ventilation, and the water bath was done under a running fume hood. The solutions were allowed to cool before 5 mL aliquots were taken from each tube. These aliquots were then diluted to 50mL for a 10x dilution for ICP-MS injection. ICP-MS was run using a Quadrupole Inductively Coupled Plasma-Mass spectrometer (PerkinElmer NexION 300D) equipped with a Cetac ASX-520 autosampler. Argon was used as the carrier gas. Rhodium was used as an internal standard. The calibration curve for ICP-MS was generated using 1 g/L of certiﬁed reference material arsenic in 2% nitric acid. Dilutions of this external standard were made for 1 ng/mL, 25 ng/mL, 50 ng/mL, 100 ng/mL, and 200 ng/mL. All external standards and rice sample dilutions were made with ultrapure water. FIGURE 1 Average Raman spectra collected from each treatment group at experimental day 5. Blue regions correspond with carotenoids, purple region corresponds with phenylpropanoids, and yellow region corresponds with cellulose. Spectrum normalized at 1440 peak indicated by the asterisk (). Frontiers in Plant Science Raman spectroscopy Given that arsenic also induces intracellular ROS production, it is reasonable to anticipate that rice would respond in a similar way as it does to light stress by utilizing its carotenoids (Hu et al., 2020). Phenylpropanoids, especially phenolic acids and ﬂavonoids, are known to increase in content during abiotic stress events due to activation of phenylpropanoid biosynthetic pathways (Sharma et al., 2019). Elevated levels of phenolic compounds can adversely affect plant growth, as observed in our experimental crops on day 9 (Cheynier et al., 2013). Functionally, these phenolic compounds possess antioxidant properties and play a vital role in regulating immune responses. Arsenic, as an exogenous source of ROS, can easily induce the biosynthetic enzymes responsible for phenylpropanoid production. In addition, researchers have identiﬁed that As can cause biochemical changes in the composition of cellulose and lignin in elm, both critical components to cell wall’s structure (Waliszewska et al., 2019). Furthermore, a slight decrease in the intensity of the 1048 cm-1 peak was noticed in the spectra acquired from the rice exposed to arsenic [Figure 2]. These spectroscopic changes point to a decrease in cellulose content upon arsenic-induced toxicity. Previous research has also shown that high levels of arsenic could cause a reduction in the cellulose content of the cell wall (Huang et al., 2021). Based on these results, we conclude that arsenic induces structural changes to the cell walls of rice plants. 04 frontiersin.org Jua´ rez et al. 10.3389/fpls.2024.1371748 FIGURE 2 Post-hoc comparison using the Tukey HSD after one-way ANOVA. Graph indicates differences in average peak intensity on experimental day 5 at 1048 cm-1, 1213 cm-1, and 1604 cm-1, corresponding to cellulose, carotenoid, and phenylpropanoid content respectively. As3+ and As5+ are statistically signiﬁcant from control and from each other, except at the 1213 cm-1 peak. FIGURE 2 Post-hoc comparison using the Tukey HSD after one-way ANOVA. Graph indicates differences in average peak intensity on experimental day 5 at 1048 cm-1, 1213 cm-1, and 1604 cm-1, corresponding to cellulose, carotenoid, and phenylpropanoid content respectively. As3+ and As5+ are statistically signiﬁcant from control and from each other, except at the 1213 cm-1 peak. High-performance liquid chromatography (RT = 14.85), pheophytin (RT = 16.52), and b-carotene (RT = 19.86) respectively (Dou et al., 2021; Higgins et al., 2022b). We observed a signiﬁcant decrease in intensity of all peaks in the samples extracted from the leaves of rice plants exposed to arsenic. Furthermore, we found that arsenate-induced toxicity caused more substantial changes in the carotenoid concentration than arsenite-induced stress. We also observed some variation in the intensity of peaks that corresponded to various carotenoids in the chromatograms of different cultivars. Based on these changes, we could conclude that Presidio suffered more from arsenic stress than the three Asian cultivars. Among the four measured As was discussed above, spectroscopic analysis of the rice leaves exposed to both arsenate and arsenite stresses indicated a decrease in the concentration of carotenoids, with a simultaneous increase in the concentration of phenylpropanoids. We used HPLC to validate these expectations as to identify the speciﬁc molecular analytes that were sensed by RS in the non-invasive detection and identiﬁcation of arsenic stress in rice (Figure 3). HPLC analyses of plant leaf extracts revealed four prominent peaks that could be assigned to lutein (RT = 12.14), chlorophyll A B FIGURE 3 HPLC results for (A) carotenoids and (B) phenylpropanoids, by cultivar and experimental condition, indicating average peak area with standard error bars. Cultivars are numbered as (1) IR64-Sub1A (2) IR154 (3) Ciherang Sub-1A (4) Presidio. Conditions are C control (III) As3+ (V) As5+. Labels indicate T-test results for the comparison of each arsenic group versus the control: NS is no signiﬁcance, * is P ≤0.05, is P ≤0.01, and * is P ≤0.001. A B FIGURE 3 HPLC results for (A) carotenoids and (B) phenylpropanoids by cultivar and experimental condition indicating average peak area with standard error bars B B FIGURE 3 HPLC results for (A) carotenoids and (B) phenylpropanoids, by cultivar and experimental condition, indicating average peak area with standard error bars. Cultivars are numbered as (1) IR64-Sub1A (2) IR154 (3) Ciherang Sub-1A (4) Presidio. Conditions are C control (III) As3+ (V) As5+. Labels indicate T-test results for the comparison of each arsenic group versus the control: NS is no signiﬁcance, * is P ≤0.05, is P ≤0.01, and * is P ≤0.001. FIGURE 3 HPLC results for (A) carotenoids and (B) phenylpropanoids, by cultivar and experimental condition, indicating average peak area with standard error bars. High-performance liquid chromatography Cultivars are numbered as (1) IR64-Sub1A (2) IR154 (3) Ciherang Sub-1A (4) Presidio. Conditions are C control (III) As3+ (V) As5+. Labels indicate T-test results for the comparison of each arsenic group versus the control: NS is no signiﬁcance, * is P ≤0.05, is P ≤0.01, and * is P ≤0.001. 05 Frontiers in Plant Science frontiersin.org Jua´ rez et al. 10.3389/fpls.2024.1371748 10.3389/fpls.2024.1371748 carotenoids, previous work has shown that lutein is the primary carotenoid detected at 830 nm (Dou et al., 2021), since chlorophyll and pheophytin are highly ﬂuorescent (Supplementary Figure 2). Coupled with our HPLC data, we can conclude that changes directly related to lutein content are responsible for the carotenoid intensity decreases we detect in rice crops. Presidio also accumulated a much greater amount of arsenic under stress in comparison to the three Asian cultivars. Still, these values align with other studies on arsenic accumulation in rice. Despite the elevated levels of arsenic found within plant tissues, rice grains typically exhibit a prevalence of lower concentrations of inorganic arsenic in contrast to organic arsenic. Furthermore, the total arsenic content within rice grains remains notably reduced in comparison to other plant tissues. Presidio also accumulated a much greater amount of arsenic under stress in comparison to the three Asian cultivars. Still, these values align with other studies on arsenic accumulation in rice. Despite the elevated levels of arsenic found within plant tissues, rice grains typically exhibit a prevalence of lower concentrations of inorganic arsenic in contrast to organic arsenic. Furthermore, the total arsenic content within rice grains remains notably reduced in comparison to other plant tissues. The chromatogram for phenylpropanoids contained over 20 peaks, so we narrowed down our analysis to the six most prominent peaks. HPLC previously done in rice has shown an increased synthesis of phenolic compounds as a response to stress in certain rice strains (He et al., 2012). We found that arsenic stress statistically increased phenylpropanoid content in at least one peak for every cultivar and visually increased at most peaks. Several peaks in the As3+ group did not show a statistically distinct response from the control. There was also much more variability in phenylpropanoid content between cultivars as compared to looking at carotenoids (Supplementary Figure 3). Discussion In comparison to carotenoids, the plants exhibit an immense diversity and number of phenylpropanoids. These molecules can be categorized according to their structure and involvement in different metabolic pathways. One of the more important pathways is the phenylpropanoid pathway (Biała and Jasiński, 2018). Lignin biosynthesis occurs through this pathway via the polymerization of three monomeric units: p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol (Fraser and Chapple, 2011). Since lignin increases during oxidative stress, we investigated this pathway to identify if it was responsible for the changes within the Raman spectra. We accomplished this by conducting HPLC on ﬁve hydroxycinnamate standards found within the pathway (cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid, and sinapinic acid) (Figure 5). Hydroxycinnamates are central intermediates in phenylpropanoid pathway that are directly converted into lignin (Ralph, 2010). To rule out other pathways, we also tested four hydroxybenzoates (gallic acid, vanillic acid, p-hydroxybenzoic acid, and protocatechuic acid), and two ﬂavonoids (quercetin and rutin) to rule out other pathways. Hydroxybenzoates are synthesized through shikimate and phenylpropanoid pathways, serving as antioxidants and structural components (Deng and Lu, 2017). In contrast, ﬂavonoids branch into distinct pathways within the overall phenylpropanoid pathway, acting as pigments and signaling molecules (Deng and Lu, 2017). High-performance liquid chromatography This indicates that certain cultivars 1) have different base levels of phenylpropanoid production and 2) have differing responses in levels of phenylpropanoid synthesis as a response to stress. This can be illustrated by looking at the peak with RT = 7.915 min, where IR64-Sub1A had little increased production because of arsenic stress; however, Ciherang-Sub1A had a greatly augmented production of phenylpropanoids. There are variations in arsenic resistance among different rice cultivars, and these variations in phenylpropanoid expression may contribute to the differing levels of resistance observed in each cultivar. Inductively coupled plasma mass spectroscopy Traditionally, ICP-MS is used to quantify the amount of arsenic found within rice crops. Here, we performed ICP-MS to validate the stress detected by RS with actual arsenic accumulation (Figure 4). This also allowed us to investigate differences in arsenic uptake by different cultivars. We found that an exceptional amount of arsenic accumulated in the arsenic-stressed rice compared to the control. Among the six prominent phenylpropanoid peaks we reported, the retention times of vanillic acid and ferulic acid corresponded FIGURE 4 ICP-MS results by cultivar and experimental condition, indicating average concentration with standard error bars. Cultivars are (1) IR64-Sub1A (2) IR154 (3) Ciherang Sub-1A (4) Presidio. Conditions are (C) control (III) As3+ (V) As5+. Labels indicate T-test results for the comparison of each arsenic group versus the control: NS is no signiﬁcance, * is P ≤0.05, is P ≤0.01, and * is P ≤0.001. FIGURE 4 ICP-MS results by cultivar and experimental condition, indicating average concentration with standard error bars. Cultivars are (1) IR64-Sub1A (2) IR154 (3) Ciherang Sub-1A (4) Presidio. Conditions are (C) control (III) As3+ (V) As5+. Labels indicate T-test results for the comparison of each arsenic group versus the control: NS is no signiﬁcance, * is P ≤0.05, is P ≤0.01, and * is P ≤0.001. 06 Frontiers in Plant Science frontiersin.org Jua´ rez et al. 10.3389/fpls.2024.1371748 FIGURE 5 Secondary metabolite pathways in Oryza sativa. Red indicates non-secondary metabolites, purple indicates hydroxycinnamics, blue indicates hydroxybenzoics, and green indicates ﬂavonoids. Bolded text indicates compounds tested, while stars indicate compounds identiﬁed. FIGURE 5 Secondary metabolite pathways in Oryza sativa. Red indicates non-secondary metabolites, purple indicates hydroxycinnamics, blue indicates hydroxybenzoics, and green indicates ﬂavonoids. Bolded text indicates compounds tested, while stars indicate compounds identiﬁed. with two speciﬁc peaks, RT = 7.315 min and RT = 8.899 min, respectively. Metabolically, ferulic acid is a precursor to the monolignol coniferyl alcohol, possibly indicating increased lignin production. Vanillic acid is not a hydroxycinnamate but is a hydroxybenzoate derived from several intermediates in the phenylpropanoid pathway. It plays a diverse protective role with strong antioxidant properties and an ability to upregulate components of the antioxidant system (Parvin et al., 2020). None of the other injected standards corresponded with any major peaks observed. To compare our HPLC ﬁndings with the observed spectra, we acquired Raman spectra from powdered forms of vanillic acid and ferulic acid (Figure 6). frontiersin.org Inductively coupled plasma mass spectroscopy Day 1 3 5 7 9 As5+ Sensitivity 92% 87% 96% 93% 98% As3+ Sensitivity 76% 74% 88% 93% 93% Selectivity 82% 84% 84% 71% 72% A B C 5 7 9 96% 93% 98% 88% 93% 93% 84% 71% 72% Day 1 3 5 7 9 As5+ Sensitivity 92% 87% 96% 93% 98% As3+ Sensitivity 76% 74% 88% 93% 93% Selectivity 82% 84% 84% 71% 72% Day 1 3 5 7 9 As5+ Sensitivity 92% 87% 96% 93% 98% As3+ Sensitivity 76% 74% 88% 93% 93% Selectivity 82% 84% 84% 71% 72% A B C FIGURE 7 Scatter plots comparing (A) HPLC and ICP-MS, (B) Raman and HPLC, and (C) Raman and ICP-MS. Diamonds represent the 1604 Raman peak, squares represent the 1632 Raman peak, circles represent the average AuC of the reported phenylpropanoid peaks. intensity were signiﬁcant as early as day 3 (Figure 2). It was noted in the control group that both carotenoid and phenylpropanoid content gradually increased over the experimental days; however, given the young age of the rice crops, this was most likely simply due to plant development. A We used PLS-DA to quantify the accuracy of RS as a diagnostic method for detecting arsenic stresses. PLS-DA is a multivariate dimensionality-reduction tool that uses a supervised machine learning algorithm to classify large sets of data (Ruiz-Perez et al., 2020). The model was trained using 40 spectra acquired from each group of plants. Next, we tested the model’s ability to differentiate the spectra collected from plants exposed to arsenite and arsenate and from the control plants. We evaluated the ability of RS to differentiate arsenic stress from the control (sensitivity) and arsenite stress from arsenate stress (selectivity). Our ﬁndings showed high reliability of RS already by day 1 of stress treatment (Table 1). At this time point, RS coupled to PLS-DA could detect arsenate and arsenite stresses with 96% and 76%, respectively. Averaged across the ﬁve time points, the model could detect the arsenic stresses with 89% accuracy. PLS-DA results also showed that RS could be used to differentiate between arsenate and arsenite stresses with 82% accuracy, although this value decreased as the experiment progressed. B B The PLS-DA model uses latent variables for its predictive power, and plotting these orthogonal factors allow us to visualize how the model classiﬁes these spectra based on their similarities and differences. Frontiers in Plant Science Inductively coupled plasma mass spectroscopy PLS-DA is a multivariate dimensionality-reduction tool that uses a supervised machine learning algorithm to classify large sets of data (Ruiz-Perez et al., 2020). The model was trained using 40 spectra acquired from each group of plants. Next, we tested the model’s ability to differentiate the spectra collected from plants exposed to arsenite and arsenate and from the control plants. We evaluated the ability of RS to differentiate arsenic stress from the control (sensitivity) and arsenite stress from arsenate stress (selectivity). Our ﬁndings showed high reliability of RS already by day 1 of stress treatment (Table 1). At this time point, RS coupled to PLS-DA could detect arsenate and arsenite stresses with 96% and 76%, respectively. Averaged across the ﬁve time points, the model could detect the arsenic stresses with 89% accuracy. PLS-DA results also showed that RS could be used to differentiate between arsenate and arsenite stresses with 82% accuracy, although this value decreased as the experiment progressed. The PLS-DA model uses latent variables for its predictive power, and plotting these orthogonal factors allow us to visualize how the model classiﬁes these spectra based on their similarities and differences. PLS-DA results showed that the sensitivity of RS in differentiating arsenic stress from the control increased from day 1 to day 9. However, we found that the selectivity of RS in differentiation between arsenite and arsenate stresses progressively decreased as the time of plant exposure to both stressors increased. By day 9, the LVA plot showed substantial overlap between the two arsenic stress groups, whereas in the control group, there was only partial overlap (Supplementary Figure 4). This decrease in the selectivity is expected since high levels of arsenic stress will ultimately lead to crop mortality, regardless of the oxidation state of arsenic. By observation, Ciherang-Sub1A and Presidio cultivars looked unhealthy and close to dying on day 9 for both arsenic stress groups, while the two other cultivars survived much better under arsenic stress. L tl i i th i ti lit f l i ll th A B C TABLE 1 PLS DA true predication rates (TPR) for each treatment and experimental time point. frontiersin.org Inductively coupled plasma mass spectroscopy Notably, the 1601 cm-1 peak observed in the spectra of the two standards closely matched the 1603 cm-1 peak in the spectra of the rice on day 9. In the spectra of ferulic acid, a secondary peak was observed at 1628 cm-1, aligning with a minor shoulder evident in the rice spectra. It is important to note that the Raman spectra of the arsenic-stressed groups exhibited non- uniformity within the phenylpropanoid region, characterized by several shoulders. This differs from the symmetric peaks observed in the control spectra. Moreover, over the 9 days of stress, the primary phenylpropanoid peak shifted from 1607 cm-1 to 1603 cm-1. This implies the involvement of multiple phenylpropanoid species contributing to the overall spectral proﬁle, and that during arsenic stress, the predominant phenylpropanoid species is likely changing. Nevertheless, the observed increases in the chromatographic peaks and the corresponding Raman spectra of the standards lead us to conclude that vanillic acid and ferulic acid signiﬁcantly contribute to the changes noticeable within the Raman spectra acquired from plant leaves. FIGURE 6 Raman spectra collected from phenylpropanoid standards of ferulic/ vanillic acid dissolved in water. Normalized by peak height. To verify the statistical signiﬁcance of changes within the Raman spectra both ANOVA (analysis of variance) and PLS-DA were performed. The 1048 cm-1, 1213 cm-1, and 1604 cm-1 peaks best demonstrated the changes in biomolecular content. At the cellulose and carotenoid peaks, the differences in intensity from the control were statistically signiﬁcant by experimental day 5; however, in the phenylpropanoid peak at 1604 cm-1, the increases in peak FIGURE 6 Raman spectra collected from phenylpropanoid standards of ferulic/ vanillic acid dissolved in water. Normalized by peak height. 07 Frontiers in Plant Science frontiersin.org 10.3389/fpls.2024.1371748 Jua´ rez et al. TABLE 1 PLS-DA true predication rates (TPR) for each treatment and experimental time point. TABLE 1 PLS-DA true predication rates (TPR) for each treatment and experimental time point. intensity were signiﬁcant as early as day 3 (Figure 2). It was noted in the control group that both carotenoid and phenylpropanoid content gradually increased over the experimental days; however, given the young age of the rice crops, this was most likely simply due to plant development. We used PLS-DA to quantify the accuracy of RS as a diagnostic method for detecting arsenic stresses. 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. Inductively coupled plasma mass spectroscopy PLS-DA results showed that the sensitivity of RS in differentiating arsenic stress from the control increased from day 1 to day 9. However, we found that the selectivity of RS in differentiation between arsenite and arsenate stresses progressively decreased as the time of plant exposure to both stressors increased. By day 9, the LVA plot showed substantial overlap between the two arsenic stress groups, whereas in the control group, there was only partial overlap (Supplementary Figure 4). This decrease in the selectivity is expected since high levels of arsenic stress will ultimately lead to crop mortality, regardless of the oxidation state of arsenic. By observation, Ciherang-Sub1A and Presidio cultivars looked unhealthy and close to dying on day 9 for both arsenic stress groups, while the two other cultivars survived much better under arsenic stress. C FIGURE 7 Scatter plots comparing (A) HPLC and ICP-MS, (B) Raman and HPLC, and (C) Raman and ICP-MS. Diamonds represent the 1604 Raman peak, squares represent the 1632 Raman peak, circles represent the average AuC of the reported phenylpropanoid peaks. C C Lastly, recognizing the impracticality of employing all three analytical methods for routine sample testing, we examined the interrelations among the results of each method utilized. Scatter plots were generated for each combination of RS, HPLC, and ICP- MS, taking into account all relevant peaks from the chromatograms and Raman spectra (Supplementary Figure 5). Notably, an increase in average phenylpropanoid concentration, as identiﬁed by HPLC, closely aligned with the arsenic bioaccumulation detected by ICP- MS (Figure 7). The rise in average phenylpropanoid concentration also strongly correlated with the increase in phenylpropanoid peak FIGURE 7 Scatter plots comparing (A) HPLC and ICP-MS, (B) Raman and HPLC, and (C) Raman and ICP-MS. Diamonds represent the 1604 Raman peak, squares represent the 1632 Raman peak, circles represent the average AuC of the reported phenylpropanoid peaks. intensity detected by RS, conﬁrming our observations highlighted in the HPLC section. In the last comparisons, we observed that the intensities of the two phenylpropanoid Raman peaks correlated 08 frontiersin.org Jua´ rez et al. 10.3389/fpls.2024.1371748 Funding The author(s) declare ﬁnancial support was received for the research, authorship, and/or publication of this article. This work was supported by Institute for Advancing Health Through Agriculture. Data availability statement The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2024.1371748/ full#supplementary-material The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Acknowledgments RS serves as a valuable tool for the non-invasive and non- destructive identiﬁcation of arsenic stress in rice crops. The spectral changes observed, including decreased carotenoid intensity and increased phenylpropanoid concentration, provide clear markers of arsenic-induced physiological alterations in the plants. The successful detection of arsenate and arsenite stresses when coupled with PLS-DA in the early stages of exposure was also validated with HPLC. We found that vanillic acid and ferulic acid contribute substantially to the spectral changes observed in the phenylpropanoid region in the Raman spectra. Overall, this research offers several insights into RS as a strategy for mitigating arsenic-related health risks in crops. This transition towards digital farming will aid agronomists in the early detection of arsenic, allowing them to prevent contaminated crops from reaching the market early on. The brief two-week timeframe of this experiment even indicates its potential promise for early screening of resistant rice germplasm. Looking forward, future studies should focus on determining RS’s limit of detection for arsenic stress in complex environments to further reﬁne its potential for usage in agriculture settings. The authors would like to thank the Center for Chemical Characterization and Analysis for their assistance with ICP-MS. Author contributions with arsenic bioaccumulation. Notably, the 1632 cm-1 peak demonstrated a stronger correlation than the 1604 cm-1 peak, suggesting that the increase in this shoulder peak serves as a superior indicator for both phenylpropanoid concentration and arsenic bioaccumulation. In addition, the relationship between phenylpropanoid concentration measured by HPLC and ICP-MS was most pronounced in the peak at RT=7.718. In contrast, the strongest correlation between HPLC and Raman spectroscopy was identiﬁed in the peak at RT=7.315, corresponding to vanillic acid. These correlations provide crucial insights into how our RS ﬁndings align with conventional analytical techniques. However, it is important to acknowledge the limitations of these ﬁndings due to our dataset size. Future studies could greatly enhance these correlations by growing rice in varying concentrations of arsenic, facilitating the development of robust calibration curves. Furthermore, considering the complex ﬁeld conditions impacting rice development, more research regarding RS’s sensitivity in detecting arsenic under these conditions must be done. IJ: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. TD: Investigation, Methodology, Writing – review & editing. SB: Investigation, Methodology, Validation, Visualization, Writing – review & editing. ES: Project administration, Supervision, Writing – review & editing. DK: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing. 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. 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https://openalex.org/W3159611499	https://www.preprints.org/manuscript/202103.0525/v1/download	English	null	Wildlife Monitoring Using a Multi-UAV System with Optimal Transport Theory	Applied sciences	2,021	cc-by	12,129	Rabiul Hasan Kabir 1 and Kooktae Lee 1* 1 Department of Mechanical Engineering, New Mexico Institute of Mining and Technology, Socorro, New Mexico 87801, USA; rabiul.kabir@student.nmt.edu, kooktae.lee@nmt.edu * Correspondence: kooktae.lee@nmt.edu; Tel.: +1-575-835-5554 1 Department of Mechanical Engineering, New Mexico Institute of Mining and Technology, Socorro, New Mexico 87801, USA; rabiul.kabir@student.nmt.edu, kooktae.lee@nmt.edu * Correspondence: kooktae.lee@nmt.edu; Tel.: +1-575-835-5554 1 Department of Mechanical Engineering, New Mexico Institute of Mining and Technology, Socorro, New Mexico 87801, USA; rabiul.kabir@student.nmt.edu, kooktae.lee@nmt.edu * Correspondence: kooktae.lee@nmt.edu; Tel.: +1-575-835-5554 Abstract: This paper addresses a wildlife monitoring problem using a team of UAVs for efﬁcient monitoring of wildlife. The state-of-the-art technology using UAVs has been an increasingly popular tool to monitor wildlife compared to the traditional methods such as satellite imagery- based sensing or GPS trackers. However, there still exist unsolved problems as to how the UAVs need to cover a spacious domain to detect animals as many as possible. In this paper, we propose the optimal transport-based wildlife monitoring strategy for a multi-UAV system, to prioritize monitoring areas while incorporating complementary information such as GPS trackers and satellite-based sensing. Through the proposed scheme, the UAVs can explore the large-size domain effectively and collaboratively with a given priority. The time-varying nature of wildlife due to their movements is modeled as a stochastic process, which is included in the proposed work to reﬂect the spatio-temporal evolution of their position estimation. In this way, the proposed monitoring plan can lead to efﬁcient wildlife monitoring with a high detection rate. Various simulation results including statistical data are provided to validate the proposed work. Keywords: Wildlife Monitoring; Multi-UAV System; Optimal Transport Keywords: Wildlife Monitoring; Multi-UAV System; Optimal Transport Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Article Efﬁcient Wildlife Monitoring using a Multi-UAV System with Optimal Transport Theory Rabiul Hasan Kabir 1 and Kooktae Lee 1* Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 unavailability of the GPS tracker depending on their locations, and partial information without details (e.g., population size, age and gender ratios, foraging strategies, etc.). g g g g g g Other technologies to monitor wildlife include camera traps [7], [8], acoustic record- ing devices [9], [10], environmental DNA monitoring for tracking community composi- tion [11], [12], and genetic monitoring for identifying individuals within populations [13]. These methods alone are not effective as they cannot cover a wide range and the installation of sensors requires some preliminary data to choose proper locations. Thus, there have been attempts to utilize the new technology using UAVs (or drones equivalently), in order to efﬁciently gather more detailed data with less time and cost. It has been reported that UAV-based sensing is an increasingly popular and promising conservation tool in ecological monitoring [14–16]. The data from UAVs can provide useful information for timely management responses [17]. The UAVs can be deployed quicker than manned airplanes [18] and are known to be less sensitive to ground survey techniques for wildlife monitoring [19]. As a consequence, many researchers and ecolo- gists applied the UAV technologies to supplementing the conventional techniques for monitoring, conservation, and management practices [19–21]. g g p Despite many advantages of UAV-based monitoring, detecting animal herds using UAVs is still a challenging problem due to the large domain size, limited energy of UAVs, and wildlife movements. Further, no systematized approaches have been developed in efforts to incorporate other complementary monitoring tools (e.g., GPS trackers) in the UAV-based monitoring. To tackle the above problem, this paper investigates an efﬁcient wildlife monitoring scheme using a multi-UAV system. We develop the optimal transport-based multi-UAV monitoring strategy that prioritizes the monitoring areas by reﬂecting partial information such as GPS trackers. The major contributions of this paper are summarized as follows. Based on the optimal transport theory, we formulate the multi-UAV monitoring problem to increase the detection rate of animal herds, particularly medium-to-large mammals having group behavior like deer. This problem setup includes which area needs to be monitored as well as how a team of UAVs cover the spacious domain collaboratively. The control algorithm for the team of UAVs is then developed under the proposed optimal transport framework. The time-varying nature of the wildlife location is incorporated into the proposed plan, dramatically increasing the detection rate. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 The proposed scheme has the potential to signiﬁcantly improve the wildlife detection rate while saving time, costs, and resources by incorporating complementary information (e.g., GPS tracker, camera traps, and acoustic recording devices) into the monitoring plan. g g p The remaining parts of this paper are organized as follows. Section 2 describes the problem to solve for efﬁcient wildlife monitoring based on the optimal transport theory. In Section 3, the animal movement modeling is brieﬂy explained. The main result for the optimal transport-based multi-UAV monitoring strategy is proposed in Section 4. Sample point generation and other monitoring strategies for performance comparison are provided in Section 5 and 6, respectively. To support our proposed works, simulation results are presented in Section 7. Finally, Section 8 concludes the paper. Notation: A set of real and natural numbers are denoted by R and N, respectively. Further, N0 = N ∪{0}. The symbols ∥· ∥and T, respectively, denote the Euclidean norm and the transpose operator. The symbol R(x, r) represents a set of points within the circle centered at x with a radius r. The symbol # indicates the cardinality of a given set. The variable t ∈N0 is used to denote a discrete-time index. 1. Introduction Over decades, biodiversity has been threatened by several factors such as land-use change and habitat fragmentation, overhunting, invasive species, and environmental change. According to [1], 25% of all mammal species are in danger due to the above factors. This necessitates informed management of wildlife to maintain biodiversity as well as to prevent the extinction of some species. Traditionally, ground-based surveys have been widely adopted to assess and monitor wildlife biodiversity, which is time- consuming, ﬁnancially expensive, and logistically challenging in remote areas [2]. Due to the high cost, surveys have not been conducted at the frequency required for proper analysis and monitoring of population trends [3]. Moreover, some areas may not be easy to collect data because of difﬁcult and inaccessible terrains [4]. As an alternative, ecologists, conservation researchers, and practitioners have uti- lized satellite imagery-based remote sensing associated with a geographic information system (GIS) for the monitoring purpose of wildlife to cope with prevailing environmen- tal challenges. Unfortunately, this type of remote sensing technology might not be ideal for accurate wildlife monitoring at the landscape level due to its obvious disadvantages: limited time to observe a certain area and low resolutions for satellite images. Moreover, persistent cloud cover may obscure the satellite remote sensing unexpectedly [5]. Deploying GPS collars on the target animals is another way to identify detailed wildlife movements. This method itself is, however, known to be costly and time- consuming with a lot of human efforts. In [6], the total cost of activities to capture animals, deploy GPS collars, and analyze data are more than $300,000 in a year only in Colorado, USA. Also, the GPS trackers used to track the animals have some drawbacks such as intermittent GPS data transmission due to the limited energy of the device, © Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 animal herds as it is in general very spacious. Moreover, UAVs have limited energy and thus, are not able to cover the entire domain because of its huge size. animal herds as it is in general very spacious. Moreover, UAVs have limited energy and thus, are not able to cover the entire domain because of its huge size. Throughout the paper, we assume that the locations of UAVs are accurately known by GPS signals. Also, it is assumed that the UAVs can detect animals during the monitoring mission via onboard image processing such as machine learning technology. Although the animal recognition and detection problem itself is another important research area for wildlife monitoring, it is out of scope in this study. Rather, we are more interested in which areas the UAVs should cover to increase the wildlife detection rate, which is a challenging problem as stated above. g g p There needs an efﬁcient wildlife monitoring strategy using a multiple UAV system to maximize the wildlife detection rate. In this study, we propose that a team of UAVs search for animal herds reﬂecting the density distribution that describes the probability of ﬁnding animals in the domain. This density distribution can be constructed from the last-received GPS tracker information or satellite images. In this case, the UAVs should spend more time on the high probability area while exploring the low probability area with less time since the probability of the given distribution indicates how likely the UAVs can ﬁnd animals. As animals do not necessarily stay at one location and move around the domain, the density distribution also needs to change for the spatio-temporal evolution of the distribution. Looking from the above perspective, the proposed wildlife monitoring strategy must address the following research questions: 1) what is the proper metric to measure the similarity between the distribution formed by the trajectories of UAVs and the given density distribution? 2) what is the control method for the team of UAVs to achieve the similarity between the two distributions? 3) how to incorporate the spatio-temporal evolution of the given density distribution for the wildlife movement into the control method? Regarding the ﬁrst question, we introduce the Optimal Transport (OT) problem. • Wasserstein distance: Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 2. Problem Description Suppose that a team of UAVs is deployed to monitor wildlife as shown in Fig. 1. Due to the time-varying nature of wildlife locations, it is not an easy task to detect animal herds using a team of UAVs even if locational information is available from the GPS trackers. The GPS trackers only provide limited information with intermittent data to save the battery. The size of the domain is another factor obstructing the detection of Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 where ψ is a probability measure, Ψ(µ, ν) indicates the collection of all probability measure with marginals µ and ν on spaces X and Y, respectively and c(x, y) = ∥x −y∥p is the Euclidean distance with pth order (p ≥1) between x ∈R2 and y ∈R2 (for two dimensional case). This Wasserstein distance describes the least amount of effort required to convert one distribution µ into another one ν. For the transportation problem in the discrete marginal case with µ and ν indi- cating particles of the given two distributions, the following linear programming (LP) formulation is equivalent to the Wasserstein distance where the given distributions are represented by the sample points. Linear Programming problem: (for p = 1) minimize πij ∑ i,j πij∥xi −yj∥ subject to πij ≥0, N ∑ j=1 πij = m(xi), i = 1, 2, . . . , M, M ∑ i=1 πij = n(yj), j = 1, 2, . . . , N, (1) (1) where xi, yj ∈R2 are the locations sample points of the ensemble (for two-dimensional scenarios), m(xi), n(yj) ∈R are some non-negative constants representing the mass or weight assigned to each particle in the ensemble. The variable πij denotes the transportation plan which indicates the amount of weight that needs to be delivered from xi to yj. Hence, the optimal transport plan π∗ ij can be interpreted as the minimum effort required to transport the mass from each xi to yj. j The Wasserstein distance in the LP form will be employed to measure the similarity between the two distributions, one from the trajectories of the UAVs and the other from the given reference distribution. For the second research question, which is how to control the UAVs to achieve the similarity, the OT-based multi-UAV exploration strategy is proposed in Section 4. For the time-varying spatial distribution case associated with the third research question, we extend our results to the spatio-temporal distribution case in Section 5. Prior to further discussions on the proposed multi-UAV exploration scheme, the animal movement modeling is discussed ﬁrst in the following section. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 Traditionally, the objective of the optimal transport is to obtain an optimal solution for a resource allocation problem [22], where the focus is to determine how a distribution can be transformed into another distribution with minimum effort. This minimum effort can be quantiﬁed using the Wasserstein distance for the continuous marginal case. This metric has been utilized in wide range of dynamical systems including system analysis [23], [24], [25] and controller synthesis [26], [27] problems. The Wasserstein distance [22] of order p can be written in the following form. • Wasserstein distance: • Wasserstein distance: Wp(µ, ν) := inf ψ∈Ψ(µ,ν) Z X×Y ∥x −y∥pdψ(x, y)\|ψ ∈Ψ(µ, ν) 1 p , Figure 1. Illustration of wildlife monitoring using a team of UAVs Wp(µ, ν) := inf ψ∈Ψ(µ,ν) Z X×Y ∥x −y∥pdψ(x, y)\|ψ ∈Ψ(µ, ν) 1 p , Figure 1. Illustration of wildlife monitoring using a team of UAVs 3. Animal Movement Modeling Among numerous different models to predict and model stochastic animal move- ment behavior, the simplest approach to explain the stochastic nature of the animal movement is the uncorrelated and unbiased random walk based on the Brownian mo- tion. In this model, the animal movement directions are assumed to be uncorrelated - the current heading direction of the animal is not inﬂuenced by the previous heading directions and unbiased - the animal movement direction is not inﬂuenced by a speciﬁc direction or location. The location of the animal at any time is simply inﬂuenced by the previous location, and the heading direction at any time is completely random. However, due to the two biological constraints related to most animals: bilateral symmetry and cephalocaudal polarization (responsible for an animal’s tendency to move forward) ac- cording to [28], this simple random walk model is unable to represent a realistic animal movement behavior. Additionally, in many realistic scenarios, the animals are inclined to go to speciﬁc locations for food, shelter, migrations, etc., which also cannot be included in the uncorrelated and unbiased random walk models. To incorporate the aforementioned biological constraints and global directional bias in the animal movement modeling, two separate random walk models were derived from the uncorrelated and unbiased random walk model: the Correlated Random Walk (CRW) and the Biased Random Walk (BRW). Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 (a) given spatial distribution (b) sampling stage (c) multi-UAV wildlife monitor- ing Figure 2. Procedure to generate the multi-UAV trajectory using the optimal transport theory (c) multi-UAV wildlife monitor- ing (a) given spatial distribution Figure 2. Procedure to generate the multi-UAV trajectory using the optimal transport theory The CRW model is developed under the assumption that there exists a correlation between consecutive heading directions of animals, which is deﬁned as ‘persistence’. The persistence term explains local directional bias for an animal since the current heading direction is biased by the previous heading angle, which ensures that the animal intends to move in the forward direction. However, there exists some uncertainty associated with the heading directions, which results in making the heading direction different from the initial heading direction and therefore, the effect of the initial heading direction decreases in time. 4. OT-based Multi-UAV Exploration: Time-Invariant Case This section presents a detailed explanation for the multi-UAV exploration strat- egy for the time-invariant distribution case. The extension to the time-varying case is provided in the next section. Given na ∈N numbers of UAVs deployed for the wildlife monitoring, the proposed exploration strategy is to determine the trajectory for the UAV k, k = 1, . . . , na in the team. The OT-based multi-agent exploration strategy is developed considering the limited energy for the agents to carry out the monitoring mission with the given reference spatial distribution. This limited energy of the UAVs also limits the total ﬂight time of the agents, which can be transformed into the total number of UAV points Ma ∈N for each agent by the speciﬁed velocity and discrete-time interval ∆t. Here, it is assumed that all agents have identical energy levels initially and therefore, the UAV points Ma is the same across all agents. Given that the agent k has Ma numbers of points, each UAV point is assumed to be uniformly distributed with the weight m(xk t ) = 1 Ma , t = 1, . . . , Ma at any discrete-time t ∈N. The weight m(xk t ) is assigned to each UAV point, describing the time-averaged behavior of the UAVs. Similar to the weights for each UAV point, the weights are uniformly assigned to each sample point in the given reference distribution. Given N ∈N numbers of sample points, each sample point has the equal weight nt=1(yj) = 1 N initially. Unlike the weight of UAV points m(xk t ), the weights for sample points are time-dependent and decrease over time. This is because a sample point closely located to the UAV position can be considered as visited and hence, the sample point will lose its weight (priority) as the UAVs explore the given domain, which is reﬂected by the time-varying weight nt(yj). This weight change for the sample points depends on the weight update law, which will be explained later in detail. Consider that there are na ∈N numbers of agents deployed for the wildlife moni- toring. In the beginning (when t = 1), all the UAV points {{xk t }M t=1}na k=1 are accumulated at the current positions {xk t=1}na k=1. • Individual animal movement model (BRW): • Individual animal movement model (BRW): zq T+1 = zq T + [rq,T+1 cos(θq,T+1) rq,T+1 sin(θq,T+1)]∆t (3) θq,T+1 = arctan uy,T+1 −zq y,T+1 ux,T+1 −zq x,T+1 ! + vq,T+1, (3) where T ∈N is discrete time, uT = [ux,T, uy,T] is the herd center location, zq T = [zq x,T, zq y,T] is the qth animal position. Also, r(·),T+1 denote speeds of herd centers and individual animals, where r(·),T+1 ∼Γ(µγ, σγ) are random variables with the gamma distribution Γ with a mean µγ and standard deviation σγ. To introduce randomness in movement directions of both herd centers and individual animals, the random variables v(·),T+1 are added to the heading directions θ(·),T+1, where v(·),T+1 ∼V(µvm, κvm) follow von Mises distribution V with a mean µvm and concentration measure κvm. The time interval between consecutive time steps is denoted by ∆t. 3. Animal Movement Modeling In the BRW model, there exists global directional bias in the animal movement directions, meaning that an animal following the BRW model will intend to move towards a speciﬁc direction or a location at all times. This directional bias can be long term (annual migration) or short term (i.e., daily foraging for food) and the speciﬁc location for the directional bias be can be either moving (i.e., herd center) or stationary (i.e., food, water, shelter). Similar to the CRW model, there will be some uncertainty regarding the movement direction at any time although the animal will have a higher probability to move towards the target location or direction. Given that there exists some persistence in the direction of the animals while moving towards a speciﬁc direction, this special form of BRW is deﬁned as the Biased-Correlated Random Walk (BCRW). Here, the animal movement direction at any time is inﬂuenced by both the previous heading direction (local directional bias) and the speciﬁc direction (global directional bias). p g The Correlated Random Walk (CRW) model has been adopted in broad literature to explain individual behavior of stochastic movement for animals, ﬁshes, insects, etc. [28–32]. In the meantime, this random walk model can hardly be utilized to replicate the group behavior of animal herds since this model cannot establish a link between individual animal movement direction and the overall herd location, which is essential for maintaining the integrity of animal herds. An animal movement strategy to explain the group behavior of animal herds was proposed in [33], where the movement of the animal group centers was modeled using BCRW and the individual animal movements were followed by either CRW or BRW, where the animals for the BRW model were biased to the herd center. This study demonstrated that the group dynamics model can explain the group-inﬂuenced behavior of animals. In this work, a simpliﬁed version of [33] is implemented, where the center of the animal herds and individual animals in the herds follow CRW and BRW with a bias toward the herd center, respectively. The implemented model helps ensure the following: The members of the animal herds are biased to move towards the herd center, which ensures herd integrity. The herd centers and the overall herd maintain a stochastic free foraging behavior. The CRW and BRW models employed in this paper to model the group behavior of animal herds are Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 doi:10.20944/preprints202103.0525.v1 • Group center movement model (CRW): • Group center movement model (CRW): uT+1 = uT + [ru,T+1 cos(θu,T+1) ru,T+1 sin(θu,T+1)]∆t (2) θu,T+1 = θu,T + vu,T+1 (2) • Individual animal movement model (BRW): • Individual animal movement model (BRW): Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Figure 3. Schematic of UAV points with their relative locations at different time steps for two agents. Initial points: {xk t=1}2 k=1, past points: {{xk t }T−1 t=2 }2 k=1, and current and future points: {{xt}Ma t=T}2 k=1 Figure 3. Schematic of UAV points with their relative locations at different time steps for two agents. Initial points: {xk t=1}2 k=1, past points: {{xk t }T−1 t=2 }2 k=1, and current and future points: {{xt}Ma t=T}2 k=1 The following assumption is provided to generalize this policy on the UAV point update. Assumption 1. Given the current UAV position of agent k, xk t=T at any time T ∈N, the weight m(xk t ), t = 1, 2, . . . , T −1, for the past UAV points is evenly distributed by 1 Ma . The undetermined future UAV points {xk t }M t=T+1 and the current UAV point of agent k are all accumulated in the current UAV position, xk t=T, which has remaining weights ∑Ma t=T 1 Ma = Ma−T+1 Ma . For notational ease, xk t=T will be replaced by xk T to indicate the position of agent k at time T when the meaning is clear. Next, we introduce the OT-based multi-UAV wildlife monitoring scheme under Assumption 1. 4.1. Methodology: A Three-Stage Approach During the monitoring mission, each agent follows the three-stage approaches: the next goal point determination, the weight update, and the weight information exchange and update stage. Each stage is explained in detail as follows. 4. OT-based Multi-UAV Exploration: Time-Invariant Case The UAVs move to new locations {xk t=2}na k=1 in the next discrete-time step t = 2 based on the proposed exploration strategy (which will be explain later in this section) and then, each of them leaves one particle at their previ- ous locations {xk t=1}na k=1 while taking all the remaining UAV points {{xk t }M t=2}na k=1 with them to the new location {xk t=2}na k=1. In this case, each of the previous UAV positions {xk t=1}na k=1 has the weight of m(xk t=1) = 1 Ma and the weight for each new UAV position is m(xk t=2) = Ma−1 Ma . The schematic for this concept is illustrated in Fig. 3. 4.1.1. Next goal point (gxk T+1) determination stage 4 (b), the sequence of sample points in the third trajectory (when l = 3) is given by {σl=3 j }3 j=1 = {y2, y1, y3}. example provided in Fig. 4 (b), the sequence of sample points in the third trajectory (when l = 3) is given by {σl=3 j }3 j=1 = {y2, y1, y3}. j j Once completed, the cost corresponding to each trajectory is calculated, where the cost function is deﬁned to determine the local-optimal trajectory for kth agent as follows. Ck(l) = ∥yσl 1 −xk T∥ nk T(yσl 1) + h ∑ j=2 ∥yσl j −yσl j−1∥ nk T(yσl j ) , (4) (4) where yσl j , j = 1, . . . , h, denote the sample points found within the circle such that σl j−1 ̸= σl j and nk T(yσl j ) is the weight information of the sample points located within the circle known to agent k. k The cost function Ck(l) in (4) is deﬁned in this way to ensure that each agent follows a trajectory with a shorter travel length in terms of the total Euclidean distance as well as that connects the sample points yj with the high weights nk T(yj) in the circle ﬁrst in order to drive the agent towards high priority sample points. g g p y p p Given the deﬁnition of the h-step trajectory from time T + 1 to T + h for agent k, xk T+1:T+h := {xk T+1, xk T+2, . . . , xk T+h}, the candidate trajectory for the agent cxk T+1:T+h(l), l = 1, 2, . . . , h!, can be obtained from the tree structure. From the candidate trajectories, the h-step local-optimal trajectory gxk T+1:T+h is determined by gxk T+1:T+h = {cxk T+1:T+h(l⋆) \| l⋆= argminlCk(l)} (5) (5) Each agent considers the ﬁrst point of the h-step local-optimal trajectory gxk T+1:T+h as the next goal point gxk T+1 in the next time step T + 1 and then, heads toward that location with the given UAV dynamics. 4.1.1. Next goal point (gxk T+1) determination stage Given that the agents are located at {xk T}na k=1 at any discrete-time step T ∈N, the agents determine the next goal position for the next time step {gxk T+1}na k=1 as following. Each agent creates a circle with the center at the current agent location xk T and the initial radius r0. The radius of the circle increases incrementally by a radius increment δ until the agent ﬁnds h number of sample points within the circle. Then, the agent generates all possible trajectories connecting the sample points found in the circle, starting from the current agent position xk T. To generate the possible trajectories, each agent creates its own tree structure representing all candidate trajectories formed by connecting the sample points in the circle starting from the current agent position xk T. For h numbers of sample points within the search circle, a total of h! trajectories can be generated by each agent in the tree structure. A schematic for the process to determine one possible trajectory is illustrated in Fig. 4 (a) and the complete tree structure is presented in Fig. 4 (b) (in this case, h = 3). The sequence of sample points in lth candidate trajectory can be denoted by σl j, j ∈ {1, 2, . . . , h}, where j indicates the sample point index and l ∈{1, 2, . . . , h!} is an index that represents a speciﬁc candidate trajectory in the tree structure. In the illustrative Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 (a) (b) Figure 4. Schematic of the next goal point gxk T+1 determination process for agent k: (a) increase the radius of the search circle until h numbers of sample points are found; (b) construct a tree associated with the found points yj and then select a particular path (red arrows) that has a minimum cost (b) (a) (a) (b) (a) Figure 4. Schematic of the next goal point gxk T+1 determination process for agent k: (a) increase the radius of the search circle until h numbers of sample points are found; (b) construct a tree associated with the found points yj and then select a particular path (red arrows) that has a minimum cost example provided in Fig. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 where πk⋆ (T+1)j denotes the optimal transport plan for agent k at time T + 1 depicting the weight distribution plan from the current agent position xk T+1 to the sample points {yj}N j=1. The optimal transport plan can be obtained from the solution of the following LP problem. subject to πk (T+1)j ≥0, N ∑ j=1 πk (T+1)j = 1 Ma , πk (T+1)j ≤min nk T(yj), 1 Ma , ∀j. (7) (7) The optimal solution πk⋆ (T+1)j for the LP problem (7) provides the information about how much weight should be distributed from 1 Ma for the new agent k position xk T+1 to the sample point weight nk T(yj) for each sample point yj. Although all the new and future UAV points {xk t }Ma t=T+1 are concentrated at the new agent position xk T+1, agent k is allowed to distribute only the assigned weight 1 Ma to the sample points {yj}N j=1. This is mainly because the future UAV points {xt}Ma t=T+2 are still undetermined and therefore, agent k can only distribute the weight for the future UAV points in the future time steps. g y g p p The ﬁrst constraint in (7) ensures that the transport plan π(T+1)j from xk T+1 to {yj}N j=1 has a non-negative value. The second constraint is included to guarantee that the law of mass conversation is satisﬁed, meaning that the total weight distributed from the new agent position xk T+1 for agent k and the weight received by the sample points {yj}N j=1, both must be the same. The last constraint guarantees that the transport plan π(T+1)j should not exceed the maximum weight capacities of the sample points and the UAV point. After calculating the optimal solution πk⋆ (T+1)j of (7), the weight of the sample points is updated by agent k using (6). Since the new UAV location xk T+1 for agent k is a single point, the analytical solution for (7) can be obtained by the following proposition. The optimal solution for the LP problem (7) is obtained by repeating πk (T+1)j⋆= min nk T(yj⋆), m(xk T+1) , where j⋆= arg min j∈{j\|nk T(yj)>0} ∥xk T+1 −yj∥ m(xk T+1) = m(xk T+1) −πk (T+1)j⋆ nk T(yj⋆) = nk T(yj⋆) −πk (T+1)j⋆ until m(xk T+1) becomes zero. Proof. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Given the new position of agent k at time T + 1, xk T+1, the optimal transport plan for agent k is to deliver the maximum permissible weight to the closest points with positive weights in order until the weight m(xk T+1) remains positive. 4.1.2. Weight update stage After arriving at a new location xk T+1, which may differ from the next goal point location gxk T+1, the agents update their own weight information nk T+1(yj) of the sample points yj from the weight update law given by nk T+1(yj) = nk T(yj) −πk⋆ (T+1)j, ∀j (6) (6) 4.2. Algorithm Then, the central agent receives the updated individual weight information nk T(yj) from other agents, updates the common weight information nT(yj) from (8), and transmits the common weight information to all agents. These procedures are performed in every time step T until the current time step T becomes Ma. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 This common weight information is transmitted to all agents at each time step. By sharing the common weight information, each UAV can know which areas are already covered by other UAVs. Thus, the team of UAVs can explore the given spacious domain efﬁciently. This common weight information is transmitted to all agents at each time step. By sharing the common weight information, each UAV can know which areas are already covered by other UAVs. Thus, the team of UAVs can explore the given spacious domain efﬁciently. 4.2. Algorithm 4.2. Algorithm The formal algorithm of the OT-based multi-UAV exploration strategy is presented in Algorithm 1. At the beginning of the exploration, all parameters are initialized as in Algorithm 1 Multi-Agent Exploration Algorithm 1: initialize xk 1, yj, Ma, N, r0, δ, h, na, T ←1 2: while T ≤Ma do 3: each agent implements the following 4: for k ←1 to na do 5: initialize circle’s radius by r ←r0 6: while #R(xk T, r) ≤h and nk T(yj) > 0 do 7: r ←r + δ 8: end while 9: calculate the cost function Ck(l) associated with all possible candidate trajec- tories cxk T+1:T+h(l) 10: obtain gxk T+1 from (5) 11: update the UAV position xk T with the given UAV dynamics with the calculated next goal position gxk T+1 12: update the individual weight nk T(yj) by (6) 13: end for 14: the central agent 15: receives information about nk T(yj) from all agents 16: updates the common weight nT(yj) from (8) 17: transmits nT(yj) to all corresponding agents 18: each agent receives nT(yj) from the central agent and nk T(yj) ←nT(yj) 19: T ←T + 1 20: end while Algorithm 1 Multi-Agent Exploration Algorithm the ﬁrst line of Algorithm 1. At any time T ≤Ma, each agent creates a circle centered at the current UAV position xk T and increases the circle radius r by δ until there are h number of sample points with positive weight in R(xk T, r), which denotes the set of sample points located within the search circle centered at xk T and radius r. Next, a tree structure is generated by each agent for all possible trajectories connecting the sample points with positive weight located in the search circle, starting from the current UAV position xk T. Then, the cost for each trajectory is calculated from (4) and the next goal position gxk T+1 is determined using (5). Once the next goal point is determined, the agent heads towards its corresponding goal points using their motion controllers and moves to a new location xk T+1. After reaching a new location, each agent distributes 1 Ma amount to weight to the sample points {yj}N j=1 and updates the weight information nk T(yj) using (6). 4.1.3. Weight information exchange and update stage Once the weight update of the sample points is completed by all agents, this infor- mation is shared with the central agent that receives all information {{nk T(yj)}N j=1}na k=1 from agents and transmits the common value to them in every time step. The weight update process for the common weight nT(yj) is provided as follows: nT(yj) = min(nk T(yj)), k = 1, 2, . . . , na (8) (8) Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 to be time-varying as well to reﬂect the time-varying nature of the animal herd locations. This section will provide the method to generate and propagate the sample points. q G p g p p g p p At any time T, let Z = {zq T}G q=1 be the tracking information containing the locations of G numbers of tracked animals obtained by the GPS trackers. If the distance between any two tracked animals is within a speciﬁc distance given as a threshold, they are considered as the same herd. Otherwise, they will be members of different herds. y Since the animal herd locations are mostly unknown, clusters of sample points need to be assigned to the herds which are determined from the available tracking information. For the animal herd with tracked animals, its distribution is given as Gaussian distribution initially. The center of each distribution is assigned to the tracked animal locations in the herd. If more than one tracked animal is in the herd, the center of the corresponding Gaussian distribution is considered as the mean of the locations of the tracked animals. The covariance of the distribution is considered as a user-deﬁned parameter. p The next step is to propagate the sample points for the estimation of the animal herds wandering around. To this end, the Correlated Random Walk model in (2) is employed to propagate each sample point, since the CRW model is associated with the drift of the animal herds. The variables uT+1, ru,T+1, θu,T+1 and vu,T+1 in (2) can be replaced by variables yj,T+1, rj,T+1, θj,T+1 and vj,T+1, followed by the sample point propagation based on (2). The sample point propagation using the CRW model alone cannot improve the performance of the monitoring as it does not incorporate an estimation correction procedure if the agents detect any animals during the monitoring mission. Hence, the center of the sample points associated with the herd of the detected animal is relocated to zq T+τ when an animal located at zq T+τ is detected by the UAV at time T + τ, where τ represents the time elapsed after the tracking information is received. After this relocation, the sample points in the distribution with the mean located at zq T+τ continue propagating using (2). Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 As the proposed method is for the centralized scheme, the sample point propagation, animal herd detection, and sample point correction are shared with all agents through communication and information sharing. Xmin = min(Xa), Ymin = min(Ya) (9) Xmax = max(Xa), Ymax = max(Ya), 5. Sample Point Generation and Propagation: Time-Varying Case 5. Sample Point Generation and Propagation: Time-Varying Case In the previous section, the OT-based multi-UAV exploration strategy is proposed for the time-invariant case, which is not appropriate for the animal herds wandering around their habitat. Therefore, the reference distribution (or the sample points) needs Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 6. Other Exploration Strategy: Lawn Mower method For the performance comparison purpose, we introduce another monitoring strat- egy - lawn mower exploration scheme, one of the most widely used methods to explore the given domain. A description of the lawn mower exploration method is provided below. In the lawn mower monitoring strategy, a single or multiple agents are tasked with exploring an area of interest uniformly in a zigzag manner. For the wildlife monitoring application, the exploration area can be determined from the tracked animal information. If multiple agents are deployed for exploration, then the exploration area is divided equally between multiple agents for independent but balanced exploration. Each agent generates equally spaced horizontal and vertical line segments to create way-points and explores the assigned region uniformly as depicted in Fig. 5. Fig. 5 (a) provides the conceptual drawing to show how the exploration area is determined and Fig. 5 (b) illustrates the way-point generation for the two-agent case. Given that G ∈N numbers of animals are being tracked and the locations of these animals {zq T}G q=1 at time T are known from the GPS trackers (presented as red triangle symbols in Fig. 5 (a)), the sets of x-coordinates and y-coordinates for these known animal locations are Xa = {z1 x, z2 x, . . . , zG x } and Ya = {z1 y, z2 y, . . . , zG y }, respectively. Then, the parameters Xmin, Ymin, Xmax, Ymax to determine the initial search area (rectangular area ABCD in Fig. 5 (a)) can be calculated by Xmin = min(Xa), Ymin = min(Ya) (9) Xmax = max(Xa), Ymax = max(Ya), (9) Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 (a) Exploration area determination (b) Waypoint generation Figure 5. Schematic for the Lawn Mower exploration strategy with two agents (a) Exploration area determination (a) Exploration area determination Figure 5. Schematic for the Lawn Mower exploration strategy with two agents where Xmin, Xmax ∈R (or Ymin, Ymax ∈R) are the minimum and maximum x-coordinates (or y-coordinates) of the initial search area, respectively. In practical scenarios, the base station for the team of UAVs may be located far away from the location of the detected animal herds by the GPS trackers. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 the exploration region for agent 1 is limited by x1 0 to x2 0 in the horizontal direction and Y′ min to Y′ max in the vertical direction and the rest of the area is assigned to agent 2. min Next, the way-points for each agent are provided in the following manner. The distance between two consecutive waypoints on the vertical line is given as dw and the spacing between two adjacent vertical line is denoted by dv. The parameters dw and dv can vary to adjust how densely the total exploration area needs to be monitored. 7. Simulations In this section, various simulation results are presented to validate the effectiveness of the proposed multi-UAV wildlife monitoring scheme. Two major factors considered as simulation parameters are the number of agents and exploration time (caused by energy limit). To compare the performance of the OT-based multi-UAV monitoring scheme with time-varying spatial distribution OT (TV-Gauss), two other exploration strategies are employed: Lawn Mower method with time-invariant uniform exploration, LM (TI-Uni), and OT-based multi-UAV monitoring strategy with time-invariant uniform distribution, OT (TI-Uni). For all simulation scenarios, the unicycle robot dynamics is considered for the UAV dynamics. A brief description of the unicycle robot dynamics is provided below. where Kθ represents the angular error gain. 7.2. Variation in the Number of Agents 7.1. Unicycle Robot Dynamics Given the UAV located at xT = [xT, yT]T with xT, yT ∈R at any time T ∈N, the UAV position for the next time step T + 1 is updated by using the following unicycle model:    xT+1 = xT + v cos(θT + ω∆t)∆t yT+1 = yT + v sin(θT + ω∆t)∆t θT+1 = θT + ∆θT, where v and ω denote the linear and angular velocity of the UAV, respectively, θT and ∆θT, respectively, indicate the heading angle and change of the heading angle for the UAV, and ∆t is the time interval between consecutive discrete-time steps. From the current location xT at time T, if the next goal point is given by gxT+1, then the positional error is deﬁned as xe = gxT+1 −xT and the required transnational velocity v to compensate the positional error can be determined by v = Kx · xe ∆t , where Kx denotes the positional error gain. Also, for the current heading angle error θe θT+1 θT, where θT+1 arctan gyT+1 −yT gxT+1 −xT , the angular velocity ω required for minimizing the heading angle error is obtained from the following equation. , g g e T+1 T, T+1 gyT+1 −yT gxT+1 −xT , the angular velocity ω required for minimizing the heading angle error is obtained from the following equation. ω = Kθ · θe ∆t Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints2 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 6. Other Exploration Strategy: Lawn Mower method When the UAVs arrive at the last updated GPS locations, the animals may not be there anymore as they may have moved to another location. Thus, the monitoring domain should be expanded considering the time delay after dispatching a team of UAVs. The expansion will be given in both horizontal and vertical directions in an unbiased manner since the animal movement directions are completely unknown. The parameters X′ min, Y′ min, X′ max, Y′ max for the expanded search area (rectangular area A’B’C’D’ in Fig. 5 (a)) can be calculated from X′ min = Xmin −fX(Xmax −Xmin) (10) Y′ min = Ymin −fY(Ymax −Ymin) X′ max = Xmax + fX(Xmax −Xmin) Y′ max = Ymax + fY(Ymax −Ymin), (10) where X′ min, X′ max ∈R (or Y′ min, Y′ max ∈R) denote the minimum and maximum value of the x-coordinates (or y-coordinates) of the expanded search area, respectively. Moreover, fX, fY ∈R are deﬁned as the expansion factors in the horizontal and vertical directions, respectively. Once the expanded search area is determined, the area is divided equally based on the number of agents na ∈N as shown in Fig. 5 (b). The initial way-point for each agent can be determined recursively as follows: xk 0 = xk−1 0 + X′ max −X′ min na , Y′ min k = 1, 2, . . . , na, (11) (11) where xk−1 0 ∈R is the x-coordinate of the initial position for (k −1)th agent. For the ﬁrst agent, x1 0 = X′ min. In this work, the total exploration area, which is the expanded search area, is partitioned vertically, meaning the range of the exploration region assigned to each agent in the vertical direction is the same as the range of the total exploration area in the same direction. Only the range of the exploration region assigned to each agent in the horizontal direction is limited, which varies from xk 0 to xk+1 0 for any agent k. For instance, Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.05 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 • No. of agents: 2, 3, 5 • No. of agents: 2, 3, 5 g • No. of simulation runs for each case: 30 • Monitoring time: 900 seconds • Time delay (traveling time from the base station to the monitoring area): 600 seconds • No. of animal herds: 9 • No. of animal herds: 9 • No. of animal herds: 9 The time delay (or equivalently the traveling time) in this context indicates the total time for the UAVs to travel from the base station to the monitoring region. The UAVs are regarded as having a monitoring mission when they arrived at the predeﬁned initial positions for the monitoring. OT (TI-Uni) is implemented to compare the performance with the proposed scheme, OT (TV-Gauss). Similar to the Lawn Mower method, an initial rectangular exploration area for OT (TI-Uni) method is determined from the animal locations obtained from the GPS trackers by (9). Next, the total exploration area is determined using (10) from the initial search area. Then, this area is ﬁlled with randomly generated sample points with uniform distribution. Based on this uniform sample point representation, multiple UAVs carry out the monitoring mission using the three-stage approach. All agents explore the monitoring area as a team, unlike the Lawn Mower method, where each agent is assigned to a pre-partitioned monitoring area. The initial animal herd center locations and the population of the herds are assumed to be identical for all scenarios, which are given as following: x (m) 300 800 400 750 150 0 500 700 200 y (m) 400 800 650 550 750 400 500 300 200 No. of animals 10 15 18 20 15 20 15 13 16 In every time step, the animal herd movements are simulated using the following distribution parameters: • ru,T+1, rk,T+1 ∼Γ(µγ = 0.4 m/s, σγ = 1 m/s) • vu,T+1 ∼V(µvm = 0, kvm = 100) • vk,T+1 ∼V(µvm = 0, kvm = 2) • −90◦≤vu,T+1 ≤90◦ • θu 0 = vu 0 ∼V(µvm = 0, kvm = 0), • ru,T+1, rk,T+1 ∼Γ(µγ = 0.4 m/s, σγ = 1 m/s) • vu,T+1 ∼V(µvm = 0, kvm = 100) • vk,T+1 ∼V(µvm = 0, kvm = 2) • −90◦≤vu,T+1 ≤90◦ • θu,0 = vu,0 ∼V(µvm = 0, kvm = 0), , • θu,0 = vu,0 ∼V(µvm = 0, kvm = 0), where the meaning of each symbol is given in Section 3. where the meaning of each symbol is given in Section 3. where the meaning of each symbol is given in Section 3. 7.2. Variation in the Number of Agents Since one of the parameters that signiﬁcantly affect the monitoring performance (detection rate) is the number of agents, we test how the different number of UAVs results in the performance variation. The simulations were carried out considering the following scenarios: • Exploration strategies: OT-based multi-UAV strategies with time-varying Gaussian (OT (TV-Gauss)) and time-invariant uniform (OT (TI-Uni)) distributions and Lawn Mower method with uniform distribution (LM (TI-Uni))) • Exploration strategies: OT-based multi-UAV strategies with time-varying Gaussian (OT (TV-Gauss)) and time-invariant uniform (OT (TI-Uni)) distributions and Lawn Mower method with uniform distribution (LM (TI-Uni))) doi:10.20944/preprints202103.0525.v1 OT (TV-Gauss) (a) T=-600 s (b) T=400 s (c) T=900 s OT (TV-Gauss) (a) T=-600 s (b) T=400 s (c) T=900 s LM (TI-Uni) (d) T=-600 s (e) T=400 s (f) T=900 s OT (TI-Uni) (g) T=-600 s (h) T=400 s (i) T=900 s . Snapshots of different monitoring strategies for the 2-agent case: (a)-(c) OT (TV-Gauss); (d)-(f) LM (TI-Uni); (g)-(i) OT ative time indicates the time to travel from the base station to the pre-speciﬁed location before the initiation of the m The monitoring mission starts at T = 1 second and continues until 900 seconds. OT (TV-Gauss) (a) T=-600 s (b) T=400 s (c) T=900 s (b) T=400 s (c) T=900 s LM (TI-Uni) (d) T=-600 s (e) T=400 s (f) T=900 s OT (TI-Uni) (g) T=-600 s (i) T=900 s (h) T=400 s (g) T=-600 s Figure 6. Snapshots of different monitoring strategies for the 2-agent case: (a)-(c) OT (TV-Gauss); (d)-(f) LM (TI-Uni); (g)-(i) OT (TI-Uni). The negative time indicates the time to travel from the base station to the pre-speciﬁed location before the initiation of the monitoring mission. The monitoring mission starts at T = 1 second and continues until 900 seconds. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 – Number of sample points: N = 3600 – Number of sample points: N = 3600 p p – Maximum number of UAV steps for each agent for monitoring: Ma = 900 p p – Maximum number of UAV steps for each agent for monitoring: Ma = 900 p p – Maximum number of UAV steps for each agent for monitoring: Ma = 900 – Initial covariance for the sample point clusters: – Initial covariance for the sample point clusters: – Initial covariance for the sample point clusters: Σ = 1000 0 0 1000 – Distribution parameters for the sample point propagation – Distribution parameters for the sample point propagation * rj,T+1 ∼Γ(µγ = 0.6 m/s, σγ = 0.05 m/s) * vj,T+1 ∼V(µvm = 0, kvm = 150) * θj,0 = vj,0 ∼V(µvm = 0, kvm = 0) – Herd threshold: 50 m – h = 5, r0 = 0.1, δ = 0.05 – h = 5, r0 = 0.1, δ = 0.05 Parameters for LM (TI-Uni) • Parameters for LM (TI-Uni) – Expansion factor for the horizontal direction, fX = 1 – Expansion factor for the vertical direction, fY = 1 – Distance between adjacent way-points, dw = 10 m – Spacing between adjacent vertical lines, dv: na = 2 na = 3 na = 5 dv (m) 120 70 40 The spacing between adjacent vertical line segments dv for LM (TI-Uni) varies with the number of agents such that the agents can cover most of their assigned monitoring regions within the given amount of time for exploration. With a higher number of agents, the area can be monitored thoroughly and therefore, dv decreases. The discrete-time interval ∆t is assumed to be 1 second for all simulation scenarios. Hence, 900 seconds exploration time corresponds to 900 robot steps for all exploration strategies, meaning that the robot positions are updated using the implemented unicycle robot dynamics in every second. y y For OT (TI-Uni), the initial UAV positions, the number of sample points N, the total number of UAV steps for each agent for exploration Ma, h, r0 and δ are the same as that for OT (TV-Gauss). Additionally, the parameters fX, fY to determine the monitoring area for OT (TI-Uni) is identical to fX, fY for LM (TI-Uni). • No. of animal herds: 9 Also, the initial agent positions for for LM (TI-Uni) are derived from (11) and the initial UAV positions for OT (TV-Gauss) and OT (TI-Uni) are given as: Also, the initial agent positions for for LM (TI-Uni) are derived from (11) and the initial UAV positions for OT (TV-Gauss) and OT (TI-Uni) are given as: Also, the initial agent positions for for LM (TI-Uni) are derived from (11) and the initial UAV positions for OT (TV-Gauss) and OT (TI-Uni) are given as: agent 1 agent 2 agent 3 agent 4 agent 5 x (m) 100 200 200 150 400 y (m) 400 600 150 400 750 The other simulation parameters for the proposed monitoring strategies are as follows. • Common parameters: – Exploration domain size: 2500 m × 3000 m – Maximum velocity of the UAVs: 30 m/s – Minimum velocity of the UAVs: 10 m/s – Angular velocity limit: 30 deg/s – Positional error gain: Kx = 0.4 – Angular error gain: Kθ = 1 – UAV sensor range to detect animals: rsensing = 15 m – Discrete-time interval: ∆t = 1 second • Parameters for OT (TV-Gauss): Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Table 1: Tracking information for OT (TV-Gauss) in Fig. 6 (a)-(c) Animal x (m) y (m) 1 -0.5765 402.5960 2 402.1108 651.829 3 296.2186 398.9142 4 297.0975 396.1902 5 151.9045 747.7304 6 199.6510 205.1129 7 503.1622 496.3184 8 404.9208 648.3867 9 305.2823 403.5791 Table 2: Estimated herd center information for OT (TV-Gauss) in Fig. 6 (a)-(c) Table 1: Tracking information for OT (TV-Gauss) in Fig. 6 (a)-(c) Table 2: Estimated herd center information for OT (TV-Gauss) in Fig. 6 (a)-(c) herd Tracked animals Estimated herd center (m) 1 3,4,9 [299.5328, 399.5612]T 2 5 [151.9045, 747.7304]T 3 1 [−0.5765, 402.5960]T 4 7 [503.1622, 496.3184]T 5 2,8 [403.5158, 650.1079]T 6 6 [199.6510, 205.1129]T for the OT (TV-Gauss) in Fig. 6 (a)-(c), the UAVs detected total 53 animals out of 142 (detection rate: 37.32%). For LM (TI-Uni), a time-invariant rectangular monitoring area is obtained from (9) and (10) by using the tracker information similar to Table 1. The agents start the monitoring mission from the locations determined by (11) after a 600 seconds time delay. The agents explored their assigned areas in a zigzag manner and ﬁnished the exploration 900 seconds after the monitoring started. The detection rate for LM (TI-Uni) in Fig. 6 (d)-(f) was 10.56%. In the case of OT (TI-Uni), the detection rate in Fig. 6 (g)-(i) was 25.35%. g g To better compare the performance of each method, the statistical results for 30 simulation runs are presented in Fig. 7, where the initial locations of detected animals were randomly generated in each run. Although it is observed that the performance of all the monitoring strategies has gradually improved by increasing the number of agents, the performance increase of LM (TI-Uni) and OT (TI-Uni) are less signiﬁcant than OT (TV-Gauss). For all three scenarios in Fig. 7, the proposed method OT (TV-Gauss) outperformed the other two methods. Notice that for all three scenarios, the UAVs had the same energy level (or alternatively the same UAV points) in the beginning for the fair comparison, however, the detection rate for the OT (TV-Gauss) method signiﬁcantly overwhelmed the other two. Thus, it is veriﬁed that the proposed method is able to efﬁciently monitor wildlife as the scheme can take into account the time-varying nature of wildlife locations in the monitoring plan and explore areas accordingly. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 As the initially detected animal locations can be a critical factor affecting the perfor- mance (detection rate), a total of 30 simulations were carried out by randomly generating their detected locations in the beginning. The snapshots of one speciﬁc simulation for three different monitoring strategies are presented in Fig. 6 as examples. This result illustrates how different the UAV trajectories are from each other. The following scenario is considered for all the simulation cases. Among a total of 142 animals in 9 herds, only 9 animals are being tracked via the GPS trackers at time T = −600 (600 seconds before the start of the monitoring mission). Since these tracker locations are the only available information, it is unknown which animal belongs to which herd and how many animals are there in each herd. The received tracker information is presented in Table 1 for the OT (TV-Gauss) method in Fig. 6 (a)-(c) For the OT (TV-Gauss) method, the estimated animal herd number and tracked animals that belong to each herd number are given in Table 2 according to the proposed policy in Section 5. We generated more numbers of sample distributions than the estimated numbers of animal herds. This is mainly because each herd location is unknown to the UAVs when they’ve arrived at the monitoring region and thus, more sample distributions with the proposed sample propagation method can better estimate the possible location of animal herds. If animals are detected by the UAV, then the sample distribution is relocated (both mean and covariance of the Gaussian) for the correction. Based on the proposed scheme Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 7.3. Variation in Exploration Time In order to investigate the effect of exploration time on the performance, other simulations were conducted with the following scenarios. • Exploration strategies: OT (TV-Gauss), OT (TI-Uni), LM (TI-Uni) • Exploration strategies: OT (TV-Gauss), OT (TI-Uni), LM (TI-Uni) • No. of simulation runs: 30 • Exploration times: 900, 1800, 3600 seconds • No. of UAVs: 3 • Time delay: 600 seconds The initial UAV positions for OT (TV-Gauss) and OT (TI-Uni) are given as Other simulation parameters are the same as the previous case except • No. of simulation runs: 30 The initial UAV positions for OT (TV-Gauss) and OT (TI-Uni) are given as Other simulation parameters are the same as the previous case except Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 (a) na = 2 (b) na = 3 (c) na = 5 Figure 7. Statistical data for the performance comparison among OT (TV-Gauss), OT (TI-Uni), and LM (TI-Uni) with the variation in the number of UAVs: (a) na = 2; (b) na = 3; and (c) na = 3. Mean values for the boxplots are indicated by the asterisk symbols (a) na = 2 (b) na = 3 (c) na = 5 (a) na = 2 Figure 7. Statistical data for the performance comparison among OT (TV-Gauss), OT (TI-Uni), and LM (TI-Uni) with the variation in the number of UAVs: (a) na = 2; (b) na = 3; and (c) na = 3. Mean values for the boxplots are indicated by the asterisk symbols OT (TV-Gauss) (a) T=-600 s (b) T=900 s (c) T=1800 s LM (TI-Uni) (d) T=-600 s (e) T=900 s (f) T=1800 s OT (TI-Uni) (g) T=-600 s (h) T=900 s (i) T=1800 s e 8. Snapshots of different monitoring strategies when the exploration time is given by 1800 seconds: (a)-(c) OT (TV-G LM (TI-Uni); (g)-(i) OT (TI-Uni). The negative time indicates the time to travel from the base station to the pre-speciﬁed lo e the initiation of the monitoring mission. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 The parameters fX, fY and dv for LM (TI Uni) are adjusted for different exploration time for the UAVs to explore most of the regions within the corresponding exploration time. p g p g p The snapshots of one simulation result when the exploration time is 1800 seconds with a time delay of 600 seconds are provided in Fig. 8, to illustrate the UAV trajectories for each case. Similar to the simulation for the variation in the number of agents, there are total of 9 tracked animals and 12 Gaussian sample point distributions are generated at the estimated herd centers and then, propagated in every time step. If an animal is detected by the UAV, a sample distribution is assigned to the detected animal herd and the center of that distribution is relocated to the detected animal’s location. For OT (TV-Gauss) in Fig. 8 (a)-(c), the three UAVs detected a total of 93 animals out of 142 (detection rate: 65.49%), whereas the detection rate for LM (TI-Uni) and OT (TI-Uni) were 28.87% and 10.56%, respectively. The statistical data for a total of 30 simulation runs are presented in Fig. 9 for three different exploration times (900, 1800, and 3600 seconds). Increasing the exploration time resulted in the decrease of the average detection rate for all monitoring strategies, which is because the domain size has increased as well with the exploration time increase. From Fig. 9, it is clearly shown that OT (TV-Gauss) outperforms the other two strategies, where the time-varying scenarios cannot be incorporated. As a result, their animal detection rates are quite low compared to the proposed scheme, OT (TV-Gauss), which demonstrates the effectiveness of the proposed method for the wildlife monitoring mission. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 agent 1 agent 2 agent 3 x (m) -100 200 500 y (m) 900 600 150 agent 1 agent 2 agent 3 x (m) -100 200 500 y (m) 900 600 150 • Common parameters: 2 – Exploration domain size: Exploration time (seconds) 900 1800 3600 Domain size ( m2) 2500×2500 3000×4000 7000×7000 3 – Discrete-time interval: ∆t = 1 second 4 • Parameters for OT (TV-Gauss): 5 – Maximum number of UAV steps for each agent for exploration: Ma = 900, 6 1800, 3600 7 • Parameters for LM (TI-Uni) 8 – Expansion factors: Exploration time (seconds) 900 1800 3600 fX, fY 1 0.7 0.5 9 – Spacing between adjacent vertical lines, dv: Exploration time (seconds) 900 1800 3600 dv (m) 70 90 200 • Common parameters: 2 – Exploration domain size: Exploration time (seconds) 900 1800 3600 Domain size ( m2) 2500×2500 3000×4000 7000×7000 3 – Discrete-time interval: ∆t = 1 second 4 • Parameters for OT (TV-Gauss): 5 – Maximum number of UAV steps for each agent for exploration: Ma = 900, 6 1800, 3600 7 • Parameters for LM (TI-Uni) 8 – Expansion factors: Exploration time (seconds) 900 1800 3600 fX, fY 1 0.7 0.5 9 – Spacing between adjacent vertical lines, dv: Exploration time (seconds) 900 1800 3600 • Common parameters: 2 – Exploration domain size: Exploration time (seconds) 900 1800 3600 Domain size ( m2) 2500×2500 3000×4000 7000×7000 3 – Discrete-time interval: ∆t = 1 second 4 • Parameters for OT (TV-Gauss): 5 – Maximum number of UAV steps for each agent for exploration: Ma = 900, 6 1800, 3600 7 • Parameters for LM (TI-Uni) 8 – Expansion factors: Exploration time (seconds) 900 1800 3600 fX, fY 1 0.7 0.5 9 – Spacing between adjacent vertical lines, dv: Exploration time (seconds) 900 1800 3600 dv (m) 70 90 200 – Spacing between adjacent vertical lines, dv: The parameters fX, fY and dv for LM (TI-Uni) are adjusted for different exploration time f th UAV t l t f th i ithi th di l ti ti The parameters fX, fY and dv for LM (TI-Uni) are adjusted for different exploration time for the UAVs to explore most of the regions within the corresponding exploration time. 7.3. Variation in Exploration Time The monitoring mission starts at T = 1 second and continues until 1800 second OT (TV-Gauss) (a) T=-600 s (b) T=900 s (c) T=1800 s (c) T=1800 s (b) T=900 s LM (TI-Uni) (d) T=-600 s (e) T=900 s (f) T=1800 s OT (TI-Uni) (g) T=-600 s (h) T=900 s (i) T=1800 s (h) T=900 s (g) T=-600 s Figure 8. Snapshots of different monitoring strategies when the exploration time is given by 1800 seconds: (a)-(c) OT (TV-Gauss); (d)-(f) LM (TI-Uni); (g)-(i) OT (TI-Uni). The negative time indicates the time to travel from the base station to the pre-speciﬁed location before the initiation of the monitoring mission. The monitoring mission starts at T = 1 second and continues until 1800 seconds. 8. Conclusion In this paper, a new wildlife monitoring strategy was proposed using a team of UAVs based on the optimal transport theory. The proposed works can incorporate complementary information such as GPS trackers into the plan, to increase the wildlife detection rate. Through the OT-based wildlife monitoring scheme, the UAV trajectories Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 (a) Exploration time = 900 s (b) Exploration time = 1800 s (c) Exploration time = 3600 s Figure 9. Statistical data for the performance comparison among OT (TV-Gauss), LM (TI-Uni) and OT (TI-Uni) with 600 seconds delay and 3 agents: boxplots for target detection rates for (a) exploration time = 900 seconds; (b) exploration time = 1800 seconds; and (c) exploration time = 3600 seconds. Mean values for the boxplots are indicated by asterisk symbols (a) Exploration time = 900 s (c) Exploration time = 3600 s (b) Exploration time = 1800 s (b) Exploration time = 1800 s (b) Exploration time = 1800 s (c) Exploration time = 3600 s (a) Exploration time = 900 s Figure 9. Statistical data for the performance comparison among OT (TV-Gauss), LM (TI-Uni) and OT (TI-Uni) with 600 seconds delay and 3 agents: boxplots for target detection rates for (a) exploration time = 900 seconds; (b) exploration time = 1800 seconds; and (c) exploration time = 3600 seconds. Mean values for the boxplots are indicated by asterisk symbols were generated enabling UAVs to collaboratively monitor the wildlife with a given priority. Moreover, the spatio-temporal evolution of animals’ locations was combined with the proposed monitoring scheme, leading to an increase in the wildlife detection rate. Numerous simulations were conducted with variation in the number of UAVs and exploration time while randomly generating the animal locations to validate the proposed method. The statistical data for numerously different scenarios demonstrated that the proposed wildlife monitoring scheme can result in high performance in terms of detection rate. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 22 March 2021 doi:10.20944/preprints202103.0525.v1 12. Valentini, A.; Taberlet, P.; Miaud, C.; Civade, R.; Herder, J.; Thomsen, P.F.; Bellemain, E.; Besnard, A.; Coissac, E.; Boyer, F.; others. Next-generation monitoring of aquatic biodiversity using environmental DNA metabarcoding. Molecular ecology 2016, 25, 929–942. 13. Gray, M.; Roy, J.; Vigilant, L.; Fawcett, K.; Basabose, A.; Cranﬁeld, M.; Uwingeli, P.; Mbu- ranumwe, I.; Kagoda, E.; Robbins, M.M. Genetic census reveals increased but uneven growth of a critically endangered mountain gorilla population. Biological Conservation 2013, 158, 230–238. 14. Dufour, S.; Bernez, I.; Betbeder, J.; Corgne, S.; Hubert-Moy, L.; Nabucet, J.; Rapinel, S.; Sawtschuk, J.; Trollé, C. Monitoring restored riparian vegetation: how can recent develop- ments in remote sensing sciences help? 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https://openalex.org/W4380484428	https://www.researchsquare.com/article/rs-2669166/latest.pdf	English	null	TNF-α activates RELA expression via TNFRSF1B to upregulate OPA1 expression and inhibit chondrogenic differentiation of human adipose stem cells	Journal of orthopaedic surgery and research	2,023	cc-by	7,503	TNF-α activates RELA expression via TNFRSF1B to upregulate OPA1 expression and inhibit chondrogenic differentiation of human adipose stem cells Jiajia Guo Medical College of Guizhou University Wang Ye Guizhou Provincial People's Hospital Xinglin Wu Guizhou Provincial People's Hospital Haifeng Huang Guizhou Provincial People's Hospital Bo Li Guizhou Provincial People's Hospital Zhijing Ren Guizhou Provincial People's Hospital Zhen Yang (  Y1234560603@163.com Guizhou Provincial People's Hospital Jiajia Guo Medical College of Guizhou University Wang Ye Guizhou Provincial People's Hospital Xinglin Wu Guizhou Provincial People's Hospital Haifeng Huang Guizhou Provincial People's Hospital Bo Li Guizhou Provincial People's Hospital Zhijing Ren Guizhou Provincial People's Hospital Zhen Yang (  Y1234560603@163.com ) Guizhou Provincial People's Hospital Results The results showed that the chondrogenic differentiation of hADSCs was inhibited in the presence of TNF-α, that optic atrophy 1 (OPA1) expression was significantly up-regulated and mitochondria were prolonged and interconnected during this process. Gene microarray and RT-qPCR data showed that the presence of TNF-α led to increased expression of TNFα receptor 2 (TNFRSF1B) and RELA during chondrogenic differentiation of hADSCs. Research Article Keywords: OPA1, mitochondrial fusion, TNFRSF1B, RELA, chondrogenic differentiation, human adipose- derived stem cells Posted Date: March 14th, 2023 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Page 1/21 Version of Record: A version of this preprint was published at Journal of Orthopaedic Surgery and Research on June 13th, 2023. See the published version at https://doi.org/10.1186/s13018-023-03846-x. Conclusions TNF-α inhibits chondrogenic differentiation of human adipose stem cells by activating RELA expression through TNFRSF1B upregulating OPA1 expression and thereby increasing mitochondrial fusion. Background Tumour necrosis factor-alpha (TNF-α), one of the pro-inflammatory cytokines mediating the local inflammatory process in joints, has an inhibitory effect on cartilage formation and has a detrimental effect on stem cell-based cartilage regeneration for the treatment of osteoarthritis (OA). However, the mechanisms behind this inhibitory effect are still poorly understood. Mitochondria are important organelles that play a vital role in maintaining the structure and function of the cell. Our study aimed to investigate the role and mechanisms of regulation of mitochondrial fusion and fission in the chondrogenic differentiation of human adipose stem cells (hADSCs) in the absence and presence of TNF- α. Methods We used flow cytometry to identify human adipose stem cells (hADSCs) immunophenotypes CD29, CD44, CD34, CD45, and HLA-DR. Alcian blue staining and Sirius red staining were used to observe the formation of proteoglycans and collagen during the chondrogenic differentiation of hADSCs, respectively. The mRNA and protein expression levels of the cartilage formation marker SOX9, type II collagen (COL2A1), and Aggrecan were measured by real-time fluorescent quantitative PCR (RT-qPCR) and western blot, respectively. The fluorescent probes MitoTracker® Red CMXRos and JC-1 were used to visualize the morphology of mitochondria and to detect mitochondrial membrane electricity (MMP) respectively. Affmetrix PrimeView™ chips for gene expression profiling. Background Page 2/21 Page 2/21 Osteoarthritis (OA) is a progressive disease involving all joint structures, with a heterogeneous syndrome of different clinical phenotypes [1]. The clinical symptoms of OA are pain and functional impairment [2] and there is a lack of effective treatment for OA. The current treatment for OA aims to reduce the progression of the disease, treat the symptoms, reduce pain and ensure as much mobility and function of the joint as possible in the late stages[3]. Cartilage tissue engineering is considered to be an emerging and effective approach to the treatment of OA[4]. Human adipose stem cells (hADSCs) are a type of stem cell with multidirectional differentiation potential isolated from adipose tissue. They can be obtained from liposuction or lipectomy by-products, are less damaging to the donor [5, 6], and have better immunomodulatory ability, making them ideal seed cells for cartilage tissue engineering[7]. Tumour necrosis factor-alpha (TNF-α) is a pro-inflammatory factor that often mediates the local inflammatory process in joints. As cartilage destruction increases, TNF-α secretion in synovial tissue and joint fluid gradually increases[8]. TNF-α has a significant inhibitory effect on articular cartilage formation. It was found that TNF-α inhibits the synthesis of SOX9, a key transcription factor required for cartilage differentiation[9], and suppresses the synthesis of cartilage matrix-type two collagen (COL2A1) [10]and Aggrecan[11]. Mitochondria are highly dynamic organelles that play a key role in a variety of biological processes in stem cells [12]. Mitochondrial fusion allows for the exchange of material and connections within the mitochondria, providing sufficient energy, mitigating oxidative damage, and maintaining the mitochondrial membrane potential (MMP)[13]. Mitochondrial fission provides sufficient numbers of mitochondria to maintain cell polarity and eliminate damaged mitochondria[14]. Mitochondrial fusion and fission are regulated by a series of evolutionarily conserved proteins and kinetically related uridine diphosphate enzymes. Optic atrophy protein (OPA1), located in the inner mitochondrial membrane, primarily mediates the fusion of the inner mitochondrial membrane[15], and dynamin-related protein 1 (Drp1) mediates mitochondrial fission[16]. Mitochondria are the main physiological source of intracellular reactive oxygen species (ROS)[17]. ROS is essential for stem cell differentiation, and extended mitochondria in stem cells maintain low ROS levels and promote self-renewal. In contrast, the shift to a fragmented state of mitochondria leads to a moderate increase in ROS levels, which can inhibit self- renewal and differentiation gene expression[18]. The mitochondrial network varies between different stem cells, pluripotent states, and directions of differentiation. Background This change in mitochondrial morphology, mediated by mitochondrial fusion and fission, is quite sensitive to environmental stimuli and is highly plastic[19]. In this study, TNF-α was used to induce cells to form an OA cell model at the cellular level to investigate the specific effects of TNF-α on mitochondrial fusion/fission during the chondrogenic differentiation of hADSCs and its mechanism, providing a new therapeutic strategy for the treatment of OA. hADSCs culture and immunophenotyping Page 3/21 We have obtained hADSCs from the by-products of liposuction in healthy humans[20]. Second-generation cells were removed from the liquid nitrogen and rapidly resuscitated and cultured in an H-DMEM medium (Pricella, China) containing 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (Sigma, USA). hADSCs were enzymatically separated using 0.25% trypsin-EDTA solution when their proliferation was above 80%, and they were then passed in a 1:3 ratio. Fourth-generation hADSCs are used for experiments. HADSCs were collected and incubated with direct-coupled antibodies CD29 (1:100; cat. no. 11-0299-42; Thermo, USA), CD44 (1:100; cat. no. 11-0441-85; Thermo, USA), CD34 (1:100; cat. no. CD34-581-01; Thermo, USA), CD45 (1:100; cat. no. 12-9459-42; Thermo, USA), and HLA-DR (1:100; cat. no. 12-9956-42; Thermo, USA) on ice for 1h. After incubation, the cells were analyzed by NovoCyte 2060R flow cytometer (ACEA Biosciences, USA). Chondrogenic 、adipogenic and osteogenic differentiation of hADSCs Page 4/21 Table 1 Induced differentiation medium preparation Induced Differentiation Medium Reagent preparation concentration Adipogenic differentiation medium 89% H-DMEM + 1% (streptomycin/penicillin) + 10% FBS + 10 ug/ml insulin + 1 µM dexamethasone(Solarbio, China) + 200 µM indomethacin + 0.5 mM 3-isobutyl-1- methylxanthine (IBMX) Chondrogenic differentiation medium 89% H-DMEM + 1% (streptomycin/penicillin) + 10% FBS + 10 ng/ml TGF-β1(MCE, USA) + 50 µg/ml Vc(Solarbio, China) + 1 µM dexamethasone + 6.25 µg/ml insulin(MCE, USA) Osteogenic differentiation medium 89% H-DMEM + 1%(streptomycin/penicillin) + 10% FBS + 1 uM dexamethasone + 10 mM beta-glycerophosphate disodium + 50 µg/ml Vc Real‑time quantified PCR Page 4/21 Table 1 Induced differentiation medium preparation Induced Differentiation Medium Reagent preparation concentration Adipogenic differentiation medium 89% H-DMEM + 1% (streptomycin/penicillin) + 10% FBS + 10 ug/ml insulin + 1 µM dexamethasone(Solarbio, China) + 200 µM indomethacin + 0.5 mM 3-isobutyl-1- methylxanthine (IBMX) Chondrogenic differentiation medium 89% H-DMEM + 1% (streptomycin/penicillin) + 10% FBS + 10 ng/ml TGF-β1(MCE, USA) + 50 µg/ml Vc(Solarbio, China) + 1 µM dexamethasone + 6.25 µg/ml insulin(MCE, USA) Osteogenic differentiation medium 89% H-DMEM + 1%(streptomycin/penicillin) + 10% FBS + 1 uM dexamethasone + 10 mM beta-glycerophosphate disodium + 50 µg/ml Vc Real‑time quantified PCR Reagent preparation concentration Page 4/21 Total RNA was extracted from the cells using the Trizol (Invitrogen, USA) reagent, and the concentration and purity of the extracted RNA were assessed using a micro-nucleic acid protein concentration meter (Bio-Sun, China) to measure absorbance values at 260 and 280 nm. PrimeScript RT(Invitrogen, USA) reagent was used for reverse transcription synthesis of cDNA. SYBR Premix Ex Taq kits (Invitrogen, USA) are used for RT-qPCR experiments. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as the housekeeping gene. The 20 µl RT-qPCR system mixture includes SYBR Green Premix (10 µl), cDNA (2 µl), forward primer (10 µm; 0.8 µl), reverse primer (10 µm; 0.8 µl), Dey Ⅱ(0.4 µl), enzyme-free water (6 µl). The reaction procedure is 30s at 95°C; 5s at 95°C and 30s at 60°C and for 40 cycles; 15s at 95°C, 30s at 60°C, and 15s at 90°C. For the primer sequences used see Table 2. The results were analyzed using the 2−ΔΔCt method. Chondrogenic 、adipogenic and osteogenic differentiation of hADSCs Chondrogenic 、adipogenic and osteogenic differentiation of hADSCs Induction of multidirectional differentiation of hADSCs using microsphere culture, hADSCs were collected in 15 ml centrifuge tubes and differentiation was induced with adipogenic differentiation, chondrogenic differentiation, and osteogenic differentiation media, respectively, with the corresponding differentiation media configurations shown in Table 1. The staining was verified after 14, 21, and 28 days of induced differentiation, respectively. At the end of the induction of differentiation, the cell microspheres were fixed in 4% paraformaldehyde (Sarvicebio, China) for 1 h. Adipogenic differentiated cells were prepared in 10 µm sections through a frozen section and suitable sections were selected to be stained first with Oil Red O dye (Sarvicebio, China) and then re-stained with hematoxylin dye (Sarvicebio, China). Chondrogenic and osteogenic differentiated cells are prepared in 5 µm thick sections by paraffin sectioning, the sections are dewaxed and placed in water. Suitable sections were selected for staining in Alcian Blue, Sirius Red, and Alizarin Red dyes(Sarvicebio, China). After the staining was completed, observation and photography were carried out using a microscope. Chondrogenic 、adipogenic and osteogenic differentiation of hADSCs Table 2 Primers used in this study Primer Sequence (5′‑3′) SOX9 Forward:CAGCGAACGCACATCAAGACG Reverse:TGTAGGTGAAGGTGGAGTAGAGGC COL2A1 Forward: CGTGGACGATCAGGCGAAAC Reverse: AAGCCAGCAAAGGCGGACAT Aggrecan Forward: ACTGCCTTCGCTGAGGTTGA Reverse: CACTGCTCATAGCCTGCTTCGT GAPDH Forward: AAGGCTGTGGGCAAGGTCAT Reverse: GGAGGAGTGGGTGTCGCTGT DNM1L Forward: CTGAGGCTGATGGCAAGTT Forward: GAAGAGCAGCGTGGGGACT OPA1 Forward: CAGACTGGAAAAAGAGGTG Reverse: CGACAAAGGTTACAATGGT RELA Forward: GAAGAGCAGCGTGGGGACT Reverse: TGCACATCAGCTTGCGAAA TNFRSF1B Forward: TGGACTGATTGTGGGTGTG Reverse: CTGGTGCCTGTGGCTGGTT W bl l i Gene expression profiling Total RNA from the cells was extracted and the concentration and purity of the RNA were checked by NanoDrop microspectrophotometer (Thermo, USA), followed by agar gel electrophoresis to determine RNA integrity and the absence of DNA contamination. After library construction and quality control, the fragmented cRNAs were added to Affmetrix PrimeView™ chips (Cnkingbio, China), hybridized and washed, and then scanned using a GeneChip 300 7G scanner (Thermo, USA). Agilent Feature Extraction software (Agilent Technologies, USA) was used to process the gene chip scans and obtain the raw data, and Gene-Spring software (Agilent Technologies, USA) was used to normalize the analysis and export the data in Excel format. The screening criteria for differentially expressed genes were Fold Change (FC) ≥ 2.0 and P < 0.05. MMP assay analysis HADSCs were inoculated in six-well plates and cell proliferation was observed at approximately 40% confluence, and the medium was replaced with the appropriate differentiation medium. At the end of differentiation, 1 ml of JC-1 staining (Beyotime, China) working solution was added, and the cells were incubated for 20 min at 37°C in a cell incubator. After incubation, the cells were observed under a fluorescent microscope and photographed. ROS detection by flow cytometry Intracellular ROS levels were detected using a reactive oxygen species kit (Beyotime, China). HADSCs were inoculated in six-well plates and observed to be at approximately 70% cell proliferation confluence when replaced with the appropriate induction differentiation medium. After induction of differentiation, the cells were resuspended in DCFH-DA solution (10 µM/L, 1 ml) diluted 1:1000 with serum-free medium and incubated for 30 min at 37°C in an incubator, and assayed and analyzed by flow cytometry at the end of incubation. Statistical analysis The experimental data were expressed as mean ± standard deviation and processed using Graphpad prism 8 software (GraphPad Software, USA). Comparisons between groups were made using independent samples t-test and one-way ANOVA. Comparisons between groups were statistically significant at P < 0.05. Western blot analysis High-performance RIPA lysate (Solarbio, China) was used for protein isolation and extraction. The BCA protein concentration assay kit (Solarbio, China) was used to determine the total protein concentration of the cells. Proteins were separated by SDS-PAGE with a protein loading volume of 30 µg per well. isolated proteins were transferred onto PVDF membranes (Millipore, USA) by electrotransfer and then mixed with proportionally diluted primary antibodies SOX9 (1:2000; cat. no. 67439-1-Ig; Proteintech, China), COL2A1 (1:2000; cat. no. 28459-1-AP; Proteintech, China), Aggrecan (1:500; cat. no. 13880-1-AP; Proteintech, China), and GAPDH (1:10000; cat. no. 60004-1-Ig; Proteintech, China) for 2 hours at room temperature in a shaker incubation. Then incubate for one hour in the corresponding mouse (1:10000; cat. no. SA00001- 1; Proteintech, China) / rabbit (1:10000; cat. no. SA00001-2; Proteintech, China) secondary antibody solution on a shaker. After incubation development exposures and photographs were taken and the results were analyzed using Image J software (National Institutes of Health, USA). Active mitochondria staining Page 5/21 Page 5/21 HADSCs were inoculated in six-well plates and when the proliferation of hADSCs was observed to be confluent to about 40%, the medium was changed to the corresponding induction medium. At the end of induction differentiation, MitoTracker® Red CMXRos (200 nM, 1 ml; Solarbio, China) staining solution was added and incubated in an incubator at 37°C for 20 min. After the incubation was completed, they were quickly observed under a fluorescent microscope and photographed. Results Page 6/21 Page 6/21 TNF-α inhibits chondrogenic differentiation of hADSCs Studies have shown that the effect of TNF-α on the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is dose-dependent, and the addition of 10 ng/ml of TNF-α reagent to the chondrogenic differentiation medium has a significant inhibitory effect on the chondrogenic differentiation of BMSCs[11]. The hADSCs were divided into two groups, the Chondro group cultured in a chondrogenic differentiation medium and the Chondro-TNF-α group cultured in a chondrogenic differentiation medium with 10 ng/ml TNF-α reagent. Both groups were induced into chondrogenic differentiation for 7, 14, and 21 days respectively. The mRNA and protein expression of SOX9, COL2A1, and Aggrecan, markers of chondrogenesis, were examined by RT-qPCR and Western blot respectively in both groups after induction of chondrogenic differentiation. The results showed that the mRNA and protein expression levels of SOX9, COL2A1, and Aggrencan were significantly upregulated in both Chondro and Chondro-TNF-α groups compared to undifferentiated (day 0 of differentiation) cells, except for Aggreccan in the Chondro-TNF-α group. The mRNA (Fig. 2A-C) and protein (Fig. 2D-G) expression levels of SOX9, COL2A1, and Aggrecan were significantly lower in the Chondro-TNF-α group than in the Chondro group at 14 and 21 days of chondrogenic differentiation. Histological structures of the Chondro group and Chondro-TNF-α group at different times of differentiation were observed using Alcian blue staining and Sirius red staining. At 14 and 21 days of chondrogenic differentiation in hADSCs, the percentage of positive areas for Alcian blue staining (Fig. 3A, B) and Sirius red staining (Fig. 3C, D) were significantly lower in the Chondro-TNF-α group than in the Chondro group. These results suggest that the presence of TNF-α during the chondrogenic differentiation of hADSCs reduces the differentiation potential of hADSCs. Characterization of hADSCs The immunophenotype of hADSCs was detected by flow cytometry. The results showed that more than 99% of the cells expressed CD29 and CD44 and less than 0.1% expressed CD34, CD45, and HLA-DR (Fig. 1A). To verify that the cells used in the experiments have a multidirectional differentiation potential, cells were induced to differentiate into chondrocytes, adipocytes, and osteocytes respectively. Cell differentiation was observed by specific staining. Alcian blue staining and Sirius red staining were used to show the chondrogenic tissue components proteoglycans and collagen respectively to determine cellular differentiation into chondrocytes. Oil Red O staining and Alizarin Red staining are primarily used to identify lipid droplets and mineralized stroma to determine cellular differentiation into adipocytes and osteoblasts respectively. All staining showed positive results (Fig. 1B), indicating that the hADSCs used in the experiments had multidirectional differentiation potential. TNF-α increases mitochondrial fusion in hADSCs during prechondrogenic differentiation TNF-α increases mitochondrial fusion in hADSCs during prechondrogenic differentiation Unlike mesenchymal stem cells (MSCs) where mitochondria fusion during adipogenic and osteogenic differentiation, MSCs undergo mitochondrial fragmentation and increased Drp1 expression during Page 7/21 Page 7/21 Page 7/21 chondrogenic differentiation[21]. The changes in mRNA expression levels of OPA1 and Drp1 during chondrogenic differentiation of hADSCs were detected by RT-qPCR. The mRNA expression levels of OPA1 in the Chondro group were significantly upregulated at day 14 of differentiation and continued to be upregulated until day 21 of differentiation, at which point OPA1 expression was significantly higher in the Chondro group than in the Chondro-TNF-α group, and the mRNA expression levels of Drp1 were highest at day 14 of differentiation and significantly higher than in the Chondro-TNF-α group; The mRNA expression levels of OPA1 and Drp1 in the Chondro-TNF-α group were significantly up-regulated at 7 days of differentiation, much higher than at other differentiation time points, and the up-regulation of OPA1 was much higher than that of Drp1, which was significantly down-regulated at 14 days of differentiation and continued to be down-regulated until 21 days of differentiation when both expression levels were lower than in undifferentiated (day 0 of differentiation) cells (Fig. 4A). To observe changes in mitochondrial morphology during chondrogenic differentiation in both groups of hADSCs, we labeled the mitochondria with a red fluorescent probe. Using fluorescence microscopy it was possible to observe that the Chondro group at 14 days of differentiation hADSCs mitochondria underwent significant fragmentation and were short and granular; the Chondro-TNF-α group at 7 days of differentiation hADSCs mitochondria underwent significant fusion and the mitochondria were extended and interconnected (Fig. 4B). TNF-α upregulates OPA1 expression by activating RELA expression through TNFRSF1B The results of the previous experiments were summarized by observing that the expression levels of OPA1, DNM1L, mitochondrial morphology, and ROS levels were significantly different in the Chondro-TNF- α group compared to the Chondro group at 7 days of chondrogenic differentiation. Therefore, we prepared hADSCs from the Chondro group and Chondro-TNF-α group into cartilage differentiation at 7 days as well as hADSCs from the control group (differentiation 0 d) for gene expression profiling. The results showed that the Chondro-TNF-α group involved up- and down-regulated differentially expressed genes (DEGs) significantly more than the Chondro group (Fig. 6A). We focused our study's interest on upregulated DEGs and screened a total of 1242 DEGs for upregulation in the Chondro-TNF-α group vs control group by subtracting the same genes from the upregulated DEGs in the Chondro group vs control group and adding the upregulated DEGs in the Chondro-TNF-α group vs Chondro group for In-depth analysis (Fig. 6B). Relevant Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analyses were performed on the screened DEGs. GO analysis showed that DEGs are mainly involved in biological processes such as "covalent chromatin modification", "histone modification", and "mRNA 3'-end processing" (Fig. 6C). KEGG analysis showed that the signaling pathways with high enrichment were mainly "Herpes simplex virus 1 infection", "T cell receptor signaling pathway", and tein export" (Fig. 6D). It was shown that TNFRSF1B stimulation synergistically increases OPA1 transcription through STAT3 and RELA binding to adjacent regions of the OPA1 promoter, which in turn upregulates OPA1 expression and increases mitochondrial fusion[25]. We measured the expression levels of TNFRSF1B and RELA in the signaling pathway of "Herpes simplex virus 1 infection" by RT-qPCR (Fig. 6E) and found that the results were consistent with the gene microarray data (Fig. 6F). suggest that TNF-α upregulates OPA1 expression by activating RELA expression through TNFRSF1B, which in turn promotes mitochondrial fusion. The presence of TNF-α reduces ROS production during the chondrogenic differentiation of hADSCs and somewhat attenuates the decrease in mitochondrial membrane potential (MMP) The presence of TNF-α reduces ROS production during the chondrogenic differentiation of hADSCs and somewhat attenuates the decrease in mitochondrial membrane potential (MMP) Intracellular ROS and MMP are closely related to mitochondrial function. Flow cytometry was used to detect changes in ROS levels during the chondrogenic differentiation of hADSCs. The results showed that the Chondro group showed the most significant increase in ROS levels at 14 days of differentiation, which was higher than that of the other differentiation time points and the Chondro-TNF-α group. The Chondro-TNF-α group showed significant downregulation of ROS levels at 7 days of differentiation and were lower than the control (0 days of differentiation) group(Fig. 5A). As differentiation continued, ROS levels increased in the Chondro-TNF-α group but were consistently lower than in the Chondro group at the same differentiation time point(Fig. 5A). MMP is required for mitochondrial inner membrane fusion[22] and paired fission is activated when MMP is eliminated[23]. Changes in ROS have a regulatory effect on MMP, with an increase in ROS causing a decrease in MMP[24]. We use the JC-1 fluorescent probe to detect MMP. the ratio of the dimer (red fluorescence) to monomer (green fluorescence) can be used to determine the level of MMP. The results showed that the Chondro group had the lowest MMP at 14 days of differentiation, which was lower than that of the other differentiation time points and the Chondro-TNF-α group(Fig. 5B, C). The Chondro-TNF-α group also showed a significant decrease in MMP at 14 days of differentiation, but not to the same extent as the Chondro group, which continued to differentiate, with no significant change in MMP in the Chondro-TNF-α group(Fig. 5B, C). These results suggest that the process of chondrogenic differentiation of hADSCs involves a moderate increase in ROS and a moderate decrease in MMP. However, the presence Page 8/21 Page 8/21 of TNF-α reduced the production of ROS during the chondrogenic differentiation of hADSCs and somewhat attenuated the decrease in MMP. Discussion As shown in our data, the presence of TNF-α significantly reduced cartilage-specific matrix synthesis and down-regulated the expression of chondrogenic marker genes. Data from several studies suggest that TNF-α significantly downregulates gene expression of SOX9, a transcription factor essential in the early stages of chondrogenesis and responsible for the subsequent inhibition of chondrocyte maturation[9, 26]. Our experimental results likewise confirm this idea, so that the inflammatory environment present in OA may lead to a lack of effectiveness or failure of defect repair. The morphology of mitochondria and their regulatory processes of fusion and division are regulated during the pre-differentiation phase of stem cells, leading to altered bioenergetic distribution during differentiation. The remodeling of the mitochondrial network is essential to the differentiation program, as these cells show a delayed or complete loss of differentiation when fusion or division is abolished. Altering mitochondrial dynamics is both a consequence and a cause of stem cell differentiation. During chondrogenesis, mitochondria are fragmented and Drp1 expression is increased, and the knockdown of Drp1 leads to a reduction in cell differentiation capacity[21]. Our experimental data also support the idea that mitochondrial fission is involved in the process of chondrogenic differentiation of hADSCs. The coordination of mitochondrial fusion and fission is a key cytoprotective mechanism for cell renewal and dynamic homeostasis under different external stresses[19]. Mitochondrial fusion may be a compensatory response to weak external stresses to promote better survival. In many cases, mitochondrial ground lengthening facilitates stress resistance[27]. In our experiments, we also found that in the presence of TNF-α, the process of chondrogenic differentiation of hADSCs tends to favor mitochondrial fusion rather than mitochondrial fission. Preliminary indications are that in the presence of TNF-α, mitochondria tend to lengthen to resist external stresses to promote better survival. Discussion There is already a lot of research data confirming that TNF-α inhibits the chondrogenic differentiation of stem cells[9, 11, 26], however, the mechanisms behind these inhibitory effects are still poorly understood. In this study, we show for the first time that the presence of TNF-α upregulates OPA1 expression during chondrogenic differentiation of hADSCs and promotes mitochondrial fusion. Our data suggest that the presence of TNF-α leads to a significant upregulation of TNFRSF1B and RELA expression during the chondrogenic differentiation of hADSCs. It has been demonstrated that TNFRSF1B activation mediates the interaction between STAT3 and RELA, coordinating the upregulation of OPA1 expression and thus improving mitochondrial fusion and function, an effect that can be abolished by downregulation of Page 9/21 STAT3 and RELA[25]. In summary, we can tentatively suggest that TNF-α can upregulate OPA1 expression through the activation of RELA expression by TNFRSF1B, which in turn increases mitochondrial fusion. In most cases, the use of stem cells for cartilage regeneration treatment OA is in an inflammatory environment, however, inflammatory environments inhibit cartilage formation and accelerate cartilage destruction[11]. This would substantially reduce the potential for chondrogenic differentiation of stem cells and the therapeutic effect. There is growing evidence that TNF-α and Interleukin-1β (IL-1β) are important pro-inflammatory mediators in OA. These cytokines play a crucial role not only in the regulation of subchondral bone resorption but also in the shift from endochondral homeostasis to catabolism[26]. As shown in our data, the presence of TNF-α significantly reduced cartilage-specific matrix synthesis and down-regulated the expression of chondrogenic marker genes. Data from several studies suggest that TNF-α significantly downregulates gene expression of SOX9, a transcription factor essential in the early stages of chondrogenesis and responsible for the subsequent inhibition of chondrocyte maturation[9, 26]. Our experimental results likewise confirm this idea, so that the inflammatory environment present in OA may lead to a lack of effectiveness or failure of defect repair. In most cases, the use of stem cells for cartilage regeneration treatment OA is in an inflammatory environment, however, inflammatory environments inhibit cartilage formation and accelerate cartilage destruction[11]. This would substantially reduce the potential for chondrogenic differentiation of stem cells and the therapeutic effect. There is growing evidence that TNF-α and Interleukin-1β (IL-1β) are important pro-inflammatory mediators in OA. These cytokines play a crucial role not only in the regulation of subchondral bone resorption but also in the shift from endochondral homeostasis to catabolism[26]. Abbreviations TNF-α Tumour necrosis factor-α hADSCs Human adipose-derived stem cells OA osteoarthritis COL2A1 type II collagen OPA1 optic atrophy 1 TNFRSF1B TNFα receptor 2 MMP mitochondrial membrane potential Drp1 dynamin-related protein 1 ROS oxygen species GO Gene Ontology KEGG, Kyoto Encyclopedia of Genes a Conclusions In conclusion, we found that mitochondria tend to fission during chondrogenic differentiation of hADSCs and that Drp1 expression is increased; however, TNF-α upregulates OPA1 expression by activating RELA expression through TNFRSF1B, which in turn increases mitochondrial fusion, leading to inhibition of chondrogenic differentiation of hADSCs (Fig. 7). Therefore, when using hADSCs for cartilage regeneration in the treatment of OA, it can be used as a therapeutic or adjuvant target by downregulating the expression of TNFRSF1B to help increase mitochondrial division and Drp1 expression required for cell differentiation. Page 10/21 Page 10/21 Declarations Ethics approval and consent to participate Ethics approval and consent to participate In the studies involving human participants, all procedures followed the ethical standards of the Ethics Committee of Guizhou Provincial People’s Hospital ({2019}064). Written informed consent was received from the donors. Consent for publication Consent for publication Not applicable. Availability of data and materials All You may access the datasets applied in this study from the corresponding authors upon reasonable request. Competing interests Page 11/21 Author details 1 Medical College of Guizhou University, Guiyang 550025, Guizhou, China; 1 Medical College of Guizhou University, Guiyang 550025, Guizhou, China; 2 Department of Orthopedics, Guizhou Provincial People's Hospital, Guiyang 550002, Guizhou, China; 2 Department of Orthopedics, Guizhou Provincial People's Hospital, Guiyang 550002, Guizhou, China; 2 Department of Orthopedics, Guizhou Provincial People's Hospital, Guiyang 550002, Guizhou, China; 3 Department of Clinical Laboratory, Guizhou Provincial People's Hospital, Guiyang 550002, Guizhou, China. 3 Department of Clinical Laboratory, Guizhou Provincial People's Hospital, Guiyang 550002, Guizhou, China. Authors' contributions Jiajia Guo and Wang Ye performed the experiment. Jiajia Guo and Xinglin Wu conducted the statistical analysis. Jiajia Guo and Bo Li re-examined the data and wrote the draft. Jiajia Guo and Haifeng Huang edited the manuscript. Zhijing Ren and Zhen Yang supervised and helped to perform the experiment. All authors read and approved the final manuscript. Acknowledgements Not applicable. Funding This work was supported by the National Natural Science Foundation of China (31660265, 31960208), Youth Fund of Guizhou Provincial People’s Hospital (GZSYQN(2015)06), Subsidy Foundation of National Natural Science Foundation of Guizhou Provincial People’s Hospital (Guizhou Science and Technology Platform (2017)5724), Science and Technology Foundation of Guizhou Province (Guizhou Science and Technology J Word (2015)2096), and Subsidy Foundation of National Natural Science Foundation of Guizhou Provincial People’s Hospital(GPPH-NSFC-2019-1). References 1. Castañeda S, Roman-Blas JA, Largo R, Herrero-Beaumont G. Osteoarthritis: a progressive disease with changing phenotypes. Rheumatology (Oxford). 2014; 53:1-3. 1. Castañeda S, Roman-Blas JA, Largo R, Herrero-Beaumont G. 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Yang F, Li B, Yang Y, Huang M, Liu X, Zhang Y, Liu H, Zhang L, Pan Y, Tian S et al. Leptin enhances glycolysis via OPA1-mediated mitochondrial fusion to promote mesenchymal stem cell survival. Int J Mol Med. 2019; 44:301-312. 27. Yang F, Li B, Yang Y, Huang M, Liu X, Zhang Y, Liu H, Zhang L, Pan Y, Tian S et al. Leptin enhances glycolysis via OPA1-mediated mitochondrial fusion to promote mesenchymal stem cell survival. Int J Mol Med. 2019; 44:301-312. Figures Page 14/21 Figure 1 Immunophenotypic and multidirectional differentiation potential of hADSCs. (A)Flow cytometric analysis of surface marker expression on MSCs. They were positive for CD44 and CD29, but negative for CD45, Figure 1 Immunophenotypic and multidirectional differentiation potential of hADSCs. (A)Flow cytometric analysis of surface marker expression on MSCs. They were positive for CD44 and CD29, but negative for CD45, CD34, and HLA-DR. (B) The hADSCs that were not induced to differentiate and those that were induced to differentiate were stained with Arsenic Blue, Sirius Scarlet, Oil Red O, and Alizarin Red, respectively. All the uninduced differentiated ones showed negative results and all the induced differentiated ones showed positive results. Scale bars=100 μm. Figure 1 Immunophenotypic and multidirectional differentiation potential of hADSCs. (A)Flow cytometric analysis of surface marker expression on MSCs. They were positive for CD44 and CD29, but negative for CD45, CD34, and HLA-DR. (B) The hADSCs that were not induced to differentiate and those that were induced to differentiate were stained with Arsenic Blue, Sirius Scarlet, Oil Red O, and Alizarin Red, respectively. All the uninduced differentiated ones showed negative results and all the induced differentiated ones showed positive results. Scale bars=100 μm. Page 15/21 Figure 2 Changes in mRNA and protein expression levels of SOX9, COL2A1, and Aggrecan, markers of Figure 2 Changes in mRNA and protein expression levels of SOX9, COL2A1, and Aggrecan, markers of chondrogenesis, during chondrogenic differentiation of hADSCs.(A-C) The mRNA expression of SOX9, COL2A1, and Aggrecan was measured by RT-qPCR after 0, 7, 14, and 21 days of chondrogenic differentiation in hADSCs. (D) Protein expression of SOX9, COL2A1, and Aggrecan was measured by western blot after 0, 7, 14, and 21 days of chondrogenic differentiation in hADSCs. (E-G) Histogram of quantitative analysis of western blot results. Mean ± SD of three independent experiments; **P < 0.0001, P < 0.001, P < 0.01, P < 0.05, ns = not significant; d: day. Figure 2 Changes in mRNA and protein expression levels of SOX9, COL2A1, and Aggrecan, markers of chondrogenesis, during chondrogenic differentiation of hADSCs.(A-C) The mRNA expression of SOX9, COL2A1, and Aggrecan was measured by RT-qPCR after 0, 7, 14, and 21 days of chondrogenic differentiation in hADSCs. (D) Protein expression of SOX9, COL2A1, and Aggrecan was measured by western blot after 0, 7, 14, and 21 days of chondrogenic differentiation in hADSCs. (E-G) Histogram of quantitative analysis of western blot results. Mean ± SD of three independent experiments; **P < 0.0001, P < 0.001, P < 0.01, P < 0.05, ns = not significant; d: day. Page 16/21 Figure 3 Formation of proteoglycans and collagen during cartilage differentiation in hADSCs. (A) Protein polyaminoglycan formation was observed with Alcian blue staining. (B) Histogram of quantitative analysis of Alcian blue staining. (C) collagen formation was observed with Sirius red staining. (D)Histogram of quantitative analysis of Sirius red staining. Mean ± SD of three independent experiments; Scale bars=100 μm; **P < 0.0001, P < 0.01, P < 0.05; ns = not significant; d: day. Figure 3 Formation of proteoglycans and collagen during cartilage differentiation in hADSCs. (A) Protein polyaminoglycan formation was observed with Alcian blue staining. (B) Histogram of quantitative analysis of Alcian blue staining. (C) collagen formation was observed with Sirius red staining. Figure 3 Figure 3 Formation of proteoglycans and collagen during cartilage differentiation in hADSCs. (A) Protein polyaminoglycan formation was observed with Alcian blue staining. (B) Histogram of quantitative analysis of Alcian blue staining. (C) collagen formation was observed with Sirius red staining. (D)Histogram of quantitative analysis of Sirius red staining. Mean ± SD of three independent experiments; Scale bars=100 μm; *P < 0.0001, P < 0.01, P < 0.05; ns = not significant; d: day. Page 17/21 Figure 4 Changes in the level of mitochondrial fusion and division during chondrogenic differentiation of hADSCs. (A) Expression of mRNA of mitochondrial fusion gene OPA1 and mitochondrial division gene DNM1L during chondrogenic differentiation of hADSCs was detected by RT-qPCR; (B) Mitochondria of hADSCs were labeled with a fluorescent dye (in red) to observe the changes in mitochondrial morphology during chondrogenic differentiation of hADSCs. Mean ± SD of three independent experiments; Scale bars=50 μm; *P < 0.000, ns = not significant; d: day Figure 4 Changes in the level of mitochondrial fusion and division during chondrogenic differentiation of hADSCs. (A) Expression of mRNA of mitochondrial fusion gene OPA1 and mitochondrial division gene DNM1L during chondrogenic differentiation of hADSCs was detected by RT-qPCR; (B) Mitochondria of hADSCs were labeled with a fluorescent dye (in red) to observe the changes in mitochondrial morphology during chondrogenic differentiation of hADSCs. Mean ± SD of three independent experiments; Scale bars=50 μm; P < 0.000, ns = not significant; d: day Page 18/21 Page 18/21 Figure 5 Changes in ROS levels and MMP during chondrogenic differentiation of hADSCs probes were mounted within entry hADSCs for ROS detection by flow cytometry. probe detects MMP with a red fluorescence from the dimer and a green fluoresce (C)The ratio of dimer to monomer, with a higher ratio indicating higher MMP and of three independent experiments; Scale bars=50μm; *P < 0.0001, P < 0.05, n Figure 5 Changes in ROS levels and MMP during chondrogenic differentiation of hADSCs. (A)DCFH-DA fluorescen probes were mounted within entry hADSCs for ROS detection by flow cytometry. (B) JC-1 fluorescent probe detects MMP with a red fluorescence from the dimer and a green fluorescence from the monomer. (C)The ratio of dimer to monomer, with a higher ratio indicating higher MMP and vice versa. Mean ± SD of three independent experiments; Scale bars=50μm; ***P < 0.0001, P < 0.05, ns = not significant; d: day Figure 5 Changes in ROS levels and MMP during chondrogenic differentiation of hADSCs. (A)DCFH-DA fluorescent probes were mounted within entry hADSCs for ROS detection by flow cytometry. (B) JC-1 fluorescent probe detects MMP with a red fluorescence from the dimer and a green fluorescence from the monomer. (C)The ratio of dimer to monomer, with a higher ratio indicating higher MMP and vice versa. Mean ± SD of three independent experiments; Scale bars=50μm; ***P < 0.0001, P < 0.05, ns = not significant; d: day Page 19/21 Page 19/21 Figure 6 Figure 6 Screening of DEGs, GO, and KEGG enrichment analysis. (A) Two-by-two comparison of gene expression profile assay data for the number of up-and down-regulated DEGs. (B) A Wayne diagram showing the screened 1242 (A+D+E+G) DEGs. (C) GO functional enrichment analysis of the screened DEGs, showing the top ten enrichment rankings, respectively. Orange indicates biological processes (BP), green indicates cellular components (CC), and dark blue indicates molecular functions (MF). (D)KEGG enrichment Figure 6 Screening of DEGs, GO, and KEGG enrichment analysis. (A) Two-by-two comparison of gene expression profile assay data for the number of up-and down-regulated DEGs. (B) A Wayne diagram showing the screened 1242 (A+D+E+G) DEGs. (C) GO functional enrichment analysis of the screened DEGs, showing the top ten enrichment rankings, respectively. Orange indicates biological processes (BP), green indicates cellular components (CC), and dark blue indicates molecular functions (MF). (D)KEGG enrichment Page 20/21 Page 20/21 analysis of screened DEGs showing the top ten KEGG pathways in terms of enrichment. The x-axis shows the enrichment score, the y-axis shows pathway names, and the point size represents the number of genes enriched in a particular pathway. (E) KEGG pathway enrichment analysis and "Herpes simplex virus 1 infection" pathway map. Red indicates up-regulated genes, dark blue indicates down-regulated genes, green indicates the presence of the gene in the species, and white indicates the absence of the gene in the species. (F) Expression levels of TNFRSF1B and RELA in the "Herpes simplex virus 1 infection" signaling pathway were measured by RT-qPCR. **P < 0.0001, ns = not significant; d: day analysis of screened DEGs showing the top ten KEGG pathways in terms of enrichment. The x-axis shows the enrichment score, the y-axis shows pathway names, and the point size represents the number of genes enriched in a particular pathway. (E) KEGG pathway enrichment analysis and "Herpes simplex virus 1 infection" pathway map. Red indicates up-regulated genes, dark blue indicates down-regulated genes, green indicates the presence of the gene in the species, and white indicates the absence of the gene in the species. (F) Expression levels of TNFRSF1B and RELA in the "Herpes simplex virus 1 infection" signaling pathway were measured by RT-qPCR. **P < 0.0001, ns = not significant; d: day Figure 7 Schematic diagram: TNF-α upregulates OPA1 expression by activating RELA expression through TNFRSF1B, which increases mitochondrial fusion and leads to inhibition of cartilage differentiation in hADSCs. Figure 7 Figure 7 Schematic diagram: TNF-α upregulates OPA1 expression by activating RELA expression through TNFRSF1B, which increases mitochondrial fusion and leads to inhibition of cartilage differentiation in hADSCs. Schematic diagram: TNF-α upregulates OPA1 expression by activating RELA expression through TNFRSF1B, which increases mitochondrial fusion and leads to inhibition of cartilage differentiation in hADSCs. Page 21/21 Page 21/21 Page 21/21
https://openalex.org/W1988231371	https://europepmc.org/articles/pmc3567202?pdf=render	English	null	Self-rated health among Greenlandic Inuit and Norwegian Sami adolescents: associated risk and protective correlates	International journal of circumpolar health	2,013	cc-by	8,540	Anna Rita Spein1, Cecilia Petrine Pedersen2, Anne Cathrine Silviken1, Marita Melhus3, Siv Eli Kvernmo4,5 and Peter Bjerregaard2 1Centre for Sami Health Research, Faculty of Health Sciences, University of Tromsø, Karasjok, Norway; 2Center for Health Research in Greenland, National Institute of Public Health, University of Southern Denmark, Copenhagen K, Denmark; 3Department of Community Medicine, Centre for Sami Health Research, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway; 4Department of Child and Adolescent Psychiatry, Division of Child and Adolescent Health, University Hospital of North Norway, Tromsø, Norway; 5Department of Community Medicine, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway bjectives. Self-rated health (SRH) and associated risk and protective correlates were investigated among tw digenous adolescent populations, Greenlandic Inuit and Norwegian Sami. Design. Cross-sectional data were collected from ‘‘Well-being among Youth in Greenland’’ (WBYG) and ‘‘The Norwegian Arctic Adolescent Health Study’’ (NAAHS), conducted during 20032005 and comprising 10th and 11th graders, 378 Inuit and 350 Sami. Methods. SRH was assessed by one single item, using a 4-point and 5-point scale for NAAHS and WBYG, respectively. Logistic regressions were performed separately for each indigenous group using a dichotomous measure with ‘‘very good’’ (NAAHS) and ‘‘very good/good’’ (WBYG) as reference categories. We simul- taneously controlled for various socio-demographics, risk correlates (drinking, smoking, violence and suicidal behaviour) and protective correlates (physical activity, well-being in school, number of close friends and adolescentparent relationship). Results. A majority of both Inuit (62%) and Sami (89%) youth reported ‘‘good’’ or ‘‘very good’’ SRH. The proportion of ‘‘poor/fair/not so good’’ SRH was three times higher among Inuit than Sami (38% vs. 11%, p50.001). Significantly more Inuit females than males reported ‘‘poor/fair’’ SRH (44% vs. 29%, p50.05), while no gender differences occurred among Sami (12% vs. 9%, p50.08). In both indigenous groups, suicidal thoughts (risk) and physical activity (protective) were associatedwith poor and good SRH, respectively. Conclusions. In accordance with other studies of indigenous adolescents, suicidal thoughts were strongly associated with poorer SRH among Sami and Inuit. The InuitSami differences in SRH could partly be due to higher ‘‘risk’’ and lower ‘‘protective’’ correlates among Inuit than Sami. The positive impact of physical activity on SRH needs to be targeted in future intervention programs. Keywords: adolescents; Arctic; indigenous; Inuit; protective factors; risk factors; Sami; self-rated health (SRH); suicidal behaviours Received: 29 September 2012; Revised: 8 November 2012; Accepted: 11 November 2012; Published: 6 February S elf-rated health (SRH) has been widely used as a measure of current health status both among and within ethnic groups and nationalities. Int J Circumpolar Health 2013. # 2013 Anna Rita Spein et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 1 Citation: Int J Circumpolar Health 2013, 72: 19793 - http://dx.doi.org/10.3402/ijch.v72i0.19793 (page number not for citation purpose) ORIGINAL RESEARCH ARTICLE ORIGINAL RESEARCH ARTICLE Self-rated health among Greenlandic Inuit and Norwegian Sami adolescents: associated risk and protective correlates Keywords: adolescents; Arctic; indigenous; Inuit; protective factors; risk factors; Sami; self-rated health (SRH); suicidal behaviours s properly cited. Citation: Int J Circumpolar Health 2013, 72: 19793 - http://dx.doi.org/10.3402/ijch.v72i0.19793 (page number not for citation purpose) Anna Rita Spein1, Cecilia Petrine Pedersen2, Anne Cathrine Silviken1, Marita Melhus3, Siv Eli Kvernmo4,5 and Peter Bjerregaard2 In Norway, the Sami people number about 60,000, and their current status is that of an indigenous people. With the exception of the Sami Highland in Finnmark County, the Sami is a numeric minority. The colonisa- tion of Sapmi (land of the Sami people) and the former assimilation policy called ‘‘Norwegianization‘‘ has con- tributed to loss of ethnic identity and language shift among many Sami, although in some geographical regions this impact has been less severe. During the last decades, a political shift towards integration has con- tributed to increased ethnic awareness and revitalisation about Sami cultural, linguistic, social, health, politically issues etc., and has also contributed to the establishment of Sami institutions within media, school, health services, art, as well as the Sami parliament in 1989 (9). y g p The impact of social change on health is more trans- parent in circumpolar populations and regions where social changes have occurred more recently, rapidly and dramatically. The Sami and Greenlandic Inuit adoles- cents are both a product of and are growing up in societies with a common history of colonisation and assimilation. In Greenland, these changes have contrib- uted to increasing alcohol dependency, youth suicides and violence (9,16). Among Sami youth, assimilation and weak ethnic identity have been associated with more substance use (12). Traditional indigenous lifestyles such as reindeer herding among Sami have been affected by increasing work-related stress including suicidal behaviour problems (12,17). Nonetheless, the colonised history and the current political and economic situa- tion are different in many ways as, for example, the Sami population is a minority in Norway, while the Greenlandic Inuit population is a majority still con- nected legally to the Danish state. These similarities and differences make it important to compare these two adolescent groups and how the social change has made an impact on their health and well-being. From a public health perspective, it is very important to understand and increase the knowledge of SRH among the two indigen- ous adolescent groups, the Sami and Inuit, as SRH is a measure of great predictive value for future poor health and death (2,18). Greenland is the world’s largest island with a total population numbering only about 57,000 of whom 90% are Inuit. Greenland was colonised by Denmark in 1721 and reforms of the Greenlandic infrastructure, industry and welfare during the 1950s and 60s were based on Danish administrative systems. Anna Rita Spein1, Cecilia Petrine Pedersen2, Anne Cathrine Silviken1, Marita Melhus3, Siv Eli Kvernmo4,5 and Peter Bjerregaard2 Across ages, ethnic minorities and indigenous people generally rate their health worse than majority peers (1). SRH is found to be an important predictor of mortality, morbidity, health service use, medical use and general well-being (25). Studies have revealed several correlates associated with SRH (6), including demographics (e.g. gender, ethnicity), educational factors (e.g. parental education), economic factors (e.g. self-rated economic position), geo- graphical aspects (e.g. urbanrural), various social cor- relates (e.g. marital status, family relationship, violence and sexual abuse) and also health-related behaviours S m w Anna Rita Spein et al. (e.g. smoking, drinking and physical activity). Long- itudinal studies have revealed that SRH is quite a stable construct during adolescence (2). educational, occupational and recreational possibilities. Greenlandic is the dominant language, but Danish is used in higher education (9). A variety of studies have investigated health behav- iour among Norwegian Sami and Greenlandic Inuit adolescents. Among young Sami, a wide range of health compromising behaviours including emotional and behavioural problems, suicidal behaviour, substance use and their relationship with ethnocultural factors have been studied (913). Generally, no differences have been found with regard to smoking, suicidal behaviour and general behavioural/conduct problems, while higher abstinence and lower drinking rates have been shown to occur among young Sami compared to the majority of Norwegians (9,12,13). A review of child and adolescent health in Greenland, primarily among Greenlandic born Inuit, concluded that negative health behaviour such as smoking and binge drinking, but also obesity, poor oral health, unhealthy diet and sexual risk behaviour frequently occurred (14). In their review, Lethi and colleagues (15) noted more suicidal problems among young Greenlandic Inuit than their Sami peers. Little research has been conducted on SRH among indigenous adolescents (1,7,8). Bombak and Bruce (1) in their review of SRH and ethnicity found that many of the factors generally associated with poorer SRH have been replicated among North American indigenous youth, including females, poor family income, poor body image and weight concerns, physical and sexual abuse, suicide attempts, substance abuse and nutritional inadequacies. In contrast, social competence, good school performance, non-smoking and having recently had a physical exam- ination were positively associated with SRH (1). Among 11-to-17-year-old Greenlanders (majority Greenlandic born Inuit), older students reported their health more negatively than younger peers (7), with a greater like- lihood of poor SRH for both youths who were bullied and those who bullied others (8). 2 (page number not for citation purpose) 2 (page number not for citation purpose) Citation: Int J Circumpolar Health 2013, 72: 19793 - http://dx.doi.org/10.3402/ijch.v72i0.19793 Anna Rita Spein1, Cecilia Petrine Pedersen2, Anne Cathrine Silviken1, Marita Melhus3, Siv Eli Kvernmo4,5 and Peter Bjerregaard2 The profound changes on everyday life and in the social structures of the commu- nities have accelerated since 1950 and most Greenlanders have experienced the transition during their own life time. In 1979 Greenland achieved Home Rule and in 2009, Self-Government, the last step before complete indepen- dence. In the process of economic, social and cultural change, the most central changes were a shift from subsistence hunting and fishing to wage earning; popula- tion movement from small villages to larger towns; and increased availability of formal education, accompanied by changing life style and eventually by changes in health status. Today the isolated populations in villages and smaller towns face the largest challenges with limited opportunities. In contrast, life in the capital reflects a contemporary Scandinavian lifestyle with a wide range of In this paper, we first examine whether Sami and Inuit adolescents rate their health differently and compare this to regional and national figures. Second, we examine gender differences in SRH for the two indigenous groups, but also with regard to ethnic differences separately for males and females. Third, we investigate the correlates 2 (pag Citation: Int J Circumpolar Health 2013, 72: 19793 - http://dx.doi.org/10.3402/ijch.v72i0.19793 Self-rated health among indigenous adolescents Table I. Study descriptions: The Norwegian Arctic Adolescent Health Study (NAAHS) and Well-Being among Youth in Greenland (WBYG) Sami (NAAHS) Inuit (WBYG) Study year 200305 200405 Invited (n)a 5877 663 Participants (n) 4880 508 Response rate (%) 83 75 Number of participating schools (n) 292b 10 Working sample of 1516-year oldsc 350 378 aIncluding both indigenous Sami and Greenlandic Inuit, but also Norwegian and Danish adolescents. bAt 116 junior high schools one or more students reported Sami origin. cNAAHS: 10th graders (98% were 16 year olds); WBYG: 10th and 11th graders. Table I. Study descriptions: The Norwegian Arctic Adolescent Health Study (NAAHS) and Well-Being among Youth in Greenland (WBYG) associated with poor versus good SRH for Sami and Inuit separately. In accordance with existing research on adolescent SRH (1,2,6,19), we included four potential risk factors (drinking, smoking, suicidal behaviour and experienced violence) and four protective factors (physical activity, well-being in school, number of friends and adolescentparent relationship) and simultaneously controlled for various socio-demographics. Sami (NAAHS) Inuit (WBYG) Study year 200305 200405 Invited (n)a 5877 663 Participants (n) 4880 508 Response rate (%) 83 75 Number of participating schools (n) 292b 10 Working sample of 1516-year oldsc 350 378 Material and methods Study samples Cross-sectional data from two school-based studies: ‘‘The Norwegian Arctic Adolescent Health Study’’ (NAAHS) and ‘‘Well-being among Youth in Greenland’’ (WBYG) were used. The NAAHS was a study on health and living conditions among indigenous Sami and non-Sami adolescents in rural and semi-rural areas and in towns in the three northernmost counties of Norway. The questionnaire included somatic health complaints, medi- cine use and health services uses, food habits, physical activity, schooling and educational plans, cultural activ- ities and traditions, sexual behaviour, as well as broader mental health issues and risk-taking behaviours including self-efficacy, stress and coping, anxiety and depression, substance use, self-harm and suicidal behaviour, bullying and sexual abuse. All 293 junior high schools in North- Norway were invited to participate in the study, of which only one school refused to take part in the study. The percentage of Sami students varied between schools with the highest number in the Sami dominated areas of Finnmark. for reason of age (1516 year olds) comparison with NAAHS. The school absentee rate for the whole WBYG sample of all 10th to 11th graders was 25% and was similar to the absentee rate caused by illness within the last 2 weeks prior to the study (22%), which is the same for this subsample. No data on the overall school absentee rate was available in the NAAHS. The sub- sample of indigenous Sami included 358 adolescents. Instruments Ethnicity y In the NAAHS eight ‘‘objective’’ questions tapped ethnicity; father’s and mother’s ethnicity and spoken language by each of the parents and grandparents, separately. All questions had answering options as Norwegian, Sami, Kven, Finnish and ‘‘Other’’, and multi- ple answers were allowed. These questions were combined into ethnic background where respondents were cate- gorised as Sami if they had answered Sami on one or more of these questions. In addition, there were ‘‘subjective’’ questions about the respondent’s own ethnicity and five questions on how they view themselves: I regard myself Norwegian/Sami/Kven/Finnish/Other. For each of the five ethnic groups, respondents were able to answer on a 4-point scale, ranging from ‘‘fully agree’’ to ‘‘fully disagree’’. There were 91 people who stated that they fully or partly agreed that they regarded themselves Sami or having Sami ethnicity, without reporting Sami ethnic- ity or language for any of the parents or grandparents. We decided to use only objective criteria to identify Sami youth to avoid misclassification of youth with very weak or no Sami affiliation. The WBYG was a study on well-being, health beha- viour and health among adolescents in Greenland. The WBYG included socio-demographic factors, family and upbringing conditions, social relations, school and leisure time factors, health behaviour, physical and mental health with a special focus on suicidal behaviour, sexual behaviour and sexual abuse. In the WBYG, students from 10 schools above the age of 14 in grade 10 and 11 were invited to participate in the study. The selected schools represented all of the different geogra- phical areas in GreenlandNorth, South, West and East. Moreover, school size and the number of adolescents from villages was taken into account so that towns with large schools were ideal for getting as many participants as possible (20). Table I summarises the two working samples, study periods and participation rates. A paper (NAAHS) and electronic (WBYG) bilingual questionnaire was avail- able in both studies (North Sami dialect and Norwegian in NAAHS and Greenlandic and Danish in WBYG) (10,13,20). The WBYG included overall 508 10th and 11th grade students. Here, only a subsample of 378 10th (N107) and 11th (N271) graders was selected In the WBYG, Inuit background was based on the question: Would you describe yourself as a Greenlander or a Dane? 3 (page number not for citation purpose Citation: Int J Circumpolar Health 2013, 72: 19793 - http://dx.doi.org/10.3402/ijch.v72i0.19793 Instruments Ethnicity We excluded those defining themselves 3 pose) 3 ose) Citation: Int J Circumpolar Health 2013, 72: 19793 - http://dx.doi.org/10.3402/ijch.v72i0.19793 3 (page number not for citation purpose) Anna Rita Spein et al. as only Danish (n12), Something else (n4) and I don’t know (n16). as only Danish (n12), Something else (n4) and I don’t know (n16). not so good/fair, good and very good SRH. Poor and not so good/fair SRH were three times higher among Inuit than Sami. Significant differences in SRH occurred among Sami and Inuit, as fewer Sami youth reported not so good SRH, while more Inuit report fair SRH (p50.001). Significant ethnic differences in SRH occurred for both males and females. These differences were due to fewer Sami reporting not so good/fair SRH when compared to Inuit counterparts; the figures were 11% vs. 41% for females [p50.001] and 9% vs. 28% for males [p50.001]. Significant ethnic differences in fair SRH occurred for both males and females as fewer Sami youth reported fair SRH than their Inuit counterparts. Sig- nificant gender differences in SRH were only found for Inuit (p50.05), but not for Sami (p50.08, Table III). Distribution of socio-demographics, risk and protective correlates by gender Distribution of socio-demographics, risk and protective correlates by gender Table IV shows that among both Sami and Inuit suicidal thoughts and attempts were reported significantly more often by females than males. Significantly more Sami males had experienced violence during the last year, while females reported significantly more poor parental econ- omy. Significantly more Inuit females than males were smokers, while Inuit females reported more difficulty in talking with their parents than males (Table IV). Inuit females were also significantly less physically active; slightly more than half of them reported seldom engaging in physical activity compared to about one third of their male counterparts (Table IV). Logistic regression analyses: socio-demographics, risk and protective correlates associated with SRH in Sami (NAAHS) and Inuit (WBYG) Table V shows the simple and multiple logistic regression analyses of correlates associated with SRH among Sami and Inuit adolescents separately. For both groups, adjusted analyses revealed that suicidal thoughts and physical inactivity were risk factors for poor SRH. Logistic regression analyses: socio-demographics, risk and protective correlates associated with SRH in Sami (NAAHS) and Inuit (WBYG) Self-rated health In the NAAHS, SRH was based on one question: What is your present state of health? with reply alternatives: poor, not so good, good and very good. In the WBYG, SHR was based on: How would you rate your health? with five reply alternatives: very poor, poor, fair, good and very good. However, no Inuit participants reported the fifth category very poor. In the logistic regression analyses, SRH was dichotomised into: (a) Poor (fair/poor) and (b) Good (very good/good) in WBYG. In NAAHS, SRH was categorised into: (a) Poor (good/not so good/poor) and (b) Good (very good). In NAAHS and WBYG, there were 9 and 11 missing values on the SRH variables, respectively. The reason for choosing different cut-offs for SRH in NAAHS and WBYG was that the distribution of SRH varied considerably between the two study samples. There were few observations in the very good, and none in the very poor category among Inuit, and few in the not so good and poor category among Sami. Distribution of socio-demographics, risk and protective correlates by indigenous group Table IV shows the distribution of the independent variables in Sami and Inuit adolescents. There were significant SamiInuit group differences for all risk and protective correlates, except for reporting of experienced violence, which occurred in about a quarter of the samples. Overall, suicidal attempts were reported twice as often by Inuit than by Sami (Table IV). Inuit adolescents found it significantly easier to talk with their parents than Sami, while significantly more Sami than Inuit reported good well-being in school. A significantly higher proportion of Sami than Inuit also reported frequent physical activity. While significantly more young Inuit were current smokers, more Inuit than Sami abstained from alcohol (Table IV). Table II shows the independent socio-demographic, risk and protective variables in NAAHS and WBYG. Also presented are the items included, their categories and final measurements. Statistical analysis To evaluate both SamiInuit and in-group differences in the distribution of covariates, Pearson’s Chi-squared test or Fischer’s exact test were used for categorical data. The cells contributing most to the statistical differences were determined by inspection of the standardised residuals. Logistic regressions were performed with SRH as the binary dependent variable, as in the majority (68%) of studies of youth SRH (6). Associations between SRH and each risk, protective and socio-demographic factors were explored by simple logistic regressions, and separately for Sami and Inuit. In line with the majority of studies of SRH (91%), we used multiple models (6) to examine the influence of each socio-demographic, risk and protective variable when mutually controlling for all other factors. A stepwise backward approach was chosen. Starting with a full model, the variable with the highest p-value was removed, and the procedure was repeated until all remaining variables were significant at the 10% level. Regardless of the significant level at each step, gender was forced into the model due to the possibility of causing any confusion. SPSS version 19 was used for statistical analysis. 4 (page number not for citation purpose) Citation: Int J Circumpolar Health 2013, 72: 19793 - http://dx.doi.org/10.3402/ijch.v72i0.19793 4 (page number not for citation purpose) Results Table V shows the simple and multiple logistic regression analyses of correlates associated with SRH among Sami and Inuit adolescents separately. For both groups, adjusted analyses revealed that suicidal thoughts and physical inactivity were risk factors for poor SRH. SRH by indigenous group and gender Table III shows that the overall proportion of Sami (N350) and Inuit (N378) adolescents reported poor, 4 ( 4 (pag l n NAAHS and WBYG ) Inuit (WBYG) NAAHS/WBYG Categories Item(s) Categories Measurement(s) oor economy verage economy ood economy ery good economy What is your mother’s (father’s) formal education? Elementary school High school Short vocational education Higher education, 34 years University education NAAHS: Good (good, very good economy) vs. Poor (poor, average economy) WBYG: Higher educational level (college, university degree) vs. Lower educational leve (elementary school, high school, short vocational education) Married/cohabiting nmarried ivorced/separated One or both are dead Other Do your parents live together? Yes No Yes (married/cohabiting/ living together) vs. No (unmarried, divorced/ separated, dead, other, or not living together) o es Have you ever seriously considered committing suicide? No Yes No vs. Suicidal thoughts (yes) vs. Suicidal attempts (Yes on suicidal thoughts and/or attempts) o es Have you ever attempted to commit suicide? No Yes o es How often do you drink alcohol? I never drink alcohol Less than monthly About once a month About once weekly Daily or almost daily Abstinence (Never drink alcohol or not drinking during the last year) vs. Moderate (Drinking monthly or less) vs. Frequent (Drinking once weekly or more) 7 times weekly 3 times weekly bout once a week 3 times monthly bout once a month few times during the ast year ave not drunk alcohol uring the last year Inuit (WBYG) NAAHS/WBYG Categories Item(s) Categories Measurement(s) er I quit casionally y Do you smoke? No Yes, but I have days where I don‘t smoke Yes, daily No (Never or non-smokers, and quitters) vs. Yes (Occasional smokers and daily smokers) y by youth y by adults h youth and adults Within the last year have you been exposed to any of the following?a Most children experience conflicts at home. Have you experienced - You were pushed and shaken in anger? - You were torn in your hair You were beaten Yes Yes No (Never, not exposed to violence within the last year, and not exposed to domestic violence (e.g. conflicts at home)) vs. Self-rated health among indigenous adolescents Self-rated health among indigenous adolescents Sami (NAAHS) Inuit (WBYG) NAAHS/WBYG Variables Item(s) Categories Item(s) Categories Measurement(s) Adolescent parent relationship If you have personal problems who could you go to?b Yes No I don‘t know How easy or difficult is it for you to talk with the following persons, when you have problems?b Very easy Easy Difficult Very difficult I don‘t have any parentsc Good (yes, very easy, easy) vs. Poor (no, difficult, very difficult) aViolence was one of ten alternatives. bParents were one of several alternatives. cThe alternative ‘‘I don’t have any parents’’ was categorized as missing. Suicidal thoughts increased the risk of poor SRH by almost four times among Sami and by about two times among Inuit. In contrast, physical activity decreased the risk of poor SRH by almost five times among Sami and two times among Inuit. Among Sami unadjusted risk correlates of poor SRH also included perception of poor family economy, experience of violence, heavy drinking and poor well-being in school. In the final multiple regression analysis, only poor parental economy was associated with poor SRH. Among Inuit, with the excep- tion of gender, an additional two correlates remained significant in the final multiple regression analysis. Being a current smoker increased the risk of poor SRH twofold, while a good parentadolescent relationship decreased the risk almost twofold. Number of friends was not related to SRH in unadjusted or adjusted analysis. 7 (page number not for citation purpose) Results Yes (Exposed to violence within the last year and/or domestic violence (conflicts at home) 4,5,. . .. How often do you exercise hard (running, soccer, something else) Every day At least once a week Less than weekly Never Frequent (1 or more times a week or every day) vs. Seldom (0 times a week, less than weekly or never) ree gree sagree agree Do you like going to school? Very well Well Fair Not so well Good (Fully or partly agree, well and very well vs. Poor (Partly or fully disagree, fair or not so well) re How many close friends do you have? No close friends at the moment 1 23 4 or more Few (None or 1) vs. Many (2 or more) Discussion This paper describes patterns of SRH among Inuit and Sami adolescents. The majority of young Sami and Inuit reported good or very good SRH. Very few reported poor health. The NAAHS is the first study to investigate SRH among Sami adolescents, while SRH has previously been assessed among Greenlandic youth (7,8). Our first aim was to compare rates of SRH among Sami and Inuit to regional and national peers, as well as to each other. The proportions of Sami youth reporting good or very good SRH (89%) and poor SRH (11%) in the NAAHS do not differ greatly from regional (Non-Sami in the NAAHS, results not shown) or national peers (2). However, this finding contrasts with data on adult Sami who have reported poorer SRH than Norwegian peers (21). Among adult Sami, poor SRH was associated with older age, female gender and lower educational and income level, but became non-significant when control- ling for discrimination (21). The differences in SRH among Sami adolescents and adults (]51 year) may be attributable to the earlier ‘‘Norwegianization’’ policy (e.g. institutional discrimination) that older age groups have experienced. Today Sami women living in Norwegian dominated areas (with greater integration and assimila- tion) that are characterised by less ethnic support reported the most unsatisfactory conditions concerning SRH (21). The 1517 year olds in the WBYG showed lower rates for good and very good health when compared to findings from the Greenlandic Health Behaviour in School-aged Children (HBSC) study (2006; 6th to 11th graders); the figures were 71% (80%) and 64% (66%) for 15-year-old males and females, respectively (7). Cross-cultural data from the Scandinavian part of the HBSC study in 2005/ 2006, using a 4-point SRH scale, found the highest rate of poor and fair SRH in Greenland (21%), followed by Norway (19%), then Denmark (14%) (22). Although most adolescents reported good SRH, there was a relatively large difference in how Inuit and 7 pose) 7 pose) 7 (page number not for citation purpose) Anna Rita Spein et al. Table III. Distribution of self-rated health in NAAHS and WBYG by gender Table III. Discussion However, violence was not significantly associated with poor SRH among Sami in the adjusted analysis, in contrast to findings by others (23). Half of the Inuit adolescents currently smoked and smokers had a more than twofold higher risk of poor SRH than non-smokers. Smoking per se may affect SRH either by causing phy- sical health problems, or being used as self-medication for mental stressors (24,25). There are three main possible explanatory factors for the higher smoking rates found among Inuit than Sami adolescents. First, Sami adoles- cents are less exposed to environmental smoking as smoking rates of adult Sami the ‘‘parental’’ generation tend to be much lower than among Greenlanders (9). Second, passive smoking is less accepted in Norway as the tobacco control policy is more extensive than in Greenland and the Norwegian efforts has also been considered less liberal in its design. For example, a smoking ban was implemented much earlier in Norway (1st of June 2004) compared to Greenland (1st of October 2010) (26). Third, smoking is highly associated with levels of education, which is currently higher among Sami than Inuit peers, as about 39% and 21% of Sami (people in Sami areas) and Greenlanders have attained high school diplomas, respectively (27,28). The second aim of the study was to describe gender differences in SRH. Although males more frequently reported good SRH than females, the crude data showed only significant gender differences in SRH among Inuit. However, gender was not a significant predictor of SRH among Inuit in the final multiple regression analysis. This finding suggests that gender differences in SRH may have been mediated by other socio-demographic, risk and protective factors, in line with the existing literature (2). Among Circumpolar indigenous adults, minimal gender differences in SRH have been found, while female gender has been negatively associated with SRH among Native American youth (1). Our third aim was to examine ethnic differences and similarities in risk and protective correlates associated with SRH. Of particular note is the almost threefold negative influence of poor socio-economic status on SRH among Sami. Generally, the Sami settlements are char- acterised by 10% and 18% lower mean income, when compared to regional and national figures, respectively (30). Higher income has been positively associated with SRH among indigenous adults (1,9). Income seems to be a more relevant factor to adolescents’ health perception than parental education and occupation (19). Discussion Distribution of self-rated health in NAAHS and WBYG by gender Samia (NAAHS) Inuita (WBYG) Male Female Totalb Male Female Totalc n162 n188 N350 n155 n223 N378 Self-rated health Very good (n, %) 64 (40) 54 (29) 118 (34) 32 (21) 32 (14) 64 (17) Good (n, %) 83 (51) 111 (59) 194 (55) 78 (50) 93 (42) 171 (45) Fair/Not so good (n, %) 15 (9) 20 (11) 35 (10) 44 (28) 91 (41) 135 (36) Poor (n, %) 0 (0) 3 (2) 3 (1) 1 (1) 7 (3) 8 (2) aSignificant SamiInuit differences in SRH, disfavouring Inuit [x2 (3, 728)77.6, **p50.001], due to fewer Sami reporting not so good SRH and more Inuit reporting fair SRH. bNon-significant gender differences in SRH among Sami [x2 (3, 350)6.71, p50.08]. cSignificant gender differences in SRH among Inuit [x2 (3, 378)10.14, p50.05]. aSignificant SamiInuit differences in SRH, disfavouring Inuit [x2 (3, 728)77.6, **p50.001], due to fewer Sami reporting not so good SRH and more Inuit reporting fair SRH. bNon-significant gender differences in SRH among Sami [x2 (3, 350)6.71, p50.08]. cSignificant gender differences in SRH among Inuit [x2 (3, 378)10.14, p50.05]. indigenous Sami and Inuit adolescents responded posi- tively. Although drinking is an inappropriate behaviour in socio-culturalreligious terms among many Sami due to the strong anti-alcohol stand of Læstadianism (9), more Inuit than Sami youth (18% vs. 11%) reported abstinence. Both Sami and Inuit adolescents have generally showed higher abstinence rates than their Norwegian and Danish peers, respectively (9,29). Frequent drinking was reported by 12% of Sami, but was not significantly associated with poor SRH in the adjusted analysis. However, drinking style was not assessed. The Greenlandic drinking style is characterised by binge drinking, which is less common in Sami adolescents than non-Sami (9,29). Sami perceived their health. Twice as many Sami as Inuit reported very good SRH (34% vs. 17%). More than two thirds (36%) of Inuit reported their health to be fair, while the corresponding not so good among Sami was 10%. These findings may partly represent significant SamiInuit differences in risk (suicidal behaviour and smoking disfavouring Inuit) and protec- tive correlates (Sami being more physically active). Nonetheless, the majority of indigenous youth did not face significant health risks. Only the prevalence of experiencing violence was similar among Sami and Inuit. 8 (page number not for citation purpose) Citation: Int J Circumpolar Health 2013, 72: 19793 - http://dx.doi.org/10.3402/ijch.v72i0.19793 Discussion Young people’s subjective perception of poor familial socio- economic status (SES) has been associated with poor An earlier review concluded that substance use prevalence was not comparable among young Sami and Inuit (15). Moderate drinking may be considered to be normal behaviour as more than three fourths of 8 (pag Self-rated health among indigenous adolescents Table IV. The distribution of independent variables in NAAHS and WBYG by gender (n, %) Table IV. Discussion The distribution of independent variables in NAAHS and WBYG by gender (n, %) p y g ( , ) Sami (NAAHS) Inuit (WBYG) Effect of gender Sami (p) Effect of gender Inuit (p) Effect of indigenous group (p) Male n162 Female n188 Total N350 Male n155 Female n223 Total N378 Socio-demographics Parents married/ cohabiting Yes 88 (55) 117 (62) 205 (59) 0.15 93 (60) 130 (59) 223 (59) 0.75a 0.88 No 73 (45) 71 (38) 144 (41) 61 (40) 92 (41) 153 (41) Socio-economic status Good 102 (65) 95 (51) 197 (57) 0.01 101 (67) 140 (66) 241 (67) 0.82a NTb Poor 56 (35) 92 (49) 148 (43) 49 (33) 72 (34) 121 (33) Risk correlates Suicidal behaviour No 119 (74) 91 (49) 210 (61) 50.001 126 (82) 98 (44) 224 (60) 50.001 50.001 Thoughts 31 (19) 67 (36) 98 (28) 15 (10) 50 (23) 65 (17) Attemptsc 10 (6) 27 (15) 37 (11) 13 (8) 73 (33) 86 (23) Alcohol use Abstinence 20 (14) 12 (8) 32 (11) 0.20 32 (21) 35 (16) 67 (18) 0.25 0.03 Moderate 103 (74) 122 (80) 225 (77) 105 (68) 153 (69) 258 (68) Frequent 16 (12) 19 (12) 35 (12) 17 (11) 35 (16) 52 (14) Current smoking No 110 (68) 120 (64) 230 (66) 0.46 85 (55) 90 (40) 175 (46) 0.005a 50.001 Yes 52 (32) 67 (36) 119 (34) 69 (44) 133 (60) 202 (54) Experienced violence No 118 (74) 154 (82) 272 (78) 0.05 128 (83) 176 (79) 304 (80) 0.23a 0.50 Yes 42 (26) 33 (18) 75 (22) 27 (17) 47 (21) 74 (20) Protective correlates Physical activity Frequent 144 (90) 154 (85) 298 (87) 0.17 106 (70) 105 (47) 211 (56) 50.001a 50.001 Seldom 16 (10) 27 (15) 43 (13) 46 (30) 117 (53) 163 (44) Well-being in school Good 142 (88) 170 (90) 312 (89) 0.50 115 (75) 170 (78) 285 (77) 0.62a 50.001 Poor 19 (12) 18 (10) 37 (11) 38 (25) 49 (22) 87 (23) Number of close friends 2 or more 153 (95) 177 (95) 330 (95) 0.87 142 (92) 193 (88) 335 (90) 0.23 0.01 0 or one friend 8 (5) 10 (5) 18 (5) 12 (8) 26 (12) 38 (10) Adolescent parent relationship Good 85 (56) 96 (52) 181 (54) 0.42 104 (78) 138 (67) 242 (71) 0.03 50.001 Poor 66 (44) 89 (48) 155 (46) 29 (22) 68 (33) 97 (29) aFisher Exact Test used. Citation: Int J Circumpolar Health 2013, 72: 19793 - http://dx.doi.org/10.3402/ijch.v72i0.19793 9 (page number not for citation purpose) Discussion There were no significant differences in the gender distribution in NAAHS and WBYG samples; [x2 (1, n728) 2.06, NS]. bNT: Not Tested. Indigenous group differences were not tested due to different socio-economic measures in NAAHS (parental financial situation) and WBYG (parental educational level). cIncludes both suicidal attempts and suicidal thoughts. The number reporting only prior suicidal attempts and not thoughts were 15 in total (4 Sami/11 Inuit). The three categories in the suicidal behaviour item are mutually exclusive. cause of death among 1524 year old indigenous males (9,16). From 1970 to 1998, suicide rates among young Sami males and females were respectively 53 and 16 per 100,000 person-years, giving a malefemale ratio of 3.5:1 (9). In the Sami dominated area of inner Finnmark County there has been a cluster of suicide (9). In Greenland during 199099, suicides among 1524 year olds amounted to 400500 for men and 100150 for women per 100,000 person-years, with a malefemale ratio of 4.3:1 (16). Suicide seems to be less frequent among young Sami than young Inuit (15). This NorwegianGreenlandic pattern has also been found with regard to suicidal thoughts among females in the SRH even when controlling for race, parental education and total household income (31). In the NAAHS, parental financial status was based on young people’s self-reported perception, while the WBYG assessed parental education. This may explain the different impact of SES on SRH among the two indigenous groups. Our main focus for the remaining discussion will be on the relationship of SRH to suicidal behaviour and physical activity, as correlations were found in both cases among Sami and Inuit. Suicidal thoughts were a strong risk correlate, increasing the odds for poor SRH twofold (Inuit) and fourfold (Sami). Suicide is a huge health problem in Arctic communities. Suicide is the leading 9 pose) 9 ose) Citation: Int J Circumpolar Health 2013, 72: 19793 - http://dx.doi.org/10.3402/ijch.v72i0.19793 9 (page number not for citation purpose) Anna Rita Spein et al. imple and multiple logistic regressions of poor vs. good self-rated health in NAAHS and WBYG p p g g p g Sami (NAAHS) Inuit (WBYG) Unadjusted (n350) Adjusteda (n265)b Unadjusted (n378) Adjusteda (n333)b OR 95% CI OR 95% CI OR 95% CI OR 95% CI Socio-demographics Parents married/ cohabiting Yes 1 Ref. 1 Ref. No 1.56 (0.99,2.48)NS 1.04 (0.68,1.59)NS Socio-economic statusc Good 1 Ref. 1 Ref 1 Ref. 10 (page number not for citation purpose) Citation: Int J Circumpolar Health 2013, 72: 19793 - http://dx.doi.org/10.3402/ijch.v72i0.19793 Ref: reference group; OR: odds ratio; CI: confidence interval; p50.001; p50.01; p50.05; NS: non significant. aEach independent variable was adjusted for all other socio-demographics, risk- and protective correlates. bNumber of cases included in the final backward logistic regression. The significance level for the variables included was set at 10%. cSocio-economic measures by parental financial situation in NAAHS and by parental educational level in WBYG. dIncludes both suicidal attempts and suicidal thoughts. The number reporting only prior suicidal attempts and not thoughts were 15 in total (4 Sami/11 Inuit). The three categories in the suicidal behaviour item are mutually exclusive. eNumber of friends was not included in the multivariate analyses. ference group; OR: odds ratio; CI: confidence interval; *p50.001; p50.01; p50.05; NS: non-significant. independent variable was adjusted for all other socio demographics risk and protective correlates Discussion Poor 2.81 (1.73,4.56) 2.74 (1.60,4.69)* 0.73 (0.46,1.15)NS Gender Male 1 Ref. 1 Ref. 1 Ref. 1 Ref. Female 1.62 (1.04,2.53)* 1.08 (0.65,1.82)NS 1.92 (1.24,2.96) 1.08 (0.63,1.84)NS Risk correlates Suicidal behaviour No 1 Ref. 1 Ref. 1 Ref. 1 Ref. Thoughts 4.57 (2.48,8.44)* 3.77 (1.96,7.25)* 2.74 (1.56,4.83)* 2.07 (1.10,3.91)* Attemptsd 4.27 (1.71,10.7)* 2.64 (0.98,7.08)NS 2.50 (1.50,4.17)* 1.66 (0.87,3.18)NS Alcohol use Abstinence 1 Ref. 1 Ref. Moderate 1.50 (0.71,3.17)NS 1.71 (0.95,3.07)NS Frequent 4.67 (1.44,15.1) 1.58 (0.73,3.41)NS Current smoking No 1 Ref. 1 Ref. 1 Ref. 1 Ref. Yes 2.39 (1.43,3.97)* 1.64 (0.92,2.91)NS 2.72 (1.76,4.22)* 2.05 (1.23,3.40)** Experienced violence No 1 Ref. 1 Ref. Yes 1.81 (1.01,3.25)* 1.07 (0.64,1.81)NS Protective correlates Physical activity Seldom 1 Ref. 1 Ref. 1 Ref. 1 Ref. Frequent 0.17 (0.06,0.50)* 0.21 (0.07,0.63) 0.41 (027,0.63)* 0.50 (0.31,0.81) Well-being in school Poor 1 Ref. 1 Ref. Good 0.35 (0.14,0.87)* 0.39 (0.14,1.11)NS Adolescentparent relationship Poor 1 Ref. 1 Ref. 1 Ref. 1 Ref. Good 0.68 (0.43,1.08)NS 0.39 (0.75,2.68)NS 0.46 (0.29,0.75)* 0.59 (0.35,0.99)* Number of friendse 01 friend 1 Ref. 1 Ref. ]2 friends 1.03 (0.38,2.81)NS 1.40 (0.71,2.75)NS Ref: reference group; OR: odds ratio; CI: confidence interval; *p50 001; p50 01; p50 05; NS: non-significant two youngest age groups (1434 year olds) in the recent ‘‘Survey of living conditions in the Arctic’’ (SLICA) study. This difference was thought to be due to differ- ences in national educational level (32). overprotection (9). Suicide of a close friend and loneli- ness were associated with suicidal behaviour among Greenlandic Inuit adolescents. Having experienced a sexual assault and problems with parents were female- specific factors, while termination of a relationship was specifically associated with Inuit male suicidal behaviour (9). Suicidal thoughts were more frequently reported among young Inuit (in particular females) from homes with poor emotional environments, alcohol problems and violence (16). Furthermore, Grossman, Milligan and Deyo (33) found in a sample of Native American adolescents that 15% reported a prior suicide attempt and that poor self- perception of health was one of several risk factors for suicide attempts. Among Sami adolescents, risk factors diverging from traditional Sami cultural norms were associated with suicide attempts, including alcohol intoxication, single-parent households and paternal Engaging in frequent physical activity decreased the odds of poor SRH about twofold (Inuit) and fivefold Citation: Int J Circumpolar Health 2013, 72: 19793 - http://dx.doi.org/10.3402/ijch.v72i0.19793 Self-rated health among indigenous adolescents in the two groups (1). Conclusion Most Sami and Inuit adolescents have good SRH and do not face health hazards such as heavy drinking, exposure to violence or suicidal behaviour problems. The poorer SRH among Inuit may partly be due to group differences in risk and protective correlates associated with SRH. Physical activity was positively associated with SRH while suicidal behaviour influenced SRH negatively in both indigenous groups. The positive impact of physical activity on SRH should be targeted in future school health promotion programs. 2. Breidablikk H-J, Meland E, Lydersen S. Self-rated health during adolescence: stability and predictors of change (Young HUNT study, Norway). Eur J Public Health. 2008;19:738. 1. Bombak AE, Bruce SG. Self-rated health and ethnicity: focus on indigenous populations. Int J Circumpolar Health. 2012; 71:18538. Limitations There are several limitations to be noted. The dependent variable SRH was not identical as the WBYG had a fifth option very poor, although no Inuit reported very poor. Also, the NAAHS had a category not so good, while the corresponding category in the WBYG was worded fair. This could have contributed to the observed skewness in SRH. Both instruments tapping SRH were non- comparative measures, although not identical with regard to rating options (SRH-4/SRH-5), and comparisons should therefore be made with some caution. However, the current limitation is believed to only have minor influence as a Swedish study found that different non- comparative measures of SRH represents parallel assess- ment of subjective health (37). Conservative figures for Inuit risk behaviours may occur due to a high school absence rate. The WBYG included the word seriously in the question about having considered suicide, which probably led to underreporting as former suicidal thoughts may in retrospect not have been considered as ‘‘serious’’. It is unknown whether the observed Sami Inuit differences in SRH reflect true differences or cultural differences in how health-related factors were understood The NAAHS was a collaborative effort between the Centre for Sami Health Research and the Norwegian Institute of Public Health. The WBYG study was a collaboration between the National Institute of Public Health, Denmark, and PAARISA (Ofﬁce of Health and Prevention Measures), the Greenland Ministry of Health and Greenland Home Rule. 3. Boardman JD. Self-rated health among U.S. adolescents. J Adolesc Health. 2006;38:4018. Discussion Potential protective cultural resilience factors such as traditional activities, spirituality, language use and healing were not included here, and these merit further research attention (38). A further limitation is that sexual abuse, parental substance use and geography, which strongly influence SRH (6), were not available for comparison here for technical reasons. A history of childhood sexual abuse has been negatively associated with SRH among Greenlandic Inuit, in parti- cular among women and those who had experienced parental substance abuse during childhood (1). Further- more, there are considerable geographical differences in living conditions and health in Greenland (9,15). Our studies were cross-sectional, in line with the majority (73%) of studies on youth SRH (6). Future studies should be longitudinal and capable of assessing whether suicidal behaviour and physical inactivity temporally precede poor SRH or vice versa. (Sami). The NAAHS survey referred to physical exercise outside school but no such distinction was made in the WBYG. In line with most studies, there were significant gender differences among Inuit only, adolescent males being more physically active than female peers (9,34,35). The differences in physical activity level among the Inuit and Sami can partly be explained by the different measures and a lower access to active leisure time offers in Greenland compared to Norway. Physical activity also positively influences self-image, family and peer relation- ships and general well-being among youth and has been inversely related to youth depression (35,36). Studies among indigenous adult groups have revealed lower leisure time physical activity when compared to majority peers, and that lower education and income are negatively associated with physical activity (9). Sami adults seem to be more physically active at work than non-Sami (9). Both poor SRH per se, and the SamiInuit differences in SRH may partly be caused by other factors outside this study that contribute to inequalities in SRH. Considerable differences in health indicators exist within the Arctic states and populations. Greenlandic Inuit are disadvantaged with regard to several health indicators when compared to Danes as Greenlanders perceive for example lower lifetime expectancy rates and higher infant mortality rates and years of life lost due to suicide, while little difference is found between the Sami and non-Sami population (9,15). Adolescent mental health care and services are relatively well staffed in Sami areas including easier access to treatment facilities, in contrast to most parts of Greenland (9). Conflict of interest and funding The authors have no conflict of interest to report. Funding has been received from the Axel Emil Søeborg Ohlsen and Spouse Else Søeborg Memorial Scholarship, the Norwegian Ministry of Health and Care Services and the Sami Parliament. Citation: Int J Circumpolar Health 2013, 72: 19793 - http://dx.doi.org/10.3402/ijch.v72i0.19793 1. Bombak AE, Bruce SG. Self-rated health and ethnicity: focus on indigenous populations. Int J Circumpolar Health. 2012; 71:18538. 2. Breidablikk H-J, Meland E, Lydersen S. Self-rated health during adolescence: stability and predictors of change (Young HUNT study, Norway). Eur J Public Health. 2008;19:738. 3. Boardman JD. Self-rated health among U.S. adolescents. J Adolesc Health. 2006;38:4018. References Javo C, Heyerdahl S, Rønning JA. Parent reports of child behaviour problems in young Sami children: a cross cultural comparison. Eur Child Adolesc Psychiatry. 2000;9:20211. 30. Statistics Norway. Sami Statistics 2010. Table 39 Income statistics for households. All of Norway and Norway North of Saltfjellet, 2007. [cited 2012 Aug 8]. Available from: http:// www.ssb.no/english/subjects/00/00/20/nos_d443_en/tab/tab_39. html 12. Kvernmo S, Heyerdahl S. Acculturation strategies and ethnic identity as predictors of behaviour problems in Arctic minority adolescents. J Am Acad Child Adolesc Psychiatry. 2003;42: 5765. 13. Turi AL, Bals M, Skre IB, Kvernmo S. Health service use in indigenous Sami and non-indigenous youth in North Norway: a population based survey. BMC Public Health. 2009;9:378. 31. Goodman E, Huang B, Schafer-Kalkhoff T, Adler NE. Perceived socioeconomic status: a new type of identity that inﬂuences adolescents’ self-reported health. J Adolesc Health. 2007;41:47987. 14. Niclasen BV, Bjerregaard P. Child health in Greenland. Scand J Public Health. 2007;35:31322. 32. Broderstad AR, Eliassen B-M, Melhus M. Prevalence of self- reported suicidal thoughts in SLICA. The survey of living conditions in the Arctic (SLICA). Glob Health Action. 2011;4:10226. 15. Lehti V, Niemela¨ S, Hoven C, Mandell D, Sourander A. Mental health, substance use, and suicidal behaviour among young indigenous people in the Arctic: a systemic review. Soc Sci Med. 2009;69:1194203. 16. Bjerregaard P, Lynge I. Suicide a challenge in modern Greenland. Arch Suicide Res. 2006;10:20920. 33. Grossman DC, Milligan BC, Deyo RA. Risk factors for suicide attempts among Navajo adolescents. Am J Public Health. 1991;18:8704. 17. Kaiser N, Sjølander P, Edin-Liljegren A, Jacobson L, Renberg ES. Depression and anxiety in the reindeer herding Sami population of Sweden. Int J Circumpolar Health. 2010;69: 3839. 34. Sallis JF, Prochaska JJ, Taylor WC. A review of correlates of physical activity of children and adolescents. 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https://openalex.org/W2566223150	https://zenodo.org/records/2024353/files/article.pdf	English	null	III.—On the Limitations of a Knowledge of Nature	Proceedings of the Aristotelian Society	1,922	public-domain	4,237	Meeting nf the Aristotelian Society at 21, Gower Street, W.C. 1, on December 5th, 1921, at 8 P.M. Meeting nf the Aristotelian Society at 21, Gower Street, W.C. 1, on December 5th, 1921, at 8 P.M. III.—ON THE LIMITATIONS OF A KNOWLEDGE OF NATURE. By JAMES JOHNSTONE. By JAMES JOHNSTONE. SOMETIME about the beginning of the eighteenth century, and during the last ten years of his lifetime, Newton spoke to a friend about his work : " I know not," he said, " how it may seem to the world but, as to myself, I seem to have been only as a boy playing on the seashore, and diverting myself in now and then finding a smoother pebble or a prettier shell than ordinary, whilst the great ocean of truth lay all undiscovered before me." These words, I take it, were, in a way, an admission of cessation of individual effort. By reason of his resolute and disciplined imagination, his genius for experiment and the mighty mathematical weapons that he had made, Newton had found a way. He thought about truth as lying there, spread out, so to speak, and waiting to be discovered and the means whereby that discovery was to be made were known to him. He knew that what he had found was only an infinitesimal part of all that was accessible by application of his methods. But there is only so much dynamic mental quality in any man, a little more or a little less as we vary from each other, and this can be " intended " to the investiga- tion of nature, the pursuit of pleasure or the acquisition of wealth, and it can be exhausted. In little over forty years Newton, like his great predecessor, Descartes, had spent his creative energy, and the undiscovered, but discoverable ocean of truth was still there. by guest on June 9, 2016 http://aristotelian.oxfordjournals.org/ d from By the time that these words were spoken the course that physical and natural science was to follow during the next century and half had already been marked out by three great men: Galileo, to an extent which has only been appreciated by F JAMES JOHNSTONE. 4 4 some of us as the result of the relativity discussions of the last few years; Descartes, by the exercise of an " inventiveness " which, as Clerk Maxwell said, " knew no bounds " ; and Newton himself, who had found the way. Throughout that century and half one seems to trace little or no really creative scientific thought but only a successful working out of the great ideas of the seventeenth century. By JAMES JOHNSTONE. From these came our conceptions of matter, inertia and force; the laws of motion; the theory of universal gravitation; the description of the Solar system; the notion of the ether as the locus and substance of physical change; the ideas of illimitable space and uniformly flowing infinite time; the Cartesian mechanism of life and the restricted theory of relativity. About the middle of the nineteenth century new ideas did come: perhaps the notion, of natural selectioa was really new—I do not know—perhaps we had in that idea, the first clear distinction between statistical and individual results—a distinction that does not yet seem fully to be realized and employed in biological science. And then, a little later, we had from the mathematical investigators, of whom J. Clerk Maxwell is the type, the germinal work that was to bring about a revolution in knowledge. by http://aristotelian.oxfordjournals.org/ Downloaded from by guest on June 9, 2016 http://aristotelian.oxfordjournals.org/ from Throughout those two centuries scientific men employed the inventions of Galileo, Descartes and Newton. Physics and natural science (which has always clung to the skirts of physics) explored the seashore, described and catalogued the pebbles and shells and now and then ventured out on the ocean. The eighteenth and nineteenth centuries elaborated the methods of the seventeenth, employing them in ways that were certainly unanticipated by Descartes and Newton- one wonders what the great French philosopher would have thought about the modern mechanistic conception of life, as it has been stated by Jacques Loeb! It has been noted that as the methods of the seventeenth century became exhausted so did the materialistic science of the nineteenth seem to approach THE LIMITATIONS OF KNOWLEDGE. 45 finality and tend to become complete and rounded-off, in a sense. Perhaps one may be quite wrong but it does appear as if the natural science of the latter third of the last century regarded its framework as sound and entirely satisfactory and one gets suggestions of that kind from some of Huxley's essays. What was the good of quarrelling about the unknow- able ? The speculative game was drawn and what was left for Science was the work of strengthening the framework and filling in the details. by gues http://aristotelian.oxfordjournals.org/ Downloaded from Perhaps this hard nineteenth century materialism had its work to do in the evolution of social and political liberty. * A. N. Whitehead, in The Cvncept of Nature, Cambridge Univemty Press, 1920, p. 73. By JAMES JOHNSTONE. It had to assert itself as a way of interpreting the meaning of the " passage of nature" and of searching out the origin and destiny of man. Medieval doctrines of social and economic privilege had to be destroyed. Perhaps that work is not yet fully accomplished and while that is so science will remain materialistic. There is still a fraudulent and grotesque spiritualism to be detected; a muddled vitalism to be replaced by something sounder and a prematurely formulated " Eugenics," that may be utilized to maintain caste and social disability, to be sifted clear from humbug. We may leave materialistic biology to these tasks. by guest on June 9, 2016 http://aristotelian.oxfordjournals.org/ d from Two centuries later than Newton a modern thinker,* writing while a revolution in scientific thought was being effected, refers to " the passage of nature which is only another name for the creative force of existence." " This operative presence " he says, " which is now urging nature forward must be sought for through- out the whole, in the remotest past as well as in the narrowest breadth of any present duration. Perhaps also in the un- realized future. Perhaps also in the future which might be, as well as in the future which will be. It is impossible to 46 JAMES JOHNSTONE. meditate on time and the mystery of the creative passage of nature without an overwhelming emotion at the limitations of human intelligence." I place this saying over against the well-known words of Newton because the two utterances illustrate very well the change in our attitude towards what is meant by scientific discovery. But I must make it clear what I mean by the "passage of nature" because this is a notion far less subtle thau that indicated by Mr. Whitehead. And I think that a candid and impartial survey of the speculative biology of the late nineteenth and twentieth centuries must force one to the recognition of a twofold passage of nature. Perhaps this is indicated even in Huxley's contrast of the cosmic and ethical processes but it is expressed, with the utmost clearness, in Bergson's vital impetus as opposed to the tendency of matter to pass into the inert condition. By JAMES JOHNSTONE. I take it that the fundamental concept of physical science is the second law of energetics—the universal augmentation of entropy—and I assume (though it is difficult to be sure) that nothing in the most modern results of mathematical relativity tends, in the least, to weaken this great conception. Nature, then, has direction, or passage, which is such that all that we recognize as physical change tends con- tiually to diminution: the Universe, regarded as a physical mechanism is one that is running down, or in Bergson's term, detending. This is the one aspect of the passage of nature. by gues http://aristotelian.oxfordjournals.org/ Downloaded from by guest on June 9, 2016 http://aristotelian.oxfordjournals.org/ d from To the biologist, however, it can only be one aspect. I am well aware that the entropy-increase law is a statistical one and that it can only hold true for organic entities which are above certain limiting magnitudes: for Maxwell's demons the law would have a double sign and the entropy of an isolated system would increase or decrease with equal probability. The biologist must recognize that, even in organic systems, entropy tends always towards augmentation but surely he misunder- stands the meanings of reproduction and adaptation if he does not see that what he calls life is the incessant attempt of certain THE LIMITATIONS OF KNOWLEDGE. 47 physico-chemical systems that we call organisms to resist the increase of entropy. There is, therefore, a passage of nature which is not that tending to inert-materiality (that is, to statistical inertia) but which is the opposite to this passage and is what one must understand by life in the physical sense. I am not sure whether we ought to insist on this two-fold passage of nature or, perhaps, regard it as a double aspect, in some way or other, of the same condition. Is life something that resists the passage of inorganic nature, or is inorganic nature already inert and extended, while living systems pass through it ? Perhaps one inclines to think about a two-fold passage because of some mental constraint that tends always to a dualism of one kind or another. By JAMES JOHNSTONE. by gues http://aristotelian.oxfordjournals.org/ Downloaded from by guest on June 9, 2016 http://aristotelian.oxfordjournals.org/ from I suppose that biologists must accept the main result of • generalized relativity: " the differentiation of the one quality of extension into time and space," but I confess that it is very difficult to do so. It seems to me that for speculative physio- logy space-time cannot be completely isotropic—the v, y, z and ^-dimensions cannot be of the same quality. I take it that our notion of space rests entirely on our degrees of freedom of bodily mobility. I can move backwards and forwards, and from side to side with equal facility but not nearly so easily can I move upwards and downwards. And the equal freedom of mobility in the x, y-plane is only possible because I can turn my body round a vertical (2;)-axis in one direction or the other with equal facility but even then the turning movement from left to right is not quite the same as that from right to left but differs in some subtle way. And, of course, the difficulty of generating the z-dimension depends on the condition that our freedom of mobility is restricted because we move in a gravitational field. Only since we have become enabled to dispense (in thought) with the gravitational field as something physically unique has our space become truly isotropic. Such as we are, however, the space-dimensions are not entirely isotropic and far less so is the ^-dimension when F 2 48 JAMES JOHNSTONE. compared with the x, y, z-ones. The quality of duration I take to be entirely different from the others and we must, I think, regard it, with Bergson, as the cumulative continuity of life. It is a passage as well as the persistence of that which, in a sense, has passed. It is life-extension but it does not seem to me to be capable of " extensive abstraction " in Mr. Whitehead's sense. * " Time " in the sense of life-extension I regard as " humped " in the " neighbourhood" of a conscious entity in somewhat the same way as Mr. Eddington regards space as being " humped " or " peaked" in the neighbourhood of a material particle. By JAMES JOHNSTONE. The passage is not a uniform one (though I confess I find it difficult to say exactly what is meant by uniformity in a durational passage).* by gue http://aristotelian.oxfordjournals.org/ Downloaded from Obviously we do not obtain the conception of a moment of organic duration by the method of extensive abstraction, for this " moment" depends on what Bergson calls the " rhythm of duration": thus the " event-particle " in the conscious life of a boy of fifteen is not the same as in the man of fifty, nor does it appear to be the same in the ephemeral insect as it is in the long-lived reptile. The matter, however, is much too difficult to be pursued here. by guest on June 9, 2016 http://aristotelian.oxfordjournals.org/ d from It is also necessary that I should deal with the purely biological conception of variability. In general we mean by a variation a deviation from a morphological type, but I generalize the notion so as to include also deviations of functioning, acting, response and mentality, perceiving no essential differ- ences between these organic activities. The organic " type," whether it be that of form, or behaviour, or mentality is, of course, only a convenient abstraction, but the general notion of " types " has brought with it the conception of variability. It is very convenient, in our description of nature, to speak of specific types and then of variations or departures from them. Observations and experiments, we say, " ought" to give unique values but for the accompanying errors of methods. So, also, THE LIMITATIONS OF KNOWLEDGE. 49 we postulate organic types which are accompanied by varia- tions in the same way that experimental results are attended by error. Then we search (rather unsuccessfully, it must be admitted) for the " cause " of variability. Obviously, this con- ception of variability is the consequence of our adoption of the logical category of determinism. Now there are organic activities that have all the appear- ance of spontaneity—whether these are truly spontaneous or not I do not argue—but there are also many activities which we call responses to events that occur " in the environment." These we can investigate and we can endeavour to establish a relation of functionality, in the mathematical sense, between the environmental " stimulus " and the organic response. By JAMES JOHNSTONE. It would be easy, I think, to make a series of such responses, beginning with tropistic ones, passing through reflexes in the decerebrate animal, reflexes in the intact one, and ending with those responses which we call " intelligent." In the various terms of such a series there will be " more or less " determinism, if one may say so without being misunderstood. At the one extreme we find (as in a tropism, or taxis) a degree of function- ality which approximates closely to the behaviour of a compass needle in a variable magnetic field, and at the other we find that apparently capricious behaviour or functioning which must be so annoying to physiologists. I suppose that there must either be determinism, or no determinism, and so I have simply to reject the validity of this concept (except as a working method, of course) even at the risk of being exposed to the dreadful accusation of throwing overboard scientific method altogether! by g http://aristotelian.oxfordjournals.org/ Downloaded from by guest on June 9, 2016 http://aristotelian.oxfordjournals.org/ from One other thing I must endeavour to make clear—the distinction between the organic variations that are called " fluctuations " by biologists and those others that are called " mutations." The former are individual and acquired while the latter are congenital and are transmitted by heredity— they are not acquired. In the lower animate fluctuations, or 50 JAMBS JOHNSTONE. acquirements, do not materially influence the process of transformism and what are of significance from that stand- point are mutations, which do lead to transformism. In man, of course, certain fluctuations persist by reason of tradition: they are not bodily variations but means of action by tools (using the term " tool" in its most general significance). Now the distinction between fluctuations and mutations is evidently one that depends upon our distinction between a racial and individual life-passage. Life is, of course, a continuous career in the morphological sense: what is discontinuous in it is the personal passage which is marked by memory, blame, merit and responsibility—sin, if you like. The mutational variation belongs then to the racial passage and it is an acquirement of this continuous life-career; the fluctuating variation arises in the discontinuous personal life-passage, or career. by guest on June 9, 2016 http://aristotelian.oxfordjournals.org/ d from I return now, after this digression, to the saying of Newton. By JAMES JOHNSTONE. The ocean of undiscovered knowledge must, to him, have been like the material' oceans explored by the voyagers of his century: they were unknown but whatever was there did not depend, in itself, in the least upon the vessels and instruments of navigation; it was only revealed by those methods. So, to Newton, physical laws were there waiting to be discovered, so to speak, but even if they were to remain undiscovered they would still be there. Without doubt he could have made most of the discoveries of the eighteenth century and perhaps those of the nineteenth up to the time of Clerk Maxwell had he been capable for a long period of that sustained intension of mind of which he spoke, for (I take it) those discoveries were implicit in the creative work of his early lifetime. But were the later physical and biological results of the later nineteenth and the twentieth century there in the same sense as were, for instance, planetary theory and tidal dynamics ? Were the quantum hypothesis of radiation and THE LIMITATIONS OF KNOWLEDGE. 51 our present-day notions of atomic structure present already in Newton's undiscovered ocean of truth ? Could these con- ceptions have been deduced by him by a sufficient intending of mind from the mathematical, physical and dynamical relationships known to him ? I take it that they could not and that only by the creative thought of Newton's nineteenth century successors did these parts of the ocean of truth actually come into existence. by gues http://aristotelian.oxfordjournals.org/ Downloaded from Otherwise it would appear that there was mental deter- minism and that Clerk Maxwell, Hertz, Planck, Einstein, J. J. Thomson and others thought as they did think because Newton's mind worked in the way it did. And nothiug in the results of modern biology seems to suggest that. I have referred already to the way in which mutations of form arise with all the appearance of spontaneity or lack of causation. It must have occurred to many biologists to attempt to predict the evolutionary career of some organic stock or other but beyond suggesting that certain specific forms are in process of extinction, or that some bodily parts of an organic species are becoming vestigial and tend to disappear nothing of the sort has, I believe, been attempted. By JAMES JOHNSTONE. No biologist has ventured to predict the appearance of a mutation—the essential step to a process of transformism. Now one admits the incredible com- plexity of the physico-chemical systems in which such muta- tions arise and we may well despair of laying bare the physical antecedents of a mutation—supposing thac there are such. But the overwhelming impression that most biologists have, in thinking about these matters at all, is that of the spontaneity of appearance of the mutation. I admit that there is evidence that environmental changes may induce mutations ; expose an organism to some environmental stimuli and mutations may arise but what we have to deal with here are active, functional adaptations of the organism, ways in which it responds to the external change. But that the particular nature of the response is a function, in the mathematical sense, of the particular by guest on June 9, 2016 http://aristotelian.oxfordjournals.org/ d from 52 JAMES JOHNSTONE. change in the environment does not seem to he established, nor do I think that it is likely. Probably we must generalize " responses" in the widest possible way. I have argued elsewhere that the most various kinds of behaviour are of essentially the same organic nature. An " adaptation," I take it, is not merely a change in colour or form that renders an animal less conspicuous to its enemies or prey, or confers upon it some useful means of finding food or shelter, or of avoiding its foes. These changes are of much significance in hypotheses of the means of transformism and so they are the things that we usually think about when we speak of adaptations. But temporary variations of functioning (such as the process of sweating when one becomes warm) are also adaptations.. So is the behaviour of a man who takes an umbrella with him on au unpromising morning, or that of the skipper of a vessel when he shortens sail in anticipation of bad weather. by gue http://aristotelian.oxfordjournals.org/ Downloaded from by guest on June 9, 2016 http://aristotelian.oxfordjournals.org/ d from Again the invention and use of a new tool is an adaptation and so also is the discovery of a new mathematical device (say tables of logarithms). By JAMES JOHNSTONE. It is quite true that some of these organic modes of behaviour are transmitted by heredity so that they become integral in the life-processes of the race while others would disappear on the death of the individual in which they are evolved. That, however, is because the one kind of adapta- tion (mutations) is characteristic of the racial life passage (it is germinal) while the other appears in the individual life-passage (it is somatic). It would disappear if it were not preserved by tradition. So I can make no essential distinction between morpho- logical or " organic" adaptations of functioning and those changes of ways of mental operation that we call scientific discoveries. The strengthening of the muscles of the fingers and wrists of a pianist; the formation of a skin callus on some part of the hand in consequence of.the persistent holding of a tool; manual dexterity in some repetitional mechanical operation; THE LIMITATIONS OF KNOWLEDGE. 53 facility in arithmetical work; the finding of some new mathe- matical relationships (say Maxwell's four thermodynamic potentials) and so on—all these seem to me to be processes that have the same significance. In each of them there is something creative or new, some means whereby the organism becomes the better able to oppose the tendency to inert-materiality. This is, of course, pure Bergsonisra: Maxwell's thermodynamic functions or the Christoffel " tensor analysis" are means of acting on nature. Sooner or later, someone endeavours to give even the most abstruse of mathematical results a " physical meaning" and sooner or later also, these results receive " appli- cations " in industry. All " intended" thinking, I take it. aims at establishing inter-connexions between events in nature, .All general discoveries are mental adaptations—something really new in organic behaviour. by guest on June 9, 2016 http://aristotelian.oxfordjournals.org/ from And if that is so we must, I think, regard Newton's ocean of truth as amorphous in structure. The relations that are to be discovered in it are only in it in the sense that they come into existence with the thought that makes the relations. Our knowledge of nature, as Eddington says, is a knowledge of form and not of content, but even the form is carved out from a nature that may have any forms—or as many as are implicit within the limitation^ of the human mind. By JAMES JOHNSTONE. At any moment in human history, then, our description of nature is complete, that is to say, what more is in it than that which we know has still to be made by us. I feel that, as I have stated this there is something paradoxical in it, but my meaning will, I trust, be plain. Finally, I return to Mr. Whitehead's saying—perhaps that which is urging nature forwards is in the future as well as in the present and past; and in the future that may be as well as in future that will be. I take this to be literally true. The impetus is certainly in the remotest past as well as in the present, since we inherit modes of acting on nature which have " passed " only in the sense that they came into existence one THE LIMITATIONS OF KNOWLEDGE. 54 after the other in " time," but which nevertheless endure in that they constitute our present life-mechanisms. The impetus is in the future that will be, surely, because many of the things that we do are done in order that some change, or condition clearly thought about but which has not yet happened, or does not yet exist, will come about as the result of our acting. So from the wolf we have bred the sheep dog and we are rearing rustless wheat and potatoes that are immune to disease. These organisms were not discovered in nature nor did they exist there in the literal sense—they were made, if my interpretation- of the meaning of mutations is a right one. The impetus is in the future that may be, because in seeking for something we make something else. There is an ideal communistic state that may be and that is ardently desired by some. In seeking to make it our present-day idealists are, without doubt, making some other society, the form of which is not discoverable. by gue http://aristotelian.oxfordjournals.org/ Downloaded from by guest on June 9, 2016 http://aristotelian.oxfordjournals.org/ from by guest on June 9, 2016 ttp://aristotelian.oxfordjournals.org/
https://openalex.org/W4361874247	https://figshare.com/articles/journal_contribution/Supplementary_Figure_S5_from_IGF2_Preserves_Osteosarcoma_Cell_Survival_by_Creating_an_Autophagic_State_of_Dormancy_That_Protects_Cells_against_Chemotherapeutic_Stress/22404182/1/files/39849995.pdf	English	null	Supplementary Figure S3 from IGF2 Preserves Osteosarcoma Cell Survival by Creating an Autophagic State of Dormancy That Protects Cells against Chemotherapeutic Stress	null	2,023	cc-by	556	0 20 40 60 80 100 10%FBS IGF2 Insulin 5-Aza-2’ -deoxycytidine (100 nM) Viable cells (% of control) 0 20 40 60 80 100 120 valproic acid (40 µM) 10%FBS IGF2 Insulin Viable cells (% of control) Supplementary Figure S5 10%FBS IGF2 0 20 40 60 80 100 Viable cells (% of control) bortezomib (10 nM) 0 20 40 60 80 100 10%FBS IGF2 Insulin 5-Aza-2’ -deoxycytidine (100 nM) Viable cells (% of control) 0 20 40 60 80 100 120 valproic acid (40 µM) 10%FBS IGF2 Insulin Viable cells (% of control) Supplementary Figure S5 10%FBS IGF2 0 20 40 60 80 100 Viable cells (% of control) bortezomib (10 nM) 0 20 40 60 80 100 10%FBS IGF2 Insu 5-Aza-2’ -deoxycytidine (100 Viable cells (% of control) 0 20 40 60 80 100 10%FBS IGF2 Insulin trichostatin A (20 nM) Viable cells (% of control) 0 20 40 60 80 100 120 valproic acid (40 µM) 10%FBS IGF2 Insu Viable cells (% of control) 0 20 40 60 80 100 120 10%FBS IGF2 Insulin phenylbutyrate (1.25 mM) Viable cells (% of control) 0 20 40 60 80 100 120 10%FBS IGF2 Insulin rapamycin (200 nM) Viable cells (% of control) metformin (1 mM) 10%FBS IGF2 Insulin 0 20 40 60 80 100 120 Viable cells (% of control) Shimizu T et al. 0 20 40 60 80 100 10%FBS IGF2 Insulin 5-Aza-2’ -deoxycytidine (100 nM) Viable cells (% of control) 0 20 40 60 80 100 120 valproic acid (40 µM) 10%FBS IGF2 Insulin Viable cells (% of control) Supplementary Figure S5 10%FBS IGF2 0 20 40 60 80 100 Viable cells (% of control) bortezomib (10 nM) Supplementary Figure S 10%FBS IGF2 0 20 40 60 80 100 Viable cells (% of control) bortezomib (10 nM) 0 20 40 60 80 100 120 Viable cells (% of control) 10 10% FBS IGF2 Insulin bafilomycin A (pM) 100 1000 10,000 0 200 400 600 800 1000 aminooxyacetate (µM) 0 20 40 60 80 100 120 Viable cells (% of control) 10% FBS IGF2 Insulin B A C 1 Viable cells (% of control) 1 Viable cells (% of control) 1 Viable cells (% of control) Vi bl ll (% f t l) Vi bl ll (% f l) 0 20 40 60 80 100 120 10%FBS IGF2 Insulin phenylbutyrate (1.25 mM) Viable cells (% of control) metformin (1 mM) 10%FBS IGF2 Insulin 0 20 40 60 80 100 120 Viable cells (% of control) Vi bl ll (% f l) 0 20 40 60 80 100 120 Viable cells (% of control) 10 10% FBS IGF2 Insulin bafilomycin A (pM) 100 1000 10,000 C C metformin (1 mM) phenylbutyrate (1.25 1 1 Viable cells (% of control) 1 1 Viable cells (% of control) 1 1 Viable cells (% of control) 1 1 Viable cells (% of control) 0 20 40 60 80 100 10%FBS IGF2 Insulin trichostatin A (20 nM) Viable cells (% of control) 0 20 40 60 80 100 120 10%FBS IGF2 Insulin rapamycin (200 nM) Viable cells (% of control) 0 200 400 600 800 1000 aminooxyacetate (µM) 0 20 40 60 80 100 120 Viable cells (% of control) 10% FBS IGF2 Insulin B A B 1 Viable cells (% of control) 1 1 Viable cells (% of control)
https://openalex.org/W4390712597	https://www.journal.unrika.ac.id/index.php/sigmateknika/article/download/4982/3536	Indonesian	null	STABILISASI TANAH LEMPUNG DENGAN DIFA SOIL STABILIZER DAN SEMEN PCC	Sigma Teknika	2,023	cc-by-sa	3,275	Sigma Teknika, Vol. 6, No.1 : 195-201 Juni 2023 E-ISSN 2599-0616 P ISSN 2614-5979 Sigma Teknika, Vol. 6, No.1 : 195-201 Juni 2023 E-ISSN 2599-0616 P ISSN 2614-5979 ABSTRAK Di daerah Bungo Tanjung – Taluk Tapang, Pasaman Barat memiliki jenis tanah yaitu lempung. Lempung merupakan jenis tanah yang memiliki konsistensi mudah berubahubah mengakibatkan daya dukung tanah rendah. Tanah lempung Bungo Tanjung – Taluk Tapang memiliki nilai CBR 3,1% yang mana nilai minimal CBR untuk tanah dasar (Subgrade) tidak boleh kurang dari 6%, oleh karena itu dilakukan perbaikan tanah dasar menggunakan Difa Soil Stabilizer dan Semen type PCC bertujuan untuk memperbaiki indeks plastisitas dan CBR tanah dasar. Setelah dilakukan penelitian di laboratorium, hasil yang didapat dari persentase semen 6% dan difa soil stabilizer 1,67%, 1,9% dan 4,26% berupa indeks plastisitas 18,79%, 17,58% dan 15,74%, serta nilai CBR 18%, 21% dan 27%. Berdasarkan hasil tersebut nilai indeks plastisitas paling baik yaitu 15,74% dengan nilai CBR yang paling baik 27% yang terdapat pada persentase campuran semen 6% dan difa soil stabillizer 4,26%. Kata Kunci : Stabilisasi, Tanah Lempung, CBR, Indeks Plastisitas, Difa Soil Stabilizer STABILISASI TANAH LEMPUNG DENGAN DIFA SOIL STABILIZER DAN SEMEN PCC Veronika1), Hendri Warman2), Bayu Andrea3) 1,2,3)Prodi Teknik Sipil, Fakultas Teknik Sipil dan Perencanaan, Universitas Bung Hatta E-mail: veronika_salmi@yahoo.com Kata Kunci : Stabilisasi, Tanah Lempung, CBR, Indeks Plastisitas, Difa Soil Stabilizer Keywords : Stabilization, Clay, CBR, Index Plasticity, Difa Soil Stabilizer 1. PENDAHULUAN Pada pengujian ini penulis melakukan stabilisasi tanah dengan menggunakan bahan tambah kimiawi berupa Difa Soil Stabilizer dan Semen PCC. 2.1 Tanah Lempung Tanah Lempung adalah partikel mineral mengandung senyawa silikat yang berdiameter kurang 5 mikrometer. Lempung mengandung leburan silikat dan/atau aluminium yang halus. Tabel 1. Sifat Umum Tanah Lempung No Parameter Nilai 1 Kadar Air 30-50% 2 Batas Cair 50 - 90% 3 Batas Plastis 25 - 40% 4 Lolos Saringan no.200 ≤ 50% 5 Kuat geser 50 - 80 kN/m2 6 Berat Volume tanah 1.75-2.10t/m3 7 Spesific Gravity (Gs) 2.68 - 2.75% 8 Kuat Tekan Bebas 0.50-1.00 kg/cm2 Sumber : Hary Christady, Mekanika Tanah 1 (2010) 2. TINJAUAN PUSTAKA Tanah merupakan suatu benda alam yang tersusun dari padatan (bahan mineral dan organik), cairan dan gas. Tanah berguna sebagai bahan bangunan pada berbagai macam pekerjaan teknik dan disamping itu tanah berfungsi sebagai pendukung pondasi dari bangunan. Tanah bisa diklasifikasikan atas dua macam, yaitu : a. Klasifikasi USCS (Unified Soil Classification System) Klasifikasi ini mengelompokkan tanah sebagai berikut : 1. Tanah butir kasar (coarse-grained-soil) yaitu tanah kerikil dan pasir kurang dari 50% berat total, contoh tanah lolos ayakan no.200. 2. Tanah berbutir halus (fine-grained-soil) yaitu tanah yang lebih dari 50% berat total, contoh tanah yang lolos ayakan no.200. a. Klasifikasi USCS (Unified Soil Classification System) Klasifikasi ini mengelompokkan tanah sebagai berikut : 1. PENDAHULUAN Dalam ilmu Teknik Sipil, salah satu hal yang diperhitungkan dalam pembangunan kontruksi ialah sifat tanah dasar serta daya dukung tanah tersebut karena tanah merupakan elemen yang berperan penting sebagai pondasi pendukung suatu kontruksi yang akan menerima beban diatasnya. 1. Ukuran butir Kerikil: bagian tanah yang lolos saringan dengan diameter 75 mm (3 inci) dan yang tertahan pada saringan No. 20 (2 mm). Lanau dan lempung: bagian tanah yang lolos saringan No. 200. g 2. Plastisitas Nama berlanau dipakai apabila bagian-bagian yang halus dari tanah mempunyai indeks plastisitas sebesar 10 atau kurang. Nama berlempung dipakai bilamana bagian-bagin yang halus dari tanah mempunyai indeks plastisitas sebesar 11 atau lebih. menerima beban diatasnya. Konstruksi jalan raya di daerah Bungo Tanjung - Taluk Tapang, Pasaman Barat berjenis lempung. Tanah lempung memiliki konsistensi yang mudah berubah- ubah, dalam keadaan kering mempunyai daya dukung tinggi dan dalam keadaan jenuh akan mempunyai daya dukung rendah akibat pengaruh air dan memiliki nilai CBR yang cenderung rendah. Nilai CBR (California Bearing Ratio) yang dikategorikan baik untuk subgrade kontruksi jalan raya berdasarkan pengujian laboratorium yaitu lebih dari 6%. Sedangkan nilai CBR tanah lempung yang penulis dapatkan dari hasil penelitian di Laboratorium memiliki nilai CBR 3,1%. Stabilisasi tanah untuk perkerasan jalan merupakan upaya memperbaiki sifat dan parameter dari tanah asli agar tanah tersebut sesuai atau memenuhi syarat untuk dipergunakan sesuai fungsinya. Pada pengujian ini penulis melakukan stabilisasi tanah dengan menggunakan bahan tambah kimiawi berupa Difa Soil Stabilizer dan Semen PCC. y Konstruksi jalan raya di daerah Bungo Tanjung - Taluk Tapang, Pasaman Barat berjenis lempung. Tanah lempung memiliki konsistensi yang mudah berubah- ubah, dalam keadaan kering mempunyai daya dukung tinggi dan dalam keadaan jenuh akan mempunyai daya dukung rendah akibat pengaruh air dan memiliki nilai CBR yang cenderung rendah. Nilai CBR (California Bearing Ratio) yang dikategorikan baik untuk subgrade kontruksi jalan raya berdasarkan pengujian laboratorium yaitu lebih dari 6%. Sedangkan nilai CBR tanah lempung yang penulis dapatkan dari hasil penelitian di Laboratorium memiliki nilai CBR 3,1%. 3. Apabila batuan (ukuran lebih besar dari 75 mm) ditemukan didalam contoh tanah yang akan ditentukan klasifikasi tanahnya, maka batuan-batuan tersebut harus dikeluarkan dahulu. Tetapi, presentasi dari batuan yang dikeluarkan harus dicatat. Stabilisasi tanah untuk perkerasan jalan merupakan upaya memperbaiki sifat dan parameter dari tanah asli agar tanah tersebut sesuai atau memenuhi syarat untuk dipergunakan sesuai fungsinya. ABSTRACT In the Bungo Tanjung – Taluk Tapang area, West Pasaman has a type of soil, namely clay. Clay is a type of soil that has a volatile consistency resulting in low soil bearing capacity. Bungo Tanjung – Taluk Tapang clay has a CBR value of 3,1% where the minimum CBR value for subgrade cannot be less that 6%, therefore the subgrade improvement using Difa Soil Stabilizer and Cement type PCC aims to improve the plasticity index and CBR of the subgrade. After doing research in the laboratory, the result obtained from the percentage of cement 6% and soil stabilizer difa 1,67%, 1,9% and 4,26% in the form of plasticity index 18,79%, 17,58% and 15,74%, and CBR values of 18%, 21% and 27%. Based on the results, the best plasticity index value is 15,74% with best CBR value of 27% which is found in the percentage of cement mixture 6% and soil stabilizer difa 4,26%. Keywords : Stabilization, Clay, CBR, Index Plasticity, Difa Soil Stabilizer 195 Sigma Teknika, Vol. 6, No.1 : 195-201 Juni 2023 E-ISSN 2599-0616 P ISSN 2614-5979 Pada sistem ini tanah diklasifikasikan atas 7 bagian, berdasarkan kriteria : Pada sistem ini tanah diklasifikasikan atas 7 bagian, berdasarkan kriteria : 2.2. Stabilisasi Tanah 1. Tanah butir kasar (coarse-grained-soil) yaitu tanah kerikil dan pasir kurang dari 50% berat total, contoh tanah lolos ayakan no.200. Pada prinsipnya stabilisasi tanah merupakan suatu penyusunan kembali butir-butir tanah agar lebih rapat dan saling mengunci. Tanah dibuat stabil agar jika ada beban yang lewat tidak terjadi penurunan (settlement). 2. Tanah berbutir halus (fine-grained-soil) yaitu tanah yang lebih dari 50% berat total, contoh tanah yang lolos ayakan no.200. p Proses stabilisasi meliputi antara lain : b. Klasifikasi AASHTO (American Association of State Highway and Transporting) 1. Penggantian tanah asli dengan tanah yang baik atau sesuai spesifikasi. 196 Sigma Teknika, Vol. 6, No.1 : 195-201 Juni 2023 E-ISSN 2599-0616 P ISSN 2614-5979 E-ISSN 2599-0616 P ISSN 2614-5979 Bagan alir dari penelitian ini adalah sebagai berikut : 2. Perbaikan gradasi butiran. 2. Perbaikan gradasi butiran. 3. Stabilisasi dengan bahan kimia. 4. Stabilisasi dengan pemadatan. Gambar 1. Bagan Alir Penelitian Tanah dapat dikatakan tidak layak atau buruk jika: Tanah dapat dikatakan tidak layak atau buruk jika: 1. Memiliki Indeks Plastisitas tinggi yang mana dengan indeks plastistisitas yang tinggi mampu berpotensi tanah mengalami pengembangan. Menurut BPSDM Perkerasan Jalan (2018) “standar dari indeks plastisitas untuk tanah dasar sebaiknya 7% dan maksimal tidak mendekati atau lebih dari 25%”. 2. Memiliki parameter daya dukung yang rendah. Parameter daya dukung yang rendah dapat dilihat dari nilai CBR tanah dasar. Berdasarkan surat edaran Manual Perkerasan Jalan (2017) “nilai CBR untuk tanah dasar tidak kurang dari 6%. Tanah dinyatakan tidak layak atau buruk untuk digunakan sebagai tanah dasar kontruksi perkerasan jalan jika memiliki nilai CBR dibawah 6% 2.3. Difa Soil Stabilizer Difa Soil Stabilizer adalah bahan aditif yang berfungsi untuk memadatkan (solidifikasi) dan menstabilkan (stabilizer) tanah secara fisik-kimia yang berupa material serbuk halus yang terdiri dari komposisi mineral anorganik yang mengandung senyawa calsiumchlorid-dihydrat dengan pH 8,24. Tabel 2. Komposisi Difa Soil Stabilizer Parameter Hasil Satuan Carbon 72,78 % Hidrogen 4,75 % Oksigen 21,11 % Nitrogen 1,36 % pH 8,24 - Sumber : PT Difa Mahakarya 4.3. Pengujian Tanah Asli 4.3. Pengujian Tanah Asli Dari hasil pengujian sifat fisik tanah asli diperoleh bahwa tanah termasuk kelompok tanah lempung tak organic atau lempung berpasir dengan symbol CL. Tabel 5. Hasil Pengujian Fisik Tanah Asli 4.4 Pengujian Sifat Mekanik Tanah Asli 1. Compaction Gambar 3. Grafik Kepadatan Tanah Asli Dari hasil pengujian sifat fisik tanah asli diperoleh bahwa tanah termasuk kelompok tanah lempung tak organic atau lempung berpasir dengan symbol CL. 4.1 Pengujian Analisa Saringan Tabel dibawah merupakan hasil analisa saringan dari tanah sampel. Tabel 3. Analisa Saringan Gambar 2. Grafik Plastisitas 3. METODE PENELITIAN 3. METODE PENELITIAN 3. METODE PENELITIAN 3.1. Lokasi Penelitian Lokasi berada di daerah Bunga Tanjung – Teluk Tapang, Kecamatan Sungai Beremas Kabupaten Pasaman Barat. Kondisi tanah pada proyek pembangunan jalan tersebut adalah tanah lempung. Gambar 1. Bagan Alir Penelitian 3.2. Bagan Alir Penelitian 197 Sigma Teknika, Vol. 6, No.1 : 195-201 Juni 2023 E-ISSN 2599-0616 P ISSN 2614-5979 Gambar 2. Grafik Plastisitas Sigma Teknika, Vol. 6, No.1 : 195-201 Juni 2023 E-ISSN 2599-0616 P ISSN 2614-5979 Sigma Teknika, Vol. 6, No.1 : 195-201 Juni 2023 E-ISSN 2599-0616 P ISSN 2614-5979 4. HASIL DAN PEMBAHASAN Gambar 2. Grafik Plastisitas Gambar 2. Grafik Plastisitas 4.2 Pengujian Atterberg Limit Tabel 5. Hasil Pengujian Fisik Tanah Asli Hasil yang diperoleh dalam pengujian atterberg limit seperti yang terlihat dalam table 4. Tabel 4. Atterberg Limit 4.4 Pengujian Sifat Mekanik Tanah Asli 4.4 Pengujian Sifat Mekanik Tanah Asli 1. Compaction Gambar 3. Grafik Kepadatan Tanah Asli Dari hasil penelitian yang didapat, maka dapat ditentukan jenis tanah yaitu sebagai berikut : Gambar 3. Grafik Kepadatan Tanah Asli 198 Sigma Teknika, Vol. 6, No.1 : 195-201 Juni 2023 E-ISSN 2599-0616 P ISSN 2614-5979 Sigma Teknika, Vol. 6, No.1 : 195-20 Juni 2023 E-ISSN 2599-0616 P ISSN 2614-5979 Pada penelitian ini diperoleh kadar air optimum (OMC) adalah 28%, dan berat volume kering maksimum pada tanah asli adalah 1,17 gr/cm3. Gambar 5. Grafik Hubungan Antara Atterberg Limit dan Campuran 5.2 Saran 1. Dikarenakan tanah dasar setiap jalan memiliki jenis tanah yang berbeda-beda, maka dianjurkan untuk melakukan pengujian selain jenis tanah Lempung 5. KESIMPULAN DAN SARAN 2. Perlu dilakukannya penelitian lanjutan dengan bahan tamb Sigma Teknika, Vol. 6, No.1 : 195-201 Juni 2023 E-ISSN 2599-0616 P ISSN 2614-5979 Sigma Teknika, Vol. 6, No.1 : 195-201 Juni 2023 E-ISSN 2599-0616 P ISSN 2614-5979 Tabel 9. Pengujian persentase Semen+Difa SS terhadap Nilai CBR Tabel 9. Pengujian persentase Semen+Difa SS terhadap Nilai CBR Tabel 9. Pengujian persentase Semen+Difa SS terhadap Nilai CBR Pasaman Barat yaitu Lempung Tak Organik atau Lempung Berpasir (Clay Low-Plasticity). 2. 2. Pada pengujian CBR laboratorium sebelum dan sesudah penambahan semen dan Difa SS, terjadi perubahan yang signifikan terhadap nilai Indeks Plastisitas dan CBR pada tanah asli. Pada pengujian ini didapatkan hasil nilai Indeks Plastisitas dan CBR sebelum dan sesudah penambahan semen dan Difa SS sebagai berikut : a. Tanah Asli didapatkan nilai Indeks Plastisitas 20,36% dan CBR 3,1% b. Tanah + 6% Semen + 1,67% Difa SS didapatkan nilai Indeks Plastisitas 18,79 dan CBR sebesar 18% c. Tanah + 6% Semen + 1,9% Difa SS didapatkan nilai Indeks Plastisitas 17,58% dan CBR sebesar 21% d. Tanah + 6% Semen + 4,26% Difa SS didapatkan nilai Indeks Plastisitas 15,74% dan CBR sebesar 27% 62 Semakin besar persentase penambahan kadar campuran mampu mengurangi potensi pengembangan serta meningkatkan daya dukung pada tanah dasar yang dapat dilihat dari nilai Indeks Plastisitas semakin menurun dan nilai CBR yang semakin tinggi sehingga memenuhi standar tanah dasar (subgrade) untuk kontruksi perkerasan jalan. Gambar 7. Grafik Nilai CBR dan Persentase Campuran Dari penelitian ini terlihat bahwa semakin besar kadar penambahan bahan campuran mampu menaikkan nilai daya dukung tanah asli, yang dilihat dari nilai CBR laboratorium semakin tinggi dan pada penelitian ini didapatkan persentase Semen 6% dan Difa SS 4,26% menjadi presentase kenaikan tertinggi dengan nilai CBR sebesar 27%. Gambar 5. Grafik Hubungan Antara Atterberg Limit dan Campuran Dari hasil penelitian tersebut penurunan terbesar terjadi pada persentase 6% semen + 4,26 Difa, dimana juga pada campuran tersebut nilai indeks plastisitasnya adalah 15,74% telah memenuhi standar dan dapat digunakan untuk tanah dasar konstruksi perkerasan jalan. 2. California Bearing Ratio (CBR) Gambar 4. Hubungan kepadatan dan Nilai CBR Tanah Asli 2. California Bearing Ratio (CBR) 4.5.2 Pengujian Sifat Mekanik Tanah Asli dengan Difa SS dan Semen PCC Tabel 8. Hasil Pengujian Kepadatan Campuran Semen dan Difa SS Terlihat dari table tersebut bahwa semakin bertambah persentase semen dan Difa SS membuat nilai kadar optimum tanah asli semakin menurun. Tabel 8. Hasil Pengujian Kepadatan Campuran Semen dan Dif SS Gambar 4. Hubungan kepadatan dan Nilai CBR Tanah Asli 4.5 Campuran Tanah dengan Difa Soil Stabilizer dan Semen PCC 4.5.1 Pengujian Sifat Fisik Tanah Asli dengan Difa SS dan Semen PCC 4.5.1 Pengujian Sifat Fisik Tanah Asli dengan Difa SS dan Semen PCC Terlihat dari table tersebut bahwa semakin bertambah persentase semen dan Difa SS membuat nilai kadar optimum tanah asli semakin menurun. Tabel 6. Hasil Pengujian Berat Jenis (Gs) Campuran Semen dan Difa SS Tabel 6. Hasil Pengujian Berat Jenis (Gs) Campuran Tabel 6. Hasil Pengujian Berat Jenis (Gs) Campuran Semen dan Difa SS Gambar 6. Grafik hubungan MDD dan Persentase Campuran Gambar 6. Grafik hubungan MDD dan Persentase Campuran Tabel 7. Hasil Pengujian Atterberg Limit Campuran Semen dan Difa SS Tabel 7. Hasil Pengujian Atterberg Limit Campuran Semen dan Difa SS Tabel 7. Hasil Pengujian Atterberg Limit Campuran S d Dif SS Gambar 6. Grafik hubungan MDD dan Persentase Campuran Dari grafik diatas tersebut dapat terlihat bahwa semakin bertambah persentase Semen dan Difa SS dapat meningkatkan nilai volume kering maksimum (MDD) pada tanah, dari hasil penelitian ini kenaikan paling tinggi didapatkan pada persentase semen 6% dan Difa SS 4,26% sebanyak 1,25%. 199 Tabel 9. Pengujian persentase Semen+Difa SS terhadap Nilai CBR Gambar 7. Grafik Nilai CBR dan Persentase Campuran Sigma Teknika, Vol. 6, No.1 : 195-201 Juni 2023 E-ISSN 2599-0616 P ISSN 2614-5979 5.1 Kesimpulan 3. ah selain semen untuk mengetahui cocok atau tidak bahan Difa SS dengan bahan tambah lainnya 3. ah selain semen untuk mengetahui cocok atau tidak bahan Difa SS dengan bahan tambah lainnya 1. Dari pengujian batas-batas konsistensi (atterberg limit) yaitu batas cair didapatkan hasil 49,56%, batas plastis 28,24% dan indeks plastisitas 21,32% yang mana dengan indeks plastisitas yang didapat pada tanah dasar di Bungo Tanjung - Taluk Tapang, Pasaman Barat ini berpotensi mengalami pengembangan dikarenakan nilai indeks plastisitas yang tinggi. Untuk nilai berat jenis pada tanah asli didapatkan 1,90%. Sedangkan pada pengujian analisa saringan analisa saringan didapatkan presentase kerikil sebanyak 1,72%, presentase pasir 40,78% dan presentase lempung 59,22%. Dari hasil pengujian ini didapatkan jenis tanah pada daerah Bungo Tanjung – Taluk Tapang, 1. Dari pengujian batas-batas konsistensi (atterberg limit) yaitu batas cair didapatkan hasil 49,56%, batas plastis 28,24% dan indeks plastisitas 21,32% yang mana dengan indeks plastisitas yang didapat pada tanah dasar di Bungo Tanjung - Taluk Tapang, Pasaman Barat ini berpotensi mengalami pengembangan dikarenakan nilai indeks plastisitas yang tinggi. Untuk nilai berat jenis pada tanah asli didapatkan 1,90%. Sedangkan pada pengujian analisa saringan analisa saringan didapatkan presentase kerikil sebanyak 1,72%, presentase pasir 40,78% dan presentase lempung 59,22%. Dari hasil pengujian ini didapatkan jenis tanah pada daerah Bungo Tanjung – Taluk Tapang, DAFTAR PUSTAKA 1] Ardi Kristiadi. & Akhmad Marzuko. 2016. Pengaruh Penambahan Bahan Additif Berupa Campuran Semen Dengan Difa SS Pada Tanah Butir Halus Terhadap Nilai CBR. Jurnal Teoritis. Yogyakarta 2] Das, Braja M. 1993. Mekanika Tanah (Prinsip- Prinsip Rekayasa Geoteknik) Jilid 2. Erlangga. Surabaya. DAFTAR PUSTAKA 1] Ardi Kristiadi. & Akhmad Marzuko. 2016. Pengaruh Penambahan Bahan Additif Berupa Campuran Semen Dengan Difa SS Pada Tanah Butir Halus Terhadap Nilai CBR. Jurnal Teoritis. Yogyakarta 2] Das, Braja M. 1993. Mekanika Tanah (Prinsip- Prinsip Rekayasa Geoteknik) Jilid 2. Erlangga. Surabaya. DAFTAR PUSTAKA 1] Ardi Kristiadi. & Akhmad Marzuko. 2016. Pengaruh Penambahan Bahan Additif Berupa Campuran Semen Dengan Difa SS Pada Tanah Butir Halus Terhadap Nilai CBR. Jurnal Teoritis. Yogyakarta 2] Das, Braja M. 1993. Mekanika Tanah (Prinsip- Prinsip Rekayasa Geoteknik) Jilid 2. Erlangga. Surabaya. DAFTAR PUSTAKA 1] Ardi Kristiadi. & Akhmad Marzuko. 2016. Pengaruh Penambahan Bahan Additif Berupa Campuran Semen Dengan Difa SS Pada Tanah Butir Halus Terhadap Nilai CBR. Jurnal Teoritis. Yogyakarta 2] Das, Braja M. 1993. Mekanika Tanah (Prinsip- Prinsip Rekayasa Geoteknik) Jilid 2. Erlangga. Surabaya. 2] Das, Braja M. 1993. 5.1 Kesimpulan Mekanika Tanah (Prinsip- Prinsip Rekayasa Geoteknik) Jilid 2. Erlangga. Surabaya. 200 Sigma Teknika, Vol. 6, No.1 : 195-201 Juni 2023 E-ISSN 2599-0616 P ISSN 2614-5979 Sigma Teknika, Vol. 6, No.1 : 195-201 Juni 2023 E-ISSN 2599-0616 P ISSN 2614-5979 Sigma Teknika, Vol. 6, No.1 : 195-201 Juni 2023 E-ISSN 2599-0616 P ISSN 2614-5979 3] Direktur Jenderal Bina Marga. 2017. Manual Desain Perkerasan Jalan. Direktorat Jenderal Bina Marga. Jakarta. 4] Direktur Jenderal Bina Marga. 2018. Perkerasan jalan. Badan Pengembangan Sumber Daya Manusia. Jakarta. 5] Hardiyatmo, Hary Christady. 2002. Mekanika Tanah I. Gadjah Mada University Press. Yogyakarta. 6] Hardiyatmo, Hary Christady. 2011. Perencanaan Perkerasan Jalan Dan Penyelidikan Tanah. Gadjah Mada University Press. Yogyakarta. 7] H di H Ch i d 2017 S bili i 5] Hardiyatmo, Hary Christady. 2002. Mekanika Tanah I. Gadjah Mada University Press. Yogyakarta. 6] Hardiyatmo, Hary Christady. 2011. Perencanaan Perkerasan Jalan Dan Penyelidikan Tanah. Gadjah Mada University Press. Yogyakarta. 7] Hardiyatmo, Hary Christady. 2017. Stabilisasi Tanah Untuk Perkerasan Jalan. Gadjah Mada University Press. Yogyakarta. 8] Iswan. Muhammad Karami. I Wayan Diana. Pengaruh Pemakaian Difa Soil Stabilizer Terhadap Daya Dukung Tanah Lempung Untuk Kontruksi Jalan. Jurnal Teoritis. Lampung. p g 9] SNI 1742:2008. 2008. Cara Uji Kepadatan Ringan Untuk Tanah. Badan Standarisasi Nasional. Bandung. 10] SNI 1744:2012. 2012. Metode Uji CBR Laboratorium. Badan Standarisasi Nasional. Bandung. 11] SNI 1964:2008. 2008. Cara Uji Berat Jenis Tanah. Badan Standarisasi Nasional. Bandung. 12] SNI 1965:2008 2008 Cara Uji Penentuan Kadar 9] SNI 1742:2008. 2008. Cara Uji Kepadatan Ringan Untuk Tanah. Badan Standarisasi Nasional. Bandung. 10] SNI 1744:2012. 2012. Metode Uji CBR Laboratorium. Badan Standarisasi Nasional. Bandung. 11] SNI 1964:2008. 2008. Cara Uji Berat Jenis Tanah. Badan Standarisasi Nasional. 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Tata Cara Klasifikasi Tanah Dan Campuran Tanah Agregat Untuk Kontruksi Jalan. Badan Standarisasi Nasional. Bandung 18] Srihandayani Susy. Aidil Abrar. Surya Indrawan. 2019. Stabilisasi Berbasis Ion Exchange Untuk Meningkatkan Daya Dukung Subgrade Di Kota Dumai. Jurnal Teoritis. Dumai. 15] SNI 3423:2008. 2008. Cara Uji Analisis Ukuran Butiran Tanah. Badan Standarisasi Nasional. Bandung. 16] SNI 6371:2015. 2015. Tata Cara Pengklasifikasian Tanah Untuk Keperluan Teknik Dengan Sistem Klasifikasi Unifikasi Tanah. Badan Standarisasi Nasional. Bandung. 17] SNI 6797-2002. 2002. Tata Cara Klasifikasi Tanah Dan Campuran Tanah Agregat Untuk Kontruksi Jalan. Badan Standarisasi Nasional. Bandung 18] Srihandayani Susy. Aidil Abrar. Surya Indrawan. 18] Srihandayani Susy. Aidil Abrar. Surya Indrawan. 2019. Stabilisasi Berbasis Ion Exchange Untuk Meningkatkan Daya Dukung Subgrade Di Kota Dumai. Jurnal Teoritis. Dumai. 201
https://openalex.org/W2323430190	https://zenodo.org/records/1531560/files/article.pdf	English	null	The Original Form of the A-Text of "Piers Plowman"	Modern language review/The modern language review	1,911	public-domain	12,859	The Original Form of the A-Text of "Piers Plowman" Author(s): R. W. Chambers Source: The Modern Language Review, Vol. 6, No. 3 (Jul., 1911), pp. 302-323 Published by: Modern Humanities Research Association Stable URL: http://www.jstor.org/stable/3712710 Accessed: 19-12-2015 21:51 UTC Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://www.jstor.org/page/ info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. Modern Humanities Research Association is collaborating with JSTOR to digitize, preserve and extend access to The Modern Language Review. http://www.jstor.org This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions I. JOHN BUTT'S ADDITION TO THE A-TEXT OF 'PIERS PLOWMAN.' Dr Henry Bradley, in the Modern Language Review for April, 1910, has called attention to the importance of the passage appended by one John Butt to the twelfth and last passus of the A-text of Piers Plowman. This passage was added during the reign of Richard II. Therefore it is, Dr Bradley points out, the earliest piece of external evidence which we possess as to the authorship of the poem. Nothing is known of John Butt, and we cannot gauge the accuracy of his information; his testimony therefore is only corroborative, not in itself conclusive, if unsupported. Yet everyone will probably agree with Dr Bradley's contention that it ought not to be rejected without consideration, and that it is worth spending some labour to ascertain, if we can, exactly what John Butt does say. y say. John Butt's addition is not always easily accessible, Professor Skeat having rejected it from his Clarendon Press edition. It seems better, therefore, to print the concluding lines of Passus xII in a note at the end of this paper, with some discussion as to what the exact limits of the passage added by John Butt may be. It is not, I fear, possible to ascertain with any certainty how many of the concluding lines are to be attributed to the original writer, and how many to John Butt. At least five theories are possible. p It would be helpful if we could decide this point; but fortunately such decision is not essential. What is important is the assertion that the seer of the visions, Will- wrought that which here is written and other works both, Of Piers the Plowman and much people also. And when this work was wrought, ere Will might espy Death dealt him a dint.... w Death dealt him a dint.... Clearly, everything turns upon the character of the manuscript to which these lines were first added. Only when we have decided that, This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions 303 R. W. CHAMBERS can we tell what is meant by 'that which here is written,' and what the 'other works' may have been which Will wrote in addition to 'that which here is written.' John Butt's lines are found in MS. Rawlinson 137 only: this MS. contains a complete A-text. 1 Dr Bradley apparently takes 'this work,' the completion of which was followed by Will's death, as signifying 'this book,' and consequently as identical with ' that which is here written.' ' When he had finished " that which is here written,"' says Dr Bradley, 'Will died.' But John Butt does not state that 'when he had finished "that which is here written," Will died.' What he says is that 'Will wrought that which here is written, and other works both, concerning Piers the Plowman and much people also. And when this work was wrought, Will died.' Surely in a fourteenth century poem, the expression 'this work' may be interpreted 'this task to which I have referred' (i.e. the writing down of ' what here is written' and the ' other works') with at least as much probability as 'this book which is here written' (to the exclusion of the 'other works '). The 'work' is the task which Will undertakes, in view of Fever's warning to address himself to 'prayers and profitable works.' This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions I. JOHN BUTT'S ADDITION TO THE A-TEXT OF 'PIERS PLOWMAN.' The prima facie interpretation of Butt's words is, then, that Will wrote the A-text as it lies before us in Rawlinson-and that he wrote other works on Piers Plowman as well. The obvious interpretation of the 'other works' would be that they are the only other works on Piers Plowman which we know to have been widely current when John Butt wrote: viz. the additional visions given in the B- or C-texts. John Butt's words should therefore be very comforting to those who are supporting the traditional view of one 'Long Will,' the author of all three texts. Oddly enough, Long Will's defenders have, up to the present, not availed themselves of John Butt's evidence. Dr Bradley's interpretation of John Butt's lines is, however, different: Now, in the first place, I think the natural inference from his statement is that his lines were appended to a MS. containing only the poem of 'Dowel': that is to say A Ix-xII. He indicates that this poem was the last of the three works written by ' Will,' the others being Piers the Plowman, and The Field of Folk (mechel puple). When he had finished 'that which is here written' Will died. If John But was correctly informed, the poems contained in the A-text, though they are obviously intended to form a continuous whole, must have been published in three instalments, comprising respectively Passus I-v, vi-vIII, and Ix-xII....It seems clear that he [John But] either did not know of the existence of the B and C texts or regarded them as spuriousl. William Langland is on trial for his life: according to Prof. Manly he is already sentenced. The accused might submit that his trial has been somewhat of the kind approved by the King and Queen of Hearts in Alice in Wonderland: sentence before verdict, and verdict before evidence. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions 1 Dr Bradley apparently takes 'this work,' the completion of which was followe by Will's death, as signifying 'this book,' and consequently as identical with ' that which is here written.' ' When he had finished " that which is here written,"' says Dr Bradley 'Will died.' Will d ed. But John Butt does not state that 'when he had finished "that which is here written," Will died.' What he says is that 'Will wrought that which here is written, and other works both, concerning Piers the Plowman and much people also. And when this work was wrought, Will died.' Surely in a fourteenth century poem, the expression 'this work' may be interpreted 'this task to which I have referred' (i.e. the writing down of ' what here is written' and the ' other works') with at least as much probability as 'this book which is here written' (to the exclusion of the 'other works '). The 'work' is the task which Will undertakes, in view of Fever's warning to address himself to 'prayers and profitable works.' The A-Text of 'Piers Plowman' It is now five years since it was asserted that the acceptance in the B-text of certain textual derangements of the A-text proved that the author of the B-text could not be identical with the author of the A-text. Some students felt surprised when, on examining the alleged proof of these textual derangements, they found that no external evidence was put forward at all: that they were avowedly unsupported by a shred of MS. authority. Yet, notwithstanding this, the derange- ment of the A-text was widely, and for some time almost universally, treated as a demonstrated fact upon which further hypothesis could safely be based; while it is nothing more than an interesting thesis, in favour of which, and also against which, there is something to be said. Then it was asserted that there were differences in dialect between the A- and B-texts which made it impossible to suppose their authors identical; and this assertion was made, not merely in publications where new and hazardous theories might legitimately be put forward, but in books like the Cambridge History of English Literature, which are supposed to speak with some measure both of authority and im- partiality. Yet here again, only the thesis is forthcoming: for any demonstration of the thesis we are still waiting. waiting. And now that a new argument against unity of authorship, based upon John Butt's words, is being brought forward, it rests again upon a thesis: that these words were appended originally to a MS. containing Dowel (Passus Ix-xn) only. Obviously, if this should turn out to be so, then the interpretation given by Dr Bradley would be natural and indeed inevitable. The 'other works of Piers Plowman and much people also' would have to be Passus I-v and vi-vII. Seeing that there would then be nothing which could be construed as a reference to the B- or C-text, although B was certainly published when Butt wrote, and possibly C also, it would appear, on Dr Bradley's assumption, that Butt's evidence tells against these being the work of 'Will,' the author of the A-text. But believers in 'Long Will' will be entitled to ask Dr Bradley for some proof of this assumption that Butt's lines were originally added to a manuscript of Dowel only. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions :204 The A-Text of 'Piers Plowman' The A-Text of 'Piers Plowman' The A-Text of 'Piers Plowman' Until proof is forthcoming they will be entitled to say, and to repeat, that John Butt's lines are actually found appended to a complete A-MS.: that, in the absence of evidence to the contrary, these lines were, presumably, originally added to a manuscript of a type similar to that in which they are extant, and that they there- fore tell in favour of the theory that Will was the author of other works 305 R. W. CHAMBERS on Piers Plowman, in addition to the A-text, and not against that theory. The burden of proof clearly rests with those who suppose that the lines were added to a manuscript of a type different from that in which we now find them. Meantime 'Long Will's' supporters are entitled to remain on the defensive, and strategically that is no doubt the securest course. But the object of this paper is not either to attack or to defend 'Long Will.' To anyone engaged in the tedious but necessary task of trying to make out the relationship of the different A-MSS., it becomes necessary to collect whatever evidence is forthcoming as to the way in which the A-text was published. Dr Bradley's interpretation of John Butt's words involves a theory as to the manner in which the text was published which does not seem to be consistent with the phenomena of MS. types and relationships, so far as one can make these out. I shall try to show: y (1) That the A-text does not lend itself to a division into instalments. (2) That, even assuming the publication in separate instalments, there are serious chronological objections to the view that the circulation in instalments was still prevalent in the reign of Richard II, when Butt added his lines. (3) That, even granting circulation in instalments at that date, the evidence of MS. relationship is opposed to the view that Butt's lines were appended to a manuscript containing Dowel only. For this evidence seems to show conclusively: y (a) That the great majority of the A-text MSS. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions The A-Text of 'Piers Plowman' do not contain a twelfth passus, and that this deficiency cannot be attributed to the mutilation either of these manuscripts individually or of a common source; that therefore the twelfth passus must have been added as an afterthought, either by the poet himself or by someone else, to the original or originals of those few manuscripts in which it is found; and that, since John Butt's lines were added to a copy containing the twelfth passus, they must be either subsequent to or contemporary with the addition of that passus, but are certainly not earlier. passus, y (b) That those MSS. which end at Passus XI, and those which contain the twelfth passus whether with or without Butt's lines, are descended ultimately from one original, and that this original contained both the Vision (Passus I-viII) and Dowel (Passus Ix, etc.), but did not contain the twelfth passus, still less John Butt's addition thereto. 20 M. L. R. VI. M. L. R. VI. The A-Text of 'Piers Plowman 306 (c) That the order of events must therefore have been first the combination of the instalments (granting the poem to have been published in instalments) into one text, and subsequently the addition of a twelfth passus and of John Butt's lines to a certain copy or to certain copies only of this combined text. (c) That the order of events must therefore have been first the combination of the instalments (granting the poem to have been published in instalments) into one text, and subsequently the addition of a twelfth passus and of John Butt's lines to a certain copy or to certain copies only of this combined text. 1 MS. Douce 323 has, instead of Passus i of Dowel, ' Primus Passus in secundo libro.' On the other hand, both Douce and Harleian 3954 conclude the A-text, not Explicit Dowel but Explicit liber petri plouman; explicit tractus de perys plowman. 2 Q i f th A t t f MS T i C ll C b R 3 14 This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions Explicit petri plouman; explicit perys plowman. 2 Quotations from the A-text are given from MS. Trin. Coll. Camb. R. 3. 14. II. THE DIVISION OF THE A-TEXT INTO SECTIONS. Firstly, before entering into the complex questions of MS. relation- ships, does the text lend itself to division into sections so distinct as to allow of their having been issued separately ? g p y For the view that Passus VI-vIII were published separately from Passus I-V there appears to be no support whatsoever in the text. They are integral parts of one and the same poem, the Vision of Piers Plowman. There is a much greater prima facie possibility that Dowel (Ix-xII) was published separately from the Vision, for in all the best A-MSS. it is carefully distinguished from the Vision proper. At the end of Passus vIIl most of the MSS. have, as we shall see, a note that 'here the Vision ends and Dowel begins.' Then the numbering of the passus begins afresh. Instead of IX, x, XI [xII] (a numbering too convenient to be now dropped, but one which is not authorized by any uncontaminated A-MS.) we have a prologue, and then Passus I, II [III] of Dowel1. This independent numbering of the passus of Dowel does not in the least prove separate publication. It does, however, suggest the possibility of it, and establishes a case for further scrutiny. But such scrutiny does not disclose any further evidence in favour of separate publication. On the contrary it shows the most intimate connection between the Vision and Dowel. The latter part of Passus vIII of the Vision is obviously intended simply to lead up to Dowel. Dowel, who has never before been mentioned, is referred to five times between vIII, 156 and vIIl, 187. We are to do such works, pat aftir oure dep day Do-wel reherse pat at pe day of dome we dede as he hi3te2. Then the Dowel poem begins with an opening which certainly does This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions 307 R. W. CHAMBERS not harmonize well with the theory that it was written for separate publication: not harmonize well with the theory that it was written for separate publication: Thus yrobid in rosset I rombide aboute Al a somer sesoun for to seke Do-wel. Thus yrobid in rosset I rombide aboute Al a somer sesoun for to seke Do-wel. Thus yrobid in rosset I rombide aboute Al a somer sesoun for to seke Do-wel. These lines are a link, connecting Dowel with the preceding Vision of Piers Plowman. They have little meaning as the opening words of an independently published poem. To what does ' thus' refer if not to the preceding poem ? And in fact 'yrobid in rosset' and 'al a somer sesoun' carry us back to the 'shroud' and the 'somer seson' of the opening of the Vision. 'Thus I roamed about to seek Dowel' carries us back to the conclusion of the Vision in which we are directed to Dowel as the one refuge against the judgment to come. This is surely a resumption of the subject at the beginning of a new section rather than the opening of an independently circulated poem. opening independently poem. But, according to Dr Bradley's interpretation of John Butt, not only was Dowel published separately from the Visio, but the Visio itself was published in two instalments, first Passus I-v, then Passus vi-vIII. In favour of this there appears, as we have seen, to be no evidence forthcoming. The difficulties, on the other hand, are greater even than those which meet us in postulating a separate publication of Dowel. For the opening of Passus vi is even more closely linked to the end of Passus v than is the opening of Dowel to Passus VIII. Had Passus vi-vIII been published in a separate instalment we might fairly expect the poet to begin: 'I have told before how I saw in a vision such and such things: I will now tell....' Instead of which Passus vi begins: Ac ]ere was fewe men so wys pat pe wei1 pider coupe But blustrid forp as bestis ouer valeis and hilles. Til late and longe pat by a lede mette Aparailid as a paynym in pilgrim wyse, etc. 1 Only one fact seems to be forthcoming at present which could be interpreted as a trace of separate circulation. Two MSS., Harleian 875 and Lincoln's Inn, break off, mutilated, shortly before the end of Passus viir is reached. In view of the frequency with which the last leaf only is missing from a MS., it has been argued that these MSS. probably ended originally with Passus vnII, and that there was therefore probably a version of the poem published containing the Vision without Dowel. If this argument could be sustained, it would tend to prove publication in instalments in the sense that the earlier passus might have been issued before the later: it would not however go to prove publication in instalments in the sense demanded by Dr Bradley's argument-viz. that the later passus, when published, were published separately, without the earlier passus preceding. But the argument cannot be sustained. Harl. 875 has indeed lost the last leaf of what is now its last section. But since Harl. 875 is apparently derived from the same archetype as Vernon, which has eleven passus, it is probable that a great deal more has been lost as well. Mr Grattan, who has collated the Lincoln's Inn MS., tells me that there is evidence that more than a single leaf has been lost there. f f i h This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions Can this be the opening of an independently published poem ? p g p y p p It may be objected that the separately published instalments did not open exactly as the present Passus vi and Passus ix do: that the lines quoted above might have been links added by the man who bound the three instalments into one, so as to make them run together more smoothly. But this way of avoiding the difficulty is, on Dr Bradley's hypothesis, impossible. For, on that hypothesis, the A-text, as we have it in the Rawlinson MS., was produced by a binding together of the instalments subsequent to John Butt's addition, which addition was made 1 Pei, T. 20-2 This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions The A-Text of 'Piers Plowman' 308 in the reign of Richard II. But by general consent the B-reviser was at work earlier than this, in the year 1376-7. And the lines in question are also found in B. Features which are common to A and to B cannot possibly be attributed to a binding together of the separate instalments which is subsequent to B: they must have characterized those in- stalments which, on Dr Bradley's hypothesis, were the original form of the poem. p We should have, then, to believe that the poem was published in three instalments, the second and third of which began 'Few knew the way thither,' and 'Thus robed in russet I roamed about.' This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions III. THE CHRONOLOGICAL DIFFICULTY. But, further, in order to make John Butt's 'what here is written' relate to Dowel only, Dr Bradley has to postulate that this circulation in instalments was still prevalent when John Butt wrote, in the reign of Richard II; that then, later, the three instalments were combined, and that hence John Butt's ending, originally intended to conclude Dowel only, came to stand at the end of the complete A-text. only, p Now there is a serious difficulty here. By general agreement. Passus I-- , at least, had been written about 1362. John Butt wrote at least fifteen and possibly as much as thirty-seven years after 1362. That the poem was then being circulated in separate sections seems unlikely, in view of the fact that no trace of this separate circulation has been preserved'. p This chronological difficulty does not admit of exact demonstration, but the more it is examined the more serious does it become. On Dr Bradley's hypothesis the different instalments in which the A-text MS., te s single There is, then, no reason for thinking that either of these MSS. concluded with Passus viii. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions 309 R. W. CHAMBERS was published must, for at least half a generation, have been copied and scattered through the land, for we know that the poem was popular; then came B, who must have formed his text by putting the three instalments together, altering and expanding them as he did so; later still, perhaps even a score of years after the B-text was issued, came John Butt, who, finding the Dowel portion circulating inde- pendently, must have added his lines to it; then, later still, the three instalments must have been bound together into one poem, and the A-text as we know it in Rawlinson and the other kindred A-MSS. formed. This A-text was so popular that it was copied and re-copied almost as widely as the B- or C-texts, one or both of which must on Dr Bradley's hypothesis have been formed earlier. But we have to believe that the separate instalments, which had the start of any combined text by some fifteen years, have all been lost, whilst some fifty of the combined texts have been preserved. This surely is very difficult. Nor can the difficulty be much reduced by arguing that, so soon as the complete A-text was in the field, scribes would cease to copy the separate instalments. A scribe had to copy what he could get, and few scribes had much choice. This is shown by the fact that the A-text, though only one-third of the B-text in bulk, was nearly as widely copied. Scribes either had not the fuller form to hand, or perhaps even in some cases preferred to copy the more brief and vigorous type. Precisely the same reasons which led scribes to copy the A-text after B had been issued would have led to the separate instalments of A, and particularly A I-v, being still copied, even after the complete A had been issued. Why is it then that no MS. is known containing A I-v, A vi-viII, or Dowel separately ? This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions IV. THE ARGUMENT FROM THE RELATIONSHIP OF THE MSS. It seems, then, exceedingly doubtful if we are justified in assuming, as Dr Bradley does, that Dowel was published separately, and was more- over being circulated in this separate form when John Butt wrote. But, assuming the independent Dowel MS. or MSS. to have existed then: is it possible that John Butt's lines were originally appended to a manuscript of this type ? a usc pt yp Of course if John Butt's lines were found in every completed MS. of the A-text, it would be possible to assume that they were joined on to Dowel before Dowel itself was joined on to the Vision. But, as we This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions The A-Text of 'Piers Plowman' 310 have seen, the lines are found in MS. Rawlinson Poet. 137 only. This MS. (R) is very closely related to two other MSS.: viz. U (Univ. Coll. Oxford, 45) and E (Trin. Coll. Dublin, D. 4. 12). These MSS. have inherited from the original from which they were all three copied a very large number of common peculiarities. very large peculiarities. R and U both have a twelfth passus: in U this breaks off imperfect at 1. 19; probably E also shared this peculiarity: but as E breaks off imperfect at vII, 45 we cannot be sure. Whether RUE all inherited John Butt's ending we cannot tell. Two leaves only have been lost from the end of U, but this would give ample room for the missing portion of Passus xII, and for John Butt's addition as well. portion , Only one other MS. contains a trace of the twelfth passus, viz. the Ingilby MS. (I) which gives that passus up to 1. 88, where it suddenly ends. The Ingilby MS. therefore has no mention of John Butt. Its conclusion at 1. 88 is apparently deliberate, as the MS. is not mutilated. Let us now turn to the most important group of MSS., consisting of T (Trin. Coll. Camb., R. 3. 14), H2 (Harl. 6041), Dig (Digby 145), W (Westminster), D (Douce 323). Here again we have a group intimately united, evidently derived from a common source. Within this group, T, H2, Dig, W, make a sub-group, being derived from a MS. in which the scribe, realizing that there was more about Piers Plowman than was contained in the A-version before him, has joined on the later portion of the C-text. p , Only one other MS. contains a trace of the twelfth passus, viz. the Ingilby MS. (I) which gives that passus up to 1. 88, where it suddenly ends. The Ingilby MS. therefore has no mention of John Butt. Its conclusion at 1. 88 is apparently deliberate, as the MS. is not mutilated. apparently , Let us now turn to the most important group of MSS., consisting of T (Trin. Coll. Camb., R. 3. 14), H2 (Harl. 6041), Dig (Digby 145), W (Westminster), D (Douce 323). Here again we have a group intimately united, evidently derived from a common source. 1 Unlikely, because the scribe throughout abstains from any such attempt to harmonize A and C. The A portion of TH2 is extraordinarily pure, and free from C influence. Apparently the scribe did not turn to his C-MS. till he had finished his transcription from A. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions 1 Indeed Dig and W have been so defaced by corruption, and interpolation from B- and C-texts that the likeness between them and TH2 is not nearly so obvious as between D and TH2. W particularly is often extraordinarily unlike TH2. But the argument in this paper would not be weakened, but slightly strengthened, even if it should be proved that W does not belong to the same group as TH2, but was formed by an independent com- bination of an A-text with Passus xi-xxii of a C-text. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions Within this group, T, H2, Dig, W, make a sub-group, being derived from a MS. in which the scribe, realizing that there was more about Piers Plowman than was contained in the A-version before him, has joined on the later portion of the C-text. p The A-portion of the manuscripts TH2DigW concludes at the end of Passus xi, with the lines: Souteris and seweris suche lewide iottis, iottis, Percen wip a pater noster pe paleis of heuene, p pater pe paleis heuene, Wipoute penaunce at here partynge in-to hei3e blisse. This, alone, is not conclusive evidence that the A-MS., from which TH2DigW are derived, ended there. It is possible, though unlikelyl, that the scribe, even though there was more in the A-MS. before him, might have broken off his transcription of the A-text at this line, because it afforded the most convenient point of juncture with the C-text. This, alone, is not conclusive evidence that the A-MS., from which TH2DigW are derived, ended there. It is possible, though unlikelyl, that the scribe, even though there was more in the A-MS. before him, might have broken off his transcription of the A-text at this line, because it afforded the most convenient point of juncture with the C-text. But in Douce we have a MS. containing an A-text only, which is clearly derived from the same original from which the A-portion of This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions 311 R. W. CHAMBERS TH2DigW is derived'. And Douce assures us that this A-text concludes with the end of Passus xI: TH2DigW is derived'. And Douce assures us that this A-text concludes with the end of Passus xI: TH2DigW is derived'. And Douce assures us that this A-text concludes with the end of Passus xI: Souters and sewers suche lewed Iottys Percen with a pater noster pe paleys of heuene With-oute penaunce at here partyng in-to heye blysse Now of jis litel book y haue makyd an ende Goddis blessyng mote he haue pat drinke wil me sende Explicit liber petri plouman. Now of jis litel book y haue makyd an ende Goddis blessyng mote he haue pat drinke wil me sende Explicit liber petri plouman. The cry for drink and the large and triumphant letters in which Explicit is written, show the scribe rejoicing in the end of his toil. I refuse to believe that this worthy and thirsty soul had found a twelfth passus in his original, but, with deliberate mendacity, failed to tran- scribe it. The tacit evidence of TH,DigW, and the express assurance of D, make it clear, then, that the original of these MSS. ended with Passus xI. If it be argued that this was not a deliberate conclusion, but was due to the loss, in some way or other, of a twelfth passus from the archetypal MS. of TH2DigWD, the fact has to be faced that other A-MSS., representing a tradition quite remote from that of TH,DigWD, also end exactly at this point. exactly point. MS. Ashmole 1468 (As) concludes: Saweris and soweris sweche lewid iottis Saweris and soweris sweche lewid iottis Mon persyn with a pater noster pe pales of heuyn Mon persyn with a pater noster pe pales of heuyn persyn pater pe pales heuyn Withoute penauns at here partyng into pe blisse of heuyn. p Amen Amen Amen p Amen Amen Amen The Ashmole MS. is of little value towards fixing the text, owing to the carelessness with which it has been copied, to its many lacunae, and to the fact that one or more of the scribes whose work it represents knew, and interpolated from, a B- or C-text. But none of these serious blemishes in the least invalidates its evidence as to the proper point where an A-MS. should stop. We want the evidence of a manuscript as remote as possible from the T group; and this evidence Ashmole gives, for it is unlike any other MS., and certainly has no special affinities with T. MS. Harleian 3954 (H3) gives an inaccurate version so corrupted in the earlier passus with B-influence, as to be worthless for the purpose This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions The A-Text of 'Piers Plowmarn 312 of fixing the A-text. But again this does not invalidate its evidence as to where the A-text should end. Mr Grattan and myself have so far been unable to trace any special affinities of Harleian 3954 to any other MS. or group of MSS. It is therefore an independent witness, and it agrees with TH2DigW, with D and with As, in concluding: With a pater noster to pe paleys of heuene pater p paleys With outyn gret penans at hys partynge to comyn to blys. p p y With outyn gret penans at hys partynge to comyn to blys. Explicit tractus de perys plowman q. heru?; Qui curn patre et spiritu sancto viuit et regnat per onznia secula seculorumn. Amen. g p y p y g y Explicit tractus de perys plowman q. heru?; Qui curn patre et spiritu sancto viuit et regnat per onznia secula seculorumn. Amen. How the VH group ended we do not know, as both Vernon and Harl. 875 break off imperfect, V in Passus xi, H in Passus vIII. p , Of the ten MSS., therefore, which it is possible to call in as evidence of the way in which the A-text ended, seven, falling into three or four groups, point to the poem having ended with Passus xI; three MSS., falling into two groups, contain the whole, or portions, of a twelfth passus. But only one of these three contains John Butt's lines. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions p Amen Amen Amen We are not free to assume that the absence of Passus xII from TH2DigWDAsH3 is due to mutilation, and this, not merely because the MSS. ending at Passus xi are seven, whilst those containing the whole or portion of a twelfth passus are only three: for a minority of MSS. might well retain an original feature. But the point is that the three MSS. containing the twelfth passus are more nearly connected with each other than are the seven which want it; the seven represent a great divergency of tradition. It is not difficult to understand how an added twelfth passus comes to be present in RUI: it would be very difficult to account for its having been lost from TH2DigWDAsH3. The same argument can be shown to apply a fortiori to the John Butt passage, present as it is in one MS. only, and wanting in eight. The poem, then, must originally have ended with Passus xI; the twelfth passus is an afterthought, added either by the poet himself or by some- one else. This was the conclusion arrived at thirty-six years ago by Professor Skeat, and I do not see that any other is possible. , a y p Again, since John Butt's lines were appended to a form of the poem containing the twelfth passus, they must have been added either at the same time as or later than the twelfth passus was added. But it can, I think, be proved that the copies to which this twelfth passus was added were copies of the complete poem, not merely of Dowel: the poem before this twelfth passus was added had already passed out of the stage of circulation in three separate instalments (assuming for the 313 R. W. CHAMBERS sake of argument that such a stage ever existed) and was being circulated as one complete whole. sake of argument that such a stage ever existed) and was being circulated as one complete whole. p For the different groups of MSS. which we have been considering are themselves intimately related. The MS. from which RUE derive, which had the twelfth passus, and the MS. from which TH,DigD derive, which certainly had not that passus, were themselves not distantly connected: they had a number of peculiarities in common, and were evidently derived in the last resort from one and the same prototype. 1 Even in this place this particular wording is not usually followed in the B- and C-texts: in those few cases where it is found, it is clear that the scribes have copied it into the B- or C-text from their knowledge of an A-MS. E.g. in MS. L1. 4. 14 in the University Library, Cambridge, we have at the end of the Vision ' Explicit visio Willelmi de petro plowman Et sequitur vita de dowell Dobett et Do-beste, secundum wytt et reson': but this is in a later hand obviously borrowed from an A-text. MS. Bodl. 851 has 'Explicit vita et visio petri plowman,' which is probably an inaccurate recollection of an A-text; for MS. Bodl. 851 is a confused MS. written under the influence of A-, B-, and C-texts. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions The A-Text of ' Piers Plowman' 314 wording (RUTH,DigDIAs) are derived from one original archetype in which this peculiarly worded note occurred, connecting the Vision with Dowel. Probably all our extant texts derive from this same archetype: but for everything that it is necessary to prove here, RUTH2DigDIAs are sufficient. From the relationship of the extant MSS. of the A-text we are entitled then to draw the following conclusions: (1) That the parent MS. from which RUTH2DigDIAs are derived contained both the Vision proper and Dowel, joined into one poem by a peculiarly worded Latin note which has survived in all its eight descendants. (1) That the parent MS. from which RUTH2DigDIAs are derived contained both the Vision proper and Dowel, joined into one poem by a peculiarly worded Latin note which has survived in all its eight descendants. (2) That John Butt's lines were not present in this MS. from which RUTH,DigDIAs are derived, since they are found in one of its descendants but are absent from the others. (2) That John Butt's lines were not present in this MS. from which RUTH,DigDIAs are derived, since they are found in one of its descendants but are absent from the others. (3) That John Butt must therefore have added his lines, later, to one of the transcripts of the parent MS. (4) That the MS. to which John Butt's lines were added must like its original have contained both the Vision and Dowel, joined by the peculiarly worded note which it had inherited from the parent MS., and which R in turn inherited from it. (5) That John Butt had therefore a complete A-text before him, and that when he speaks of 'what is here written' he must refer to the complete A-text. These appear to be the results which legitimately follow from the known facts of MS. relationships. I do not deny that extraordinary complications, cross-copying, and contamination, often throw us out of our reckoning, and that the most legitimately drawn conclusions may be wrong. Thus it is conceivable that the John Butt passage has no business where it is found, in MS. Rawlinson 137, at all, and that therefore the conclusions legitimately drawn from its presence there are misleading. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions p Amen Amen Amen But this prototype contained not only the Vision of Piers Plowman proper (Passus I-vIII) but also Dowel (Passus Ix-xI). For the same Latin rubric, by which the Dowel poem is connected to the Vision, is inherited by RUTH,DigD from their common prototype, in a common form, with only unessential variants: (Explicit hic visio Willelmi de Petro Plou3man: et hic incipit) Vita de Do-wel, Do-bet et Do-best secundum wyt et resoun. Moreover, this peculiarly worded note is, so far as we can trace, common to all the A-MSS. H, L (Lincoln's Inn) and E break off before this point is reached. H, has no rubric here; I have not yet collated W up to this point, and, as the other members of the group to which W belongs are agreed, it appeared to be hardly worth while going to Eaton Hall to procure a reading which, in any case, would not materially influence the argument. g Of the rest all have a note: and, in it, all except the University College and the Vernon MSS. describe Dowel as the Vita de dowel: and all except Ashmole and Vernon further define it as secundum wyt et resoun. Now the usual title is either Dowel or the Visio de Dowel; I know of no other place than this in the A-, B- or C-texts either where the Dowel is called the Vita, or where it is explained as being secundum wyt et resoun: there may be instances, but they are certainly very exceptional' exceptional . Now it is not conceivable that this peculiar wording could have been hit upon by scribes, engaged independently in fitting together into one poem the hitherto separate instalments. It must be that those MSS. which possess either one or both of these peculiarities of This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions The A-Text of ' Piers Plowman' The A-Text of ' Piers Plowman' Assuming for the sake of argument that there were manuscripts containing Dowel only, it would not be inconceivable that the Butt passage was originally added to a manuscript of this type, and that it was not present in the parent of the Rawlinson MS. at all: that the scribe who wrote the Rawlinson MS. knew the Dowel MS. and inserted the passage from it into his Rawlinson: that then the Dowel MS. was lost, and a puzzle thus left to mislead future students. Such things do happen. Take, for example, the new lines of Juvenal discovered in the 315 R. W. CHAMBERS Oxford MS. some eleven years ago. They had no business in a manu- script of the type of that in which they were found: and the best way of accounting for them is by supposing that the scribe who wrote this MS. had access not only to the comparatively commonplace MS. which he copied, but also to a very ancient MS. of another type, all trace of which has now disappeared: that at this point he turned from his copy, and inserted these lines from the more ancient MS., and thus produced a text which, whilst quite ordinary in its other features, preserves lines not known in any MS. of its own, or indeed of any other, family. y , y y There is always the remote possibility of some extraordinary cross copying: and in the case of the Juvenal MS. the phenomena of the text have compelled many, if not most scholars, to assume that such cross copying has taken place'. py g p But in the case of the Butt passage we have no tittle of evidence to lead us to suppose that this has happened. The obvious fact is that we find the lines in MS. Rawlinson 137. There is a remote possibility that they might have been interpolated there from some other MS. of a quite different type: but in the absence of evidence for this we must base our theories upon the obvious fact, and not upon the remote possibility. 1 See the Classical Review, xvii, 394; Gott. gel. Anzeiger, 1899, p. 895. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions V. THE ' OTHER WORKS.' Dr Bradley's theory has, I submit, to meet and successively overcome three obstacles: firstly, that the form in which the second and third of the postulated instalments open, does not suggest independent publica- tion, but the reverse: secondly, that if our extant A-text was put together from such instalments as late as the reign of Richard II, we might reasonably expect very considerable traces of such instalments to have survived: and thirdly that, even assuming the independent Dowel in the time of Richard II, the known facts of MS. relationship would not allow us to suppose that John Butt's lines were added to it, except by postulating cross copying of a most extraordinary character. Unless we postulate such cross copying I cannot see how we can avoid the conclusion that John Butt's lines were added to a complete A-text. co p ete Still less do I see why we should wish to avoid this conclusion. For even if we had not the evidence of MS. relationships, the most straightforward interpretation of John Butt's statement, that Will wrote 'what here is written,' would be that he wrote what lies before This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions The A-Text of 'Piers Plowman' The A-Text of 'Piers Plowman' 316 us written in the MS., where we find the lines, 'Will wrought what here is written [i.e. the A-text] and other works both concerning Piers the Plowman and much people also. And when this labour was concluded, he died.' us written in the MS., where we find the lines, 'Will wrought what here is written [i.e. the A-text] and other works both concerning Piers the Plowman and much people also. And when this labour was concluded, he died.' The only other works concerning Piers Plowman which we know to have been extant when Butt wrote, in the reign of Richard II, are (1) the bulky additions forming Passus xI--xx of the B-text; and, towards the end of the reign, (2) the same in revised form forming Passus xIII-xxIII of the C-text, and (3) the Crede. , ( ) Now the essential characteristic of the B-additions to the poem is the prominent part which Piers the Plowman plays in them. In the original Dowel Piers the Plowman is never so much as mentioned. V. THE ' OTHER WORKS.' We have seen that, although the opening of Dowel assumes that Piers Plowman precedes, the author did not regard Dowel as part of the Vision of Piers Plowman. For he called its sections i, ii of Dowel, not x, xi of the Vision of Piers Plowman. But when we come to the B-text it is different. Here the formula is 'Passus nonus (decimus) de visione, et primus (secundus) de Dowel.' And this difference in title corresponds to a difference in the subject matter of Dowel in the B-text compared with Dowel in A. The search for Dowel as we have it in A passes in B into a search for Piers Plowman. Not all at once. We have Imaginative: then the dinner in which Conscience, Clergy, Patience, Reason, Will and the doctor sit down together; then Hawkyn the active man-and here for the first time Piers is again mentioned (xIII, 237). More and more frequent become the references to Piers and to the help which he alone can give (xv, 190, 193, 206: xvi, 18). At the last mention: Piers pe plowman! quod I po, and al for pure ioye pat I herde nempne his name anone I swouned after And laye longe in a lone dreme. Piers pe plowman! quod I po, and al for pure ioye pat I herde nempne his name anone I swouned after And laye longe in a lone dreme. Piers pe plowman! quod I po, and al for pure ioye f Piers pe plowman! quod I po, and al for pure ioye pat I herde nempne his name anone I swouned after A d i l p p quod po, pure ioye pat I herde nempne his name anone I swouned after A d i l pat nempne And laye longe in a lone dreme Piers himself comes to the dreamer: Piers himself comes to the dreamer: And atte laste me pou3te And atte laste me pou3te pat Pieres pe plowman al pe place me shewed. d atte laste me pou3te pat Pieres pe plowman al pe place me shewed. From this point Piers is the poet's theme, for the remaining nineteen From this point Piers is the poet s theme, for hundred lines of the Vision. Piers vanishes: p poet s , hundred lines of the Vision. V. THE ' OTHER WORKS.' Piers vanishes: And I awaked pere-with and wyped myne eygheii pere with wyped myne eyg And after piers pe plowman pryed and stared. piers pe plowman p yed Estwarde and westwarde I awayted after faste A d 3 d forth as i i t t Estwarde and westwarde I awayted after faste And 3ede forth as an ydiote in contre to aspye 3ede ydiote aspye After Pieres pe plowman many a place I sou3te. (xvi, 167--171.) (xvi, 167--171.) following (1) John Butt 'saw these saws busily alleged by James, Jerome and Job.' Jerome, so far as I know, is never quoted in A or B, though he is alluded to in B xix, 265. If Butt had the A-text before him, he would have seen saws alleged from James and Job: if Dowel, then from Job only. Dr Bradley's contention is that we ought not, without proof, to assume John Butt to be talking rubbish; and to assume that he had an A-text before him makes better sense of his talk than to assume Dowel only. (2) The supposition that Butt's lines were added to the lost archetype of RUE agrees excellently with the chronological data. R and U belong to the early fifteenth century: the lost archetype, intermediate between this period and the first publication of the A-text about 1362 would probably belong to the reign of Richard II: and John Butt, as we know, wrote in that reign. (3) The fact that there were several different versions of Piers Plowman was much better understood in the late fourteenth and early fifteenth centuries than has generally This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions pp y 1 Some slight corroboration of the view expressed above may perhaps be drawn from the following considerations: wrote in t at reign. (3) The fact that there were several different versions of Piers Plowman was much better understood in the late fourteenth and early fifteenth centuries than has generall him only. (2) The supposition that Butt's lines were added to the lost archetype of RUE agrees excellently with the chronological data. R and U belong to the early fifteenth century: the lost archetype, intermediate between this period and the first publication of the A-text about 1362 would probably belong to the reign of Richard II: and John Butt, as we know, wrote in that reign o ow g (1) John Butt 'saw these saws busily alleged by James, Jerome and Job.' Jerome, so far as I know, is never quoted in A or B, though he is alluded to in B xix, 265. If Butt had the A-text before him, he would have seen saws alleged from James and Job: if Dowel, then from Job only. Dr Bradley's contention is that we ought not, without proof, to assume John Butt to be talking rubbish; and to assume that he had an A-text before him makes better sense of his talk than to assume Dowel only This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions 317 R. W. CHAMBERS The search for Piers Plowman-faithful, humble and laborious humanity, for the time identified with that perfect manhood in which the Son of God clothed himself-culminates in the eighteenth passus, which Skeat has finely named 'The Vision of the Triumph of Piers the Plowman': the story of how the Saviour clad 'in Piers armes in his helme and in his haberioun' overcame death. But from the point where the dreamer awakes, with the Easter bells ringing in his ears, we have a return to the earlier conception of Piers 'the ideal of conduct' labouring to build up the Church. But Piers' work is attacked by Pride, Covetousness, Sloth, Flattery, and laid waste, for all that Conscience can do: and Conscience becomes a pilgrim to walken as wyde as al pe worlde lasteth To seke Piers pe plowman. Now we may call all this 'the B-addition': but that is not the language of the fourteenth century. I do not know that John Butt could have described these extraordinary visions much better than in the words he has used--'other works concerning Piers the Plowman and much people also.' He could not have described them as the Vision of Dowel, Dobet and Dobest, for that name belongs equally to Passus IX-xII of the A-text, from which he is seeking to distinguish the 'other works.' The interpretation of 'what is here written' as the A-text and the ' other works' as the B-additions presents, then, no difficulties: whilst Dr Bradley's interpretation of 'what is here written' as Dowel and the ' other works' as the Vision is open to the objections that it forces us to assume a type of MS.-an independent Dowel-for the existence of which we have no evidence, and to assume that John Butt's lines were added to a MS. of this type, and that Dowel with this addition was subsequently combined with the Vision, though the legitimate conclusion from the evidence appears to be the contrary'. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions pp 1 Some slight corroboration of the view expressed above may perhaps be drawn from the following considerations: This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions The A-Text of 'Piers Plowman' The A-Text of 'Piers Plowman' John Butt's statement then amounts to this: (1) That Will, so far from being'a creation of the Muse' wrote 'what here is written' in MS. Rawlinson 137: viz. the complete A-text: that he also wrote 'other works concerning Piers the Plowman,' which, so far as our knowledge goes, can best be explained as the B-additions. In both these assertions John Butt confirms, and is confirmed by, tradition. (2) That Will was already dead in the reign of Richard II: he cannot, therefore, be the author of Richard the Redeless (Mum Sothsegger). Here again John Butt is in harmony with tradition, as this poem was first attributed to Langland in the nineteenth century. (2) That Will was already dead in the reign of Richard II: he cannot, therefore, be the author of Richard the Redeless (Mum Sothsegger). Here again John Butt is in harmony with tradition, as this poem was first attributed to Langland in the nineteenth century. In conclusion I have to thank Dr Bradley for having read through this paper in an earlier draft, and for having suggested modifications which add greatly to any force the arguments may carry. I have also to thank Mr Grattan for his criticism, and for certain MS. readings with which he has supplied me. been supposed. We have seen that the scribe of the lost MS. from which TH2DigW derive, when he had got to the end of his A-text, turned to a C-text and added the matter which he found there. T is a very early MS., and has even been attributed by Dr M. R. James to the fourteenth century. The common ancestor being older, belongs almost certainly to the reign of Richard II. We can, then, point to a scribe in the reign of Richard II who, having an A-MS. before him, clearly understood that besides 'what was there written' there were ' other works' concerning Piers the Plowman: and pro- ceeded to add them. We have in this an excellent precedent for the interpretation of John Butt's words given above. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions 318 This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions THE EXTENT OF JOHN BUTT'S ADDITION. It is exceedingly difficult to say where John Butt's lines begin. For in none of the A-MSS. does the A-text attain to anything that can be regarded as a satisfactory or final conclusion. ega ded y We have seen that most MSS. end with Passus XI. But this can never have been intended for anything more than a temporary halting place. True, the words 'parting,' 'bliss of heaven' strike a note common in conclusions. But it is not possible that the poet can have meant the poem to close here. Will has been sent, in his search for Dowell, to Clergy and Scripture, representing different forms of learning. To them he roundly expresses his conviction that learning is idle. Man's lot is predestined: the most learned, Solomon and Aristotle, are damned: Clergy was never commended by Christ's mouth: Austin, the most learned doctor, commended the unlearned rather than great clerks. been supposed. We have seen that the scribe of the lost MS. from which TH2DigW derive, when he had got to the end of his A-text, turned to a C-text and added the matter which he found there. T is a very early MS., and has even been attributed by Dr M. R. James to the fourteenth century. The common ancestor being older, belongs almost certainly to the reign of Richard II. We can, then, point to a scribe in the reign of Richard II who, having an A-MS. before him, clearly understood that besides 'what was there written' there were ' other works' concerning Piers the Plowman: and pro- ceeded to add them. We have in this an excellent precedent for the interpretation of John Butt's words given above. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions 319 R. W. CHAMBERS Now all this is obviously meant to lead up to a reply, by or on behalf of Clergy and Scripture, such as we get in the B-text1. It must have been intended from the first that the other side of the case should be put. Meantime the poem was issued in a form concluding with Passus xI. To some copies, as we have seen, a twelfth passus was added: but this is not satisfactory. The poet is reproached for his disrespectful words, but no attempt is made to refute them. 'I have done my devoir,' says Clergy, 'to teach thee Dowel...and if I knew that thou wouldest work accordingly I would answer all thy questions.' But Clergy does not answer Will's furious onslaught. He hardly seems to have heard it. Scripture scornfully bids Clergy answer the questioner no further, for he came not in order'to lerne to Dowel.' But Scripture also does not answer Will's objections. She merely bids her helpmeet be still, exactly as in Passus XI Dame Study had commanded her husband Wit to tell the poet no more. Clergy is as much under the control of his wife as Wit had been. Wit had become confounded, could say no more, and drew him aside: Clergy creeps into a cabin, draws the door after him and bids Will do well, or evil if he will, whichever he likes. The poet appeals to Dame Scripture just as before he had appealed to Dame Study, promising in each case 'to be her man.' In each case the shrew relents and directs the poet to her cousin, who is to teach him more. In the last case the cousin is Kind Wit, who is lodging with Life; Scripture called a young clerk Omnia probate who is to lead Will to the borough Quod bonum est tenete where Kind Wit dwells, and bid Kind Wit show Dowel to Will. So they set out together. they together. It is at this point that Prof. Manly supposes John Butt to have begun. He points out, with justice, that John Butt's words, 'for he medleth of making he made this end' imply an addition of some length, something more than the twelve or nineteen lines which Skeat would allow. 1 The manner in which the end of the A-text leads up to the beginning of the B-text is one of the arguments in favour of unity of authorship. On this point the most interesting and able article by Dr Otto Mensendieck in the Journal of English and Germanic Philology, Ix, 404ff., should be consulted. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions But the whole twelfth passus is quite short: including Butt's un- doubted addition it contains only 112 lines. Why may not the whole of it be John Butt's end? Butt was clearly a person of no great ability, who possessed the knack of writing tolerable alliterative verse. The lines summarized above (xii, 1-54), with their strongly imitative This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions The A-Text of ' Pers Plowman' 320 cast, are what we might expect from such a writer. There is no chronological difficulty; for the fact that there is a phrase and an idea common to this twelfth passus of A and to the B-text, does not prove it earlier than B1. It would be conceivable that Butt put together this twelfth passus from one or two vague recollections of the B-continuation and by more deliberate borrowing from the eleventh passus of A, which lay before him. y If however we are to attribute the twelfth passus in part to the author of Dowel and in part to John Butt it is difficult to know where to make the division. At least four places are possible. Professor Manly makes John Butt begin at 1. 55, where Will and Kind Wit set out together. Another change is, I think, indicated at 1. 89. Prof. Skeat has suggested 1. 99, 'Wille wiste thurgh in-wit.' Originally Prof. Skeat suggested 1. 106, 'And so bad lohan but.' suggested , If Prof. Manly is right, the passus must originally have broken off imperfect; for 1. 54, so far from concluding anything, marks the beginning of another adventure, Will's visit to Kind Wit and Life. This is, of course, no argument against Prof. Manly's view; presumably the end was abrupt, or John Butt would not have felt it necessary to round it off by adding his lines. In seeking the place where the original Passus xII left off we are not justified in demanding that it shall make a satisfactory conclusion to the A-text. satisfactory John Butt certainly managed to imitate the style of his original fairly well: very well, if he wrote all that Prof. Manly attributes to him; and it is not easy to find differences in language and metre which will enable us to decide exactly where his addition begins. 1 If, with Dr Mensendieck, we interpret B xi, 414, as a reference to A xII, 35, etc., then clearly this opening of the twelfth passus of A must be earlier than the B-text. But the reference may, I think, be to B xi, 404-5. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions y g A study of the subject-matter may be more helpful. Line 55, where Prof. Manly supposes John Butt to begin, shows us Omnia probate and Will setting out together to seek Kind Wit. But before Will reached his journey's end, he met with many marvels. First he was greeted by Hunger, who like himself, was on the way to find Life: 'though Kind Wit help, I shall fell that freke in a few days.' 'I would follow thee,' said Will, and Hunger consented; but Will ate so much of Hunger's broken scraps that he lagged behind, and was hailed by Fever-' a knave with a confessor's face, lean and wretched, with legs full small.' Fever, too, was on the way to Life, bearing a message from his Duke, Death. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions 321 R. W. CHAMBERS I ant masager of dep * men haue I tweyne * g dep tweyne pat on is called Cotidian * a courrour of oure hous T i d i k g dep tweyne pat on is called Cotidian * a courrour of oure hous T i d i k pTercian pat oper trewe drinkeres bope pTercian pat oper trewe drinkeres bope. pat oper p We hain letteres of lyf he shal his lyf ty[n]e F l) ti d k d di y y ty[n]e Fro dep l)at is oure duk ? swyche dedis we brynge 'If I might,' said Will, 'I would follow in thy track.' g , , y 'Nay, Will,' said Fever, 'thou fallest into a snare if thou followest me; so live and work according to Dowel, that thou mayest reach Paradise at last.' Will knew that these were profitable words, so he made haste and wrought what is written here, and other works con- cerning Piers the Plowman and much people also: and when this work was wrought, Death dealt him a dint, and drove him to the earth, and he is closed under clay: Christ have his soul. And so John Butt prayed, when he saw these saws quoted concerning James, Jerome and Job: and because he is something of a poet he made this ending. Now God save King Richard and the lords who love him: Mary pray for us: Christ save us. Amen. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions Where Fever dismisses the poet, an essential change comes over the story of the search for Dowel. The subject of Dowel was first introduced when, at the end of Passus VIII, the poet saw in Dowel the one help in that judgment which must follow death. Through four passus Will, in his search for Dowel, has been sent from one allegorical character to another, has received much good advice, but has got no nearer his goal: and now when he is hastening to seek Dowel at the dwelling of Life, and is passed on the way by Hunger and Fever, who are also seeking Life, we seem to be leading up to a conclusion which shall emphasize the familiar moral, how, faced by death and judgment, Life can find support and consolation in nothing but Dowel, his good deeds. Kind Wit will not help him, Hunger says as much: but we should expect to learn how, dwelling in the burgh of quod bonumn est tenete, and knowing Dowel familiarly, Life has nothing to fear from Hunger, Fever, or Death. familiarly, nothing Hunger, , Suddenly, when Fever turns the poet aside from his quest, all is changed. At this point the subject of the poem ceases to be Will's search for Dowel, or the struggle between Life and Death, and is directed to the personality of Will himself-his writings, his death, the prayer for his soul. p aye The matter does not, of course, admit of proof: but the point where John Butt takes up his pen is surely very likely to be identical with that where the reader's thoughts are thus suddenly, and with some violence, diverted from the search for Dowel to the fate of Will. v o e ce, Now it is precisely at this point that the Ingilby MS. stops. Where 21 M. L. R. VI. M. L. R. VI. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions The A-Text of 'Piers Plowman' 322 our third authority for this twelfth passus, the University College MS., may have ended, we do not know, as, owing to a mutilation, it breaks off quite near the beginning of the passus. 1 Ingilby simply stops: no Explicit is added. But, as pointed out above, this abrupt ending cannot be used as an argument that something has been lost. Presumably John Butt found the twelfth passus abrupt. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions The Ingilby MS., however, is not mutilated: the scribe has left the last written page blank all but one line: two subsequent leaves have been nearly all torn out, but they clearly contained no more of the story of Dowel. The Ingilby scribe must have stopped at this line because his copy stopped1. Of course his copy may have been mutilated: but it would be an odd coincidence that it should have been mutilated just at the very line where there is a break in the thought in the Rawlinson MS. so marked as to lead us to conjecture that the original poem may have stopped there. It is a far easier hypothesis to suppose that the original poet stopped where Ingilby makes him stop', and that those lines which are found in Rawlinson but are not found in Ingilby (viz. 89-117) constitute John Butt's addition. At the sanme time it must be remembered that this is only a probability: it does not admit of demonstration. p obab y There is, -however, also a clear break in the continuity at 1. 99, and again at 1. 106. Prof. Skeat's original view, that John Butt's lines begin ' And so bad John Butt,' and that the preceding lines, in which Will is killed off, are not John Butt's at all, has still some supporters. , , pp Five points have been indicated where it is conceivable that the original poet stopped. Diligent search would probably reveal five more. Such theories, unless backed up by some external evidence, are not usually convincing. The only places for which we have MS. authority for stopping are at the end of Passus XI, or at xiI, 88. For this reason, unless strong evidence is forthcoming, I should be inclined to believe either that Butt wrote the whole of Passus xiI, or that he wrote only from 1. 89 onwards. only The text of the twelfth passus, froml the point where Ingilby stops, is given below, from Rawlinson. 'Nay, wil!' quod ?at wy3th ' wend ]ou no ferther y, q ] 90 But lyue as p]is lyf . is ordeyned for the: pe tomblest wip a trepget ? 3if Pou my tras folwe And mannes mer]e wrou3J no mor * }an he deseruyp here Whil his lyf and his lykhame . lesten togedere. This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions y , 115 God saue hem sound ? by se and by land; Marie moder and may ? for man ]ou by-seke; pat barn bryng vs to blys - pat bled vp-on Pe rode! Amen. R. W. CHAMBERS. LONDON LONDON 21-2 This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions 323 R. W. CHAMBERS And Per-fore do after do-wel ? whil Pi dayes duren ? Pi dayes 95 pat pi play be plentevous ? in paradys with aungelys. pou shalt be lau3th into ly3th * with loking of an eye So Pat J~ou werke pe word ] pat holy wryt techep And be prest to preyeres and profitable werkes. Wille [wiste] purgh in-wit J ou wost wel p]esope, [ ] purgh p]e p , 100 pat pis speche was spedelich and sped hin wel faste, And wrou3the pat here is wryten ? and over werkes boiPe Of peres Pe plowman and mechel puple al-so; And whan Pis werk was wrou3t ? ere wille my3te a-spie, DeJ delt him a dent ? and drof him to Pe erJe, ? , 105 And is closed vnder clom ? crist haue his soule. And so bad Iohan but busily wel ofte, When he saw Pes sawes busyly a-legged By Iames and by Ierom by Iop and by opere, And for he medlep of makyng ? he made Pis ende. ? , 105 And is closed vnder clom ? crist haue his soule. And so bad Iohan but busily wel ofte, When he saw Pes sawes busyly a-legged By Iames and by Ierom by Iop and by opere, And for he medlep of makyng ? he made Pis ende. p y g ? s 110 Now alle kenzne creatures . Jat cristene were euere, God for his goudnesse ? gif hem swyche happes, To lyue as P]at lord lyky] ]Pat lyf in hem putte. Furst to rekne Richard- kyng of P]is rewme, And alle lordes pat louyn him ? lely in herte, p y g 110 Now alle kenzne creatures . Jat cristene were euere, God for his goudnesse ? gif hem swyche happes, To lyue as P]at lord lyky] ]Pat lyf in hem putte. Furst to rekne Richard- kyng of P]is rewme, And alle lordes pat louyn him ? lely in herte, pat louyn ? lely e e, 115 God saue hem sound ? by se and by land; Marie moder and may ? for man ]ou by-seke; pat barn bryng vs to blys - pat bled vp-on Pe rode! Amen. Explicit do-Wel. Nornen scriptoris tilot plenus alnoris. p ouy ? This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 192.75.12.3 on Sat, 19 Dec 2015 21:51:52 UTC All use subject to JSTOR Terms and Conditions
https://openalex.org/W2801926645	https://journals.plos.org/ploscompbiol/article/file?id=10.1371/journal.pcbi.1006121&type=printable	English	null	Identifying robust hysteresis in networks	PLOS computational biology/PLoS computational biology	2,018	cc-by	15,373	Editor: Lingchong You, Duke University, UNITED STATES Editor: Lingchong You, Duke University, UNITED STATES RESEARCH ARTICLE Identifying robust hysteresis in networks Toma´sˇ Gedeon1, Bree Cummins1, Shaun Harker2, Konstantin Mischaikow2 1 Department of Mathematical Sciences, Montana State University, Bozeman, Montana, United States of America, 2 Department of Mathematics, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America RESEARCH ARTICLE * gedeon@math.montana.edu (TG); mischaik@math.rutgers.edu (KM) * gedeon@math.montana.edu (TG); mischaik@math.rutgers.edu (KM) Received: September 21, 2017 Accepted: April 3, 2018 Published: April 23, 2018 To summarize our understanding of how genes, their products and other cellular actors interact with each other, we often employ networks to describe their interactions. How- ever, networks do not fully specify how the underlying biological system behaves in differ- ent conditions, nor how such response evolves in time. We present a new modeling and computational approach that allows us to compute and collect summaries of network dynamics for large sets of parameter values. We can then search these summaries for all observed behavior. We illustrate our approach on networks that govern entry to the cell cycle in humans and yeast. We rank networks based on how robustly they exhibit the experimentally observed behavior of hysteresis. We find similarities in network structure of the best ranked networks in yeast and humans, which are not explained by a common ancestry. Our approach provides a tool linking network structure and the behavior of the underlying system. Copyright: © 2018 Gedeon 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. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. The computer codes used to reproduce the results in the paper are stored in two code repositories. The first repository is the DSGRN project https://github.com/shaunharker/DSGRN. This is an open-source project which, as of writing, is hosted on the code-sharing website GitHub at \url{https://github.com/shaunharker/DSGRN}. The version utilized for this paper is 1.0.0. The second repository is the supplemental for this paper https://github.com/shaunharker/2017-DSGRN- IdentifyingRobustHysteresisInNetworks and Abstract We present a new modeling and computational tool that computes rigorous summaries of network dynamics over large sets of parameter values. These summaries, organized in a database, can be searched for observed dynamics, e.g., bistability and hysteresis, to dis- cover parameter regimes over which they are supported. We illustrate our approach on sev- eral networks underlying the restriction point of the cell cycle in humans and yeast. We rank networks by how robustly they support hysteresis, which is the observed phenotype. We find that the best 6-node human network and the yeast network share similar topology and robustness of hysteresis, in spite of having no homology between the corresponding nodes of the network. Our approach provides a new tool linking network structure and dynamics. RESEARCH ARTICLE Identifying robust hysteresis in networks Toma´sˇ Gedeon1, Bree Cummins1, Shaun Harker2, Konstantin Mischaikow2 1 Department of Mathematical Sciences, Montana State University, Bozeman, Montana, United States of America, 2 Department of Mathematics, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America OPEN ACCESS Citation: Gedeon T, Cummins B, Harker S, Mischaikow K (2018) Identifying robust hysteresis in networks. PLoS Comput Biol 14(4): e1006121. https://doi.org/10.1371/journal.pcbi.1006121 Author summary Received: September 21, 2017 Accepted: April 3, 2018 Published: April 23, 2018 Copyright: © 2018 Gedeon 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. Identifying robust hysteresis in networks relative ease compared to the parameters governing these interactions. If the premise of a pre- dictive relationship holds, then the network approach to complex regulation is highly advanta- geous, since the phenotype of the cell encoded in its dynamics can be deduced only from the interaction data. houses the code which is used to reproduce the above results. This, again, is open-source. houses the code which is used to reproduce the above results. This, again, is open-source. Funding: The work of SH and KM was partially supported by grants NSF-DMS-1125174, 1248071, 1521771 and a DARPA contracts HR0011-16-2-0033 and FA8750-17-C-0054, and NIH grant R01 GM126555-01. The work of TG was partially supported by NSF grants DMS-1226213, 1361240, DARPA contracts D12AP200025 and FA8750-17-C-0054 and NIH R01 grants 1R01AG040020-01 and R01 GM126555-01. The work of BC was partially supported by DARPA D12AP200025 and FA8750-17-C-0054, USDA 2015-51106-23970 and NIH R01 GM126555-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The firm bridge between network structure and the dynamics of the corresponding nonlin- ear system remains elusive for the fundamental reason that it cannot exist in the suggested gen- erality. The dynamics will always depend on the state of the cell, which in the models is represented by the parameters and initial data. Some partial results in terms of motif theory have been suggested [1], but these are limited to small networks and their applicability to the dynamics of larger networks is questionable [5, 6]. Furthermore, there is currently no mathematical theory that suggests that understand- ing of dynamics of a small motif that is embedded in a larger network informs our knowledge of the dynamics of the larger network. In fact, the classical theory of dynamical systems lacks tools that describe dynamics when parameters are unmeasured, or, if measured, carry large uncertainty. In this paper we report on a new approach [7–9] referred to as Dynamic Signatures Gener- ated by Regulatory Networks (DSGRN) that provides a queryable global characterization of dynamics over large regions of parameter space. This is based on a new, still developing, computationally efficient perspective of nonlinear dynamics [10–12]. The philosophy of this approach has already seen applications in other settings [13–16]. Introduction In cell biology, the power of a network model as an organizational principle of complex regula- tion rests on the premise that there is a predictive relationship between the network structure and the network dynamics [1–4]. A network model only requires specifying the character of the interactions between genes, proteins and signaling molecules, which can be inferred with 1 / 23 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 Novel features of DSGRN include the following: (i) DSGRN does not use an explicit functional form for the nonlineari- ties governing the dynamics, (ii) the decomposition of parameter space reflects the representa- tion of the nonlinear dynamics, and (iii) the decomposition of parameter space is determined by information local to each node of the regulatory network, and this local determination is computed a priori. Competing interests: The authors have declared that no competing interests exist. For the sake of clarity we discuss DSGRN in the specific, but important biological context of resettable bistability and hysteresis, especially as they relate to cell cycle restriction point dynamics. A key decision for each cell is when to replicate DNA and initiate proliferation. This deci- sion is based on multiple factors, but once the process has started DNA replication must be finished. Therefore the influence of these factors must be uncoupled at the moment of the decision, called the restriction point of the cell cycle [17–19]. The requirement of irreversibility and decoupling suggest that phenotypically a bistable switch may underlie the restriction point. The simplest model of a bistable switch involves a hysteresis curve as indicated in Fig 1(a) where the curve indicates the equilibria for a differential equation _x ¼ f ðx; lÞ and λ is a parameter. Parameter space naturally divides into three intervals, low λ and high λ for which there exists a single stable fixed point denoted Off and On, respectively, and medium values of λ for which there exist two stable fixed points (B). Assume the system is in the On state. In the setting of Fig 1(a) or 1(b) if the value of λ is decreased by a sufficient amount (beyond the left hash mark) then the internal dynamics of the system will drive it to the Off state. This is not the case in the setting of Fig 1(c). Observe that the global structure indicated in Fig 1(a) allows for the occurence of hysteresis, i.e. the abil- ity to repeatedly reset the system from On to Off and from Off to On by changing the value of λ and a region of parameter space, the medium values of λ, at which the direction of of the change in λ (increasing or decreasing) determines whether the system is in the on or off state. Identifying robust hysteresis in networks Fig 1. Hysteresis and resettable bistability. Solid (dashed) lines indicate stable (unstable) equilibria for fixed values of input, Off = Low Equilibrium, B = Bistability, and On = High Equilibrium. Resettable bistability: When input signal is withdrawn from a bistable system, and the system resets to the low equilibrium (filled circle). Hysteresis: In addition to resettable bistability, when signal is increased, system goes to high equilibrium (circle). (a) Hysteresis; (b) resettable bistability, but not hysteresis. (c) No resettable bistability. https://doi.org/10.1371/journal.pcbi.1006121.g001 Fig 1. Hysteresis and resettable bistability. Solid (dashed) lines indicate stable (unstable) equilibria for fixed values of input, Off = Low Equilibrium, B = Bistability, and On = High Equilibrium. Resettable bistability: When input signal is withdrawn from a bistable system, and the system resets to the low equilibrium (filled circle). Hysteresis: In addition to resettable bistability, when signal is increased, system goes to high equilibrium (circle). (a) Hysteresis; (b) resettable bistability, but not hysteresis. (c) No resettable bistability. https://doi.org/10.1371/journal.pcbi.1006121.g001 https://doi.org/10.1371/journal.pcbi.1006121.g001 experimental data [20, Fig 2b] leads to a blurred version (with measurement on the vertical axis presented using a logarithmic scale) of the simple single valued curve of Fig 1. With this in mind, the hysteresis phenomenon detected by DSGRN consists of identifying well defined regions that contain the attractors associated with Off and On states. As is made clear in the Materials and Methods section, whether these attractors are stable fixed points or not depends on details of the particular differential equation used in the model. A network that may be responsible for the restriction point dynamics in mammalian cells was suggested by [21] and then further elaborated by Yao et al. [4]. The essential elements of the restriction point network is a family of E2F transcription factors which are sequestered in a heterodimer by Rb in non-proliferating cells in G1 phase. Release of E2F by phosphorylation of Rb results in initiation of S phase of the cell cycle. The principal controls of Rb are cyclin/kinase complexes CycD/Cdk4,6 and CycE/Cdk2. CycD/Cdk4,6 is up-regulated by Myc which responds to the cell growth; the initial phosphory- lation of Rb by CycD/Cdk4,6 releases E2F, which up-regulates the second kinase CycE/Cdk2, which then completes the phosphorylation of Rb and finishes the release of E2F [4, 17–19, 21]. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 While this simple model of a bistable switch provides intuition for the analysis performed in this paper, experimental data leads us to entertain the possibility that the dynamics of switches in biological systems may be more complex. For example, the lac operon is among the most carefully studied regulatory networks that exhibits bistability. Associated PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 2 / 23 Identifying robust hysteresis in networks Fig 2. DSGRN for toggle switch. (a) Regulatory network for the toggle switch. (b) Thresholds {θ2,1, θ1,2} divide phase space, (0, 1)2, into four 2-dimensional domains (black dots) and 1-dimensional walls separating the domains (circles). (c) For each choice of parameters, given a domain there is a well-defined direction of dynamics at walls that defines the state transition graph (see Fig 6 for the derivation of this dynamics). State transition graph for parameters in regions 5 and 9 of the parameter graph. The Morse graph for 9 consists of a single node FP(0, 1) where FP indicates that the node is terminal in the Morse graph (a trivial statement for this simple example) and that the vector representation of the associated region is (0, 1). The Morse graph for 5 contains two nodes FP(0, 1) and FP(1, 0). (d) Visual interpretation of SQL DSRGN database for toggle switch organized as a parameter graph with explicit description of parameter domains (inequalities in bottom of each square) and Morse graphs (top part of each square) valid over corresponding parameter domain. Dashed boxes indicate elements of PG(¬1) = {G1, G2, G3}. htt //d i /10 1371/j l bi 1006121 002 Fig 2. DSGRN for toggle switch. (a) Regulatory network for the toggle switch. (b) Thresholds {θ2,1, θ1,2} divide phase space, (0, 1)2, into four 2-dimensional domains (black dots) and 1-dimensional walls separating the domains (circles). (c) For each choice of parameters, given a domain there is a well-defined direction of dynamics at walls that defines the state transition graph (see Fig 6 for the derivation of this dynamics). State transition graph for parameters in regions 5 and 9 of the parameter graph. The Morse graph for 9 consists of a single node FP(0, 1) where FP indicates that the node is terminal in the Morse graph (a trivial statement for this simple example) and that the vector representation of the associated region is (0, 1). The Morse graph for 5 contains two nodes FP(0, 1) and FP(1, 0). (d) Visual interpretation of SQL DSRGN database for toggle switch organized as a parameter graph with explicit description of parameter domains (inequalities in bottom of each square) and Morse graphs (top part of each square) valid over corresponding parameter domain. Dashed boxes indicate elements of PG(¬1) = {G1, G2, G3}. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 Since one of the hallmarks of cancer is sustained proliferation in cells that are immune to external signals that would prevent proliferation in normal cells, it is not surprising that dysre- gulation of this network is observed in the majority of cancers [22]. For recent comprehensive reviews on the connection between retinoblastoma protein (Rb), a key member of this net- work, and cancer, see [23–26]). This system exhibits resettable bistability [4] if, as the growth factor input is reduced to zero, bistability vanishes and the cell returns to a non-proliferating phenotype with E2F sequestered, e.g. in Fig 1 as λ is reduced the system moves from bistability to the monostable state Off. Observe that resettable bistability, and hysteresis are physiological phenomena that can only be expressed via an understanding of global dynamics over paths in parameter space. Yao et. al. [4] executed a modeling study of the mammalian cell cycle restriction point with the goal of identifying “the basic gene circuit underlying resettable Rb-E2F bistable switch by the criterion of robustness” where robustness is defined in terms of the ability to maintain functionality against perturbations. Note that even an idealized description of the restriction point network has multiple variables and a multitude of parameters. Thus, from the mathematical perspective to rigorously carry out the program proposed in [4] requires PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 3 / 23 Dynamic Signatures Generated by Regulatory Networks (DSGRN) We view DSGRN as an algorithm; the input is a regulatory network and the output is a query- able database of the global dynamics for all parameters. This algorithm is based on four essen- tial concepts: • a Regulatory Network, the input; • a Regulatory Network, the input; • the State Transition Graph, a finite representation of the dynamics; • the Morse Graph, a compact queriable representation of the dynamics at a given parameter value; and • the Morse Graph, a compact queriable representation of the dynamics at a given parameter value; and • the Parameter Graph, an explicit finite representation of parameter space. The DSGRN database consists of the parameter graph along with an association of a valid Morse graph to each node of the parameter graph. Before turning to the Rb-E2F network we use the toggle switch, which consists of two con- stitutively expressed repressors that repress each other, in an attempt to focus on the general philosophy and novel concepts associated with DSGRN. For more detailed descriptions see the Methods section and [8]. The regulatory network of the toggle switch has the form of Fig 2(a). DSGRN requires that a regulatory network be a network for which each node has at least one outgoing edge and there is at most one edge from one node to another node. To each node n in regulatory network, DSGRN associates a real value, e.g., concentration, xn 0 and a parameter γn > 0 representing the rate of degradation of xn. To each edge m ! n (denoting activation) or m a n (denoting repression) in a regulatory network, DSGRN assigns three parameters ℓn,m, un,m and θn,m where ℓn,m and un,m represent low and high levels of growth of xn, respectively, (in particular ℓn,m < un,m) that are determined by the value of xm rel- ative to the threshold θn,m. Observe that for a regulatory network with N nodes and E edges the dimension of the space of parameters is D = N + 3E and is a subset of [0, 1)D. To construct a state transition graph DSGRN uses the thresholds θn,m to decompose the phase space into rectangular regions (see Fig 2(b)). DSGRN assigns a vertex to each region (solid dot) and to each face between regions (circle). Identifying robust hysteresis in networks There are similarities between DSGRN and a variety of other approaches. To the best of our knowledge the novel aspects of DSGRN are that we (1) approximate continuous system by a discrete system (the state transition graph) and (2) via our computations we obtain knowledge about the global dynamics for all parameters associated with the model. Other approaches, for instance CPSS (Continuous parameter space search) [27] chose a particular nonlinearity which in turn determines the parameter space. A query based on exis- tence of stable equilibria is established, regions of parameter space are non-uniformly sampled, and the differential equation is integrated at the chosen parameter values to identify existence or lack of existence of equilibria. In contrast DSGRN searches for attracting regions (a more robust concept than stable equilibria) and thus the set of nonlinearities for which the computations are provably valid is much larger [7]. Furthermore, there is no sampling of parameter space, instead the dynamics is reported for all possible parameter values. https://doi.org/10.1371/journal.pcbi.1006121.g002 mathematical and efficient computational techniques capable of addressing at least three fundamental challenges: mathematical and efficient computational techniques capable of addressing at least three fundamental challenges: mathematical and efficient computational techniques capable of addressing at least three fundamental challenges: 1. identify the existence of all parameters exhibiting bistability with minimal knowledge of the functional form of nonlinearities; 2. identify the existence of curves in parameter space over which resettable bistability or hys- teresis take place; and 3. provide a systematic quantification of the extent to which a particular regulatory network is capable of exhibiting these features. The aim of this paper is to demonstrate that DSGRN is capable of meeting these challenges for moderate sized networks. As is discussed in detail below, DSGRN provides information about the global dynamics for all parameter values, and to the best of our knowledge, is unique in these capabilities. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 4 / 23 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 Dynamic Signatures Generated by Regulatory Networks (DSGRN) Furthermore, each solid dot is labeled as a vector where the n-th entry indicates the number of thresholds θm,n with values less than xn for any xn in the associated region. For each fixed set of parameter values the state transition graph represents the dynamics of the regulatory network via a directed graph based on the PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 5 / 23 Identifying robust hysteresis in networks above mentioned vertices. As is shown in Fig 2(c) the edges go from circles to solid dots, from solid dots to circles, and potentially there are self edges on solid dots. Fig 2(c) shows that the edges in a state transition graph are parameter dependent. above mentioned vertices. As is shown in Fig 2(c) the edges go from circles to solid dots, from solid dots to circles, and potentially there are self edges on solid dots. Fig 2(c) shows that the edges in a state transition graph are parameter dependent. Since the size of the state transition graph grows rapidly with the size of the regulatory net- work, DSGRN stores a minimal representation of the global dynamics using a Morse graph, which is an acyclic directed graph (see Fig 2(d)). Morse graphs are capable of encoding poten- tially complicated dynamics (see [16]), however, since the focus of this paper is on robust switch behavior, we restrict our discussion accordingly. In the state transition graph the most natural representative of a stable fixed point is a vertex with a unique out edge that is a self edge. In the associated Morse graph this is a terminal vertex labeled FP. Furthermore, since only solid dots have self edges, we identify each such terminal vertex by the vector label of the associated solid dot. The directed edges in the state transition graph are determined by affine multilinear inequalities involving the parameters. These inequalities are defined at each node in the regula- tory network RN according to the out edges, in edges, and the logic governing the interaction of in edges. For each node n in the regulatory network the inequalities are organized via a node-graph PG(n) where edges indicate change of a single inequality. For the toggle switch the node-graphs and their associated inequalities are PGð1Þ : ju12 < g1y21j jl12 < g1y21 < u12j jg1y21 < l12j; PGð2Þ : ju21 < g1y12j jl21 < g1y12 < u21j jg2y12 < l21j: A node in PG(n) is called low (high) if all the associated ℓ(u) values are less (greater) than all the associated γθ values. The remaining nodes are called intermediate nodes. A node in PG(n) is called low (high) if all the associated ℓ(u) values are less (greater) than all the associated γθ values. The remaining nodes are called intermediate nodes. Identifying robust hysteresis in networks remaining vertices along the lifted path exhibit either a unique terminal node FP = p or (p, ) bistability, where stands for arbitrary FP. remaining vertices along the lifted path exhibit either a unique terminal node FP = p or (p, ) bistability, where stands for arbitrary FP. • hysteresis between p and q if there exists a path in PG(n) from low (high) to high (low) nodes that exhibits both resettable (p, q) bistability to p and resettable (p, q) bistability to q. • hysteresis between p and q if there exists a path in PG(n) from low (high) to high (low) nodes that exhibits both resettable (p, q) bistability to p and resettable (p, q) bistability to q. We demonstrate the biological relevance of DSGRN on the synthetic toggle switch imple- mented in Gardner et. al. [28] using Lac repressor lacI and a temperature sensitive phage λ repressor (cIts), with externally supplied IPTG as a control. Since IPTG binds directly to the Lac repressor and inactivates it, IPTG effectively lowers the available concentration of the Lac repressor. Let x1 represent the concentration of cIts and x2 represent the concentration of lacI. The state where cIts (x1) is fully expressed and lacI (x2) is repressed is designated as an ON state, and the state where x1 is low and x2 is high is designated as an OFF state. In the DSGRN database (Fig 2(d)) FP(1, 0) and FP(0, 1) correspond to ON and OFF, respectively. Increase in the con- centration of IPTG decreases the values of ℓ12 and u12 and thus is quantified by a path in PG(1) from the high node to the low node. Visual inspection of Fig 2(d), shows that G2 is the only subgraph in PG(¬1) that exhibits (ON, OFF) bistability. Furthermore, moving monotonically along the path defined by G2 from node 4 to node 6 results in hysteresis between ON and OFF states. Thus the DSGRN database analysis suggests that if the toggle switch is operating at the bistable regime (node 5), then suf- ficiently strong IPTG treatment phenotype leads to FP(1, 0), which represents the ON state. This agrees with the experimental observations [28]. In the parameter graph there are parameter nodes, called inessential parameter nodes, for which at the associated parameter values there is a node n in the regulatory network such that the inequalities that determine the state transition graph do not vary as a function of xn. At inessential parameter nodes the network dynamics is identical to that of a subnetwork of the regulatory network. In the computations presented in the remainder of the paper we only con- sider essential subgraphs EPG(¬n) PG(¬n), i.e. subgraphs of the parameter graph that do not contain any inessential parameter nodes. The parameter graph for a regulatory network is the product of the node-graphs, i.e. N PG ¼ QN n¼1 PGðnÞ. A graphical representation of the parameter graph of the toggle switch is given in Fig 2(d). Observe that the inequalities associated with each node provides a subdivi- sion of parameter space. For the toggle switch the parameter graph provides a representation of parameter space, which is an unbounded region of (0, 1)8, via nine regions. Furthermore, it is guaranteed that for every parameter in a given region the associated state transition graph is the same, and therefore, the Morse graph is constant over each region. Because the parameter graph is a product graph, if we fix a node n in the regulatory net- work, then we can decompose the parameter graph into a collection of subgraphs each one of which is isomorphic to PG(n). We denote this collection of subgraphs by PG(¬n). As shown in Fig 2(d), for the toggle switch, PG(¬1) = {G1, G2, G3}. The fact that any G 2 PG(¬n) is isomor- phic to PG(n) implies that a path in PG(n) defines a path in G. We call the path in G the lift of the path in PG(n). We now describe implemented queries to the DSGRN database that are relevant to the anal- ysis of switching behavior. A Morse graph exhibits (p, q) bistability if it contains terminal nodes FP = p and FP = q. Fix a node n in the regulatory network and let G be a element of PG(¬n). The subgraph G exhibits: • (p, q) bistability if there is a vertex in G such that the associated Morse graph exhibits (p, q) bistability. • resettable (p, q) bistability to p if there is a path in PG(n) from a low (or high) node to an intermediary node such that for the lifted path in G, the Morse graph associated to the lift of the low (high) node has unique terminal node FP = p, the Morse graph associated to the lift of the intermediate node exhibits (p, q) bistability, and the Morse graphs associated with the Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 6 / 23 Restriction point in mammalian cell cycle The E2F-Rb network regulates the restriction point of the mammalian cell cycle, i.e. the point where the progression through the cell cycle decouples from the growth signals [4, 17–19]. The E2F-Rb network exhibits two essential phenotypes: when the growth signal is absent, the tran- scription factor E2F is sequestered in the heterodimer with Rb. This is the quiescent state (QS). On the other hand, when the growth signal is present at high level, E2F disassociates from the E2F-Rb dimer and activates numerous downstream processes. This proliferative state (PS) ini- tiates entry into S-phase of the cell cycle. Yao et. al. [4] executed a modeling study of the mammalian cell cycle restriction point with the goal of identifying “the basic gene circuit underlying resettable Rb-E2F bistable switch by the criterion of robustness” where robustness is defined in terms of the ability to maintain functionality against perturbations. In particular, they start with a large network from [17, 18] that, as is indicated in Fig 3(a), they coarse-grain into a system with three nodes and a variety of possible edges. By considering connected subnetworks of Fig 3(a) they construct a library of 768 mathematical models of networks. They assume that interactions between the nodes are governed by Hill-functions, thus producing a model consisting of a three dimensional system of ordinary differential equation model with up to 26 parameters. To evaluate the models they generate 20,000 parameter sets by randomly sampling from reasonable parameter ranges for PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 7 / 23 Identifying robust hysteresis in networks Fig 3. 3 node E2F-Rb networks. (a) 3 node network with potential edges from [4] with signal S acting on MD. (b) Histogram showing 24 regulatory networks expressing full path hysteresis between QS and PS. Five regulatory networks show full path hysteresis in 100% of EPG(¬MD). (c) Histogram showing 27 regulatory networks expressing partial path hysteresis between QS and PS. (d) Histogram showing 25 regulatory networks expressing full path resettable (QS,PS) bistability to QS. (e) Histogram showing 35 regulatory networks expressing full path resettable (QS,PS) bistability to QS. (f)-(j) Five networks that exhibit full path hysteresis along all appropriate paths in EPG(¬MD) These networks are also the top five networks in partial path hysteresis in descending order (f),(g)-(i), (j). Network (f) is also highest in prevalence of partial path resettable bistability, but does not show any full path resettable bistability. Identifying robust hysteresis in networks insufficient to sample dynamics at distinct values of the input variable S as a proxy for pres- ence of these phenomena. We now show that DSGRN can efficiently replicate the efforts of [4]. We begin with a dis- cussion of how it avoids the above mentioned mathematical concerns. First, DSGRN allows one to identify any point in parameter space with a node in the associated parameter graph; in turn, each node in the parameter graph is identified with a region of parameter space for which the dynamics can be described via a Morse graph. Therefore, with DSGRN one does not restrict the analysis to finite collections of parameters; the analysis is valid for all parameter val- ues. Second, the DSGRN analysis is based on representing dynamics via state transition graphs and, for the purposes of this paper, interpreting the dynamics via terminal nodes in the Morse graph. The terminal nodes represent regions in phase space that are trapping regions for broad classes of nonlinearities [7]. Hence, with DSGRN one is not restricted to a specific analytic representation of the dynamics. Third, as is demonstrated in the toggle switch example, paths through the parameter graph represent continuous paths through parameter space, thus ques- tions of resettable bistability or hysteresis can be rigorously addressed. In addition, because there are finitely many lifts of parameter paths in the parameter graph we can quantify the number of lifts for which the desired switching phenomenon does occur. There are 49 regulatory subnetworks of Fig 3(a), such that every node has at least one out- edge, and there is no more than one edge from one node to another. For each of these subnet- works we compute EPG(¬MD), where node MD is singled out since the input signal S impacts the network at the node MD. Varying the input of the network corresponds to a path in EPG (¬MD). We assume that a monotone change in the strength of the signal S acts monotonically on MD, but we do not necessarily assume that we know the range of S. This leads us to con- sider two cases. In the first case we assume that the network parameters are “aligned” with the range of the signal. In this case as S ranges from its lowest value to its highest value, MD moves from lowest quantifiable level, i.e. Restriction point in mammalian cell cycle Full results for all networks can be find in S1 Table, where the five networks (f)-(j) have numbers 24,39,46,26 and 22. https://doi.org/10.1371/journal.pcbi.1006121.g003 Fig 3. 3 node E2F-Rb networks. (a) 3 node network with potential edges from [4] with signal S acting on MD. (b) Fig 3. 3 node E2F-Rb networks. (a) 3 node network with potential edges from [4] with signal S acting on MD. (b) Histogram showing 24 regulatory networks expressing full path hysteresis between QS and PS. Five regulatory networks show full path hysteresis in 100% of EPG(¬MD). (c) Histogram showing 27 regulatory networks expressing partial path hysteresis between QS and PS. (d) Histogram showing 25 regulatory networks expressing full path resettable (QS,PS) bistability to QS. (e) Histogram showing 35 regulatory networks expressing full path resettable (QS,PS) bistability to QS. (f)-(j) Five networks that exhibit full path hysteresis along all appropriate paths in EPG(¬MD) These networks are also the top five networks in partial path hysteresis in descending order (f),(g)-(i), (j). Network (f) is also highest in prevalence of partial path resettable bistability, but does not show any full path resettable bistability. Full results for all networks can be find in S1 Table, where the five networks (f)-(j) have numbers 24,39,46,26 and 22. https://doi.org/10.1371/journal.pcbi.1006121.g003 https://doi.org/10.1371/journal.pcbi.1006121.g003 each of the 26 parameters. Each of the 768 networks is given a score based on the percentage of this collection of parameters at which the differential equations exhibits a particular switching characteristic like resettable bistability or hysteresis. There are several mathematical objections that can be made to this procedure. First, if one were to generate the random parameters by insisting on at least two independent choices for each parameter, then one would need to consider 226 7 × 107 parameter sets. It is insufficient to sample dynamics at distinct parameter values as a proxy for its prevalence without a priori bounds on the sensitivity of this dynamics to changes in the parameters. Second, it is not clear how well a Hill function approximates the nonlinear behavior of the system. Third, as indicated in the introduction, the phenomena of resettable bistability and hysteresis are both a function of a continuous change of parameters. Therefore it is 8 / 23 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 Identifying robust hysteresis in networks monotonically with respect to the signal S, this implies that we can without loss of generality assume that the signal acts in a monotone fashion on the unique node that MD acts on. Of course, whether we assume that the signal acts on this node in a monotone increasing or decreasing manner depends on whether the out edge from MD represents activation or inhibition. For the regulatory subnetworks of Fig 3 we order the nodes by (MD, RP, EE). We interpret the quiescent and proliferative states of the restriction point network in terms of dynamic sig- natures of DSGRN. In the quiescent state QS, E2F is sequestered in the heterodimer with Rb and therefore the levels of free E2F are low. In the 3-node network in Fig 3(a) free E2F is repre- sented as EE. Following [4] we associate the quiescent state with low levels of EE and the prolif- erative state with high levels of EE. In the DSGRN database the precision to which we can search for attractors is limited by the number of thresholds. In other words, we can identify if coordinates associate with an attractor are bounded between consecutive threshold values of that variable, but any finer identification requires choice of a particular nonlinear differential equation and a choice of particular parameter values. The number of thresholds of a variable is determined by the number of edges emanating from a node that corresponds to the variable, since each such edge is associated to a single distinct threshold. We characterize quiescent state QS as a minimal mode in the Morse graph with labeling FP(, , 0). Similarly, the proliferative state PS is characterized by high levels of free E2F (represented by EE), and therefore a PS phenotype will be represented by an attractor that has the EE coor- dinate above at least one threshold. Since the number of thresholds of EE changes depending on the subnetwork that we analyze we characterize this attractor as a minimal mode in the Morse graph with labeling FP(, , m), for some m 1. We are now in a position to compute statistics that measure the robustness of the subnet- works with regard to the phenotypic behaviors of resettable bistability and hysteresis. being below all the thresholds associated with MD, to the highest quantifiable level, i.e. being above all the thresholds associated with MD. In terms of paths in EPG(¬MD), this is associated with a full path, that is, any path that starts at the parameter node where all outputs of the node MD are below all thresholds of the nodes MD connects to, and finishes at the parameter node where all outputs of the node MD are above all thresholds of the nodes MD connects to. In the second case we do not assume that the range of the signal S is matched to range of MD. In terms of paths in EPG(¬MD) this is modeled by a partial path, i.e. any subpath of the full paths. Note that the extreme case of a partial path is a constant path, that is, a path consist- ing of a single node in EPG(¬MD). Physically, this implies that the variance of the input signal is not sufficiently large to impact the dynamics of the regulatory network. We remark that given a regulatory network it is straightforward to determine the collection of monotone full and partial paths of EPG(¬MD); however, the total number of paths is net- work dependent. It is important to note that in order to exhibit resettable bistability (hysteresis) the parame- ter graph EPG(¬MD) must contain monotone paths with at least two (three) nodes, respec- tively. This, in turn, implies that the parameter graph for MD must contain at least two (three) nodes. Therefore networks where MD has a single outgoing and no incoming edge cannot exhibit hysteresis under our approach. We view this as a technical failing of DSGRN induced by our insistence on only using coarse measurements defined in terms of the thresholds. To circumvent this problem, we note that the assumption that in the regulatory network MD has a single outgoing and no incoming edge implies that while MD talks to the network, it is not affected by the network. In particular, returning to our assumption that MD behaves PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 9 / 23 Identifying robust hysteresis in networks top 8 in full path resettable bistability and among top 10 in full path hysteresis, but not among the top eight in either partial path hysteresis or resettable bistabilty. It is worth contemplating why the results using DSGRN do not agree exactly with the results of [4] (aside from the obvious fact that we only consider networks that involve all three nodes) and the biological significance of these differences. There are at least three fundamental differences in the approaches. 1. As indicated earlier the results of [4] are based on sampling of parameter space via 20,000 sets of parameter values, whereas DSGRN is computed over all associated parameter values. Thus, it is not surprising that there is not complete agreement with respect to the orderings. What is not clear is which result is more biologically relevant for this particular problem. It is easy to imagine that [4] has under-sampled parameter space. However, since DSGRN examines all of parameter space, it also possible that our count includes parameter values that are not biologically relevant. However, this concern is also applicable to the work of [4]. 2. The bistability measurement of [4] is a measurement of particular parameter values at which bistability occurs. Similarly, resettable bistability is a measure of pairs of sets parame- ter values. DSGRN identifies paths through parameter space such that along the path there exist appropriately ordered sets of parameter values at which bistability and monostability occur. If one assumes that the signal acting on the systems takes on continuous values, then DSGRN provides a more appropriate model on which to count. 3. In [4] the dynamics is modeled using Hill function nonlinearities. One of the parameters in this model is the value of the exponent. Since [4] chooses the parameter values at random, some fraction of the sampled systems involve relatively low values of the exponent, which may be a significant feature of the biochemical reactions. DSGRN is based on switching functions and thus from a functional perspective is close to Hill functions with high values of the exponent, and in this sense, DSGRN is sampling from a more restrictive subset of parameter values. At the same time since DSGRN does not depend on the precise form of the Hill function, it captures the dynamics of a wider variety of nonlinearities. In particular given a subnetwork, we define its prevalence of full path resettable bistability and prevalence of full path hysteresis to be the number of full paths in EPG(¬MD) that exhibit resettable (QS, PS) bistability to QS and hysteresis normalized by the number of full paths in EPG(¬MD), respectively. Similarly, we define prevalence of partial path resettable bistability and prevalence of partial path hysteresis to be the number of partial paths in EPG(¬MD) that exhibit resettable (QS, PS) bistability to QS and hysteresis normalized by the number of partial paths in EPG(¬MD), respectively. Note that although a full path is also a partial path, the differ- ent normalizations do not allow one to make a priori conclusions on the relative values of full path prevalence and partial path prevalence for either phenotype. These numbers provide different information about the networks. As indicated above, prevalence based on full paths assesses the ability to achieve a given phenotype when the input range matches the range of MD, while prevalence based on partial paths provides information about the behavior of the network where no assumption is made about these ranges. Fig 3(b)–3(e) provides histograms indicating the number of subnetworks of Fig 3(a) for which there is positive prevalence of full and partial path hysteresis and resettable bistability. Focussing on the full path hysteresis, there are five networks (Fig 3(f)–3(j)) that exhibit hyster- esis for every full path in EPG(¬MD). More generally, the networks (Fig 3(f)–3(j)) are also the top five networks with respect to prevelance of partial path hysteresis. Network in Fig 3(f) shows partial path hysteresis in 20% of paths, networks Fig 3(g)–3(i) in 16.66%, and Fig 3(j) in 15.49% of all the partial paths. Fur- thermore, all the networks Fig 3(f)–3(j) are among the top 8 networks that exhibit the greatest prevalence for partial path resettability. Interestingly, none of these networks rank among the top 8 networks for full path resettable bistability and one of them, network in Fig 3(h), has no full path with resettable bistability. The best two subnetworks found in [4] appear among the PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 10 / 23 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 5 node restriction point networks Search for the best 5 node E2F-Rb network. (a) Full 5 node E2F-Rb network with input S. We test 12 Fig 4. Search for the best 5 node E2F-Rb network. (a) Full 5 node E2F-Rb network with input S. We test 12 subnetworks, listed in Table (b), top, for robustness of partial path and full path resettable bistability and hysteresis. (b) Each of the 12 subnetworks contains the unmarked edges in (a). In addition, we either add one of the edges 7 or 8, and/ or a subset of the pair of edges (2a, 2b). The second through fifth columns list the percentage of subgraphs in EPG (¬MD) satisfying indicated query. Top three results in each column are emphasized. Note that top three networks in first three queries agree. While the very best network under full path resettable bistability is the same as for full path hyesteresis, none of the top three networks for full path hysteresis has either edge 7 or the edge 8. https://doi org/10 1371/journal pcbi 1006121 g004 Fig 4. Search for the best 5 node E2F-Rb network. (a) Full 5 node E2F-Rb network with input S. We test 12 subnetworks, listed in Table (b), top, for robustness of partial path and full path resettable bistability and hysteresis. (b) Each of the 12 subnetworks contains the unmarked edges in (a). In addition, we either add one of the edges 7 or 8, and/ or a subset of the pair of edges (2a, 2b). The second through fifth columns list the percentage of subgraphs in EPG (¬MD) satisfying indicated query. Top three results in each column are emphasized. Note that top three networks in first three queries agree. While the very best network under full path resettable bistability is the same as for full path hyesteresis, none of the top three networks for full path hysteresis has either edge 7 or the edge 8. https://doi org/10 1371/journal pcbi 1006121 g004 partial and full path hysteresis and partial path resettable bistability. The network that is in top three in every category is network that does not have either edge 7, nor 8 and does not have either 2a, nor 2b. On the other hand, full path resettable bistability orders networks differently. The top net- work is still the same. g 4. Search for the best 5 node E2F-Rb network. (a) Full 5 node E2F-Rb network with input S. We test 12 5 node restriction point networks The 3-node networks of the previous section were derived as simplification of [4, Fig 1(A)]. With this in mind we return to [4, Fig 1(A)] and consider less radical simplifications that result in the 5-node networks indicated in Fig 4(a). In particular, we replace the MD node by two nodes Myc and CycD representing cyclin/kinase complex CycD/Cdk4,6, with the assumption that Myc up-regulates CycD, and the EE node by E2F and CycE, representing cyclin/kinase complex CycE/Cdk2, with the assumption that E2F up regulates CycE. The node Rb represents free and active form of Rb proteins. We also include all the potential edges from Fig 3(a) with appropriate modifications of beginning and ending nodes. We repeat the bistability, resettable bistability and hysteresis queries from the previous section on EPG(¬MD) for the regulatory network subnetworks indicated in Fig 4(b). The unlabeled edges are included in all computations with additional edges listed in first column of table in Fig 4(b). We organize the networks into three groups: networks that have edge 7, networks that have edge 8, and networks that have neither. The prevalence of partial path and full path hysteresis, as well as partial path and full path resettable bistability is indicated in the columns of Fig 4(b). In each column we highlight top three or four values. Note that the best three networks are the same under the measures of PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 11 / 23 Identifying robust hysteresis in networks Fig 4. Search for the best 5 node E2F-Rb network. (a) Full 5 node E2F-Rb network with input S. We test 12 subnetworks, listed in Table (b), top, for robustness of partial path and full path resettable bistability and hysteresis. (b) Each of the 12 subnetworks contains the unmarked edges in (a). In addition, we either add one of the edges 7 or 8, and/ or a subset of the pair of edges (2a, 2b). The second through fifth columns list the percentage of subgraphs in EPG (¬MD) satisfying indicated query. Top three results in each column are emphasized. Note that top three networks in first three queries agree. While the very best network under full path resettable bistability is the same as for full path hyesteresis, none of the top three networks for full path hysteresis has either edge 7 or the edge 8. https://doi.org/10.1371/journal.pcbi.1006121.g004 Fig 4. Identifying robust hysteresis in networks Fig 5. Comparison between human and yeast networks. (a) The best 5-node network from Fig 4(b) that exhibits the most robust full path and partial path hysteresis. (b) Cell cycle initiation network from yeast (START network) [29, 30] exhibits robust resettable bistability in 23.81% of the full paths 12.8%. Because the networks in (a) and (b) only differ by a node with a single input and single output (Myc), the networks in (a) and (b) will give the same results in our analysis. (c) Best 5-node network from Fig 4(b) with added p27 shows full path resettable bistability in 6.43% and full path hysteresis in 0.35% of the corresponding parameter paths. Fig 5. Comparison between human and yeast networks. (a) The best 5-node network from Fig 4(b) that exhibits the t b t f ll th d ti l th h t i (b) C ll l i iti ti t k f t (START t k) [29 30] Fig 5. Comparison between human and yeast networks. (a) The best 5-node network from Fig 4(b) that exhibits the most robust full path and partial path hysteresis. (b) Cell cycle initiation network from yeast (START network) [29, 30] exhibits robust resettable bistability in 23.81% of the full paths 12.8%. Because the networks in (a) and (b) only differ by a node with a single input and single output (Myc), the networks in (a) and (b) will give the same results in our analysis. (c) Best 5-node network from Fig 4(b) with added p27 shows full path resettable bistability in 6.43% and full path hysteresis in 0.35% of the corresponding parameter paths. Fig 5. Comparison between human and yeast networks. (a) The best 5-node network from Fig 4(b) that exhibits the most robust full path and partial path hysteresis. (b) Cell cycle initiation network from yeast (START network) [29, 30] exhibits robust resettable bistability in 23.81% of the full paths 12.8%. Because the networks in (a) and (b) only differ by a node with a single input and single output (Myc), the networks in (a) and (b) will give the same results in our analysis. (c) Best 5-node network from Fig 4(b) with added p27 shows full path resettable bistability in 6.43% and full path hysteresis in 0.35% of the corresponding parameter paths. https://doi.org/10.1371/journal.pcbi.1006121.g005 2b. We assume that in this network S acts monotonically on Myc, which in turn acts monoton- ically on CycD, which acts monotonically on Rb. Thus, as is described above, our analysis of this network reduces to the analysis of the 3 node network involving only the nodes Rb, CycE, and E2F with repressive input on Rb. This reduced 3 node network is identical to the similarly reduced yeast START network, therefore the numbers in the corresponding rows in table in Fig 4(b) are identical. We conclude that the START network matches the subnetwork of the mammalian E2F-Rb network, that most robustly exhibits both full path resettable bistability and hysteresis. The similarity of E2F-Rb network and yeast START network led to a hypothesis that what is main- tained in the process of evolution is network structure and function [31]. Our results support this hypothesis, since the core subnetwork exhibiting most robustly the expected phenotype of a switch is common between E2F-RB mammalian and yeast START networks. 5 node restriction point networks Existence of edges 7 and 8 is still undesirable, but a network with the edge 2a, a network with edge 2b alone, and some networks with both 2a and 2b rank very highly. The subtle difference between partial path and full path resettable bistability stems from the fact that full path resettable bistability requires that there are only two Morse graph types along the entire path: either the bistable state and the terminal state to which the bistable state resets. In the context of E2F-Rb network this corresponds to a set of states (QS, . . ., QS, . . ., B,. . ., B) where B is a bistable state (QS, PS). A full path hysteresis does not correspond to a full path resettable bistability, since there are different states at the ends of the path. In E2F-Rb networks the full hysteresis corresponds to states (QS, . . ., QS, . . ., B, . . .,B, . . .,PS, . . .PS). However, a full path hysteresis gives rise to a partial path resettable bistability by considering only first half of the full path. For comparison, we study the yeast cell cycle initiation network (START), see Fig 5(b) [29, 30]. The START network of the budding yeast cell cycle has the same topology as E2F-Rb net- works, yet there is no homology among the protein and transcription factors in the two net- works [29, 31]. A transcription factor SBF is sequestered by Whi5 during G1. The cell growth leads to accumulation of cyclin/kinase complex Cln3/Cdk1 which phosphorylates Whi5 and as a result, releases SBF from the complex. Released SBF promotes expression of another cyclin Cln2, which is part of a cyclin/kinase complex Cln2/Cdk1. This complex in turn finishes phosphorylation of Whi5 and completes the release of SBF [30, 32, 33]. The analogy with the mammalian restriction point network in Fig 4(a) is striking. The results for the START network are in the table in Fig 4(b). We note that the best of the 12 subnetworks of mammalian E2F-Rb network is the network that does not have edge 7,8, 2a or PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 12 / 23 Discussion We present a new modeling and computational platform that can rapidly compute rigorous summaries of dynamics over large regions of parameter space. The description of the dynam- ics is fine enough to distinguish attractors with different expression levels, find bistability, resettable bistability and hysteresis; while not the focus of this paper, DSGRN can also find oscillatory behavior [9]. We validate our approach using the toggle switch and the E2F-Rb network responsible for mammalian restriction point dynamics. In particular, in contrast to the approach adopted by Yao [4] to investigate the the prevalence of resettable bistability in the parameter space of 3 node networks based on Hill type models by running simulations of the Hill type models at 20,000 fixed parameters, we use DSGRN to search for resettable bistability and hysteresis over the entire parameter space. We quantify the prevalence of these two phenotypes by counting paths connecting regions of the parameter space along which dynamics shifts from quiescent state to bistability (for resettable bistability phenotype), or from quiescent state through bistability to proliferative state (for hysteresis phenotype). These paths represent the response of the network to an exter- nal input S. Since we do not necessarily assume that the network parameters are aligned with the range of the signal S, we count both full paths, that represent a full range of the network response and partial paths, that are subpaths of the full paths. The computational efficacy of DSGRN allows us to perform the same calculations on sev- eral 5-node networks that include explicit representations of CycD/Cdk4,6 and CycE/Cdk2, and on a 4 node START network in the yeast that is functionally and structurally similar to the E2F-Rb network, as well as 6-node network which includes CycD/Cdk4,6, CycE/Cdk2 and p27. We are not aware of any other approach which computes a description of the global dynamics of a 6-dimensional system over the entirety of a 39-dimensional parameter space. Our computations show that out of the twelve 5-node subnetworks of the network Fig 4(a) the one that most robustly shows both full path hysteresis and resettable bistability does not have edges 2a,2b, 7 or 8. This is the network that is, apart of the presence of Myc, identical to the START network in yeast. Both show full path hysteresis in 12.8% of full paths and 23.81% of full paths of the relevant parameter graph. Identifying robust hysteresis in networks Being part of of a control network that provides ability to arrest cell cycle, p27 provides capabil- ities that are not captured by the assessment of the robustness of the switch. Being part of of a control network that provides ability to arrest cell cycle, p27 provides capabil- ities that are not captured by the assessment of the robustness of the switch. We also note, that when we tested this network for (QS,PS), rather than the (PS,QS), hyster- esis and resettable bistability, we found there are no parameter paths that exhibit either hyster- esis, or resettable bistability, either partial or full path. This suggests that, while not as robust as in its subnetworks, the phenotype of the switch from QS to PS is an important aspect of the network design. Restriction point network with p27 Starting with the best 5-node network, we add effects of kinase inhibitor p27, which prevents activation of cyclinE/Cdk2 complex (CycE) (see Fig 5(c)). p27 is sequestered by the cyclinD/ Cdk2,4 (CycD) complex, as well as tagged for ubiquitination by CycE. Increased levels of p27 protein typically result in arrest in G1 [34]. Using the regulatory network of Fig 5(c) we measure prevalence of (QS, PS) resettable bista- bilty and hysteresis in EPG(¬Myc). The p27 network still shows resettable bistability and hyster- esis, see Fig 4(b), but at the levels below the levels observed in the best 5 node network Fig 5(a) and the yeast START network Fig 5(b). Since p27 is a part of other cellular control mechanisms responsible for initiation of cellular arrest, and we have not included these in our network, there is no reason to believe that addition of p27 should make the switching phenotype of the E2F-Rb more robust. Therefore we do not expect that the prevalence of hysteresis and reset- table bistability in p27 network will be higher than in the the best 5 node network in Fig 5(a). PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 13 / 23 DSGRN We provide a brief description of the steps involved in the DSGRN computations but refer the reader to [8] for complete details. A regulatory network is a finite directed graph with edges annotated by j ! i or j a i (but not both) indicating up and down regulation of i by j, respectively. We allow vertices to have zero or more in-edges and one or more out-edges. Each node i with more than one incoming edge has an assigned logic, expressed in terms of and or or statements of the inputs, that describes how the information is processed. The passage from a regulatory network to a state transition graph is dependent upon the parameter values γ, ℓ, u and θ described earlier and based on the following constructions. The passage from a regulatory network to a state transition graph is dependent upon the parameter values γ, ℓ, u and θ described earlier and based on the following constructions. Observe that fixed threshold values {θn, k}, give rise to an explicit decomposition of the phase space [0,1)N into N-dimensional cells (see Fig 2(b)). To avoid degenerate cells we assume that for all j 6¼ k Observe that fixed threshold values {θn, k}, give rise to an explicit decomposition of the phase space [0,1)N into N-dimensional cells (see Fig 2(b)). To avoid degenerate cells we assume that for all j 6¼ k yn;j 6¼ yn;k; n ¼ 1; . . . ; N: ð1Þ ð1Þ These are called the n-threshold inequalities. Under this assumption the number of N- dimensional cells is always the same, independent of the particular choice of parameters. Fur- thermore, since the boundaries of the N-dimensional cells are determined by the thresholds, we can label any cell κ by an integer vector κ = (κ1, . . ., κN) by setting κn to be the number of thresholds θj, n below the xn component of an arbitrary point x 2 κ. The vertices of the state transition graph are the N-dimensional cells and their N −1-dimensional faces. As indicated earlier there are three types of edges in the state transition graph: (i) from an N-dimensional cell to one of its N −1-dimensional faces, (ii) from an N −1-dimensional face to an N-dimensional cell of which it is a face, or (iii) a self edge on an N-dimensional cell. Identifying robust hysteresis in networks few nodes [1]. The hope is that methods like those presented in this paper may be used to directly probe dynamics of larger networks. few nodes [1]. The hope is that methods like those presented in this paper may be used to directly probe dynamics of larger networks. DSGRN Recall that N-dimensional cell has a self edge if and only if there it has no edges of type (i). The choice of edges of type (i) and (ii) is determined by the parameter dependent inequalities, called the n-field inequalities, As indicated earlier there are three types of edges in the state transition graph: (i) from an N-dimensional cell to one of its N −1-dimensional faces, (ii) from an N −1-dimensional face to an N-dimensional cell of which it is a face, or (iii) a self edge on an N-dimensional cell. Recall that N-dimensional cell has a self edge if and only if there it has no edges of type (i). The choice of edges of type (i) and (ii) is determined by the parameter dependent inequalities, called the n-field inequalities, 0 6¼ gnyjk;n þ LnðkÞ; k ¼ 0; 1; and n ¼ 1; . . . ; N ð2Þ ð2Þ ð2Þ Discussion This indicates significant robustness of the switch-like behavior. Wang et.al. [29] pose the question: which mechanisms may be responsi- ble for converting a relatively small change in total Cln3 into a large effect in switching on SBF in START network? Super-sensitivity, and cooperativity were mentioned as potential explana- tions. Our approach allows for the analysis of the the dynamics of networks with 4-7 nodes and the results do not seem to be immediately explainable by properties of its smaller subnet- works with 2 or 3 nodes. The robustness of the observed dynamics may be an emergent prop- erty of the entire network. This conclusion presents a challenge to the current paradigm of motifs, that assumes that we can achieve understanding of complex networks by studying smaller, accessible motifs with Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 14 / 23 Identifying robust hysteresis in networks Fig 6. (a) Field equations for toggle switch. (b) Parameter values that are associated with parameter node 5. (c) Evaluation of field inequalities at four 1-dimensional faces (walls) of domains based on the given parameter value. Arrows indicate direction of dynamics induced by inequalities. https://doi org/10 1371/journal pcbi 1006121 g006 Fig 6. (a) Field equations for toggle switch. (b) Parameter values that are associated with parameter node 5. (c) Evaluation of field inequalities at four 1-dimensional faces (walls) of domains based on the given parameter value. Arrows indicate direction of dynamics induced by inequalities. https://doi.org/10.1371/journal.pcbi.1006121.g006 and) of the step functions s n;k. Observe that Λn is constant on any N-dimensional cell κ. We denote this value by Λn(κ). Because of the biological interpretation of the parameter values the quantity γnxn + Λn(κ) indicates the growth rate of species xn. In the xn direction the N-dimensional cell κ is typically bounded by two N −1-dimensional faces whose xn coordinate values are threshold values that we label as θjk,n, k = 0,1. Observe that (2) indicates whether an initial condition on the N −1-dimensional face θjk,n flows into or out of the N-dimensional cell κ. In the former case the state transition graph edge is of type (i) and the latter case the state transition graph edge is of type (ii). Fig 6 indicates the application of these ideas in the context of the toggle switch. To give proper perspective to our transition from a regulatory network to a state transition graph it is important to recall the notion of a switching system ð3Þ _xn ¼ gnxn þ LnðxÞ; n ¼ 1; . . . ; N; ð3Þ introduced by Glass and Kaufmann [35, 36]. In particular, while it is absolutely clear that switching systems motivate our modeling approach, our results are not limited to this particu- lar system of differential equations. For the analysis performed in this paper we focus on the existence of FP(κ) where the N-cell κ has an edge of type (iii). Consider any system of differen- tial equations _x ¼ f ðxÞ where the vector field f points in the same direction on the boundary of κ as Λ. Under the induced dynamics, there exists a compact attractor in the interior of κ. Of course, in the special case that f(x) = Λ(x), i.e. explained below. As indicated earlier parameters are assigned to each edge. These parameters are used to define a step function according to the rules: As indicated earlier parameters are assigned to each edge. These parameters are used to define a step function according to the rules: if k ! n; then sþ n;kðxkÞ ¼ ( ‘n;k if xk < yn;k; un;k if xk > yn;k; and if k a n; then sn;kðxkÞ ¼ ( un;k if xk < yn;k; ‘n;k if xk > yn;k: Since by assumption ℓn, k < un, k, the implied biological interpretation is that sþ n;k (sn;k) models activation (repression) of the n-th species by the k-th species. For node n, a function Λn is defined in terms of sums (if the logic indicates or) and products (if the logic indicates PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 15 / 23 Identifying robust hysteresis in networks The state transition graph grows rapidly with respect to the number of nodes and edges in the regulatory network. Furthermore, as is shown in [39] even switching systems can exhibit complicated dynamics and thus it is not surprising that the dynamics represented by the state transition graph can also be complicated. Thus, to construct a finite queryable representation of the global dynamics requires a coarse description of the dynamics. To compress this information we construct the associated Morse graph that captures the essential recurrent and nonrecurrent dynamics. A recurrent component in a state transition graph is a maximal subgraph that contains at least one edge and for any two vertices (perhaps the same) there exists a path from one to the other. The recurrent components of the state transition graph make up the vertices of the Morse graph. Since any recurrent component must contain at least one N-dimensional cell, we label each vertex in the Morse graph by the collection of labels of all the N-dimensional cells in the associated recurrent component of the state transition graph. Observe that there is a natural partial ordering on the vertices of the Morse graph; given two recurrent components Mi and Mj in the state transition graph we set Mi > Mj if there is a path in the state transition graph from Mi to Mj. The focus of this paper is on the existence of FPs, thus we restrict our attention to minimal vertices of the Morse graph derived from recurrent components consisting of a single vertex in the state transition graph. However, in general a vertex in the Morse graph can arise from a recurrent component of the state transition graph that captures complex, even chaotic, dynam- ics. Thus, the DSGRN is capable of encoding a wide variety of different dynamical structures. We now turn to a discussion of the parameter graph. A nonempty region defined by particular instantiations of the n-threshold inequalities (1) and the n-field inequalities (2), e.g. yn;j > <yn;k and 0> < gnyjk;n þ LnðkÞ, over all n = 1, . . ., N is called a parameter cell. The collection of all parameter cells decomposes parameter space. For a network node n which has a single external input S and no other incoming edges from the network, we consider n-field inequalities 0> < gnyjk;n þ S. that we have a switching system, then there exists a unique stable fixed point in the interior of κ. In summary, the results obtained via DSGRN are not only applicable to switching systems, but to a much large class of nonlinear models (see [7] for a detailed discussion in planar systems). In the switching system literature the closest to our perspective are papers [37, 38] that emphasize combinatorial aspects of the dynamics of switching systems. However, the emphasis is on analysis of a particular state transition graph, rather than consideration of the entire col- lection of transition graphs that can be generated by a given network. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 16 / 23 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 that we label as the integers UO i ≔f0; 1; . . . ; Tig. We say that a function f : Ini ! UO i is monotone if a < b implies f(a) f(b). The node of a parameter graph PG(i) is ωk ≔(gk, Ok) where Ok is a particular order of thresholds Θi and gk is a monotone map gk : Ini ! U Ok i : Two nodes (gk, Ok) and (gl, Ol) in PG(i) are joined by an edge, if the orders Ok and Ol Two nodes (gk, Ok) and (gl, Ol) in PG(i) are joined by an edge, if the orders Ok and Ol differ by transposition of exactly one inequality; or, if Ok = Ol and there is exactly one choice of z 2 Ini such that \|gk(z) −gl(z)\| = 1. Note that these two choices correspond to a single difference in a choice of inequality in (1) and (2), respectively. It is important to note that DSGRN database represents the dynamics not just of the net- work itself, but also the dynamics of all its subnetworks. This significantly increases the size of the parameter graph and we often chose to compute the essential subgraph of the parameter graph that only contains parameter nodes at which all network edges are essential. L Ci j ≔fðb1; b2Þ 2 Ini Ini j b1 and b2 differ in exactly the jth coordinateg: Identifying robust hysteresis in networks and θnj. Therefore, there are 2Sn possible input values to vertex vn: and θnj. Therefore, there are 2Sn possible input values to vertex vn: flnj1; unj1g flnjSn; unjSn g that we represent by binary numbers Inn ≔f0; 1g Sn under the mapping lnjk ! 0, unjk ! 1. We view Inn as a partially ordered set where the order is inherited from the embedding of Inn to RSn. We define PGð:jÞ ≔ G ¼ n PGðjÞ j n a vertex in Y n i¼1;i6¼j PGðiÞ ( ) : The collection of out-edges from vi correspond to a collection of thresholds Yi ≔fyl1;i; yl2;i; . . . ; ylTi ;ig. Every ordering O of the set Θi defines Ti + 1 states of vi: ð0; yl1;iÞ; ðyl1;i; yl2;iÞ; . . . ; ðylTi ;i; 1Þ that we label as the integers UO i ≔f0; 1; . . . ; Tig. that we label as the integers UO i ≔f0; 1; . . . ; Tig. that we label as the integers UO i ≔f0; 1; . . . ; Tig. If node n has k out edges and hence k-thresholds yn;1; . . . ; yn;jk there are k + 1 such selections for each fixed threshold order given by n-threshold inequalities (1). Observe that for each parameter value in a given parameter cell the associated state transition graph will be identical, and therefore, to each parameter cell we can assign a unique Morse graph. The parameter graph is a graph where each vertex corresponds to a nonempty parameter cell. There is an edge between two vertices if and only if all but one of the inequalities that define the corresponding parameter cells are the same. The parameter graph is always a product of parameter graphs associated to each vertex of the regulatory network. To see this observe that the choice of an instantiation of n-threshold inequalities is independent on the choice of an instantiation of k-threshold inequalities for n 6¼ k, and therefore the set of all such instantiations decomposes as a product PG ¼ Y N n¼1 PGðnÞ ð4Þ ð4Þ where N is the number of vertices in the regulatory network. However, it is important to realize that the choices of n-threshold inequalities are not independent of each other when there are multiple thresholds (associated with out edges vertex n) and/or multiple in edges. To describe the form of PG(n) for vertices with more than one input and/or one output requires additional notation. Given a vertex vn in a regulatory network let Sn > 0 be number of inputs (in-edges) and Tn > 0 be the number of outputs (out-edges). Every edge vj ! vn has three parameters, lnj, unj, 17 / 23 17 / 23 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 DSGRN database for E2F-Rb networks The main biological phenotypes of the E2F-Rb network are quiescent state (QS) and prolifer- ative state (PS). We have identified them in DSGRN as minimal nodes in Morse graphs with labels FP(,. . .,,0) and FP(,. . .,,m), for some m 1, respectively. We now describe the more complicated phenotypes of resettable bistability and hysteresis that are function of the external input S. Resettable bistability and hysteresis in restriction point networks As noted in (4) the parameter graph PG is a product of parameter graphs PG(i) that corre- spond to individual nodes i of the network. The input S enters the network at a particular node, which in E2F-Rb networks is either node MD, or the node Myc; for the START network it is the node representing Cln3. We call this the receiving node R. Increasing S correspond to a path in the parameter graph PG(R). As mentioned in the previous subsection, if R has k out edges, and no in-edges, the parameter graph PG(R) has k + 1 elements that are linearly ordered. Parameter graphs for R with multiple in- and out edges are much more complicated [8] but they always form a partially ordered set with the unique lowest node and the highest node. The lowest parameter node in PG(R) is the parameter node where all outputs of the node R are below all thresholds of the nodes R connects to; this corresponds to the situation where R is sub-threshold to all of its outputs and so it is always off. The highest parameter node in PG(R) is the parameter node where all outputs of the node R are above all thresholds of the nodes R connects to; this corresponds to the situation where R is super-threshold to all of its outputs and so it is always on. We call an path in a parameter graph PG(R) a full path if starts at lowest parameter node and finishes at the highest parameter node. A full path in PG(R) represents the effect of varying the input S through the entire response range of R: the input S moves R from being sub-threshold to being super-threshold with respect to all its downstream nodes. Since the range of the input signal S does not have to be always matched to the range of R, we will also consider partial paths that are subpaths of the full paths. Note that these include constant paths. Constant paths correspond to input that has no measurable impact on the dynamics of the network. We will search for partial path hysteresis and resettable bistability, as well for full path hysteresis and resettable bistability. Identifying robust hysteresis in networks A parameter node p is essential if it is i-essential for all i = 1, . . ., n. The essential parameter graph is a subgraph of the parameter graph induced on essential parameter nodes. Ci j ≔fðb1; b2Þ 2 Ini Ini j b1 and b2 differ in exactly the jth coordinateg: Definition 0.1. Fix a network RN(V, E), parameter node p and an edge vj ! vi 2 E with associated threshold θij. Assume that θij is between states k and k + 1 in Uj. We say that the edge vj ! vi is 1. input-essential if 9ðb1; b2Þ 2 Ci j such that gi(b1) 6¼ gi(b2); and 1. input-essential if 9ðb1; b2Þ 2 Ci j such that gi(b1) 6¼ gi(b2); and 2. output-essential if 9b1,b2 2 Inj such that gj(b1) k, gj(b2) k + 1. 2. output-essential if 9b1,b2 2 Inj such that gj(b1) k, gj(b2) k + 1. In other words, an edge vj ! vi is input-essential if it provides a non-constant input to the target node vi, and an edge vj ! vi output-essential if if some values of Inj drive vj above θij and some drive it below θij. j Definition 0.2. Fix a network RN(V, E). A parameter node p is i -essential if 1. every in-edge of vi is input-essential; and 1. every in-edge of vi is input-essential; and 2. every out-edge of vi is output-essential. 2. every out-edge of vi is output-essential. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 18 / 23 Identifying robust hysteresis in networks minimal node in the quiescent state QS. We then check that a Morse graph at least one other lifted node B is (QS, PS)-bistable. Finally, we check that there is at least one lifted path from B to lifted low node along which the Morse graph has either minimal node QS, or exhibits (QS, ) bistability. To check for hysteresis in G, in addition to checking for resettable (QS, PS) bistability to QS, we also check for (QS, PS) bistability to PS, where G is required to have a unique minimal node in the proliferative state PS at the lifted high node of PG(R). We make occasionally two modifications to this search. When the network node R that receives the input S has only single out-edge to a node Q, the node R only transmits the input information S to the rest of the network via Q. In such a situation, we search for bistability and resettable bistability in PG(Q) instead of PG(R). Since for networks considered in this paper, S always activates R, if the edge from R to Q is also activating, then we search for the resettable bistability and hysteresis in PG(Q) as described above for PG(R). However, if the edge from R to Q is repressing, then high value of input S causes a high level of repression and hence low value of expression of node Q, and low value of S causes high value of expression of the node Q. Therefore our search for resettable bistability and hysteresis in PG(Q) interchanges the roles of highest and lowest node of PG(Q) compared to their roles in PG(R). For example, for full path resettable bistability we check if the Morse graph in G at the lifted highest node in PG(Q) has a unique minimal node in the quiescent state QS. We then check that a Morse graph at least one other lifted node B is (QS, PS)-bistable. Finally, we check that there is at least one lifted path from B to lifted high node along which the Morse graph has either minimal node QS, or exhibits (QS, ) bistability. Similar modifications are made in search for hysteresis. If there is multiple nodes in a row that only transmit the input S, for instance S ! R ! Q ! P ! . . . we repeat the above procedure and query hysteresis and resettable bistability along paths in PG(W) where W is the first node in the pathway which has either more than one input, or more than one output. Resettable bistability and hysteresis in restriction point networks We perform these searches in the parame- ter graph PG ≔PGðRÞ EPGð:RÞ; where we take only essential parameter values for all non-receiving nodes, but allow non- essential parameter nodes for the receiving nodes of the network. Fix a node G in the parame- ter graph EPG(¬R); that is, we fix all parameter inequalities for all non-receiving nodes. To completely characterize a parameter node in the parameter graph, and hence the parameter domain in the parameter space, we need to select inequalities for the node R. For each such a choice of a node in PG(R) together with the fixed choice of a node in EPG(¬R), there is a well- defined Morse graph. where we take only essential parameter values for all non-receiving nodes, but allow non- essential parameter nodes for the receiving nodes of the network. Fix a node G in the parame- ter graph EPG(¬R); that is, we fix all parameter inequalities for all non-receiving nodes. To completely characterize a parameter node in the parameter graph, and hence the parameter domain in the parameter space, we need to select inequalities for the node R. For each such a choice of a node in PG(R) together with the fixed choice of a node in EPG(¬R), there is a well- defined Morse graph. We search for resettable bistability in G by checking for the existence of a lifted path, either partial or full, in G that satisfies the properties listed in the Results section. In particular, for full path we check if the Morse graph in G at the lifted lowest node in PG(R) has a unique Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 19 / 23 Code availability In order to ensure our results may be reproduced we adhere to the following recipe: (1) We release our code under an open-source license, (2) We host our code on a publicly available site using version-control (i.e. history tracking), (3) We give the version numbers of the code used to produce the result, (4) We provide instructions for installing and running the code, and (5) We produce digital object identifiers (DOIs) of the versioned code for use in bib- liographical entries. The computer codes used to reproduce the results in this paper are stored in two code repositories. The first repository is the DSGRN project [41]. This is an open- source project which, as of writing, is hosted on the code-sharing website GitHub at https:// github.com/shaunharker/DSGRN. The version utilized for this paper is 1.0.0. The second repository is the supplemental for this paper [40] and houses the code (which relies on DSGRN) which is used to reproduce the above results. This again is open-source and is hosted at https://github.com/shaunharker/2017-DSGRN-IdentifyingRobustHysteresisInNetworks. The version utilized for this paper is 1.0.3. The DOIs for these can be found in the references. Supporting information S1 Table. Summary of all 3 node networks. We provide results for full path and partial path resettable bistablility and hysteresis for all 49 three node subnetworks. The first column con- tains the network number (referenced in Fig 3), second column a picture of the network, and columns 3-6 prevalence of partial path hysteresis, partial path resettable bistability, full path hysteresis and full path resettable bistability. The last column is time it took to complete the computation in each row. Identifying robust hysteresis in networks Table 1. Computational time. CPU time to compute all four resettable bistability and hysteresis queries for various networks. Network with edges: time (s) 2a 21.87266 2a 2b 344.0229 2a 2b 7 224277.6 2a 2b 8 232487.9 2a 7 8638.32 2a 8 7733.29 2b 18.44617 2b 7 13359.98 2b 8 12681.72 7 546.7937 8 246.005 neither7 nor 8 3.467421 7 p27 4366.87 Yeast START 2.895762 https://doi.org/10.1371/journal.pcbi.1006121.t001 https://doi.org/10.1371/journal.pcbi.1006121.t001 Performance The computations demonstrated in this paper can in principle be obtained by computing and subsequently querying a dynamical database for the network of interest. In particular, the data- base provides a lookup table allowing us to obtain the annotated Morse graph corresponding to each parameter graph node. We may use this lookup table as a subroutine as we search the subgraphs according to the algorithms in the previous section. On the other hand, we found that this approach adds technical complexities to scaling to high performance computing. When scaling to many simultaneous cores, it becomes necessary to handle many database requests in parallel in an IO-efficient manner. While this appears to be a solvable technical problem, for our purposes it is easier to sidestep these complexities by not using a database, but rather computing the Morse graphs as needed. In other words, we simulate the database query by recomputing the Morse graph. This renders the majority of disk access unnecessary. With this approach, performing the searches described in the section Resettable bistability and hysteresis in restriction point networks, for each node in PG(R) is an example of an embarassingly parallel problem. That is, it can be trivially broken into pieces which may be solved in parallel. Our implementation exploits this and was run on a high per- formance computing cluster (see Table 1), which spread the roughly 10 million seconds of total compute time over approximately 800 cores, so results were available within a few hours [40]. We note that there is no obstacle to further scaling with this technique. It would also be possible to scale down: we could run these computations on a present-day laptop computer (using four CPU cores) in about a month. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1006121 April 23, 2018 20 / 23 References 1. Alon U. An introduction to systems biology. Chapman & Hall/CRC; 2007. 2. Ma W, Trusina A, El-Samad H, Lim WA, Tang C. Defining Network Topologies that Can Achieve Bio- chemical Adaptation. Cell. 2009; 138(4):760–773. https://doi.org/10.1016/j.cell.2009.06.013 PMID: 19703401 3. Shah NA S C. Robust Network Topologies for Generating Switch-Like Cellular Responses. 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https://openalex.org/W3192630753	https://www.nature.com/articles/s41419-021-04026-7.pdf	English	null	DEPDC1B regulates the progression of human chordoma through UBE2T-mediated ubiquitination of BIRC5	Cell death and disease	2,021	cc-by	8,002	INTRODUCTION of 529 amino acids, which includes an N-terminal DEP domain and a C-terminal Rho-GAP (GTPase activating protein)-like domain [10]. The DEP domain is a spherical domain of about 90 amino acids, which plays a role in the localization of dielectric membranes [11]. The Rho-GAP domain is involved in Rho GTPase signal transduction, such as RAC, Cdc42, and Rho, regulating cell movement, growth, differentiation, cytoskeleton reorganization, and cell cycle process [12]. The membrane binding of DEP domain enables DEPDC1B to interact with G-protein-coupled receptors and membrane phospho- lipids required for Wnt signal transduction [13]. Furthermore, DEPDC1B can coordinate debonding events and cell cycle progres- sion during mitosis [14]. Figeac et al. supported that DEPDC1B is a key regulator of mouse and human myoblast proliferation [15]. In addition, DEPDC1B is required for a variety of malignancies, such as breast cancer [11, 16], non-small cell lung cancer [17], oral cancer [18], prostate cancer [19], melanoma [20], and glioblastoma [21]. In pancreatic cancer or prostate cancer, DEPDC1B could promote migration and invasion through Rac1/PAK1 signaling [22, 23]. In bladder cancer, DEPDC1B is a tumor promotor through targeting SHC1 [24]. Accordingly, we found that DEPDC1B played a promoting role in the progression of a variety of cancers. Because our research group is dedicated to the investigation of chordoma, we wondered the role of DEPDC1B in chordoma. Therefore, the biological function and potential molecular mechanism of DEPDC1B in chordoma were explored in this study. of 529 amino acids, which includes an N-terminal DEP domain and a C-terminal Rho-GAP (GTPase activating protein)-like domain [10]. The DEP domain is a spherical domain of about 90 amino acids, which plays a role in the localization of dielectric membranes [11]. The Rho-GAP domain is involved in Rho GTPase signal transduction, such as RAC, Cdc42, and Rho, regulating cell movement, growth, differentiation, cytoskeleton reorganization, and cell cycle process [12]. The membrane binding of DEP domain enables DEPDC1B to interact with G-protein-coupled receptors and membrane phospho- lipids required for Wnt signal transduction [13]. Furthermore, DEPDC1B can coordinate debonding events and cell cycle progres- sion during mitosis [14]. Figeac et al. supported that DEPDC1B is a key regulator of mouse and human myoblast proliferation [15]. In addition, DEPDC1B is required for a variety of malignancies, such as breast cancer [11, 16], non-small cell lung cancer [17], oral cancer [18], prostate cancer [19], melanoma [20], and glioblastoma [21]. 1Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, No. 119 Nansihuan Xilu, Beijing 100070, China. 2Department of Orthopaedic Surgery, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, No. 1111 Xianxia Road, Shanghai 200336, China. 3Department of Orthopaedic Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, No. 2000 Jiangyue Road, Shanghai 200127, China. 4These authors contributed equally: Liang Wang, Liang Tang. ✉email: wuzhen@bjtth.org; scoliosis@126.com ARTICLE OPEN DEPDC1B regulates the progression of human chordoma through UBE2T-mediated ubiquitination of BIRC5 Chordoma is a rare bone malignancy with a high rate of local recurrence and distant metastasis. Although DEP domain-containing protein 1B (DEPDC1B) is implicated in a variety of malignancies, its relationship with chordoma is unclear. In this study, the biological role and molecular mechanism of DEPDC1B in chordoma were explored. The function of DEPDC1B in chordoma cells was clariﬁed through loss-of-function assays in vitro and in vivo. Furthermore, molecular mechanism of DEPDC1B in chordoma cells was recognized by RNA sequencing and Co-Immunoprecipitation (Co-IP) assay. The malignant behaviors of DEPDC1B knockdown chordoma cells was signiﬁcantly inhibited, which was characterized by reduced proliferation, enhanced apoptosis, and hindered migration. Consistently, decreased expression of DEPDC1B suppressed tumor growth in xenograft mice. Mechanically, DEPDC1B affected the ubiquitination of baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) through ubiquitin-conjugating enzyme E2T (UBE2T). Simultaneous downregulation of BIRC5 and DEPDC1B may exacerbate the inhibitory effects of chordoma. Moreover, BIRC5 overexpression reduced the inhibitory effects of DEPDC1B knockdown in chordoma cells. In conclusion, DEPDC1B regulates the progression of human chordoma through UBE2T-mediated ubiquitination of BIRC5, suggesting that it may be a promising candidate target with potential therapeutic value. Cell Death and Disease (2021) 12:753 ; https://doi.org/10.1038/s41419-021-04026-7 Cell Death and Disease (2021) 12:753 ; https://doi.org/10.1038/s41419-021-04026-7 Received: 16 November 2020 Revised: 12 July 2021 Accepted: 12 July 2021 www.nature.com/cddis Cell clone formation assay The MUG-Chor1 cells were inoculated at a density of 1000 cells/well on a 6-well plate for 14 days and washed with PBS buffer. After that, the cells were ﬁxed by 4% paraformaldehyde (SIGMA, Cat. No. P6148) of 1 mL for 60 min, stained with 500 μL Giemsa (Tripod biotechnology, Cat. No. KGA229) for 20 min, washed and dried by ddH2O. Finally, the cells clone was photographed and counted. Xenograft mouse tumor model Xenograft mouse tumor model The 4-week-old female BALB/c nude mice (Lingchang Biotechnology, Shanghai, China) were divided into two groups (shDEPDC1B and shCtrl). After the MUG-Chor1 cells (with or without knockdown of DEPDC1B) were digested by trypsin, the concentration of 1E + 7 cells/ mL was maintained. Tumor growth was observed after subcutaneous injection of 200 μL of cells into the right forearm of mice for 5–7 days. Subsequently, the anesthetized mice were intraperitoneally injected with 0.7% pentobarbital sodium at 10 μL/g and placed in a living body imager for imaging and data preservation. In addition, tumor size and mice weight were measured every other day. After 21 days, the mice were euthanized and the tumors were weighed and photographed. Finally, the tumor tissues were extracted from mice and incubated with antibody Ki67 (Table S2) for IHC staining. All procedures involving mice and experimental protocols were approved by the Institutional Animal Care and Use Committees of Shanghai Jiao Tong University School of Medicine. (Co IP) assay After the U-CH1 and MUG-Chor1 cells were lysed, the protein was obtained and the concentration was measured by BCA protein detection kit (Thermo Fisher Scientiﬁc, California, USA). The protein with equal amounts was subjected to SDS-PAGE, transferred to nitrocellulose membranes and incubated with primary antibodies (Table S2) overnight at 4 °C. After washed by TBST, the membranes were probed with secondary antibodies. Finally, Millipore Immobilon Western Chemiluminescent HRP Substrote kit (Millipore, Cat. No. RPN2232) was used to color rendering and Chemiluminescent imager (GE, Cat. No. AI600) observation. The protein–protein interaction was analyzed by Co-IP assay and the experimental procedures were performed as previously described [27]. Transwell assay y U-CH1 and MUG-Chor1 cells with a density of 5 × 104 cells/well were incubated in the well-hydrated chamber (3422 corning). The inner chamber contained 100 μL of serum-free medium and the external chamber contained 600 μL 30% FBS. After the cell suspension was diluted with serum-free medium, the cells were added to each chamber for 24 h cultivation. After the migrating cells were ﬁxed with 4% formaldehyde, stained with Giemsa. Finally, the cells were observed under the ﬂuorescence microscope and photographed to estimate the migration capacity. Cell counting assay The MUG-Chor1 cells were cultured on a 96-well plate with a density of 2000 cells per well. After that, the cells were counted by Celigo (Nexcelom) every day for 5 days. Accordingly, the cell proliferation curve was drawn by counting the number of GFP cells in each scanning oriﬁce plate. L. Wang et al. L. Wang et al. 2 Our study showed that the malignant behavior of the chordoma cells after DEDDC1B knockdown was signiﬁcantly inhibited. Mechanically, DEPDC1B affected the ubiquitination of baculoviral inhibitor of BIRC5 through UBE2T. Simultaneous downregulation of BIRC5 and DEPDC1B may exacerbate the inhibitory effects of chordoma. Meanwhile, BIRC5 overexpression reduced the inhibi- tory effects of DEPDC1B knockdown in chordoma cells. Therefore, the signiﬁcant breakthrough that DEPDC1B regulates the progres- sion of human chordoma through UBE2T-mediated ubiquitination of BIRC5 may provide a valuable target for molecular therapy of chordoma patients. MTT cell proliferation assay The U-CH1 and MUG-Chor1 cells were inoculated at a density of 1000 cells/ well and cultured overnight on a 96-well plate. On the next day, 20 μL of 5 mg/mL MTT (Genview, Cat. No. JT343) was added, the culture medium was completely absorbed 4 h later. After 100 μL DMSO was added to dissolved Formazan and crystallized. The absorption value was determined at 490 nm by enzyme micro-plate reader (Biotek Cat. No. Elx800) for 5 consecutive days. Cell apoptosis analysis by ﬂow cytometry (FACS) p p y y y y ( ) After the U-CH1 and MUG-Chor1 cells were continuously cultured in 6-well plates for 7 days, the cell precipitates were washed by precooled D-Hanks (pH = 7.2–7.4) and 1× buffer solution in turn. Subsequently, the resuspen- sion cells were precipitated and stained with Annexin V-APC (eBioscience, Cat. No. 88-8007-74) for 10–15 min. Finally, the apoptosis rate was analyzed and calculated by ﬂow cytometry (Millipore, Cat. No. IX73). The human chordoma cell lines U-CH1 and MUG-Chor1 were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained the atmosphere of 37 °C with 5% CO2. The cells were cultured in DMEM (Corning, Cat. No. 10-013-CVR) supplemented with 10% fetal bovine serum (FBS) (Ausbian, Cat. No. VS500T) and puromycin (Gibco, Cat. No. A11138-003). Notably, U-CH1 is the ﬁrst conﬁrmed chordoma cell line with cellular genetic aberrations typical of chordomas [25]. MUG-Chor1 is markedly similar to chordomas and can grow steadily, which can be served as an optimal chordoma model in vitro [26]. Lentiviral shRNA vector construction and cell infection First, the RNA interference (RNAi) against DEPDC1B (shDEPDC1B-1: GCT GCTAGATTGGTAACGTTT; shDEPDC1B-2: GAGGCCAATGTAGAAGAGATA; shDEPDC1B-3: CAGCTATGAAGTGTTTGGCAA) or BIRC5 (shBIRC5-1: ATGCAC TTCAGACCCACTTAT; shBIRC5-2: CGGCCTTTCCTTAAAGGCCAT; shBIRC5-3: GGCCAACTGCCATCCTGGAAA) and corresponding negative control (shCtrl: 5′-TTCTCCGAACGTGTCACGT-3′) were designed and synthesized. These sequences were ligated to BR-V-108 lentivirus vectors (Shanghai Bioscienceres, Co., Ltd) that carried the green ﬂuorescent protein (GFP), respectively. After that, the target sequences with great knockdown effects were screened out by WB. These lentiviruses were used to infect U-CH1 and MUG-Chor1 cells and viewed under a ﬂuorescence microscope (Olympus). RNA isolation and qPCR The RNA of U-CH1 and MUG-Chor1 cells was extracted with the Trizol reagent (Sigma, Cat. No. T9424-100m) and reverse-transcribed into cDNA by Hiscript QRT Supermix (Vazyme, Nanjing, China, Cat. No. R123-01). Subsequently, the mixed reaction solution composed of cDNA, corre- sponding primers (Table S1) and SYBR premix (Vazyme) was performed to qPCR. Finally, GAPDH was used as an internal reference and the relative mRNA levels were estimated by 2−ΔΔCt. Wound-healing assay y U-CH1 and MUG-Chor1 cells were cultured in the 96-well plate with a density of 5 × 104 cells/well. After the low concentration of serum medium was replaced the next day, the scratches were formed by nudging upward at the center of the lower end of the 96-well plate with a scratch meter. At 0, 24, and 48 h of cell migration, the width of scratch area was measured and the migration ability was analyzed. Human apoptosis antibody array y y The concentrations of 43 human apoptotic markers in the MUG-Chor1 cells (with or without knockdown of DEPDC1B) were detected simultaneously using human apoptosis antibody array-membrane (Abcam, Cat. No. ab134001). After the cell protein was obtained, the product instructions were followed to detect the differential expression of the groups of shCtrl and shDEPDC1B. INTRODUCTION In pancreatic cancer or prostate cancer, DEPDC1B could promote migration and invasion through Rac1/PAK1 signaling [22, 23]. In bladder cancer, DEPDC1B is a tumor promotor through targeting SHC1 [24]. Accordingly, we found that DEPDC1B played a promoting role in the progression of a variety of cancers. Because our research group is dedicated to the investigation of chordoma, we wondered the role of DEPDC1B in chordoma. Therefore, the biological function and potential molecular mechanism of DEPDC1B in chordoma were explored in this study. Chordoma is a rare, slow-growing primary bone malignancy that originates from primitive notochordal tissue [1, 2]. In addition, chordomas have neither obvious early symptoms nor any external manifestations and are usually diagnosed at advanced [3]. Chordomas do not respond to conventional radiotherapy or cytotoxic chemotherapy, and surgery is the primary treatment option [4, 5]. Unfortunately, the complex anatomy of the spine and the relatively large tumor volume make resection technically challenging, resulting in a high rate of local recurrence and distant metastasis [4]. As a result, traditional treatment options are far from adequate [6]. In recent years, with the vigorous development of molecular biology, molecular targeted therapy is gradually being applied. For example, the nuclear expression of brachyury, a key transcription factor for notochord development, can be used as a classic diagnostic marker for chordoma [7, 8]. Moreover, a variety of potential molecular targets for chordoma were identiﬁed, such as platelet-derived growth factor receptor β, PI3K/mTOR, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and tyrosine kinase. However, the objective response of these molecular targeting drugs is not satisfactory [9]. Therefore, identiﬁcation of new potential mole- cular targets is essential for patients with chordoma. g The DEP Domain-Containing Protein 1B (DEPDC1B) is located on human chromosome 5q12 and encodes a 61 kDa protein consisting Received: 16 November 2020 Revised: 12 July 2021 Accepted: 12 July 2021 Ofﬁcial journal of CDDpress RNA sequencing Affymetrix human GeneChip PrimeView combined with Affymetrix Scanner 3000 was performed to elaborate the molecular mechanism. Accordingly, the volcano plot and hierarchical clustering of the MUG-Chor1 cells (with or without knockdown of DEPDC1B) were presented by the differentially expressed genes (DEGs) with criterion of \|Fold Change\| ≥2 and false Cell Death and Disease (2021) 12:753 Knockdown of DEPDC1B inhibits the malignant behaviors of chordoma cells in vitro First of all, WB results showed that the protein level of DEPDC1B was highly expressed in U-CH1 and MUG-Chor1 cells (Fig. S1A). Subsequently, the cells with knockdown of DEPDC1B were applied to detect the alterations of biological behavior. As illustrated in Fig. S1B, more than 80% of U-CH1 and MUG-Chor1 cells with GFP indicated the high infection efﬁciency. Moreover, the signiﬁcant downregulation of the RNA (Fig. S1C) and protein (Fig. S1D) levels of DEPDC1B in U-CH1 and MUG-Chor1 cells consistently suggested that DEPDC1B was successfully knocked down. As a consequence, the effects of alteration of DEPDC1B expression on chordoma cells was investigated in vitro. The results of MTT detection showed that the OD490 value of shDEPDC1B group was remarkably lower than that of the shCtrl L. Wang et al. 3 Fig. 1 Knockdown of DEPDC1B inhibits cell proliferation and migration, promotes apoptosis in chordoma cells. A Cell proliferation of U-CH1 and MUG-Chor1 cells with or without knockdown of DEPDC1B was evaluated by MTT assay. B Flow cytometry analysis based on Annexin V-APC staining was utilized to detect cell apoptotic ratio for U-CH1 and MUG-Chor1 cells. C, D Cell migration of U-CH1 and MUG- Chor1 cells with or without knockdown of DEPDC1B was evaluated by Transwell assay (D) and wound-healing assay (E). The presented results were representative of experiments repeated at least three times. Data were represented as mean ± SD. P < 0.01, P < 0.001. Fig. 1 Knockdown of DEPDC1B inhibits cell proliferation and migration, promotes apoptosis in chordoma cells. A Cell proliferation of U-CH1 and MUG-Chor1 cells with or without knockdown of DEPDC1B was evaluated by MTT assay. B Flow cytometry analysis based on Annexin V-APC staining was utilized to detect cell apoptotic ratio for U-CH1 and MUG-Chor1 cells. C, D Cell migration of U-CH1 and MUG- Chor1 cells with or without knockdown of DEPDC1B was evaluated by Transwell assay (D) and wound-healing assay (E). The presented results were representative of experiments repeated at least three times. Data were represented as mean ± SD. P < 0.01, *P < 0.001. discovery rate (FDR) < 0.05. Furthermore, the signiﬁcant enrichment of DEGs in canonical pathway, and diseases or functions were investigated based on ingenuity pathway analysis (IPA). group (P < 0.001), which revealed that the downregulation of DEPDC1B led to the decrease of U-CH1 and MUG-Chor1 cells viability (Fig. 1A). Furthermore, the apoptosis percentage of shDEPDC1B group was signiﬁcantly increased than that of shCtrl group (P < 0.001), indicating that the knockdown of DEPDC1B increased the apoptosis susceptibility of chordoma cells (Fig. 1B). Interestingly, the scratch distance of the shDEPDC1B group was obviously shorter than that of the shCtrl group between 0 and 48 h (P < 0.001) (Fig. 1C). Consistently, the migration fold change of shDEPDC1B group was indeed lower than that of shCtrl group (P < 0.001), which further veriﬁed that the downregulation of DEPDC1B expression can inhibit the ability of cell migration (Fig. 1D). Taken together, the above results demonstrated that the downregulation of DEPDC1B can inhibit the malignant behaviors of chordoma cells by weakening viability, enhancing apoptosis, and reducing migration. Statistical analysis l l Statistical analyses were accomplished by SPSS 19.0 with GraphPad Prism 8.0 software, the data were presented as the mean ± standard deviation. The independent Student’s t test was used to analyze the statistical signiﬁcance between different groups and P < 0.05 was considered statistically signiﬁcant. Simultaneous downregulation of BIRC5 and DEPDC1B exacerbates the inhibitory effects of chordoma y The biological behavior of BIRC5 and DEPDC1B in MUG-Chor1 was elucidated by loss-of-function assays. Firstly, the protein level of BIRC5 was highly expressed in U-CH1 and MUG-Chor1 cells, which was similar to DEPDC1B (Fig. S3A). Secondly, the protein of shBIRC5-1 group was downregulated most signiﬁcantly, which was used to construct cell model of knockdown of BIRC5 (Fig. S3B). Moreover, MUG-Chor1 cells not only had high infection efﬁciency (Fig. S3C), but also the protein level of BIRC5 and DEPDC1B was signiﬁcantly downregulated (Figure S3D), which indicated that BIRC5 and DEPDC1B was knocked down success- fully. Notably, shBIRC5 was downregulation of BIRC5, shBIRC5 + shDEPDC1B was simultaneous downregulation of BIRC5 and DEPDC1B in MUG-Chor1 cells. As a result, the results showed a signiﬁcant inhibition in the progression of BIRC5-knocked-down MUG-Chor1 cells, such as inhibited proliferation (P < 0.001) (Fig. 5A), decreased clones (P < 0.001) (Fig. 5B), enhanced apoptosis (P < 0.001) (Fig. 5C) and hindered migration (Fig. 5D, E). Besides, simultaneous downregulation of BIRC5 and DEPDC1B could exacerbate the inhibitory effects of chordoma cell progression, which was characterized by inhibiting proliferation (P < 0.001, fold change = −3.3) (Fig. 5A), reducing clone formation (P < 0.001, fold change = −7.0) (Fig. 5B), stimulating apoptosis (P < 0.001, Knockdown of DEPDC1B suppresses tumor growth in mice The effects of DEPDC1B on chordoma regulation was further elucidated by mouse xenograft model. The total bioluminescent intensity of mice was clearly presented, which reﬂected that the tumor load of shDEPDC1B group was obviously weaker than that of the control group (P < 0.01) (Fig. 3A). In addition, the size and volume of xenografts were measured and calculated throughout the animal experiments for 21 days, suggesting the signiﬁcantly slower growth rate and smaller ﬁnal tumor volume of shDEPDC1B group than the shCtrl group (P < 0.01) (Fig. 3B–D). Additionally, the results of immunohistochemical detection in the tumor tissues showed that the expression of Ki67 in shDEPDC1B group was lower than that in shCtrl group (P < 0.05) (Fig. 3E), indicating that downregulation of DEPDC1B can inhibit tumor proliferative activity in mice. Collectively, knockdown of DEPDC1B suppressed tumor growth of chordoma in vivo. Knockdown of DEPDC1B regulates apoptosis-related factors and AKT, ERK, RHOA/ROCK cascades BIRC5 was the most relevant to the proliferative phenotype of chordoma cells. As a consequence, BIRC5 was preliminarily identiﬁed as a downstream target of DEPDC1B in chordoma cells. On the other hand, upon the treatment of CHX (0.2 mg/mL, protein synthesis inhibitor), the protein stability of BIRC5 in shCtrl and shDEPDC1B chordoma cells was examined, indicating that DEPDC1B silencing induced a decrease of BIRC5 protein stability (Fig. 4C). Notably, the effects of DEPDC1B knockdown on BIRC5 protein stability could be partially eliminated by the treatment of MG-132 (20 μM), an inhibitor of proteasome (Fig. 4D), suggesting that DEPDC1B may regulate BIRC5 through ubiquitin-proteasome system (UPS) [28, 29]. Subsequently, we evaluated the regulation of DEPDC1B on ubiquitination of BIRC5 and the results indicated that DEPDC1B downregulation distinctly promotes BIRC5 ubiquitination (Fig. 4E). Considering that our previous study showed the regulation of BIRC5 ubiquitination by ubiquitin-conjugating enzyme E2T (UBE2T) (data not shown), we further explored the interaction between UBE2T and DEPDC1B. As shown in Fig. 4F, the direct interaction between DEPDC1B and UBE2T make us believe that DEPDC1B may inﬂuence UBE2T-mediated ubiquitination of BIRC5 through interacting UBE2T. Fig. 2 Knockdown of DEPDC1B regulates apoptosis-related factors and AKT, ERK, RHOA/ROCK cascades. A Human apoptosis antibody array analysis was performed in MUG-Chor1cells with or without DEPDC1B knockdown. B We tested the protein expression of AKT, p-AKT, ERK, p-ERK, RHOA, p-RHOA, ROCK1 after the expression of DEPDC1B decreased. Data was shown as mean ± SD. P < 0.05. knockdown of DEPDC1B contributed to downregulation of p-AKT, p-ERK, p-RHOA, and ROCK1 expression (Fig. 2B). As a consequence, knockdown of DEPDC1B could regulate the apoptosis-related factors and AKT, ERK, RHOA/ROCK cascades. Knockdown of DEPDC1B regulates apoptosis-related factors and AKT, ERK, RHOA/ROCK cascades Notably, the effects of DEPDC1B knockdown on BIRC5 protein stability could be partially eliminated by the treatment of MG-132 (20 μM), an inhibitor of proteasome (Fig. 4D), suggesting that DEPDC1B may regulate BIRC5 through ubiquitin-proteasome system (UPS) [28, 29]. Subsequently, we evaluated the regulation of DEPDC1B on ubiquitination of BIRC5 and the results indicated that DEPDC1B downregulation distinctly promotes BIRC5 ubiquitination (Fig. 4E). Considering that our previous study showed the regulation of BIRC5 ubiquitination by ubiquitin-conjugating enzyme E2T (UBE2T) (data not shown), we further explored the interaction between UBE2T and DEPDC1B. As shown in Fig. 4F, the direct interaction between DEPDC1B and UBE2T make us believe that DEPDC1B may inﬂuence UBE2T-mediated ubiquitination of BIRC5 through interacting UBE2T. resulted in DEGs in MUG-Chor1 cells, with 858 upregulated genes and 878 downregulated genes, which were presented in the hierarchical clustering (Fig. 4A). Furthermore, the signiﬁcant enrichment of DEGs in canonical pathway as well as diseases and functions were investigated based on IPA, suggesting that ‘colorectal cancer metastasis signaling, Wnt/β-catenin signaling, Wnt/ca+ pathway’ (Fig. S2A) and cell proliferation, death and other functions (Fig. S2B) would be affected by DEPDC1B. Subsequently, the DEGs with the most signiﬁcant alterations were selected by PCR (Fig. S2C) and WB (Fig. 2D). Figure S2E showed the interaction network based on IPA, in which DEPDC1B can affect BIRC5, EGFR, RHOU, and others. According to the results of protein interaction analysis (https://www.string-db.org/ cgi/network?taskId=bVGGIYt5GuUjsessionId=bVQ88ddIyQRY.), y we found that DEPDC1B and BIRC5, EGFR, and RHOU both interact and regulate each other (Fig. S2F). Therefore, only these three genes were selected for subsequent experiments. Next, lentivirus was used to deliver shRNAs into MUG-Chor1 cells to knockdown these genes, respectively. As shown in the Fig. 4B, the inhibitory effect of shBIRC5 group on proliferation was the most signiﬁcant compared with other groups (P < 0.001). BIRC5 was the most relevant to the proliferative phenotype of chordoma cells. As a consequence, BIRC5 was preliminarily identiﬁed as a downstream target of DEPDC1B in chordoma cells. we found that DEPDC1B and BIRC5, EGFR, and RHOU both interact and regulate each other (Fig. S2F). Therefore, only these three genes were selected for subsequent experiments. Next, lentivirus was used to deliver shRNAs into MUG-Chor1 cells to knockdown these genes, respectively. As shown in the Fig. 4B, the inhibitory effect of shBIRC5 group on proliferation was the most signiﬁcant compared with other groups (P < 0.001). Knockdown of DEPDC1B regulates apoptosis-related factors and AKT, ERK, RHOA/ROCK cascades Additionally, the effect of DEPDC1B on apoptosis-related factors was revealed by human apoptosis antibody array membrane. We found that knockdown of DEPDC1B resulted in downregulation of Bcl-2, CD40, HSP27, IGF-1sR, Livin, Survivin, XIAP and upregulation of Caspase3, sTNF-R1 in MUG-Chor1 cells (P < 0.05) (Fig. 2A). As DEPDC1B was previously described as involved in signaling by Rho-GTPases and by G-proteins [13, 14], the expression of MAPK/AKT and RHOA/ROCK cascades related proteins was explored. The results indicated that Cell Death and Disease (2021) 12:753 L. Wang et al. 4 Fig. 2 Knockdown of DEPDC1B regulates apoptosis-related factors and AKT, ERK, RHOA/ROCK cascades. A Human apoptosis antibody array analysis was performed in MUG-Chor1cells with or without DEPDC1B knockdown. B We tested the protein expression of AKT, p-AKT, ERK, p-ERK, RHOA, p-RHOA, ROCK1 after the expression of DEPDC1B decreased. Data was shown as mean ± SD. P < 0.05. 4 resulted in DEGs in MUG-Chor1 cells, with 858 upregulated genes and 878 downregulated genes, which were presented in the hierarchical clustering (Fig. 4A). Furthermore, the signiﬁcant enrichment of DEGs in canonical pathway as well as diseases and functions were investigated based on IPA, suggesting that ‘colorectal cancer metastasis signaling, Wnt/β-catenin signaling, Wnt/ca+ pathway’ (Fig. S2A) and cell proliferation, death and other functions (Fig. S2B) would be affected by DEPDC1B. Subsequently, the DEGs with the most signiﬁcant alterations were selected by PCR (Fig. S2C) and WB (Fig. 2D). Figure S2E showed the interaction network based on IPA, in which DEPDC1B can affect BIRC5, EGFR, RHOU, and others. According to the results of protein interaction analysis (https://www.string-db.org/ cgi/network?taskId=bVGGIYt5GuUjsessionId=bVQ88ddIyQRY.), we found that DEPDC1B and BIRC5, EGFR, and RHOU both interact and regulate each other (Fig. S2F). Therefore, only these three genes were selected for subsequent experiments. Next, lentivirus was used to deliver shRNAs into MUG-Chor1 cells to knockdown these genes, respectively. As shown in the Fig. 4B, the inhibitory effect of shBIRC5 group on proliferation was the most signiﬁcant compared with other groups (P < 0.001). BIRC5 was the most relevant to the proliferative phenotype of chordoma cells. As a consequence, BIRC5 was preliminarily identiﬁed as a downstream target of DEPDC1B in chordoma cells. On the other hand, upon the treatment of CHX (0.2 mg/mL, protein synthesis inhibitor), the protein stability of BIRC5 in shCtrl and shDEPDC1B chordoma cells was examined, indicating that DEPDC1B silencing induced a decrease of BIRC5 protein stability (Fig. 4C). DEPDC1B affects the BIRC5 ubiquitination through UBE2T in chordoma cells The molecular mechanism of chordoma regulated by DEPDC1B was preliminarily explored. Firstly, knockdown of DEPDC1B Cell Death and Disease (2021) 12:753 L. Wang et al. 5 Fig. 3 Knockdown of DEPDC1B inhibits tumor growth in mice xenograft models. A The total bioluminescent intensity of tumors in shCtrl group and shDEPDC1B group. B The volume of tumors in shCtrl group and shDEPDC1B group was measured after post-injection. C The average weight of tumors in shCtrl group and shDEPDC1B group. D Images of mice and tumors in shCtrl group and shDEPDC1B group. E The Ki67 staining of tumor tissues in shCtrl group and shDEPDC1B group. Data were represented as mean ± SD. P < 0.05, *P < 0.01. Fig. 3 Knockdown of DEPDC1B inhibits tumor growth in mice xenograft models. A The total bioluminescent intensity of tumors in shCtrl group and shDEPDC1B group. B The volume of tumors in shCtrl group and shDEPDC1B group was measured after post-injection. C The average weight of tumors in shCtrl group and shDEPDC1B group. D Images of mice and tumors in shCtrl group and shDEPDC1B group. E The Ki67 staining of tumor tissues in shCtrl group and shDEPDC1B group. Data were represented as mean ± SD. P < 0.05, P < 0.01. fold change = 14.1) (Fig. 5C) and repressing migration (P < 0.001, >70%) (Fig. 4D, E). Unsurprisingly, the results of the Transwell experiment were consistent with these trends (Fig. 6C). Therefore, overexpression of BIRC5 can reduce the inhibitory effect of DEPDC1B knockdown on the malignant behaviors of chordoma cells. BIRC5 overexpression attenuates the inhibitory effects of DEPDC1B knockdown in chordoma cells To fully demonstrate the effects of DEPDC1B and BIRC5 in chordoma cells, the functional recovery assays was conducted. Accordingly, the MUG-Chor1 cells with simultaneously upregu- lated BIRC5 and downregulated DEPDC1B (BIRC5 + sh DEPDC1B) were established (Fig. S4A–C). Notably, NC(OE+KD) group was the MUG-Chor1 cells infected with negative control lentivirus; BIRC5+NC-shDEPDC1B group was the overexpression of BIRC5; shDEPDC1B+NC-BIRC5 group was the cells with low expression of DEPDC1B. As illustrated in Fig. 6A, compared with NC (OE+KD) group, cell proliferation was weakest in shDEPDC1B+NC-BIRC5 group (P < 0.05), and strongest in BIRC5+NC-shDEPDC1B group (P < 0.01); The BIRC5+shDEPDC1B group can attenuate the inhibitory effect of cell proliferation (P < 0.05). Meanwhile, apoptosis was the weakest in the BIRC5+NC-shDEPDC1B group (P < 0.05). On the contrary, shDEPDC1B+NC-BIRC5 group of apoptosis was the strongest (P < 0.001) (Fig. 6B). The apoptosis of BIRC5+shDEPDC1B group was higher than that of BIRC5+NC- shDEPDC1B group (P < 0.001), but weaker than shDEPDC1B+NC- BIRC5 group (P < 0.01). There was no doubt that the cell migration rate was the highest in the BIRC5+NC-shDEPDC1B group and the lowest in the shDEPDC1B+NC-BIRC5 group compared with NC(OE+KD) group (Fig. 6C, S5A). The BIRC5 +shDEPDC1B group can reduce the inhibitory effect of shDEPDC1B+NC-BIRC5 group in chordoma cells (P < 0.05). DISCUSSION A The PrimeView Human Gene Expression Array was performed to identify the differentially expressed genes (DEGs) between shDEPDC1B and shCtrl groups of MUG-Chor1 cells. B The effects of knockdown of RHOU, BIRC5, and EGFR on proliferation of MuG-Chor1 cells were examined. C Protein levels of BIRC5 in MUG-Chor1 cells with or without DEPDC1B knockdown following 0.2 mg/mL CHX treatment for 0–12 h. D Levels of BIRC5 proteins in MUG-Chor1 cells with or without DEPDC1B knockdown following MG-132 treatment for indicated times (12 h). E The lysates of MUG-Chor1 cells were immunoprecipitated and WB was performed to examine the ubiquitination of BIRC5. F Co-IP analysis of interaction of DEPDC1B and UBE2T in MUG-Chor1 cells. The presented results were representative of experiments repeated at least three times. Data were represented as mean ± SD. P < 0.001. which could inhibit Caspases and prevents cell death [34, 35]. Not surprisingly, the molecular mechanism of chordoma cell apoptosis induced by DEPDC1B knockdown is complex, which required the participation of a series of apoptosis-related factors. and molecular mechanism of BIRC5 in chordoma have not been determined. The present study clariﬁed that DEPDC1B affected the ubiquitination of BIRC5 through UBE2T. Ubiquitination is a widespread post-translational modiﬁcation that mediates the localization, metabolism, function, regulation, and degradation of proteins in cells [47]. The UPS is composed of ubiquitin, ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, ubiquitin ligase E3, proteasome and its substrates. Studies have shown that UPS plays a central role in regulating protein levels and activities, cell cycle, gene expression, response to oxidative stress, cell survival, proliferation and apoptosis, which is closely related to the onset of cancers and cardiovascular diseases [48]. Moreover, UBE2T is a member of the E2 family in the UPS [49]. Recently, Yin et al, reported that UBE2T promoted radiation resistance of non-small cell lung cancer through ubiquitin-mediated FOXO1 degradation [50]. Our study clariﬁed that DEPDC1B affected the ubiquitination of BIRC5 through UBE2T, leading to dysregulation of gene expression. Further- more, simultaneous downregulation of BIRC5 and DEPDC1B Importantly, apoptosis repeat-containing 5 (BIRC5) was considered to be the downstream target of DEPDC1B involved in the progression of chordoma. Previous study reported that BIRC5 is a member of IAP family, which is a mitotic spindle checkpoint gene and encodes Survivin protein. Additionally, BIRC5 acts as a bifunctional regulator of apoptosis inhibition and cell cycle progression [36, 37]. DISCUSSION In this study, the unique role and potential molecular mechanism of DEPDC1B in chordoma were recognized. Speciﬁcally, knock- down of DEPDC1B inhibits the malignant behavior of chordoma cells in vitro, such as reduction of proliferation, induction of apoptosis, and inhibition of migration. As expected, downregula- tion of DEPDC1B suppressed growth of chordoma in vivo. In particular, the knockdown of DEPDC1B resulted in not only a signiﬁcant upregulation of Caspase3, sTNF-R1, but also down- regulation of Bcl-2, CD40, HSP27, IGF-1sR, XIAP, Livin, Survivin in chordoma cells. In addition, apoptosis involves both internal and external pathways, in which the endogenous pathway is mediated by an anti-apoptotic protein Bcl-2 (B-cell lymphoma-2) that integrates death and survival signals [30]. CD40 interacts with CD40 ligand (CD40L) to regulate apoptosis according to different membrane localization [31]. HSP27 is a molecular chaperone with the ability to interact with a large number of proteins, which could regulate apoptosis by interacting with components involved in Caspase activation and apoptosis [32]. Moreover, XIAP, an X-linked inhibitor of apoptosis, exerts a strong inhibitory effect on apoptosis depending on its unique ability to bind Caspases [33]. As members of the inhibitors of apoptosis (IAP) family, Livin and Survivin are abnormally expressed in the progression of cancers, Cell Death and Disease (2021) 12:753 Fig. 4 DEPDC1B affects the BIRC5 ubiquitination through UBE2T in chordoma cells. A The PrimeView Human Gene Expression Array was performed to identify the differentially expressed genes (DEGs) between shDEPDC1B and shCtrl groups of MUG-Chor1 cells. B The effects of knockdown of RHOU, BIRC5, and EGFR on proliferation of MuG-Chor1 cells were examined. C Protein levels of BIRC5 in MUG-Chor1 cells with or without DEPDC1B knockdown following 0.2 mg/mL CHX treatment for 0–12 h. D Levels of BIRC5 proteins in MUG-Chor1 cells with or without DEPDC1B knockdown following MG-132 treatment for indicated times (12 h). E The lysates of MUG-Chor1 cells were immunoprecipitated and WB was performed to examine the ubiquitination of BIRC5. F Co-IP analysis of interaction of DEPDC1B and UBE2T in MUG-Chor1 cells. The presented results were representative of experiments repeated at least three times. Data were represented as mean ± SD. *P < 0.001. L. Wang et al. L. Wang et al. 6 Fig. 4 DEPDC1B affects the BIRC5 ubiquitination through UBE2T in chordoma cells. Cell Death and Disease (2021) 12:753 DISCUSSION Moreover, BIRC5 is highly expressed in tumors, including cancer cells and tumor stem cells, whose expression is associated with the differentiation, proliferation, invasion and metastasis of tumor cells [38–41]. In recent years, considerable evidence suggested that abnormal expression of BIRC5 is involved in the progression of various cancers, including lung, breast, colon, pancreatic, and prostate cancers [42–46]. Although abnormal expression of BIRC5 is signiﬁcantly associated with tumor progression, the exact role Cell Death and Disease (2021) 12:753 L. Wang et al. 7 Fig. 5 Knockdown of BIRC5 deepens the effects on chordoma cells by DEPDC1B knockdown. After lentivirus shCtrl, shBIRC5 or shBIRC5 + shDEPDC1B infected with MUG-Chor1 cells, they were subjected to the detection of proliferation (A), clone formation (B), apoptosis (C) and migration (D, E). The presented results were representative of experiments repeated at least three times. Data were represented as mean ± SD. P < 0.05, P < 0.01, P < 0.001. Fig. 5 Knockdown of BIRC5 deepens the effects on chordoma cells by DEPDC1B knockdown. After lentivirus shCtrl, shBIRC5 or shBIRC5 + shDEPDC1B infected with MUG-Chor1 cells, they were subjected to the detection of proliferation (A), clone formation (B), apoptosis (C) and migration (D, E). The presented results were representative of experiments repeated at least three times. Data were represented as mean ± SD. P < 0.05, P < 0.01, P < 0.001. may exacerbate the inhibitory effects of chordoma cells. Moreover, overexpression of BIRC5 can reduce the inhibitory effect of DEPDC1B knockdown on the malignant behaviors of chordoma cells. 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Int J Oncol. 2012;40 DISCUSSION After lentivirus NC(OE+KD), BIRC5+NC-shDEPDC1B, shDEPDC1B+NC-BIRC5, or BIRC5+sh DEPDC1B infected with MUG-Chor1 cells, they were subjected to the detection of proliferation (A), apoptosis (B), and migration (C, D). The presented results were representative of experiments repeated at least three times. Data were represented as mean ± SD. P < 0.05, P < 0.01, P < 0.001. may exacerbate the inhibitory effects of chordoma cells. Moreover, overexpression of BIRC5 can reduce the inhibitory effect of DEPDC1B knockdown on the malignant behaviors of chordoma cells. In conclusion, DEPDC1B affected the ubiquitination of BIRC5 through UBE2T. Simultaneous downregulation of BIRC5 and DEPDC1B may exacerbate the inhibitory effects of chordoma. Moreover, BIRC5 overexpression reduced the inhibitory effects of Fig. 6 Overexpression of BIRC5 reduces the inhibitory of DEPDC1B knockdown on the malignant behaviors of chordoma cells. After lentivirus NC(OE+KD), BIRC5+NC-shDEPDC1B, shDEPDC1B+NC-BIRC5, or BIRC5+sh DEPDC1B infected with MUG-Chor1 cells, they were subjected to the detection of proliferation (A), apoptosis (B), and migration (C, D). The presented results were representative of experiments repeated at least three times. Data were represented as mean ± SD. P < 0.05, P < 0.01, *P < 0.001. may exacerbate the inhibitory effects of chordoma cells. Moreover, overexpression of BIRC5 can reduce the inhibitory effect of DEPDC1B knockdown on the malignant behaviors of chordoma cells. may exacerbate the inhibitory effects of chordoma cells. Moreover, overexpression of BIRC5 can reduce the inhibitory effect of DEPDC1B knockdown on the malignant behaviors of chordoma cells. In conclusion, DEPDC1B affected the ubiquitination of BIRC5 through UBE2T. Simultaneous downregulation of BIRC5 and DEPDC1B may exacerbate the inhibitory effects of chordoma. Moreover, BIRC5 overexpression reduced the inhibitory effects of Cell Death and Disease (2021) 12:753 L. Wang et al. 8 DEPDC1B knockdown in chordoma cells. DEPDC1B regulates the progression of human chordoma through UBE2T-mediated ubiqui- tination of BIRC5, which may be a promising candidate target in molecular therapy of chordoma patients. 25. Scheil S, Bruderlein S, Liehr T, Starke H, Herms J, Schulte M, et al. Genome-wide analysis of sixteen chordomas by comparative genomic hybridization and cyto- genetics of the ﬁrst human chordoma cell line, U-CH1. Genes Chromosomes Cancer. 2001;32:203–11. 26. Rinner B, Froehlich EV, Buerger K, Knausz H, Lohberger B, Scheipl S, et al. Establishment and detailed functional and molecular genetic characterisation of 26. Rinner B, Froehlich EV, Buerger K, Knausz H, Lohberger B, Scheipl S, et al. COMPETING INTERESTS The authors declare no competing interests. The authors declare no competing interests. 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons. org/licenses/by/4.0/. FUNDING STATEMENT Not applicable. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Not applicable. ETHICS STATEMENT All procedures involving mice and experimental protocols were approved by the Institutional Animal Care and Use Committees of Shanghai Jiao Tong University School of Medicine. Cell Death and Disease (2021) 12:753 © The Author(s) 2021, corrected publication 2022 AUTHOR CONTRIBUTIONS 23. Li Z, Wang Q, Peng S, Yao K, Chen J, Tao Y, et al. The metastatic promoter DEPDC1B induces epithelial-mesenchymal transition and promotes prostate cancer cell proliferation via Rac1-PAK1 signaling. Clin Transl Med. 2020;10:e191. This program was designed by Xiaodong Zhu. The experiments were operated by Ruijun Xu. The data collection and analysis were conducted by Yanbin Liu. The manuscript was produced by Liang Tang and Yanghu Lu, which was checked and revised by Xiaodong Zhu and Liang Tang. All the authors have conﬁrmed the submission of this manuscript. 24. Lai CH, Xu K, Zhou J, Wang M, Zhang W, Liu X, et al. DEPDC1B is a tumor promotor in development of bladder cancer through targeting SHC1. Cell Death Dis. 2020;11:986. Cell Death and Disease (2021) 12:753 Cell Death and Disease (2021) 12:753 L. Wang et al. 9 FUNDING STATEMENT Not applicable. ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41419-021-04026-7. Correspondence and requests for materials should be addressed to Z.W. or X.Z. Reprints and permission information is available at http://www.nature.com/ reprints Reprints and permission information is available at http://www.nature.com/ © The Author(s) 2021, corrected publication 2022 Cell Death and Disease (2021) 12:753
https://openalex.org/W2254079930	https://europepmc.org/articles/pmc4724130?pdf=render	English	null	Clinicopathological analysis in patients with neuroendocrine tumors that metastasized to the brain	BMC cancer	2,016	cc-by	6,133	* Correspondence: jiroaki@gmail.com 1Department of Neurosurgery, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan Full list of author information is available at the end of the article Akimoto et al. BMC Cancer (2016) 16:36 DOI 10.1186/s12885-015-1999-x Akimoto et al. BMC Cancer (2016) 16:36 DOI 10.1186/s12885-015-1999-x Open Access Clinicopathological analysis in patients with neuroendocrine tumors that metastasized to the brain Jiro Akimoto1, Hirokazu Fukuhara1, Tomohiro Suda1, Kenta Nagai1, Megumi Ichikawa1, Shinjiro Fukami1, Michihiro Kohno1, Jun Matsubayashi2 and Toshitaka Nagao2 © 2015 Akimoto et al. 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: A neuroendocrine tumor (NET) can develop anywhere in the body, but is mainly found in the pancreas, gastrointestinal tract, and lungs. This report is a retrospective study of the clinicopathological features of NET patients with brain metastasis whose tissue diagnosis was made at our hospital. Methods: Patients with brain metastasis evidenced by clinical records and images were accumulated among 302 patients in whom tissue diagnosis of NETs was made at our hospital between 2008 and 2013. In the patients, the primary lesion, pathological classification, pattern of metastasis, details of treatment, and outcomes were analyzed. Results: Brain metastasis was observed in 31 patients (10.3 %). The primary lesion was in the lungs in 26 patients (83.9 %), and the mammary glands, esophagus, and uterus in 1 patient each. Primary lesions were unknown in 2 patients, including 1 patient in whom NETs were detected in the lymph nodes alone. Pathological classification of the primary lesion was NET Grade 2 (Ki-67: 3 to 20 %) in 3 patients and neuroendocrine carcinoma (NEC, Ki-67: ≥21 %) in 26 patients. The median period from onset of the primary lesion up to diagnosis of brain metastasis was 12.8 months, and the brain lesion preceded brain metastasis in 6 patients. Ten patients had a single metastasis whereas 21 patients had multiple metastases, but no characteristics were observed in their images. Brain metastasis was extirpated in 10 patients. Stereotactic radiotherapy alone was administered in 6 patients, and brain metastasis was favorably controlled in most of the patients with coadministration of cranial irradiation as appropriate. The median survival period from diagnosis of brain metastasis was 8.1 months, and the major cause of death was aggravation of the primary lesion or metastatic lesions in other organs. Conclusion: Most of NET patients with brain metastasis showed the primary lesion of NEC in the lungs, and they had multiple metastases to the liver, lymph nodes, bones, and so forth at the time of diagnosis of brain metastasis. The guidelines for accurate diagnosis and treatment of NETs should be immediately established based on further analyses of NET patients with brain metastasis. Keywords: Neuroendocrine tumor, Metastatic brain tumor, Large cell neuroendocrine tumor, Small cell carcinoma, Radiotherapy, Outcomes Keywords: Neuroendocrine tumor, Metastatic brain tumor, Large cell neuroendocrine tumor, Small cell carcinoma, Radiotherapy, Outcomes © 2015 Akimoto et al. Correspondence: jiroaki@gmail.com 1Department of Neurosurgery, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan Full list of author information is available at the end of the article © 2015 Akimoto et al. 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. Background 26 patients, stomach in 20 patients, mammary glands in 20 patients, uterus in 18 patients, lymph nodes in 17 pa- tients, and duodenum in 12 patients. Other sites of onset were the thymus gland in 8 patients, esophagus in 6 patients, ileum in 5 patients, liver in 5 patients, colon in 3 patients, pharynx in 3 patients, paranasal sinus in 3 patients, kidneys in 2 patients, urinary tract in 2 patients, and ovaries in 2 patients. A neuroendocrine tumor (NET) is characterized by pro- duction and secretion of peptide hormones, amines, or the presence of secretory granules. The term “carcinoid” was used for a long time because patients with a primary lesion in the gastrointestinal tract were mainly examined and the course was relatively favorable [1]. However, recent research has shown that NETs are not limited to the gastrointestinal tract and originate from dispersed endocrine cells that are widely distrib- uted all over the body. They develop in all organs, and there are not only patients who present a favorable clinical pathology, but many of them also have a ma- lignant course. The World Health Organization (WHO) therefore attempted to grade NETs based on systematic clinical pathological classification, but classifications with different criteria were proposed for NETs originating from the favored sites (pancreas and gastrointestinal tract) and NETs originating from the lungs. Discussions are still continuing concerning the validity of each classification and establishment of standard criteria, but a standard pathological entity is not yet available. We then searched medical charts and imaging findings of the 302 patients carrying a diagnosis of NETs and extracted 31 patients (10.3 %) in whom brain metastasis was diagnosed based on either medical charts or imaging findings. In the 31 patients, their clinical images, primary lesion, pathological diagnosis, imaging findings, treat- ment, and outcomes were retrospectively analyzed. This study was conducted in accordance with the Helsinki Declaration and was approved by the Ethical Review Board of Tokyo Medical University Hospital. Each patient signed a written informed consent form that was approved by our institutional Committee on Human Rights in Research for the publication of their data. With advances in pathological diagnosis of NETs, neu- rosurgeons are increasingly experiencing patients with brain metastasis from tumors diagnosed as NETs [2–7]. Background However, few reports examined brain metastasis of NET patients [2] and therefore, guidelines should be prepared for oncologists based on findings from clinical images and pathology as well as treatment and outcomes of brain metastasis of NETs, and then taking appropriate measures. Abstract 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. Akimoto et al. BMC Cancer (2016) 16:36 Page 2 of 8 Page 2 of 8 Results (Table 1) Clinical features The age of the 31 patients ranged between 34 and 78 years (median: 68 years old) and 19 of them were men and 12 of them were women. The primary lesion in 6 patients (19.4 %) was detected by close examination after hospitalization following the onset of intracranial hypertension, disorientation, or local neurological symptoms such as paralysis and cerebellar symptoms, due to brain metastasis. The primary lesion remained unknown in 1 patient in spite of repeated close systemic examinations. We accumulated cases of NET patients with brain metastasis at our hospital and performed clinical patho- logical analysis. The results are reported below. Methods Of the remaining 25 patients in whom brain metasta- sis was diagnosed during treatment of the primary le- sion, brain metastasis was confirmed in 17 patients by imaging, which was performed due to the occurrence of local neurological symptoms or intracranial hyperten- sion, whereas 8 patients did not show definite neuro- logical symptoms and brain metastasis was confirmed by imaging screening. The period from definitive diagnosis of primary lesion up to confirmation of brain metastasis ranged between 0 and 33.4 months (median: 12.8 months) (Fig. 1). The terms related to NETs, including “carcinoid”, “endo- crine”, “neuroendocrine”, “small cell carcinoma” and “large cell neuroendocrine carcinoma (LCNEC)”, were searched among histopathological diagnoses of all neoplasm speci- mens obtained during surgical procedures at Tokyo Medical University Hospital during the 6 years between January 2008 and December 2013. As a result, 302 pa- tients were confirmed carrying a diagnosis of NETs. The diagnoses consisted of carcinoid in 75 patients, endocrine in 47 patients, neuroendocrine in 149 patients, small cell carcinoma in 30 patients, and LCNEC in 26 patients. They also included duplications of search terms, and final diagnosis of LCNEC or small cell carcinoma following the initial diagnosis of carcinoma with neuro-endocrine differ- entiation. The age of patients ranged between 21 and 91 years (median: 64.5 years old), and there were 182 men and 120 women. The primary site of NETs was the lungs in 103 patients, rectum in 37 patients, pancreas in Primary lesion and pathological diagnosis y p g g The primary lesion was in the lungs in 26 (83.9 %) of 31 patients. Other organs containing the primary lesion in- cluded the mammary glands, esophagus, and uterus in 1 patient each, and unknown in 2 patients, including 1 pa- tient in whom NETs were detected in the lymph glands alone. The final pathological diagnosis of lung NETs was Page 3 of 8 Page 3 of 8 Page 3 of 8 Akimoto et al. BMC Cancer (2016) 16:36 Table 1 Clinical pathological images of a patient with brain metastasis originating from NETs confirmed by pathological diagnosis N (cases) Neuroendocrine tumor (NET) 302 Brain metastasis 31 (10.3 %) Age 34-78 (Median: 68) Gender Male 19 Female 12 Origin Lung 26 (83.9 %) Breast 1 Esophagus 1 Lymph node 1 Uterus 1 Unknown 1 Pathology LCNEC 11 (35.5 %) Small cell carcinoma 12 (38.7 %) Carcinoma with neuroendocrine differentiation 8 (25.8 %) Grade (Ki-67) 2 (3-20 %) 3 3 (≥21 %) 28 (90.3 %) Imaging Single/nodular type 7 Single/cystic type 3 Multiple/nodular type 10 Multiple/cystic type 3 Multiple/nodular and cystic type 8 Treatment Surgery 2 Surgery + WBRT 7 Surgery + GK 1 Surgery + WBRT + GK 1 WBRT 11 GK 6 WBRT + GK 1 Chemotherapy 1 Best supportive care 1 N number, LCNEC large cell neuroendocrine tumor, WBRT whole brain radiotherapy, GK gamma knife glands was diagnosed as having adenocarcinoma with neuroendocrine differentiation, the patient with the pri- mary lesion in the esophagus as having squamous cell carcinoma with neuroendocrine differentiation, and the patient with the primary lesion in the uterus as having LCNEC. A diagnosis of small cell carcinoma was made in the patients with no primary lesion other than in the lymph nodes or with an unknown primary lesion. Accord- ing to the grading by the percentage of Ki-67 positively stained cells, 3 patients (all of them had NETs originating from the lungs) were Grade 2 (Ki-67: 3 to 20 %) and 28 patients (90.3 %) were Grade 3 (Ki-67: ≥20 %). Treatment When onset of brain metastasis was confirmed, optimal therapy was chosen and performed based on compre- hensive evaluation of the age, performance status (PS), imaging findings, and clinical course of the primary lesion. The lesion that caused symptoms was extirpated as completely as possible in principle, and postoperative cranial irradiation or stereotactic radiotherapy was con- comitantly performed as necessary. Details of treatment varied with each patient. For example, the gamma knife was used for a lesion with 3 or fewer small tumors to alleviate symptoms, and cranial radiotherapy alone for a lesion with 4 or more tumors. In addition, treatment was switched to best supportive care at the onset of brain metastasis due to poor PS caused by lesions in other organs. Eventually, the main treatments included craniotomy plus cranial irradiation in 7 patients, cranial irradiation alone in 11 patients, and gamma knife alone in 6 patients. Imaging diagnosis (Fig. 2) As a result of examination of patterns of metastasis by contrast-enhanced T1-weighted MRI, 7 patients were classified as having the single/nodular type, 3 patients as having the single/cystic type, 10 patients as having the multiple/nodular type, 3 patients as having the multiple/ cystic type, and 8 patients as having the multiple/nodular and cystic type. Therefore, no unique imaging patterns characteristic of NET patients with brain metastasis were observed. All the patients demonstrating single brain me- tastasis had NETs originating from lung, but the patients from non-lung (breast, esophagus, uterus, lymph node) NETs demonstrated multiple brain metastases. Outcomes N number, LCNEC large cell neuroendocrine tumor, WBRT whole brain radiotherapy, GK gamma knife The follow-up period after treatment of metastatic brain tumors was from 1 to 34 months (median: 5.8 months), and 29 of 31 patients were deceased by the time of completion of examinations. Analysis by the Kaplan- Meier survival curve revealed a median survival period of 8.1 months (Fig. 3). Of the 29 deceased patients, only large cell neuroendocrine carcinoma (LCNEC) in 10 pa- tients, small cell carcinoma in 10 patients, and carcin- oma with neuroendocrine differentiation in 6 patients. The patient with the primary lesion in the mammary Akimoto et al. BMC Cancer (2016) 16:36 Page 4 of 8 Time (months) Detection of metastasis to brain (%) Fig. 1 Period from tissue diagnosis of the primary neuroendocrine tumor until diagnosis of brain metastasis in 31 patients ig. 1 Period from tissue diagnosis of the primary neuroendocrine tumor until diagnosis of brain metastasis in 31 patients mediastinum at the right S2 (Fig. 4a). The result of transbronchoscopic lung biopsy (TBLB) led to the diagnosis of LCNEC. Contrast-enhanced head MRI was conducted because he presented with headache, diplopia, and disorientation during maintenance chemotherapy with cisplatin and CPT-11 (camptothecin 11). A solid tumor was observed in the pineal body, together with obstructive hydrocephalus. A nodular tumor was observed in the cerebellar parenchyma in the vicinity of the superior medullar velum, which was diagnosed as a metastatic brain tumor of the multiple/nodular type 11.7 months after the pathological diagnosis of the lungs (Fig. 4b). Biopsy for the pineal body tumor and a third ventriculostomy for hydrocephalus were performed by neuroendoscopy. The isolated tissues were comprised of a dense proliferation of large epithelial tumor cells with severe necrotic degeneration, and synaptophysin and chromogranin G were positive and diffused in the cytoplasm. The percentage of Ki-67 positive cells was 35 % and the patient’s lesion was diagnosed as brain metastasis originating from Grade 3 LCNEC (Fig. 4c-f). The 1 patient died due to central nervous system symptoms caused by uncontrollable brain metastasis. The other patients died because of aggravation of their general condition due to the primary lesion or metastasis to the liver, adrenal gland, lymph nodes, or bones (including the spine), observed at the time of confirmation of brain metastasis. Both of the patients who survived until com- pletion of examinations had the primary lesion in the lungs. Outcomes Of these 2 patients, 1 patient who was on chemo- therapy due to poorly differentiated carcinoma with neuroendocrine differentiation of the single/nodular type was favorably controlled for 34 months with the gamma knife. The other patient who was on chemotherapy for LCNEC developed brain metastasis of the multiple/ nodular type, but was favorably controlled for 10 months by the gamma knife. Presentation of typical cases A patient with no notable medical history and no history of smoking presented with an abnormal shadow of the chest detected at a medical checkup. A pulmonary CT scan showed a small lesion on the A B C D Fig. 2 Imaging diagnosis patterns. a single/nodular type. b single/cystic type. c multiple/nodular type. d multiple/cystic type D A B A D C B Fig. 2 Imaging diagnosis patterns. a single/nodular type. b single/cystic type. c multiple/nodular type. d multiple/cystic type Akimoto et al. BMC Cancer (2016) 16:36 Page 5 of 8 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 Time (months) Overall survival (%) Fig. 3 Survival period after diagnosis of metastatic brain tumor Overall survival (%) Fig. 3 Survival period after diagnosis of metastatic brain tumor abdomen, endoscopy of the upper and lower gastrointestinal tract, and whole-body FDG-PET. The brain tumors were favorably controlled in the meantime and PS was maintained at 0 or 1 (Fig. 5g, h). However, generalized malaise rapidly increased at around 14 months after brain surgery and the patient died 16 months after surgery while receiving treatment at home. The primary lesion remained unknown. patient complained of pain in the posterior cervical region, weakness in the arms, and paresthesia after postoperative cranial irradiation at 30 Gy, and close examination showed metastasis to the cervical vertebra at C3, and the vertebral body and vertebral arch at C6 and C7 (Fig. 4g). Neither the patient nor his family wished to continue treatment. Therefore, the patient was switched to best supportive care at 2.9 months after brain surgery, and died 1 month later due to respiratory failure caused by rapid enlargement of pulmonary lesions. Discussion g p y A patient with no notable medical history and no history of smoking visited our hospital with a complaint of sudden onset of left hemiplegia. Contrast-enhanced head MRI showed multiple tumors with cysts in the right posterior lobe and the parietal lobe (Fig. 5a, b). The tumor in the right parietal lobe was considered to be the cause of the hemiplegia. Therefore, craniotomy was conducted for total resection of the tumor. He received postoperative cranial irradiation at 30 Gy and received ambulatory discharge. Images revealed isolated tissues comprised of pattern-less dense proliferation of small spindle-shaped tumor cells. The cytoplasm of tumor cells was strongly positive for synaptophysin, CD56, TTF1, and CK7 (Fig. 5c-f). Small cell carcinoma originating from the lungs was suspected, although the primary lesion was not identified in spite of examinations by contrast- enhanced whole-body CT scan, gallium scintigraphy, ultrasonography of the thyroid gland and the In 1907, NETs in the small intestine were described as “carcinoid” by Oberndorfer, because the clinical course was relatively favorable and they were pathologically differentiated [1]. Since then, carcinoid has been the description of the disease group for more than 90 years. It was later considered that NETs should be treated as clinically malignant tumors and the term “carcinoid” was eliminated from the WHO classification in 2000. Sub- sequently, NETs were categorized into five types, namely NET Grade 1, NET Grade 2, NEC (large cell or small cell), mixed adeno-neuroendocrine carcinoma (MANEC), and hyperplastic and neoplastic lesions, in the 2010 WHO classification according to the grading by the Ki-67 label- ing index [8]. However, the classification of the pancre- atic/gastrointestinal NETs (where the research is the most advanced) is slightly different from that of lung NETs which are sometimes still called “carcinoid”, and the standard pathological classification is yet to be achieved. NETs are also clinically classified as follows: 1) func- tional or non-functional NETs, depending on the onset Akimoto et al. BMC Cancer (2016) 16:36 Page 6 of 8 C A C D E F A B G Fig. 4 a Chest CT scan shows a small lesion (arrow) on the mediastinum at right S2. b Sagittal contrast-enhanced T1-weighted head MRI reveals two tumors in the pineal body and the cerebellum accompanied by obstructive hydrocephalus. c HE stain image of neuroendoscopic biopsy tissues (original magnification, × 200). d anti-synaptophysin antibody. Discussion (Rabbit polyclonal, Cell Marque, CA, USA) stain image (original magnification, × 200). e anti-chromogranin A antibody (Rabbit polyclonal, Dako, Glostrup, Denmark) stain image (original magnification, × 200). f Ki-67 (MIB-1, Dako, Glostrup, Denmark) stain image (original magnification, × 200). g Bone scintigraphy shows multiple metastases to the cervical vertebrae D E F B G A B C D E F Fig. 4 a Chest CT scan shows a small lesion (arrow) on the mediastinum at right S2. b Sagittal contrast-enhanced T1-weighted head MRI reveals two tumors in the pineal body and the cerebellum accompanied by obstructive hydrocephalus. c HE stain image of neuroendoscopic biopsy tissues (original magnification, × 200). d anti-synaptophysin antibody. (Rabbit polyclonal, Cell Marque, CA, USA) stain image (original magnification, × 200). e anti-chromogranin A antibody (Rabbit polyclonal, Dako, Glostrup, Denmark) stain image (original magnification, × 200). f Ki-67 (MIB-1, Dako, Glostrup, Denmark) stain image (original magnification, × 200). g Bone scintigraphy shows multiple metastases to the cervical vertebrae F E F C C D E D Fig. 4 a Chest CT scan shows a small lesion (arrow) on the mediastinum at right S2. b Sagittal contrast-enhanced T1-weighted head MRI reveals two tumors in the pineal body and the cerebellum accompanied by obstructive hydrocephalus. c HE stain image of neuroendoscopic biopsy tissues (original magnification, × 200). d anti-synaptophysin antibody. (Rabbit polyclonal, Cell Marque, CA, USA) stain image (original magnification, × 200). e anti-chromogranin A antibody (Rabbit polyclonal, Dako, Glostrup, Denmark) stain image (original magnification, × 200). f Ki-67 (MIB-1, Dako, Glostrup, Denmark) stain image (original magnification, × 200). g Bone scintigraphy shows multiple metastases to the cervical vertebrae of NETs in Caucasian patients was the lungs/bronchus in 31.9 %, small intestine in 17.7 %, unknown in 13.5 %, and rectum in 12.3 %, whereas that in African American patients was the rectum in 27 %, small intestine in 21 %, and lungs/bronchus in 18.3 %. Thus, a slight ethnic difference was observed [9]. Also in the percentage of NETs against all types of tumors in each site of origin, NETs accounted for the highest (1.42 %) in the lungs/ bronchus in Caucasian patients, whereas NETs accounted for the highest (1.65 %) in the rectum in African American patients [9]. The clinical pathology of pancreatic NETs has been most intensively studied. Discussion c HE image of the isolated parietal lobe tumor tissues (original magnification, × 200). d anti-synaptophysin antibody stain image (original magnification, × 200). e anti-CD56 antibody (1B6, Leica, Newcastle, UK) stain image (original magnification, × 200). f anti-TTF-1 (8G7G3/1, Dako, Glostrup, Denmark) antibody stain image (original magnification, × 200). g, h Horizontal contrast-enhanced T1-weighted head MRI after cranial irradiation therapy NETs [10, 11]. The description of brain, cardiac, and ovarian metastasis in the article stated that brain metas- tasis from primary lung NETs accounted for 45 % to 71 % of patients with the median period of 1.5 years from the diagnosis of the primary lesion until confirm- ation of brain metastasis [8]. Lymph node metastasis and liver metastasis were present in 75 % and 50 % of the patients with brain metastasis [11], respectively, which were comparable to the results of our present study. Approximately 1.4 % of metastatic brain tumors were NETs, whereas the incidence of brain metastasis from NETs was 1.5 % to 5 % [11]. The incidence of primary lung NETs was higher in our present study and that would be the cause of slightly higher incidence in brain metasta- sis at 10.3 %. A report that summarized 24 patients was the highest number of patients and only 57 patients were reported during the 46 years between 1962 and 2007 [11]. Accumulation of 31 patients during 6 years in this study is therefore unsurpassed in terms of analysis of brain metas- tasis from NETs. The report of ENET [11] did not contain a definite description of pathological findings and grading. 103 patients (34.1 %), rectum in 37 patients (12.3 %), and pancreas in 26 patients (8.6 %), which were almost comparable with the site of onset in Caucasian patients, and the number of patients with NETs in the pancreas was higher in Japanese patients. Brain metastasis was observed in 31 patients (10.3 %) and it was interesting that 26 (83.9 %) of them were metastasis from the pri- mary lung NETs. Primary brain metastasis is mainly caused by primary lung NETs whereas pancreatic/gastro- intestinal NETs are suggested to reach the brain as the final distal site of metastasis via metastasis to the liver, lymph node, lungs, or other organs. Brain metastasis from NETs has been frequently re- ported [2, 4–7] although standardized pathological ana- lysis, diagnosis, and treatment have not been established. Discussion However, pancreatic NETs accounted for 4 % of all types of the NETs and for approximately 0.3 % of all pancreatic cancers [9], being a very rare disease. Regarding the metastasis of NETs to other organs, distant metastasis was observed in 20 % to 30 % of patients at the time of diagnosis. of characteristic hormonal symptoms; 2) foregut origin (lungs/bronchus, thymus, stomach, pancreas, duodenum), midgut origin (jejunum, ileum, appendix), and hindgut origin (colon, rectum), depending on the site of embryo- logical development; 3) whether or not associated with inherited diseases (including multiple endocrine neoplasia type 1, von Hippel-Lindau disease, neurofibromatosis type 1, and tuberous sclerosis) [8]. Therefore, the clinical and pathological definition of NETs is still complicated. The National Cancer Institute’s Surveillance, Epidemi- ology, and End Results (SEER) Program (1993 to 2004) searched all registered patients using the International Classification of Disease (ICD) codes such as carcinoid, neuroendocrine carcinoma, and LCNEC, and extracted 17,321 patients with NETs [9]. The incidence of NETs among all tumors was 4.44 % in Caucasians and 6.5 % in African Americans, and male patients were slightly pre- dominant; the incidence increased by 37 % in Caucasians and 40 % in African Americans compared with the stat- istical data between 1993 and 1997 [9]. The site of onset Using the search words of SEER [9], we extracted 302 patients with NETs who were diagnosed at our hospital over the past 6 years. The site of onset was the lungs in Akimoto et al. BMC Cancer (2016) 16:36 Page 7 of 8 C D E F A B G H Fig. 5 a, b Horizontal contrast-enhanced T1-weighted head MRI shows multiple tumors with cysts in the right occipital lobe and the parietal lobe. c HE image of the isolated parietal lobe tumor tissues (original magnification, × 200). d anti-synaptophysin antibody stain image (original magnification, × 200). e anti-CD56 antibody (1B6, Leica, Newcastle, UK) stain image (original magnification, × 200). f anti-TTF-1 (8G7G3/1, Dako, Glostrup, Denmark) antibody stain image (original magnification, × 200). g, h Horizontal contrast-enhanced T1-weighted head MRI after cranial irradiation therapy C D E F A B G H A H B G E D C C F F D E Fig. 5 a, b Horizontal contrast-enhanced T1-weighted head MRI shows multiple tumors with cysts in the right occipital lobe and the parietal lobe. Acknowledgments The authors wish to thank Dr. Edward Barroga and the native English- speaking medical editors from the Department of International Medical Communications at Tokyo Medical University for editing and reviewing the English manuscript. References 2. Mallory GW, Fang S, Giannini C, Van Gompel JJ, Parney IF. Brain carcinoid metastases: outcomes and prognosis. J Neurosurg. 2013;118:889–95. Authors’ contributions JA, HF, TS, KN, MI, and SF made substantial contributions in the conception and design of the study. JA, JM, and TN participated in data collection, analysis, and interpretation. JA and MK drafted and carefully revised the manuscript for important intellectual content. All authors have read and approved the final manuscript. All authors take public responsibility for appropriate portions of the content and agree to be accountable for all aspects of the work. Received: 16 February 2015 Accepted: 11 December 2015 Received: 16 February 2015 Accepted: 11 December 2015 Received: 16 February 2015 Accepted: 11 December 2015 Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Discussion The European Neuroendocrine Tumor Society (ENETS) in 2012 published the consensus guidelines for the diag- nosis and treatment of neuroendocrine tumors for each site of onset [10]. During preparation of the guidelines, the ENETS advisory board and experts have published the analytical results on metastasis to other organs from Page 8 of 8 Page 8 of 8 Akimoto et al. BMC Cancer (2016) 16:36 Page 8 of 8 Our report may be the first to suggest that most of the patients consist of Grade 3 LCNEC and Grade 3 small cell cancer. Our report may be the first to suggest that most of the patients consist of Grade 3 LCNEC and Grade 3 small cell cancer. Abbreviations ENETS: European Neuroendocrine Tumor Society; ICD: International Classification of Disease; LCNEC: large cell neuroendocrine carcinoma; MANEC: mixed adeno-neuroendocrine carcinoma; NET: neuroendocrine tumor; PS: performance status; SEER: Surveillance, Epidemiology, and End Results; TBLB: transbronchoscopic lung biopsy; WHO: World Health Organization. To the best of our knowledge, no report described detailed treatment and outcomes for brain metastasis in NET patients. When limited to lung NETs, brain metas- tasis occurs in 30 % or more of the LCNEC patients and in 24 % of the small cell carcinoma patients [3, 12]. In the present study, the strategy of surgery and radio- therapy for the patients was determined based on the symptoms, PS, and imaging findings (number of brain metastases), and such action would be the standard concept for metastatic brain tumors at present. On the other hand, prophylactic cranial irradiation is also a possible option for small cell lung cancer [3, 12, 13]. The median survival period from diagnosis of brain metastasis was 8.1 months in the present study, and it was not greatly different from that of brain metastasis in all lung cancer patients [13]. Lately, various therap- ies such as combination chemotherapy, EGFR tyrosine kinase inhibitors (EGFR-TKI), and angiogenesis inhibitors have become available for brain metastasis originating from lung cancer [13, 14]. Development of optimal ther- apy is expected in addition to surgery and radiotherapy for brain metastasis originating from NETs based on the accumulation of findings of new chemotherapies and molecular targeted therapies. Author details 1D f 1Department of Neurosurgery, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan. 2Department of Anatomic Pathology, Tokyo Medical University, Tokyo, Japan. 13. Chi A, Komaki R. Treatment of Brain Metastasis from lung cancer. Cancers. 2010;2:2100–37. References 1. Oberndorfer S. Karzinoide tumoren des dünndarms. Frankf Z Pathol. 1907;1: 425–429. 1. Oberndorfer S. Karzinoide tumoren des dünndarms. Frankf Z Pathol. 1907;1: 425–429. 1. Oberndorfer S. Karzinoide tumoren des dünndarms. Frankf Z Pathol. 1907;1: 425–429. 1. Oberndorfer S. Karzinoide tumoren des dünndarms. Frankf Z Pathol. 1907;1: 425–429. 2. Mallory GW, Fang S, Giannini C, Van Gompel JJ, Parney IF. Brain carcinoid metastases: outcomes and prognosis. J Neurosurg. 2013;118:889–95. 3. Sun JM, Ahn MJ, Ahn JS UMSW, Kim H, Kim HK, Choi YS, et al. Chemotherapy for pulmonary large cell neuroendocrine carcinoma: similar to that for small cell lung cancer or non-small cell lung cancer? Lung Cancer. 2012;77(2):365–70. 4. Terada T. Pulmonary large cell neuroendocrine carcinoma diagnosed in a brain metastasis. Int J Clin Exp Pathol. 2012;5(2):159–62. 5. Tsai HJ, Wu CC, Tsai CR, Lin SF, Chen LT, Chang JS. Second Cancers In Patients with Neuroendocrine Tumors. PLoS One. 2013;8(12):e86414. 6. Tsugu A, Yoshiyama M, Matsumae M. Brain metastasis from large cell neuroendocrine carcinoma of the urinary bladder. Surg Neurol Int. 2011;2:84. 7. Zeichner SB, Cusnir M, Francavilla M, Hirzel A. Typical Bronchial Carcinoid Metastasizing to the Brain: A Case Presentation. Case Rep Oncol. 2011;4:602–10. 8. Vinik AI, Woltering EA, Warner RR, Caplin M, O’Dorisio TM, Wiseman GA, et al. NANETS Consensus Guidelines for the Diagnosis of Neuroendocrine Tumor. Pancreas. 2010;39(6):713–34. 9. Hauso O, Gustafsson BI, Kidd M, Waldum HL, Drozdov I, Chan AK, et al. Neuroendocrine Tumor Epidemiology: Contrasting Norway and North America. Cancer. 2008;113:2655–64. 10. European Neuroendocrine Tumor Society. Available from URL. http://www. enets.org. Accessed 12 Feb 2015. 11. Pavel M, Grossman A, Arnold R, Parren A, Kaltsas G, Steinmüller T, et al. ENETS Consensus Guidelines for the Management of Brain, Cardiac and Ovarian Metastases from Neuroendocrine Tumors. Neuroendocrinology. 2010;91:326–32. 12. Kim SJ, Kim JW, Han SW, Oh DY, Lee SH, Kim DW, et al. Biological characteristics and treatment outcomes of metastatic or recurrent neuroendocrine tumors; tumor grade and metastatic site are important for treatment strategy. BMJ Cancer. 2010;10:448. doi:10.1186/1471-2407-10-448. 13. Chi A, Komaki R. Treatment of Brain Metastasis from lung cancer. Cancers. 2010;2:2100–37. 14. D’Antonio C, Passaro A, Gori B, Del Signore E, Migliorino MR, Ricciardi S, et al. Bone and brain metastasis in lung cancer: recent advances in therapeutic strategies. Ther Adv Med Oncol. 2014;6(3):101–14. 2. Mallory GW, Fang S, Giannini C, Van Gompel JJ, Parney IF. Brain carcinoid metastases: outcomes and prognosis. J Neurosurg. 2013;118:889–95. 14. D’Antonio C, Passaro A, Gori B, Del Signore E, Migliorino MR, Ricciardi S, et al. Bone and brain metastasis in lung cancer: recent advances in therapeutic strategies. Ther Adv Med Oncol. 2014;6(3):101–14. Conclusion 3. Sun JM, Ahn MJ, Ahn JS UMSW, Kim H, Kim HK, Choi YS, et al. Chemotherapy for pulmonary large cell neuroendocrine carcinoma: similar to that for small cell lung cancer or non-small cell lung cancer? Lung Cancer. 2012;77(2):365–70. Approximately 10 % of NET patients developed brain metastasis at slightly longer than 1 year after diagnosis, and metastasis originating from LCNEC or small cell carcinoma occurred in most of them. Local recurrence or metastasis, or metastasis to another organ were often present at the time of diagnosis of brain metastasis, and the treatment options for brain metastasis varied depend- ing on the symptoms due to brain metastasis, PS, and metastatic images. Most of the metastases were controlled by applying active therapy to the brain lesion, but the mean survival period was 8 months from diagnosis of brain metastasis, mainly because of aggravation of the pri- mary lesion or metastasis to other organs. Standardization of clinical pathological interpretations of NETs has been attempted in recent years, and such reports are rapidly increasing. Nevertheless, few reports have been published concerning the pathology of brain metastasis of NETs, and the findings of our clinicopathological analysis of this pathology were limited to the experience of our institu- tion. We hope for standardization of clinical and patho- logical interpretation of NETs and the establishment of optimal guidelines for diagnosis and treatment of brain metastasis originating from NETs by accumulating more information on this disease. 4. Terada T. Pulmonary large cell neuroendocrine carcinoma diagnosed in a brain metastasis. Int J Clin Exp Pathol. 2012;5(2):159–62. brain metastasis. Int J Clin Exp Pathol. 2012;5(2):159–62. 5. Tsai HJ, Wu CC, Tsai CR, Lin SF, Chen LT, Chang JS. Second Cancers In Patients with Neuroendocrine Tumors. PLoS One. 2013;8(12):e86414. 6. Tsugu A, Yoshiyama M, Matsumae M. Brain metastasis from large cell neuroendocrine carcinoma of the urinary bladder. Surg Neurol Int. 2011;2:84. 7. Zeichner SB, Cusnir M, Francavilla M, Hirzel A. Typical Bronchial Carcinoid Metastasizing to the Brain: A Case Presentation. Case Rep Oncol. 2011;4:602–10. 8. Vinik AI, Woltering EA, Warner RR, Caplin M, O’Dorisio TM, Wiseman GA, et al. NANETS Consensus Guidelines for the Diagnosis of Neuroendocrine Tumor. Pancreas. 2010;39(6):713–34. f brain metastasis. Int J Clin Exp Pathol. 2012;5(2):159–62. 5. Tsai HJ, Wu CC, Tsai CR, Lin SF, Chen LT, Chang JS. Second Cancers In Patients with Neuroendocrine Tumors. PLoS One. 2013;8(12):e86414. 6. Tsugu A, Yoshiyama M, Matsumae M. 11. Pavel M, Grossman A, Arnold R, Parren A, Kaltsas G, Steinmüller T, et al. ENETS Consensus Guidelines for the Management of Brain, Cardiac and Ovarian Metastases from Neuroendocrine Tumors. Neuroendocrinology. 2010;91:326–32. 10. European Neuroendocrine Tumor Society. Available from URL. http://www. enets.org. Accessed 12 Feb 2015. Conclusion Brain metastasis from large cell neuroendocrine carcinoma of the urinary bladder. Surg Neurol Int. 2011;2:84. 7. Zeichner SB, Cusnir M, Francavilla M, Hirzel A. Typical Bronchial Carcinoid Metastasizing to the Brain: A Case Presentation. Case Rep Oncol. 2011;4:602–10. 8. Vinik AI, Woltering EA, Warner RR, Caplin M, O’Dorisio TM, Wiseman GA, et al. NANETS Consensus Guidelines for the Diagnosis of Neuroendocrine Tumor. Pancreas. 2010;39(6):713–34. 9 H O G f BI Kidd M W ld HL D d I Ch AK l 9. Hauso O, Gustafsson BI, Kidd M, Waldum HL, Drozdov I, Chan AK, et al. Neuroendocrine Tumor Epidemiology: Contrasting Norway and North America. Cancer. 2008;113:2655–64. 9. Hauso O, Gustafsson BI, Kidd M, Waldum HL, Drozdov I, Chan AK, et al. Neuroendocrine Tumor Epidemiology: Contrasting Norway and North America. Cancer. 2008;113:2655–64. 10. European Neuroendocrine Tumor Society. Available from URL. http://www. enets.org. Accessed 12 Feb 2015. 11. Pavel M, Grossman A, Arnold R, Parren A, Kaltsas G, Steinmüller T, et al. ENETS Consensus Guidelines for the Management of Brain, Cardiac and Ovarian Metastases from Neuroendocrine Tumors. Neuroendocrinology. 2010;91:326–32. 12. Kim SJ, Kim JW, Han SW, Oh DY, Lee SH, Kim DW, et al. Biological characteristics and treatment outcomes of metastatic or recurrent neuroendocrine tumors; tumor grade and metastatic site are important for treatment strategy. BMJ Cancer. 2010;10:448. doi:10.1186/1471-2407-10-448. 13. Chi A, Komaki R. Treatment of Brain Metastasis from lung cancer. Cancers. 2010;2:2100–37. 14. D’Antonio C, Passaro A, Gori B, Del Signore E, Migliorino MR, Ricciardi S, et al. Bone and brain metastasis in lung cancer: recent advances in therapeutic strategies. Ther Adv Med Oncol. 2014;6(3):101–14.
https://openalex.org/W2807168095	https://europepmc.org/articles/pmc5780476?pdf=render	English	null	Gamma-interferon exerts a critical early restriction on replication and dissemination of yellow fever virus vaccine strain 17D-204	npj vaccines	2,018	cc-by	9,974	INTRODUCTION Despite being safe and effective, the mechanisms for attenua- tion and immunogenicity of 17D remain elusive. The 17D vaccine strain was derived by Max Theiler in 1930s through serial passaging wild-type (WT) YFV strain Asibi in tissue culture, resulting in loss of viscerotropism and neurotropism in non- human primates.9 Multiple substrains of 17D, including 17D-204, 17D-213, and 17DD have been generated and used for vaccina- tion,10 with similar efﬁcacy.11 An average of 48 nucleotide and 20 amino acid changes distinguish 17D from its parent Asibi strain.12,13 The attenuation of 17D is attributed to genetic differences in both structural and non-structural proteins, but the role of speciﬁc mutations in the attenuation phenotype are not well characterized. Lee et al. suggested that a positive-charge mutation in the 17D envelope (E) protein that leads to enhanced glycosaminoglycan binding, contributes to attenuation, but attenuation likely involves multiple 17D loci.14 It was recently demonstrated that E protein mutations allow 17D to enter susceptible cells through clathrin-independent pathways, leading to enhanced antiviral response in vitro.15 We recently reported that both neutralizing antibody and CD4+ T cells are important in 17D-mediated protection from lethal WT YFV infection in a viscerotropic disease model.16 However, questions remain regard- ing the speciﬁc virus-host interactions that are modiﬁed by the 17D attenuating mutations and the mechanisms through which 17D induces highly protective immune responses. Understanding of the mechanisms of 17D attenuation will promote informed Yellow fever virus (YFV) is the prototypical member of the Flavivirus genus of the Flaviviridae family, which includes many other important arthropod-borne pathogens such as dengue, Zika, West Nile, and Japanese encephalitis viruses. YFV is estimated to cause 200,000 cases of disease and 30,000 mortalities annually. Historically, YFV has caused devastating outbreaks in North and South America, Africa, and Europe but is currently endemic to sub- Saharan Africa and South America. However, a recent YF outbreak in Angola led to atleast 300 deaths1 and 10 symptomatic traveler- associated cases in China,2 suggesting that YFV is still a problem in the modern world and threatening to expand geographically.3 Mosquito abatement programs and the live-attenuated YFV vaccine strain 17D remain the primary strategies for controlling YF outbreaks.4 The YFV vaccine strain 17D is arguably one of the most effective vaccines ever developed. www.nature.com/npjvaccines ARTICLE OPEN Gamma-interferon exerts a critical early restriction on replication and dissemination of yellow fever virus vaccine strain 17D-204 L. K. Metthew Lam 1, Alan M. Watson1, Kate D. Ryman1 and William B. Klimstra 1 Live attenuated viruses are historically among the most effective viral vaccines. Development of a safe vaccine requires the virus to be less virulent, a phenotype that is historically arrived by empirical evaluation often leaving the mechanisms of attenuation unknown. The yellow fever virus 17D live attenuated vaccine strain has been developed as a delivery vector for heterologous antigens; however, the mechanisms of attenuation remain elusive. The successful and safe progress of 17D as a vaccine vector and the development of live attenuated vaccines (LAVs) to related ﬂaviviruses requires an understanding of the molecular mechanisms leading to attenuation. Using subcutaneous infection of interferon-deﬁcient mouse models of wild type yellow fever virus (WT YFV) pathogenesis and 17D-mediated immunity, we found that, in the absence of type I IFN (IFN-α/β), type II interferon (IFN-γ) restricted 17D replication, but not that of WT YFV, by 1–2 days post-infection. In this context, IFN-γ responses protected 17D-infected animals from mortality, largely restricted the virus to lymphoid organs, and eliminated viscerotropic disease signs such as steatosis in the liver and inﬂammatory cell inﬁltration into the spleen. However, WT YFV caused a disseminated infection, gross liver pathology, and rapid death of the animals. In vitro, IFN-γ treatment of myeloid cells suppressed the replication of 17D signiﬁcantly more than that of WT YFV, suggesting a direct differential effect on 17D virus replication. Together these data indicate that an important mechanism of 17D attenuation in vivo is increased sensitivity to IFN-γ stimulated responses elicited early after infection. npj Vaccines (2018) 3:5 ; doi:10.1038/s41541-017-0039-z Received: 26 September 2017 Revised: 27 November 2017 Accepted: 30 November 2017 1Center for Vaccine Research, Department of Microbiology and Molecular Genetics, University of Pittsburgh, 3501 Fifth Avenue, Pittsburgh, PA 15261, USA Correspondence: William B. Klimstra (klimstra@pitt.edu) Kate D. Ryman is Deceased INTRODUCTION A single dose of 17D results in seroconversion in 95% of vaccinees within a week and can offer essentially life-long immunity against YFV infections.5,6 While the safety record of 17D is exemplary, severe adverse events do occur with low frequency, and these can involve both viscerotropic and neurotropic manifestations of virus disease. Regardless, due to the overall safety and effectiveness of 17D, multiple candidate vaccine vectors have been created using its genetic backbone and are currently being tested as a delivery system for antigens of other ﬂaviviruses or other pathogens.7,8 Received: 26 September 2017 Revised: 27 November 2017 Accepted: 30 November 2017 1Center for Vaccine Research, Department of Microbiology and Molecular Genetics, University of Pittsburgh, 3501 Fifth Avenue, Pittsburgh, PA 15261, USA Correspondence: William B. Klimstra (klimstra@pitt.edu) Kate D. Ryman is Deceased Published in partnership with the Sealy Center for Vaccine Development Received: 26 September 2017 Revised: 27 November 2017 Accepted: 30 November 2017 Published in partnership with the Sealy Center for Vaccine Development Published in partnership with the Sealy Center for Vaccine Development IFNγ attenuates YFV vaccine strain 17D-204 in vivo LKM Lam et al. IFNγ attenuates YFV vaccine strain 17D-204 in vivo LKM Lam et al. 2 design of live attenuated versions of other ﬂaviviruses and vaccines derived from other virus types. The regional lymph node draining the site of infection (DLN) is one of the earliest sites for ﬂavivirus replication and important for seeding viremia and dissemination.19 At 1 dpi, 17D-204 virus was recovered from the DLN in both AB6 and AGB6 mice. In the absence of the IFN-γ response, 17D-204 virions were more abundant as early as at 1 dpi (Fig. 1c). Serum viremia could be observed by 2 dpi; however, no signiﬁcant difference between serum viremia was observed in AB6 versus AGB6 at 2 dpi (Fig. 1d). 17D-204 virions were detectable in bone marrow aspirate and spleen at 2 dpi only in the absence of IFN-γ response, but by 4 dpi, high virus titers were observed in the spleen, bone aspirate, non- draining LN, adrenal glands, kidneys, and heart in both types of mice (Fig. 1e, f and S1), although, higher virus titers were observed in AGB6 mice in most of these tissues. Interestingly, infectious virus particles were recovered from liver and brain in AGB6 mice but never with AB6 mice (Fig. 1g-h). INTRODUCTION IFN-γ restricted virus replication and dissemination early during infection and enhanced 17D-204 virus clearance late after infection. Antiviral gene induction and cytokine production was also inﬂuenced by the presence or absence of type II IFN signaling. Importantly, we found that 17D-204 was more sensitive than WT virus strain Angola71 to the antiviral state induced by IFN-γ in vitro in speciﬁc myeloid cell subtypes likely important to YFV pathogenesis. This is suggestive that IFN-γ sensitivity is a primary attenuation mechan- ism for 17D in vivo. Finally, 17D-204 infection resulted in liver stress and histological abnormalities in the brains of AGB6 but not AB6 mice, indicating that both viscerotropic and neurotropic diseases occurred in the absence of IFN-γ signaling and revealing a possible application for the AB6 model in understanding vaccine-associated viscerotropic and neurotropic adverse events. y Because 17D is known to cause viscerotropic and neurotropic infection in vaccine-associated severe adverse events (SAE) cases,21,22 we sought to determine if tissue pathology could be detected after 17D infection in the absence of an IFN-γ response. We investigated tissue pathology at 4 dpi, which is the peak of viral replication in most visceral organs in both AB6 and AGB6 mice and 11 dpi, when the AGB6 animals displayed neurologic signs before succumbing to disease. At 4 dpi, hematoxylin and eosin (H&E) stained spleen sections (Fig. 2a) of 17D-204-infected AB6 and AGB6 mice were indistinguishable from mock-infected animals. In contrast, spleens of Angola71-infected mice displayed loss of splenic architecture and increase in inﬂammatory inﬁltrates, similar to our previous report.19 Despite the lack of titerable virus in the spleen at 12 dpi (Fig. 1f), spleens of 17D-204-infected AGB6 had increased numbers of inﬂammatory inﬁltrates and exhibited a loss of splenic architecture (Fig. 2a inset). Immunostaining of spleen sections from infected AB6 and AGB6 mice demonstrated the presence of YFV antigens in F4/80+ cells and cells in the outer marginal zone (Fig. 2b) at 4 dpi at a similar level. H&E-stained liver sections revealed microsteatosis occurring only in the liver of 17D- 204-infected AGB6 mice at 4 dpi (Fig. 2c) a time at which virions were detectable in the liver (Fig. 1g), and thus, indicative of a viscerotropic phase of 17D-204 infection in the absence of IFN-γ responses. Moreover, YFV antigen could be detected in some F4/ 80+ cells in the liver of infected AGB6 mice, suggesting that Kupffer cells were infected in these animals (Fig. 2d). INTRODUCTION In the presence of IFN-γ responses, virus clearance was observed in most organs by 8 dpi, but virions were still detected in all sampled organs in AGB6 mice at this time. Despite lacking both type I and type II IFN, virus clearance was observed in most of the non-CNS tissues in AGB6 mice by 12 dpi, before they succumbed to disease. In contrast, virus replication in brain increased through 12 dpi (up to 106 PFU/ g) in the AGB6 mice (Fig. 1h), corresponding to the time when neurological signs were ﬁrst observed (Fig. 1b). Overall, in the presence of IFN-γ, 17D-204 replication was controlled as early as 1 dpi. Some dissemination of virus still occurred in AB6 mice, but the peak viral load was signiﬁcantly lower than that of AGB6 mice. Virus tropism was also restricted by IFN-γ, most notably from liver and brain, and virus clearance was observed in most tissues by 8 dpi in AB6 mice. In the absence of IFN-γ responses, virus clearance occurred later and only in non-CNS tissues. y To assess the speciﬁc host interactions affected by 17D attenuating mutations, we have used a pathophysiologically relevant mouse model to study host-pathogen interactions of YFV. Many WT ﬂaviviruses, including YFV, replicate extremely poorly in mice with a functioning type I IFN system, precluding study of the roles of other host factors in their pathogenesis. Flaviviruses are more effective at antagonism of human type I IFN signaling than the analogous mouse responses.17,18 Indeed, mice deﬁcient in the type I interferon (IFN-α/β) receptor (AB6) are susceptible to viscerotropic disease and lethality after subcuta- neous (s.c.) infection with wild-type YFV strains Asibi and Angola71.19 Interestingly, s.c. infection of 17D in AB6 mice, which mimics vaccination, does not cause discernable disease and results in life-long immunity against challenge of WT virus strain Angola71.16 However, mice lacking both type I and type II IFN receptors (AGB6) are susceptible to lethal infection by 17D by either subcutaneous or intra-peritoneal routes.14,19,20 The fact that the additional deﬁciency of type II IFN (IFN-γ) receptors renders 17D infection lethal in AB6 mice suggests that the type II IFN system plays a critical role in attenuation of 17D in vivo. y p y To begin to understand the effects of IFN-γ on 17D, we have compared infection of AB6 and AGB6 mice. INTRODUCTION Correspond- ing to the time of neurological signs in 17D-204-infected AGB6 mice (11 dpi, Fig. 1b), we observed an increase in immune inﬁltration to the cerebral cortex (Fig. S2a) and virus infected cells in the cerebral cortex and cerebellum of the brain (Fig. S2b-c), indicative of neurotropic disease. No such phenomena were detectable in 17D-204-infected AB6 animals. Overall, our histology data demonstrates that IFN-γ restricts 17D-204 dissemination and protects mice from viscerotropic and neurotropic diseases. Published in partnership with the Sealy Center for Vaccine Development RESULTS IFN-γ attenuates 17D-204 but not virulent YFV in vivo To explore the role of IFN-γ, we compared pathogenesis of 17D- 204 in AB6 and AGB6 mice. Groups of 6-week-old AB6 and AGB6 mice were infected subcutaneously with 104 PFU of YFV 17D-204 or Angola71 in both of the hind limb footpads. The virulent WT strain Angola71 was used as control. As reported previously,16,19 Angola71-infected mice experienced severe weight loss and disease, requiring euthanasia (AST 7.5 +/−0.6 dpi), whereas 17D- 204 was uniformly non-lethal in AB6, and 17D-204-infected AGB6 mice experienced severe weight loss and eventually succumbed to disease (AST 10.75 +/−0.5 dpi) (Fig. 1a). Both 17D-204-infected AB6 and AGB6 had footpad swelling. In addition, 17D-204-infected AGB6 displayed signs of brain infection and neurologic disease including hind-limb paralysis at the late stage of infection before euthanasia (Fig. 1b). IFN-γ controls virus replication in vivo and protects mice from viscerotropic and neurotropic diseases Because IFN-γ inﬂuences survival of mice after 17D-204 infection, we hypothesized that virus replication is reduced and restricted in the presence of IFN-γ. Thus, we compared 17D-204 virus replication kinetics in different tissues in the AB6 and AGB6 mice. In parallel, tissues from 17D-204-infected wild-type (C57BL/6) mice were harvested but no infectious virus was recovered from any tissue sampled (Fig. 1c–h). Published in partnership with the Sealy Center for Vaccine Development npj Vaccines (2018) 5 IFNγ attenuates YFV vaccine strain 17D-204 in vivo LKM Lam et al. Fig. 1 IFN-γ attenuates 17D-204 in vivo. Survival (a) and symptoms (b) of diseases of YFV-infected mice were monitored daily. c–h 17D-204 replication kinetics in vivo. c Popliteal draining lymph node, d serum, e 1 mL bone marrow aspirate, f spleen, g liver, h brain. Data in (a) are analyzed with log-rank test. Data in c–h are presented in geometric mean ± 95% CI, 1 out of 3 independent experiments is shown. (p < 0.05; p < 0.01; p < 0.005; *p < 0.001; multiple t-test, corrected by Holm–Sidak method. 17D-204, n ≥6, Angola71, n = 4) 3 Fig. 1 IFN-γ attenuates 17D-204 in vivo. Survival (a) and symptoms (b) of diseases of YFV-infected mice were monitored daily. c–h 17D-204 replication kinetics in vivo. c Popliteal draining lymph node, d serum, e 1 mL bone marrow aspirate, f spleen, g liver, h brain. Data in (a) are analyzed with log-rank test. Published in partnership with the Sealy Center for Vaccine Development RESULTS Data in c–h are presented in geometric mean ± 95% CI, 1 out of 3 independent experiments is shown. (p < 0.05; p < 0.01; p < 0.005; **p < 0.001; multiple t-test, corrected by Holm–Sidak method. 17D-204, n ≥6, Angola71, n = 4) Cytokine induction by 17D-204 is reduced in the absence of IFN-γ signaling and TNFα) more rapidly than in 17D-infected AB6 or AGB6 mice (Fig. 3), possibly associated with the severe viscerotropic disease and rapid death of the WT virus-infected animals.19 The cytokines that are more signiﬁcantly upregulated in 17D-204-infected AB6 but not AGB6 mice, i.e. IL-12, MCP-1, MIG, and IP-10, are all IFN-γ- inducible, suggestive of a central role for IFN-γ in protecting AB6 mice from 17D-204 infection through cytokine induction. Similar to our observations, in adult human male vaccinees receiving YFV vaccine sub-strain 17DD, serum levels for MCP-1, MIG, IP-10, and IFN-γ were reported to be elevated.24 IFN-γ is central to activation of T cells and macrophages, and induces production of proinﬂammatory cytokines.23 Therefore, we hypothesized that the absence of IFN-γ would impact cytokine production during 17D infection. To this end, serum cytokine levels in infected mice were measured at various times post- infection using the Cytokine 20-plex Mouse Panel (Fig. 3). In AB6 mice infected with 17D-204, serum levels of IFN-γ, IL-12p40, MIG, MCP-1, and IP-10 were elevated transiently versus uninfected controls on day 4 pi, corresponding to the peak spleen virus titer (Fig. 1f). While AGB6 mice also exhibited a slight increase in serum IL-12 and MCP-1 levels on day 4pi, serum IFN-γ levels were highly elevated in comparison with control or AB6 mice on this day and at days 8 and 12pi. Wild-type YFV-infected AB6 mice produced higher levels of serum cytokines (IFNγ, MIG, MCP-1, IL-4, IL-5, IP-10, Antiviral gene induction is limited in 17D-204-infected AGB6 mice IFN-γ has been shown to upregulate and activate cellular and humoral immunity,25,26 but it can also be directly antiviral versus YFV in many cell types through upregulation of genes that encode antiviral effector proteins such as IFN-stimulated genes (ISGs).27 npj Vaccines (2018) 5 npj Vaccines (2018) 5 Published in partnership with the Sealy Center for Vaccine Development IFNγ attenuates YFV vaccine strain 17D-204 in vivo LKM Lam et al. Fig. 2 17D-204 causes viscerotropic abnormalities in the absence of IFN-γ. RESULTS Original magniﬁcation = 40x, n = 4 recently documented replication of 17D in NK1.1+ cells in the absence of IFN-α/β and IFN-γ signaling.29 Since IFN-γ inhibition of 17D-204 occurs at early times after infection of AGB6 mice, we attempted to determine if differences in tissue-speciﬁc expression of IFN-γ or ISGs could be detected in the absence of IFN-γ. Because we observed inhibition of 17D-204 replication and dissemination before 4 dpi, we examined IFN-γ transcript induction from 1 dpi to 4 dpi. Robust upregulation of the IFN-γ gene was observed as early as 2 dpi in lymph nodes but not spleens of both AB6 and AGB6 mice (Fig. S3a). Using ﬂow cytometry (Fig. 4a, b), we observed induction of IFN-γ in NK1.1+ cells in the DLN, similar to a previous report.28 Despite the lack of IFN-γ induction in the spleen (Fig. S3a), various antiviral genes including IGTP, IFIT1, and IFIT2 were upregulated in the spleens of 17D-204-infected AB6 mice more robustly than that of AGB6 mice, indicative of IFN-γ-dependent gene induction and likely a systemic effect of IFN-γ production (Fig. S3b). Since IFN-γ inhibition of 17D-204 occurs at early times after infection of AGB6 mice, we attempted to determine if differences in tissue-speciﬁc expression of IFN-γ or ISGs could be detected in the absence of IFN-γ. Because we observed inhibition of 17D-204 replication and dissemination before 4 dpi, we examined IFN-γ transcript induction from 1 dpi to 4 dpi. Robust upregulation of the IFN-γ gene was observed as early as 2 dpi in lymph nodes but not spleens of both AB6 and AGB6 mice (Fig. S3a). Using ﬂow cytometry (Fig. 4a, b), we observed induction of IFN-γ in NK1.1+ cells in the DLN, similar to a previous report.28 Despite the lack of IFN-γ induction in the spleen (Fig. S3a), various antiviral genes including IGTP, IFIT1, and IFIT2 were upregulated in the spleens of 17D-204-infected AB6 mice more robustly than that of AGB6 mice, indicative of IFN-γ-dependent gene induction and likely a systemic effect of IFN-γ production (Fig. S3b). 17D-204 is more sensitive to an IFNγ-induced antiviral state than WT-YFV Based on our observations that (1) AGB6 mice succumbed to 17D- 204 infection but not AB6 mice (Fig. 1a); (2) 17D-204 replicated to higher titer and disseminated faster in AGB6 mice than AB6 (Fig. 1c–h); (3) IFN-γ induction in infected AB6 and AGB6 mice occurred as early as 2 dpi (Fig. RESULTS H&E sections (a–c) antibody-stained frozen sections (b, d) of spleen (a, b) and liver (c, d) were presented. a Spleen sections from 17D-204-infected animals were indistinguishable at 4 dpi, whereas Angola71- infected animals display loss of white pulp and red pulp architecture and increase in inﬁltrating macrophages and neutrophils (inset) at 4 dpi. At 11 dpi, 17D-204 infected AGB6 has increased immune inﬁltration and extramedullary hematopoiesis (inset) but not AB6 mice. b YFV antigen can be detected in the spleen at 4 dpi in both AB6 and AGB6 mice in the red pulp and outer marginal zone area. c 17D-204-infected AB6 mice do not display major histological changes but infected AGB6 mice and Angola71-infected animals had microsteatosis (inset) at 4 dpi. In AGB6 mice, microsteatosis only occurs transiently at 4 dpi and resolved by 11 dpi. Antibody-stained frozen liver sections (d) revealed that YFV antigen could only be detected in infected AGB6 mice at 4 dpi but not AB6 mice, parallel to titer data in Fig. 1g. Original magniﬁcation = 40x, n = 4 LKM Lam et al. 4 Fig. 2 17D-204 causes viscerotropic abnormalities in the absence of IFN-γ. H&E sections (a–c) antibody-stained frozen sections (b, d) of spleen (a, b) and liver (c, d) were presented. a Spleen sections from 17D-204-infected animals were indistinguishable at 4 dpi, whereas Angola71- infected animals display loss of white pulp and red pulp architecture and increase in inﬁltrating macrophages and neutrophils (inset) at 4 dpi. At 11 dpi, 17D-204 infected AGB6 has increased immune inﬁltration and extramedullary hematopoiesis (inset) but not AB6 mice. b YFV antigen can be detected in the spleen at 4 dpi in both AB6 and AGB6 mice in the red pulp and outer marginal zone area. c 17D-204-infected AB6 mice do not display major histological changes but infected AGB6 mice and Angola71-infected animals had microsteatosis (inset) at 4 dpi. In AGB6 mice, microsteatosis only occurs transiently at 4 dpi and resolved by 11 dpi. Antibody-stained frozen liver sections (d) revealed that YFV antigen could only be detected in infected AGB6 mice at 4 dpi but not AB6 mice, parallel to titer data in Fig. 1g. Published in partnership with the Sealy Center for Vaccine Development RESULTS S3); (4) Angola71-infected AB6 mice produced higher levels of serum IFN-γ than 17D-infected AB6 (Fig. 3), yet virus infection was not controlled in the Angola71- infected mice; (5) Angola71-related mortality was delayed in AB6 versus AGB6 mice (Fig. 1a); and (6) reports that IFN-γ inhibited ﬂavivirus infection in vitro,30,31 we hypothesized that an IFN-γ- induced antiviral state can inhibit YFV replication in vitro and in vivo and that enhanced sensitivity to the IFN-γ antiviral state is a prominent attenuation mechanism of 17D. To test this hypothesis, we compared virus replication of 17D-204 and Angola71 in various relevant cell types treated with IFN-γ or IFN-α/β. Consistent with the hypothesis, we observed a dose-dependent inhibition of 17D- 204 and Angola71 virus replication by IFN-γ in bone marrow- derived macrophages (BMMΦ) and dendritic cells (BMDC) derived from C57BL/6 mice (Fig. 5 and S5-6). In addition, 17D-204 was signiﬁcantly more inhibited than Angola71 by an equivalent dose of IFN-γ or IFN-α/β. Since basal replication, which may be different To conﬁrm the role of NK cells in IFN-γ production in vivo, we performed antibody-mediated depletion of NK1.1+, CD4+-, or CD8+-T cells in 17D-204-infected mice. In both AB6 and AGB6 mice, NK1.1+ cell depletion led to a reduction in serum IFN-γ levels at 4 dpi (Fig. 4c), supporting the ﬂow cytometry data identifying NK1.1+ cells as a primary source of IFN-γ. Depletion of CD8+ T cells in AGB6, but not AB6, animals also led to reduction in serum IFN-γ levels. NK1.1+ cell depletion also resulted in enhanced 17D-204 replication in AB6 mice (Fig. 4d), likely due to less IFN-γ production. However, depletion of NK1.1+ cells resulted in reduced virus titer in AGB6 mice. The latter may be due to the Published in partnership with the Sealy Center for Vaccine Development npj Vaccines (2018) 5 IFNγ attenuates YFV vaccine strain 17D-204 in vivo LKM Lam et al. 5 Fig. 3 Cytokine response of 17D-204-infected animal. Serum cytokines IFN-γ (a), IL-12 (b), MCP-1 (c), MIG (d), IL-5 (e), IL-6 (f), IP-10 (g), and TNFα (h) were quantiﬁed using Cytokine 20-Plex Mouse Panel bead-based assay. n = 3 (17D-204) or 5 (Angola71). Data are presented in geometric mean ± 95% CI. Statistical comparisons are determined between 17D-204-infected AB6 and AGB6 mice. (p < 0.05, p < 0.01, p < 0.005; multiple t-test, corrected by Holm–Sidak method) Fig. 3 Cytokine response of 17D-204-infected animal. DISCUSSION Despite the success and widespread use of the 17D YFV vaccine strains, the molecular mechanisms for attenuation of 17D remain unclear. One major restriction on understanding YF pathogenesis has been the lack of pathophysiologically relevant and cost- effective small animal models for studying YFV pathogenesis and attenuation. Previously, we reported that mice lacking type I interferon receptor (IFNAR-/-) are susceptible to subcutaneous (s.c.) infection of wild-type YFV and display signs of viscerotropic disease similar to human YF, whereas 17D is avirulent in this model system.19 In addition, 17D-infection leads to protective immunity against wild-type YFV challenge.16 However, we and others have reported that 17D is virulent in mice lacking both type I and type II IFN receptors (IFNAGR-/-), suggesting that type II IFN is a critical attenuation factor for 17D in vivo. In this study, we investigated the role of type II IFN in controlling replication of 17D in vitro and in vivo. Our results suggest that type II IFN restricts 17D-204 replication and spread in vivo. Furthermore, 17D-204 is more sensitive to the antiviral activities of IFN-γ than wild-type YFV in myeloid, but not ﬁbroblastic, cells in vitro. p p In this report, we examined the role of type II IFN in attenuation of 17D in vivo. Consistent with other studies of 17D vaccination in mice28 and non-human primates (NHP)36, we observed an early induction of IFN-γ after 17D-204 vaccination in our mouse model. Such early induction of IFN-γ resulted in contemporaneous restriction of 17D-204 in AB6 mice. In both AB6 and AGB6 mice, NK1.1+ cells were critical for IFN-γ production; however in AGB6, CD8+ T cells (CTL) were also involved. Lymphocyte activation is driven by antigen abundance and the high virus titers in AGB6 mice may have contributed to a more robust CTL activation and IFN-γ production than in AB6 mice. In addition, a recent report has shown that CTL and NK cells can be infected by 17D in the absence of STAT1. In the report29, deletion of STAT1 in the hematopoietic compartment rendered mice susceptible to intra- venous 17D infection, and enhanced 17D replication in leukocytes from both myeloid and lymphoid lineages was detected in the spleen and circulation. Thus, we speculate that infection of, both CTL and NK cells leads directly or indirectly to IFN-γ secretion. RESULTS Serum cytokines IFN-γ (a), IL-12 (b), MCP-1 (c), MIG (d), IL-5 (e), IL-6 (f), IP-10 (g), and TNFα (h) were quantiﬁed using Cytokine 20-Plex Mouse Panel bead-based assay. n = 3 (17D-204) or 5 (Angola71). Data are presented in geometric mean ± 95% CI. Statistical comparisons are determined between 17D-204-infected AB6 and AGB6 mice. (p < 0.05, p < 0.01, p < 0.005; multiple t-test, corrected by Holm–Sidak method) compartments by type II IFN. In the absence of IFN-γ response, 17D-204 infection in AGB6 mice rapidly disseminated to tissues beyond DLN, and bone marrow. In addition to overall higher viral titers, we observed signs of liver stress and recovered infectious virus particles in the liver on 4 dpi, indicative for a viscerotropic phase of 17D-204 infection with the additional absence of IFN-γ responses. Despite viral clearance in the peripheral tissues, virus accumulated to high titer in the brain associated with signs of neurological disease including immune cell recruitment to the brain and paralysis. Taking together our data and those of other reports,19,20 we propose that 17D-infected AGB6 mice could be used to model neurotropic SAEs associated with 17D vaccination. Despite the presence of a brief viscerotropic phase of 17D-204 infection in AGB6 mice, we did not observe cytokine storm comparable to Angola71-infected AB6 mice or extensive damage to the visceral organs (which can be found in viscerotropic SAE patients32), except the spleen late during infection. These data indicate involvement of additional or alternative factors, such as host genetics33,34 and/or 17D genetic variants,35 both of which have been proposed as sources of SAE. between the viruses, can potentially induce different levels of IFN- α/β in cultured cells and alter sensitivity measurements, we performed the same experiments using cells that lack the ability to produce (IRF3x5x7−/−cells) or respond to (AB6 cells) IFN-α/β (Fig. 5b-c). Cells derived from AGB6 mice, which cannot respond to either type of IFN, were also included as negative controls (Fig. 5d). While 17D-204 and Angola71 multiply to higher titers in IRF3x5x7−/−and AB6 BMMΦ than BMMΦ derived from C57BL/6 mice, 17D-204 is still signiﬁcantly more inhibited by IFN-γ than Angola71. Interestingly, we did not observe signiﬁcant differences between the viruses to either IFN-α/β or IFN-γ treatment in immortalized mouse embryonic ﬁbroblasts (MEFs) (Fig. 5e-f), suggesting that 17D-204 and Angola71 are differentially inhibited by IFNγ-induced antiviral state in a cell type-speciﬁc manner. Published in partnership with the Sealy Center for Vaccine Development DISCUSSION Data are presented in mean ± SD in b, c and geometric mean ± 95% CI in d (p < 0.05, p < 0.01, p < 0.005, **p < 0.001; multiple t-test, corrected by Holm–Sidak method) Whereas in AB6 mice, the presence of IFN-γ response partially protects T cells directly through inducing antiviral state and/or indirectly through control of virus titer, leaving NK cells as the major IFN-γ producer. neurotropic vaccine strain) are possible candidates for inﬂuencing IFN-γ sensitivity.40 For M-L36F, substitution of the analogous amino acid in Japanese encephalitis virus results in a mutant deﬁcient in virion maturation and virus particle production.41 NS4B from various ﬂaviviruses is an antagonist of IFN signaling and inhibits STAT1 translocation.42 It is possible that these mutations, alone or in combination with other 17D mutations, could inﬂuence sensitivity to IFN-γ. However, this remains to be investigated. Although IFN-γ exerts an early effect on viral replication and dissemination, peripheral clearance is also observed in AGB6 mice by 12 dpi, which is suggestive of a type II IFN-independent viral clearance mechanism. The nature of this response also needs to be determined as it may be important in minimizing pathological consequences of 17D immunization such as SAEs. Despite the lack of both type I and type II IFN responses, some antiviral genes were upregulated in our model, especially in the DLN and spleen. These genes may be induced directly without type I or type II IFN through RIG-I and MDA5,43 toll-like receptors,44 or type III IFN,45 which may aid eventual viral clearance from the periphery even in AGB6 mice. However, it was recently reported that mice lacking both type I and type III IFN receptors had higher 17D titers in the brain but not other tissues when compared to While the role IFN-γ plays in inducing protective immunity against 17D requires further study, our data suggest one mechanism by which IFN-γ controls 17D is through inducing a direct antiviral response, especially in macrophages and DCs. Cell type-speciﬁc effects of IFN-γ have been previously observed. DISCUSSION By comparing 17D-204 pathogenesis in AB6 and AGB6 mice, we found 17D-204 replication was largely restricted to lymphoid npj Vaccines (2018) 5 npj Vaccines (2018) 5 IFNγ attenuates YFV vaccine strain 17D-204 in vivo LKM Lam et al. Fig. 4 NK cells are important for IFN-γ production. a, b IFN-γ production in draining lymph nodes cells at 3 dpi. Single cell suspensions were prepared for ﬂow cytometry analysis to identify cell types producing IFN-γ. (n = 6, 1 out of 3 independent experiments is shown; see Fig. S4 for gating strategy.) c, d Depletion of NK cell led to lower serum IFN-γ levels and enhanced viral replication. c ELISA quantiﬁcation of serum IFN-γ at 4 dpi. d Serum virus titer at 4 dpi. Data are presented in mean ± SD in b, c and geometric mean ± 95% CI in d (p < 0.05, p < 0.01, p < 0.005, **p < 0.001; multiple t-test, corrected by Holm–Sidak method) LKM Lam et al. 6 Fig. 4 NK cells are important for IFN-γ production. a, b IFN-γ production in draining lymph nodes cells at 3 dpi. Single cell suspensions were prepared for ﬂow cytometry analysis to identify cell types producing IFN-γ. (n = 6, 1 out of 3 independent experiments is shown; see Fig. S4 for gating strategy.) c, d Depletion of NK cell led to lower serum IFN-γ levels and enhanced viral replication. c ELISA quantiﬁcation of serum IFN-γ at 4 dpi. d Serum virus titer at 4 dpi. Data are presented in mean ± SD in b, c and geometric mean ± 95% CI in d (p < 0.05, p < 0.01, p < 0 005 **p < 0 001; multiple t test corrected by Holm Sidak method) Fig. 4 NK cells are important for IFN-γ production. a, b IFN-γ production in draining lymph nodes cells at 3 dpi. Single cell suspensions were prepared for ﬂow cytometry analysis to identify cell types producing IFN-γ. (n = 6, 1 out of 3 independent experiments is shown; see Fig. S4 for gating strategy.) c, d Depletion of NK cell led to lower serum IFN-γ levels and enhanced viral replication. c ELISA quantiﬁcation of serum IFN-γ at 4 dpi. d Serum virus titer at 4 dpi. Published in partnership with the Sealy Center for Vaccine Development DISCUSSION In mice, IFN-γ-induced antiviral state inhibits murine cytomegalo- virus more robustly in macrophages than in MEF.37 Murine DCs and macrophages also respond to IFN-γ differently.38 Furthermore, the maturation state of macrophages affects the binding of STAT1 to IFN-γ-activated promoter sites (GAS).39 Clearly, further work is needed to elucidate the role of speciﬁc IFN-γ induced antiviral effectors in suppression of 17D replication, in particular, the speciﬁc mutations in 17D responsible for the increased IFN-γ sensitivity need to be identiﬁed and how these mutations increase susceptibility to individual antiviral effectors. Other than muta- tions in E protein that confer increased heparan sulfate binding,14 the effects of mutations in 17D have yet to be characterized. The mutations M-L36F and NS4B-I95M that differentiate between WT strains and attenuated strains (17D substrains and French npj Vaccines (2018) 5 IFNγ attenuates YFV vaccine strain 17D-204 in vivo LKM Lam et al. 7 Fig. 5 17D-204 is more sensitive to IFN-γ-induced antiviral states than wild-type strain Angola71 in myeloid cells. Indicated cell types were treated with designated IFN 12 h prior to infection with 17D-204 or Angola71 at MOI = 0.1. (n = 6, 1 out of 3 independent experiments is shown.) At 48 hpi, supernatants were harvested and infectious virus particles were quantiﬁed by focus forming assay on Vero cells. a–d Bone marrow-derived macrophages and e, f mouse embryonic ﬁbroblasts from various strains of mice. Foci titers (FFU/mL) were normalized to that of untreated cells, and data are presented in mean percentage ± SD. Corresponding foci titers are available in Fig. S5. (p < 0.05, p < 0.01, p < 0.005, **p < 0.001; multiple t-test, corrected by Holm–Sidak method) Fig. 5 17D-204 is more sensitive to IFN-γ-induced antiviral states than wild-type strain Angola71 in myeloid cells. Indicated cell types were treated with designated IFN 12 h prior to infection with 17D-204 or Angola71 at MOI = 0.1. (n = 6, 1 out of 3 independent experiments is shown.) At 48 hpi, supernatants were harvested and infectious virus particles were quantiﬁed by focus forming assay on Vero cells. a–d Bone marrow-derived macrophages and e, f mouse embryonic ﬁbroblasts from various strains of mice. Foci titers (FFU/mL) were normalized to that of untreated cells, and data are presented in mean percentage ± SD. Corresponding foci titers are available in Fig. S5. Ethics statement Animals were maintained and procedures were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council. Protocols 1103456 and 14033545 were approved by the University of Pittsburgh’s IACUC committee. Approved euthanasia criteria were based on weight loss and morbidity. DISCUSSION (p < 0.05, p < 0.01, p < 0.005, ***p < 0.001; multiple t-test, corrected by Holm–Sidak method) mice lacking IFNAR alone, suggestive of a neuroprotective role for type III IFN.46 human vaccination. In contrast, the lack of an IFN-γ response renders 17D virulent and leads to viscerotropic and neurologic disease and eventual death, potentially similar to human SAEs. Enhanced sensitivity to IFN or particular ISGs has been documen- ted in several arbovirus LAVs.50–52 Thus, the deliberate creation of an IFN-γ sensitive phenotype may be a productive approach to rational design of new LAV candidates against arboviruses. yp One potential criticism of a mouse model lacking type I IFN response is the lack of viral control and impaired induction of adaptive immunity. However, evidence suggests infection of these mice recapitulates important aspects of YFV infection and vaccination, namely, viscerotropic disease during WT virus infection and long-term protective immunity after 17D vaccina- tion.16,19 Indeed, robust, protective B and T cell responses to 17D are present in these animals.16 In human vaccinees, the presence of type I IFN does not prevent 17D replication and establishment of serum viremia or mild reactions at the site of vaccination. Whereas in C57BL/6 mice, the presence of type I IFN suppresses 17D replication to the point that replicating viruses cannot be detected by conventional assays, and infected mice do not have local reactions such as footpad swelling after infection. This suggests that YFV is more resistant to, or more capable of suppressing, the human type I IFN system than the murine counterpart. In fact, dengue and Zika virus are more effective in antagonizing human innate immune signaling molecules than murine homologs.17,18,47 Together, these observations suggest that the type I IFN receptor-deﬁcient mouse is a pathophysiolo- gically relevant model that is valuable in exploring the immuno- genicity, pathogenicity, and attenuation mechanisms of different YFV strains. However, type I IFN likely also has an important role in controlling YFV in humans.48,49 Cells lines Vero (ATCC-CCL-81), Huh7 (Charles M. Rice, The Rockefeller University), and mouse embryonic ﬁbroblasts (MEFs, derived based on53) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 0.05 mg/mL streptomycin, 0.29 mg/mL L-glutamine, and 1 mM sodium pyruvate unless otherwise speciﬁed. All cell incubation were performed at 37 °C in 5% CO2 unless otherwise speciﬁed. g These studies have revealed that sensitivity of 17D to murine IFN-γ-mediated response protects IFNAR-/- mice from disease and promotes the eventual development of robust and long-lived protective immunity against wild-type YFV challenge16 similar to Published in partnership with the Sealy Center for Vaccine Development npj Vaccines (2018) 5 Mouse experiments AB6 and AGB6 mice were bred in-house. C57BL/6 mice were purchased from Charles River. Groups of randomized 6-week-old sex-matched mice were infected subcutaneously with 1 × 104 PFU or mock at the hind-limb footpad after isoﬂurane anesthesia. Weight and swelling of footpad were monitored daily for at least 21 days. Animal experiments were not blinded. To harvest tissues, anesthetized mice were bled at submandibular vein and then sacriﬁced with isoﬂurane overdose, followed by cardiac perfusion with virus diluent. Blood were collected in Microtainer serum separator tube (BD, Cat.: 365967) and serum were obtained by centrifugation at 13523g for 5 min at 4 °C. Tissues were stored in virus diluent or Tri Reagent- LS (Invitrogen) at −80 °C until further usage. For histology, mice were perfused with virus diluent and 4% paraformaldehyde (PFA), followed by 24 h ﬁxation in 4% PFA at 4 °C, prior to processing for sectioning. For antibody depletion experiments, 3 doses of 75 μg of mouse IgG2a (C1.18.4) or NK1.1 (PK136, BioXCell) or 3 doses of 150 μg of CD4 (GK1.5) or CD8 (2.43) at −3, −1, and 1 dpi were injected to animals by intraperitoneal route. IgG2a, CD4, and CD8 antibodies were generated from the corresponding hybridomas (ATCC) using CELLine bioreactors (Argos Technologies) according to manufacture’s protocol and puriﬁed with ammonium sulfate precipitation as described previously.54 IFN-γ ELISA ELISA for IFN-γ was performed using the ELISA Ready-Set-Go kit (eBioscience) according to manufacture directions. Virus stocks Stocks of 17D-204 and Angola71 were generated from infectious clones by electroporating in vitro transcribed RNA into Vero cells as described previously.16 Virus-containing supernatant were harvested after incubation Published in partnership with the Sealy Center for Vaccine Development npj Vaccines (2018) 5 IFNγ attenuates YFV vaccine strain 17D-204 in vivo LKM Lam et al. IFNγ attenuates YFV vaccine strain 17D-204 in vivo LKM Lam et al. 8 Generation of bone marrow-derived cells Popliteal (draining) lymph node were harvested from infected animals at 3 dpi, minced, and strained through 70 μm cell strainer (Falcon). Single cell suspensions were cultured in media – RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin, 0.05 mg/mL streptomycin, 20 μM β-mercap- toethanol (Sigma), and 5 μg/mL Brefeldin A (Tonbo bioscience) for 6 h. Cells were stained as previously described.16 Antibody and dyes used in this study were: Fixable Viability dye UV Blue (eBioscience), anti-mouse CD16/32 (93, eBioscience), APC-Cy7-anti-CD8 (2.43, Tonbo bioscience), PerCP-Cy5.5-anti-CD4 (GK1.5, Tonbo bioscience), APC-anti-NK1.1 (PK136, Tonbo bioscience), PE-anti-γδTCR (GL3, eBioscience), and FITC-anti-IFN-γ (XMG1.2, Tonbo bioscience). FITC-rat IgG1 (MOPC-21, Tonbo bioscience) is used as isotype control. Stained cells were ﬁxed in 1% PFA and analyzed using BD LSRFortessa (BD Bioscience) and FlowJo software (Tree Star). Bone marrow-derived macrophages (BMMΦ) and dendritic cells (BMDC) were generated as described previously.19 After 6 days of maturation, DCs were collected from supernatant and macrophages were scraped from plates. Cells were washed, counted, and seeded on U-bottom 96-well plates for DC or 24-well plates for macrophages, followed by treatment with IFNs and virus infection. Histology for 4 (Angola71) or 7 days (17D-204), followed by clariﬁcation by centrifugation at 875g for 30 min and stored at −80 °C. Virus stock titers were quantiﬁed by plaque assay on Huh7. Viruses were diluted to appropriate titer using virus diluent—PBS supplemented with 1% donor bovine serum, 100 U/mL penicillin, and 0.05 mg/mL streptomycin. For Hematoxylin & Eosin (H&E) staining, PBS- and PFA-perfused mouse organs were harvested. Sectioning and staining were performed by the Histology Core at the McGowan Institute of Regenerative Medicine at the University of Pittsburgh. For immunostaining, PBS-perfused mouse organs were harvested fresh, sunk in 30% sucrose at 4 °C, and frozen in O.C.T. medium. For Angola71-infected mice, PFA perfusion and ﬁxation were performed prior to sucrose treatment. Frozen tissues were stored at −80 °C until cryosection. 7 μm (spleen and liver) or 20 μm (brain) sections were permeabilized in 100% methanol at −20 °C, rehydrated in PBS with 0.05% Tween20 (PBST), antigen retrieved by proteinase K digest, and blocked in 2% BSA, 1:200 anti-CD16/32, 5% normal rat serum and goat serum, followed by incubation in M.O.M. reagent (VectorLab). YFV proteins were stained using heat-inactivated immune sera from immunized AB6 mice at 21 dpi or normal serum as isotype control at 1:50 dilution, followed by AlexaFluor488- or AlexaFluor594-conjugated goat-anti-mouse antibody. For staining liver, tissues sections were treated with normal mouse serum prior to incubation in M.O.M. reagent. Speciﬁc cell types were probed with antibodies against F4/80 (BM8, Tonbo Bioscience), CD169 (MOMA-1, Acris Antibodies, Cat.: SM066B), LYVE-1 (Goat polyclonal, R&D systems, Cat.: AF2125), and GFAP (Chicken polyclonal, Abcam, Cat.: Ab4674). Stained slides were mounted on DAPI in SlowFade Gold (Invitrogen) and imaged using FluoView 1000 confocal microscope (Olympus). Plaque assay Huh7 cells were infected with serially diluted inoculum for 1 h. For quantiﬁcation of virus in mouse organs, tissues were homogenized in sterile pestles (Bel-Art), and the virus-containing supernatant were clariﬁed by centrifugation at 13523g for 15 min at 4 °C and used as inoculum. Infected Huh7 were overlaid with media supplemented with 0.5% carboxymethocellulose (CMC, high viscosity, Sigma). At 4 dpi (virus stocks) or 5 dpi (tissues), plaques were visualized by staining with 0.5% crystal violet in 2% PFA. Cytokine and gene expression analyses Cytokine and gene expression analyses Cytokine and gene expression analyses Serum cytokines were quantiﬁed by the Cytokine 20-plex Mouse Panel (Invitrogen) according to manufacturer protocol. To analyze gene expression in different organs, RNA from tissues was isolated using Tri reagent-LS (Invitrogen) following the manufacturer protocol. Polyacryl carrier was added to serum and lymph node samples only. Reverse transcription of 100 ng of puriﬁed RNA was performed using random hexamer and TaqMan reverse transcription reagent (AB) under the following condition: 25 °C for 10 min, followed by extension at 48 °C for 30 min, and inactivation of enzymes at 95 °C for 5 min. Quantitation of 18S cDNA and antiviral genes was performed in Maxima Probe qPCR Master Mix (Thermo) and Maxima SYBR Green/Rox qPCR mix, respectively. Fluorescent intensity data were collected on a 7900HT real-time PCR system (AB). Thermocycling conditions were as follow: denaturing and polymerase activation at 95 °C for 10 min, followed by 40 cycles of denaturing at 95 °C for 15 s and extension at 60 °C for 1 min with ﬂuorescent intensity data collection. An additional melting curve cycle was added for quality controls for antiviral genes. Primers and probes used in PCR were listed in Table S1. Virus infection Mouse embryonic ﬁbroblasts (MEFs), macrophages, and dendritic cells were treated with various concentrations of IFN-γ (Peprotech) or IFN-α4/β (made in-house as described previously55) for 12 h. Media containing IFNs were removed prior to infection with YFVs at designated MOI for 1 h. Cells were washed three times after infection. For BMDC, cells were pelleted at 219g for 5 min at 4 °C between washes. Infected cells were maintained in their corresponding media. Supernatant was harvested daily for focus- forming assay. Focus-forming assay 4 1 × 104 Vero cells were seeded on 96-well plate, followed by infection with serially diluted virus inoculum. After 1 h infection, cells were washed and overlaid with media supplemented with 0.5% CMC for 96 h. Cell monolayers were washed in PBS and ﬁxed in 4% PFA for 24 h. Fixed cells were washed and permeabilized with 100% methanol at −20 °C for 10 min, followed by washes in PBS containing 0.05% tween20 (PBST). Nonspeciﬁc protein binding was blocked by incubation in PBST with 1% BSA and 5% normal goat serum for 1 h at room temperature. YFV proteins were stained using heat-inactivated immune sera from immunized AB6 mice at 21 dpi at 1:100 dilution for 24 h at 4 °C, followed by probing with HRP-goat anti- mouse antibody for 1 h at room temperature. Foci were visualized by 3,3’- diaminobenzidine as substrate (ThermoFisher, cat.: 34065). Statistical analysis All data were analyzed with GraphPad Prism software. Log-rank test was performed on survival studies. 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Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 28. Neves, P. C. C., Santos, J. R., Tubarão, L. N., Bonaldo, M. C. & Galler, R. Early IFN- gamma production after YF 17D vaccine virus immunization in mice and its association with adaptive immune responses. PLoS ONE 8, e81953 (2013). ACKNOWLEDGEMENTS 19. Meier, K. C., Gardner, C. L., Khoretonenko, M. V., Klimstra, W. B. & Ryman, K. D. A mouse model for studying viscerotropic disease caused by yellow fever virus infection. PLoS Pathog. 5, e1000614 (2009). We thank Chelsea Maksin for management of mouse colonies and Matthew Dunn for excellent technical assistance. We would like to acknowledge Lori Walton from the Histology Core at the McGowan Institute of Regenerative Medicine at the University of Pittsburgh. This study was funded by R01 AI081886 from the National Institute of Allergy and Infectious Diseases to K.D.R. and W.B.K. Microscopy work were performed in Center for Biologic Imagining in the University of Pittsburgh supported by NIH grant 1S10OD019973-01. 20. Thibodeaux, B. A. et al. A small animal peripheral challenge model of yellow fever using interferon-receptor deﬁcient mice and the 17D-204 vaccine strain. Vaccine 30, 3180–3187 (2012). 21. Breugelmans, J. G. et al. Adverse events following yellow fever preventive vac- cination campaigns in eight African countries from 2007 to 2010. Vaccine 31, 1819–1829 (2013). 22. de Menezes Martins, R., da Luz Fernandes Leal, M. & Homma, A. Serious adverse events associated with yellow fever vaccine. Hum. Vaccin. Immunother. 11, 2183–2187 (2015). 9 AUTHOR CONTRIBUTIONS All authors designed the research and analyzed the data; L.K.M.L. and A.M.W. performed research. L.K.M.L, A.M.W., and W.B.K. wrote the manuscript. All authors read and approved the ﬁnal manuscript version. 23. Lin, F.-C. & Young, H. A. The talented interferon-gamma. Adv. Biosci. Biotechnol. 4, 6–13 (2013). 24. Campi-Azevedo, A. C. et al. Subdoses of 17DD yellow fever vaccine elicit equivalent virological/immunological kinetics timeline. Bmc. Infect. Dis. 14, 391 (2014). ADDITIONAL INFORMATION 25. Finkelman, F. D., Katona, I. M., Mosmann, T. R. & Coffman, R. L. IFN-gamma regulates the isotypes of Ig secreted during in vivo humoral immune responses. J. Immunol. Baltim. Md 1950 140, 1022–1027 (1988). Supplementary information accompanies the paper on the npj Vaccines website (https://doi.org/10.1038/s41541-017-0039-z). Supplementary information accompanies the paper on the npj Vaccines website (https://doi.org/10.1038/s41541-017-0039-z). 26. Schoenborn, J. R. & Wilson, C. B. Regulation of interferon-gamma during innate and adaptive immune responses. Adv. Immunol. 96, 41–101 (2007). Data availability statement Data availability statement Data availability statement All relevant data from this study are available from the authors. Published in partnership with the Sealy Center for Vaccine Development Published in partnership with the Sealy Center for Vaccine Development npj Vaccines (2018) 5 IFNγ attenuates YFV vaccine strain 17D-204 in vivo LKM Lam et al. IFNγ attenuates YFV vaccine strain 17D-204 in vivo REFERENCES Zika virus targets human STAT2 to inhibit type i interferon sig- naling. Cell Host Microbe 19, 882–890 (2016). 55. Yin, J., Gardner, C. L., Burke, C. W., Ryman, K. D. & Klimstra, W. B. Similarities and differences in antagonism of neuron alpha/beta interferon responses by vene- zuelan equine encephalitis and sindbis alphaviruses. J. Virol. 83, 10036–10047 (2009). 48. Querec, T. D. et al. Systems biology approach predicts immunogenicity of the yellow fever vaccine in humans. Nat. Immunol. 10, 116–125 (2009). 48. Querec, T. D. et al. Systems biology approach predicts immu yellow fever vaccine in humans. Nat. Immunol. 10, 116–125 (200 49. Gaucher, D. et al. 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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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons. org/licenses/by/4.0/. 50. Gardner, C. L., Burke, C. W., Higgs, S. T., Klimstra, W. B. & Ryman, K. D. 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https://openalex.org/W4388008387	https://www.nature.com/articles/s41598-023-45903-9.pdf	English	null	Serum oxalate concentration is associated with coronary artery calcification and cardiovascular events in Japanese dialysis patients	Scientific reports	2,023	cc-by	9,501	Serum oxalate concentration is associated with coronary artery calcification and cardiovascular events in Japanese dialysis patients OPEN Yoko Nishizawa 1,2, Satoshi Miyata 2, Mai Tosaka 1, Eriko Hirasawa 1, Yumi Hosoda 1, Ai Horimoto 1, Kiyotsugu Omae 1, Kyoko Ito 3, Nobuo Nagano 3, Junichi Hoshino 4 & Tetsuya Ogawa 1,3 Coronary artery calcification (CAC) is associated with cardiovascular disease (CVD). CAC might contain calcium oxalate, and a high serum oxalate (SOx) concentration is associated with cardiovascular mortality in dialysis patients. We assessed the associations between SOx and CAC or CVD events in Japanese hemodialysis patients. This cross-sectional and retrospective cohort study was done in 2011. Seventy-seven hemodialysis patients’ Agatston CAC score was measured, and serum samples were collected. SOx concentrations were measured in 2021 by using frozen samples. Also, new-onset CVD events in 2011–2021 were retrospectively recorded. The association between SOx concentration and CAC score ≥ 1000, and new-onset CVD events were examined. Median SOx concentration and CAC score were 266.9 (229.5–318.5) µmol/L and 912.5 (123.7–2944), respectively. CAC score ≥ 1000 was associated with SOx [adjusted odds ratio (OR) 1.01, 95% confidence interval (CI), 1.00–1.02]. The number of new-onset CVD events was significantly higher in patients with SOx ≥ median value [hazard ratio (HR) 2.71, 95% CI 1.26–6.16]. By Cox proportional hazard models, new-onset CVD events was associated with SOx ≥ median value (adjusted HR 2.10, 95% CI 0.90–4.91). SOx was associated with CAC score ≥ 1000 and new-onset CVD events in Japanese hemodialysis patients. Cardiovascular disease (CVD) is a major complication of end-stage renal disease (ESRD)1. Vascular calcifica- tion, especially coronary artery calcification (CAC), is associated with CVD in ESRD patients2,3. Especially, “very high” CAC individuals, defined as Agatston CAC score4 ≥ 1000, are increasingly recognized as higher risk for CVD events and mortality5–7. A large body of evidence suggests that dysregulation of calcium and phosphate homeostasis, which are factors related to mineral and bone disorder in chronic kidney disease patients, has direct effects on vascular smooth muscle cells and promotes vascular calcification8. Indeed, vascular calcification in ESRD patients is formed from hydroxyapatite and calcium phosphate, both of which contain phosphate9,10. Phosphate binders and other treatments are available for the control of mineral and bone disorders11; however, even after these treatments, the prevalence of vascular calcification is still higher in ESRD patients than in the population with normal kidney function. p p y Studies in non-ESRD patients12,13 and ESRD patients14 indicate that CAC contains calcium oxalate crys- tals. www.nature.com/scientificreports www.nature.com/scientificreports Baseline patient characteristics Of the 77 patients that underwent the voluntary atherosclerosis checkup, 17 had normal SOx concentrations and were excluded from the study. There were no missing values except for ankle-brachial index of two cases. Of the remaining 60 patients, 41 (68.3%) were male, mean age was 63.1 ± 11.9 years, and median dialysis dura- tion was 87.6 (44.1–152.2) months. Twenty-one patients (35%) had diabetes mellitus and 10 (16.7%) had CVD. Median SOx, Agatston CAC score, and major artery calcification volume were 266.9 (229.5–318.5) µmol/L, 912.5 (123.7–2943.8), and 7.0 (3.0–18.4) cm3, respectively; these data were right skewed. When the subjects were divided into two groups according to CAC score ≥ 1000, baseline CVD, serum phosphate, low-density lipopro- tein (LDL) cholesterol, and major artery calcification volume were significantly higher in the CAC ≥ 1000 group. Table 1 provides the baseline patient characteristics. www.nature.com/scientificreports/ suggests that controlling SOx could be a novel strategy to reduce the risk of CVD in ESRD patients. However, the mechanism of how oxalate affects CVD is currently unknown, limiting the rational development of an effective therapeutic approach. p pp Here, we conducted analyses to examine the association between SOx and CAC and the association between SOx and CVD events in Japanese hemodialysis patients. First, we conducted a cross-sectional analysis to under- stand more about the relationship between high SOx concentration and CAC ≥ 10005–7. Then, we conducted a retrospective cohort study to understand more about the relationship between SOx and CVD events. Serum oxalate concentration is associated with coronary artery calcification and cardiovascular events in Japanese dialysis patients OPEN Oxalate is an organic acid abundant in plants such as spinach, where it acts as a controller of calcium concentration15. However, in humans, it is a waste product16. Urine is the major excretion pathway of oxalate17, meaning that patients with decreased kidney function also show elevated serum oxalate (SOx) concentrations, which can be 20–100 times normal in ESRD patients18. SOx is a small molecule, and its serum concentration can be reduced by approximately 90% per hemodialysis session, although it can easily rebound to its pre-dialysis level; for example, at only 2 h after hemodialysis treatment, SOx can already be back at its mid-dialysis level19. Excess SOx combines with calcium to form calcium oxalate crystals that are deposited in various tissues; for example, myocardium, renal tubules, and interstitium20,21. A recent study has reported a relationship between high SOx concentration and CVD mortality, especially in dialysis patients with high SOx concentrations22. This 1Department of Medicine, Tokyo Women’s Medical University Adachi Medical Center, 4‑33‑1, Kohoku, Adachi, Tokyo 123‑8558, Japan. 2Teikyo University Graduate School of Public Health, Itabashi, Tokyo, Japan. 3Kidney Disease and Dialysis Center, Hidaka Hospital, Hidaka‑kai, Takasaki, Gunma, Japan. 4Department of Nephrology, Tokyo Women’s Medical University, Shinjuku, Tokyo, Japan. email: nishizawa.youko@twmu.ac.jp \| https://doi.org/10.1038/s41598-023-45903-9 Scientific Reports \| (2023) 13:18558 www.nature.com/scientificreports/ Cross‑sectional analysis of the association between SOx and vascular calcificationi yi To examine the association between SOx and vascular calcification, we performed a logistic regression analysis. The patients were stratified into those with CAC score < 1000 and those with CAC score ≥ 1000. All of the patients with CVD (n = 10) were in the CAC score ≥ 1000 group; those patients were excluded from this analysis, leaving 50 patients in the analysis group. Table 1. Baseline patient characteristics. Continuous variables are reported as mean ± standard deviation for normally distributed data or median (inter-quartile range) for non-normally distributed data. Discrete variables are expressed as numeral (percentage). The subjects were divided into two groups according to CA score, and statistical significance was tested by using Welch’s t-test for normally distributed data, the Wilcoxo rank sum test for non-normally distributed data, and Fisher’s exact test for discrete variables. A two-tailed P value of < 0.05 was considered to indicate statistical significance. CAC coronary artery calcification, HDL hig density lipoprotein, LDL low-density lipoprotein, SOx serum oxalate concentration. Welch’s t-test; †Fisher’s exact test; ‡the Wilcoxon rank sum test. Cross‑sectional analysis of the association between SOx and vascular calcificationi All (n = 60) CAC < 1,000 (n = 33) CAC ≥ 1,000 (n = 27) P Age (years) 63.1 ± 11.9 61.8 ± 13.1 64.7 ± 10.4 0.335 Male 41 (68.3%) 19 (57.6%) 22 (81.5%) 0.057† Hemodialysis duration (months) 87.6 (44.1, 152.2) 87.4 (42.1, 138.8) 96.9 (47.5, 165.2) 0.637‡ Cardiovascular disease 10 (16.7%) 0 (0%) 10 (37%) < 0.001† Diabetes mellitus 21 (35%) 12 (36.4%) 9 (33.3%) 1.000† Body mass index (kg/m2) 21.5 (20.6, 23.2) 21.4 (20.4, 22.9) 21.5 (20.9, 23.5) 0.440‡ Ankle-brachial index 1.2 (1.1, 1.2) 1.2 (1.1, 1.2) 1.2 (1.1, 1.2) 0.789‡ Albumin-adjusted calcium (mg/dL) 9.0 ± 0.5 9.0 ± 0.5 9.0 ± 0.4 0.940* Serum phosphate (mg/dL) 5.2 (4.6, 5.7) 4.9 (4.5, 5.2) 5.5 (4.7, 6.1) 0.020‡ Intact parathyroid hormone (pg/mL) 176.2 (152.1, 245.4) 171.8 (152.2, 211.7) 188.4 (156.3, 255.8) 0.352‡ Alkaline phosphatase (U/mL) 259.1 (205.5, 318.8) 264.3 (212.5, 346.0) 251.2 (197.4, 282.7) 0.178‡ Magnesium (mg/dL) 2.6 ± 0.3 2.6 ± 0.3 2.5 ± 0.3 0.174* Triglyceride (mg/dL) 107.3 (70.6, 148.9) 94.3 (58.5, 144.8) 111.3 (96.7, 154.4) 0.099‡ LDL cholesterol (mg/dL) 86.2 ± 21.6 81.0 ± 21.0 92.7 ± 21.5 0.038* HDL cholesterol (mg/dL) 40.7 (32.8, 52.6) 44.2 (34.9, 54.6) 36.8 (29.7, 51.0) 0.115‡ Uric acid (mg/dL) 7.1 ± 0.9 7.1 ± 0.8 7.2 ± 1.0 0.548* Beta-2 microglobulin (µg/L) 27.1 (23.0, 29.2) 27.3 (21.2, 29.2) 27.0 (23.3, 29.1) 0.876‡ Blood urea nitrogen (mg/dL) 60.1 ± 10.2 59.0 ± 8.6 61.4 ± 11.9 0.397* Hemoglobin (g/dL) 10.7 (10.3, 11.0) 10.9 (10.5, 11.1) 10.4 (10.0, 10.9) 0.023‡ Serum albumin (g/dL) 3.7 (3.6, 4.0) 3.7 (3.6, 4.0) 3.7 (3.6, 4.0) 0.994‡ Calcium carbonate intake 49 (81.7%) 27 (81.8%) 22 (81.5%) 1.000† Vitamin D medication 26 (43.3%) 11 (33.3%) 15 (55.6%) 0.117† Statin intake 10 (16.7%) 5 (15.2%) 5 (18.5%) 0.742† SOx (μg/L) 266.9 (229.5, 318.5) 256.1 (228.2, 290.1) 280.4 (251.3, 355.5) 0.081‡ Major artery calcification volume (cm3) 7.0 (3.0, 18.4) 3.6 (1.6, 8.6) 12.2 (6.1, 31.3) < 0.001‡ All (n = 60) CAC < 1,000 (n = 33) CAC ≥ 1,000 (n = 27) P Age (years) 63.1 ± 11.9 61.8 ± 13.1 64.7 ± 10.4 0.335* Male 41 (68.3%) 19 (57.6%) 22 (81.5%) 0.057† Hemodialysis duration (months) 87.6 (44.1, 152.2) 87.4 (42.1, 138.8) 96.9 (47.5, 165.2) 0.637‡ Cardiovascular disease 10 (16.7%) 0 (0%) 10 (37%) < 0.001† Diabetes mellitus 21 (35%) 12 (36.4%) 9 (33.3%) 1.000† Body mass index (kg/m2) 21.5 (20.6, 23.2) 21.4 (20.4, 22.9) 21.5 (20.9, 23.5) 0.440‡ Ankle-brachial index 1.2 (1.1, 1.2) 1.2 (1.1, 1.2) 1.2 (1.1, 1.2) 0.789‡ Albumin-adjusted calcium (mg/dL) 9.0 ± 0.5 9.0 ± 0.5 9.0 ± 0.4 0.940* Serum phosphate (mg/dL) 5.2 (4.6, 5.7) 4.9 (4.5, 5.2) 5.5 (4.7, 6.1) 0.020‡ Intact parathyroid hormone (pg/mL) 176.2 (152.1, 245.4) 171.8 (152.2, 211.7) 188.4 (156.3, 255.8) 0.352‡ Alkaline phosphatase (U/mL) 259.1 (205.5, 318.8) 264.3 (212.5, 346.0) 251.2 (197.4, 282.7) 0.178‡ Magnesium (mg/dL) 2.6 ± 0.3 2.6 ± 0.3 2.5 ± 0.3 0.174* Triglyceride (mg/dL) 107.3 (70.6, 148.9) 94.3 (58.5, 144.8) 111.3 (96.7, 154.4) 0.099‡ LDL cholesterol (mg/dL) 86.2 ± 21.6 81.0 ± 21.0 92.7 ± 21.5 0.038* HDL cholesterol (mg/dL) 40.7 (32.8, 52.6) 44.2 (34.9, 54.6) 36.8 (29.7, 51.0) 0.115‡ Uric acid (mg/dL) 7.1 ± 0.9 7.1 ± 0.8 7.2 ± 1.0 0.548* Beta-2 microglobulin (µg/L) 27.1 (23.0, 29.2) 27.3 (21.2, 29.2) 27.0 (23.3, 29.1) 0.876‡ Blood urea nitrogen (mg/dL) 60.1 ± 10.2 59.0 ± 8.6 61.4 ± 11.9 0.397* Hemoglobin (g/dL) 10.7 (10.3, 11.0) 10.9 (10.5, 11.1) 10.4 (10.0, 10.9) 0.023‡ Serum albumin (g/dL) 3.7 (3.6, 4.0) 3.7 (3.6, 4.0) 3.7 (3.6, 4.0) 0.994‡ Calcium carbonate intake 49 (81.7%) 27 (81.8%) 22 (81.5%) 1.000† Vitamin D medication 26 (43.3%) 11 (33.3%) 15 (55.6%) 0.117† Statin intake 10 (16.7%) 5 (15.2%) 5 (18.5%) 0.742† SOx (μg/L) 266.9 (229.5, 318.5) 256.1 (228.2, 290.1) 280.4 (251.3, 355.5) 0.081‡ Major artery calcification volume (cm3) 7.0 (3.0, 18.4) 3.6 (1.6, 8.6) 12.2 (6.1, 31.3) < 0.001‡ Table 1. Table 1. Baseline patient characteristics. Continuous variables are reported as mean ± standard deviation for normally distributed data or median (inter-quartile range) for non-normally distributed data. Discrete variables are expressed as numeral (percentage). The subjects were divided into two groups according to CAC score, and statistical significance was tested by using Welch’s t-test for normally distributed data, the Wilcoxon rank sum test for non-normally distributed data, and Fisher’s exact test for discrete variables. A two-tailed P value of < 0.05 was considered to indicate statistical significance. CAC coronary artery calcification, HDL high- density lipoprotein, LDL low-density lipoprotein, SOx serum oxalate concentration. Welch’s t-test; †Fisher’s exact test; ‡the Wilcoxon rank sum test. www.nature.com/scientificreports/ In a risk pre- diction model predicting CAC score ≥ 1000, we used the five selected factors (i.e., SOx, male, serum phosphate, intact parathyroid hormone, and LDL cholesterol); the area under the receiver operating characteristic (ROC) curve was 0.87 (95% CI 0.76–0.98), and sensitivity and specificity were 81.8% and 76.5%, respectively (Fig. 1).i ( ), y pi y , p y ( g ) In a univariate analysis examining associations with major artery calcification volume, variables with a P value < 0.2 were SOx, age, male, albumin-adjusted calcium, HDL cholesterol, beta-2 microglobulin, serum albumin, and calcium carbonate intake (Table 3). In a multivariate analysis using these selected variables after variable selection, SOx (β = 0.03, 95% CI 0.00–0.06, P = 0.084), age (β = 0.26, 95% CI 0.05–0.47, P = 0.018), and beta-2 microglobulin (β = 0.55, 95% CI 0.00–1.09, P = 0.048) were selected as factors associated with major artery calcification volume (Table 3). Retrospective cohort analysis of the relationship between SOx and new‑onset CVD events During the 10-year observation period, seven patients (11.7%) were censored due to transfer to another dialysis center, three patients (5.0%) were censored due to kidney transplantation, and nine patients (15.0%) died before experiencing a CVD event. A total of 29 (48.3%) patients experienced new-onset CVD events. We divided the subjects into two groups using the median SOx value of 266.9 µmol/L. Table 2. Association between SOx and coronary artery calcification score ≥ 1000 by logistic regression analysis. In the analysis, we divided subjects into two groups: those with CAC score < 1000 and those with CAC score ≥ 1000. A logistic regression analysis was performed for the association between SOx and CAC. The explanatory variable was SOx, and the covariates were factors that are reported to be associated with vascular calcification8,25,32,33 or CVD29,34–36. A univariable analysis was performed first, and factors with a two-tailed P value < 0.2 were used for a multivariable analysis. For variable selection, we used Akaike’s information criteria with stepwise backward elimination. A two-tailed P value of < 0.05 was considered to indicate statistical significance. aCa albumin-adjusted calcium, AIC Akaike’s information criteria, ALP alkaline phosphatase, β2MG beta-2 microglobulin, BUN blood urea nitrogen, BMI body mass index, CaCO3 calcium carbonate, CI confidence interval, HD hemodialysis, HDL-C high-density lipoprotein cholesterol, iPTH intact parathyroid hormone, LDL-C low-density lipoprotein cholesterol, SOx serum oxalate concentration. www.nature.com/scientificreports/ www.nature.com/scientificreports/ In a univariate analysis examining associations with CAC score ≥ 1000, variables with P value < 0.2 were SOx concentration, male, body mass index (BMI), serum phosphate, intact parathyroid hormone, triglyceride, LDL cholesterol, high-density lipoprotein (HDL) cholesterol, uric acid, and blood urea nitrogen (Table 2). In a multivariate analysis using these selected variables, SOx [odds ratio (OR) 1.01, 95% confidence interval (CI) 1.00–1.02, P = 0.168], male (OR 6.90, 95% CI 1.10–77.5, P = 0.065), serum phosphate (OR 2.82, 95% CI 1.17–8.94, P = 0.033), intact parathyroid hormone (OR 1.01, 95% CI 1.00–1.01, P = 0.058), and LDL cholesterol (OR 1.04, 95% CI 1.00–1.10, P = 0.055) were selected as factors associated with CAC score ≥ 1000 (Table 2). In a risk pre- diction model predicting CAC score ≥ 1000, we used the five selected factors (i.e., SOx, male, serum phosphate, intact parathyroid hormone, and LDL cholesterol); the area under the receiver operating characteristic (ROC) curve was 0.87 (95% CI 0.76–0.98), and sensitivity and specificity were 81.8% and 76.5%, respectively (Fig. 1). In a univariate analysis examining associations with major artery calcification volume, variables with a P value < 0.2 were SOx, age, male, albumin-adjusted calcium, HDL cholesterol, beta-2 microglobulin, serum albumin, and calcium carbonate intake (Table 3). In a multivariate analysis using these selected variables after variable selection, SOx (β = 0.03, 95% CI 0.00–0.06, P = 0.084), age (β = 0.26, 95% CI 0.05–0.47, P = 0.018), and beta-2 microglobulin (β = 0.55, 95% CI 0.00–1.09, P = 0.048) were selected as factors associated with major artery calcification volume (Table 3). In a univariate analysis examining associations with CAC score ≥ 1000, variables with P value < 0.2 were SOx concentration, male, body mass index (BMI), serum phosphate, intact parathyroid hormone, triglyceride, LDL cholesterol, high-density lipoprotein (HDL) cholesterol, uric acid, and blood urea nitrogen (Table 2). In a multivariate analysis using these selected variables, SOx [odds ratio (OR) 1.01, 95% confidence interval (CI) 1.00–1.02, P = 0.168], male (OR 6.90, 95% CI 1.10–77.5, P = 0.065), serum phosphate (OR 2.82, 95% CI 1.17–8.94, P = 0.033), intact parathyroid hormone (OR 1.01, 95% CI 1.00–1.01, P = 0.058), and LDL cholesterol (OR 1.04, 95% CI 1.00–1.10, P = 0.055) were selected as factors associated with CAC score ≥ 1000 (Table 2). Cross‑sectional analysis of the association between SOx and vascular calcificationi Baseline patient characteristics. Continuous variables are reported as mean ± standard deviation for normally distributed data or median (inter-quartile range) for non-normally distributed data. Discrete variables are expressed as numeral (percentage). The subjects were divided into two groups according to CAC score, and statistical significance was tested by using Welch’s t-test for normally distributed data, the Wilcoxon rank sum test for non-normally distributed data, and Fisher’s exact test for discrete variables. A two-tailed P value of < 0.05 was considered to indicate statistical significance. CAC coronary artery calcification, HDL high- density lipoprotein, LDL low-density lipoprotein, SOx serum oxalate concentration. Welch’s t-test; †Fisher’s exact test; ‡the Wilcoxon rank sum test. Table 1. Baseline patient characteristics. Continuous variables are reported as mean ± standard deviation for normally distributed data or median (inter-quartile range) for non-normally distributed data. Discrete variables are expressed as numeral (percentage). The subjects were divided into two groups according to CAC score, and statistical significance was tested by using Welch’s t-test for normally distributed data, the Wilcoxon rank sum test for non-normally distributed data, and Fisher’s exact test for discrete variables. A two-tailed P value of < 0.05 was considered to indicate statistical significance. CAC coronary artery calcification, HDL high- density lipoprotein, LDL low-density lipoprotein, SOx serum oxalate concentration. *Welch’s t-test; †Fisher’s exact test; ‡the Wilcoxon rank sum test. https://doi.org/10.1038/s41598-023-45903-9 Scientific Reports \| (2023) 13:18558 \| www.nature.com/scientificreports/ New-onset CVD events occurred in 19/30 (63.3%) patients with SOx greater than or equal to the median value, whereas new-onset CVD events occurred Univariable analysis Multivariable analysis Variable selection (AIC) Odds ratio 95% CI P Odds ratio 95% CI P Odds ratio 95% CI P SOx (µmol/L) 1.01 1.00–1.02 0.038 1.01 1.00–1.03 0.099 1.01 1.00–1.02 0.168 Age (years) 1.00 0.95–1.05 0.854 Male 3.44 0.91–17.02 0.089 24.29 1.14–1672.3 0.076 6.90 1.10–77.5 0.065 HD duration (months) 1.00 1.00–1.01 0.235 Diabetes mellitus 0.95 0.27–3.20 0.941 BMI (kg/m2) 1.12 0.95–1.36 0.196 1.38 0.94–2.25 0.128 Ankle-brachial index 0.80 0.01–92.26 0.923 aCa (mg/dL) 1.36 0.43–4.63 0.604 Phosphate (mg/dL) 3.40 1.48–10.04 0.011 2.95 0.84–14.0 0.127 2.82 1.17–8.94 0.033 iPTH (pg/dL) 1.01 1.00–1.01 0.054 1.01 1.00–1.03 0.025 1.01 1.00–1.01 0.058 ALP (U/mL) 1.00 0.99–1.00 0.773 Magnesium (mg/dL) 0.46 0.05–3.62 0.467 Triglyceride (mg/dL) 1.01 1.00–1.02 0.105 0.98 0.96–1.00 0.116 LDL-C (mg/dL) 1.03 1.00–1.06 0.088 1.07 1.01–1.14 0.031 1.04 1.00–1.10 0.055 HDL-C (mg/dL) 0.96 0.92–1.01 0.126 0.95 0.83–1.04 0.337 Uric acid (mg/dL) 1.60 0.79–3.39 0.198 2.85 0.55–20.2 0.239 β2MG (µg/L) 1.08 0.95–1.27 0.277 BUN (mg/dL) 1.04 0.98–1.11 0.190 0.92 0.79–1.04 0.211 Hemoglobin (g/dL) 0.77 0.32–1.62 0.519 Serum albumin (g/dL) 1.11 0.12–9.93 0.927 CaCO3 intake 3.56 0.54–70.37 0.260 Vitamin D medication 1.40 0.41–4.70 0.585 Statin intake 0.75 0.10–3.94 0.744 https://doi.org/10.1038/s41598-023-45903-9 Scientific Reports \| (2023) 13:18558 \| www.nature.com/scientificreports/ Figure 1. Receiving operating curve for predicting Agatston coronary artery calcification score ≥ 1000. In a risk prediction model for predicting CAC score ≥ 1000, we used five factors obtained from an earlier variable selection (i.e., SOx, male, serum phosphate, intact parathyroid hormone, and LDL cholesterol). The area under the receiver operating characteristic curve was 0.87 (95%CI 0.76–0.98), and sensitivity and specificity were 81.8% and 76.5%, respectively. AUC area under the curve, CAC coronary artery calcification, CI confidence interval, LDL low-density lipoprotein, SOx serum oxalate concentration. Figure 1. Receiving operating curve for predicting Agatston coronary artery calcification score ≥ 1000. In a risk prediction model for predicting CAC score ≥ 1000, we used five factors obtained from an earlier variable selection (i.e., SOx, male, serum phosphate, intact parathyroid hormone, and LDL cholesterol). The area under the receiver operating characteristic curve was 0.87 (95%CI 0.76–0.98), and sensitivity and specificity were 81.8% and 76.5%, respectively. AUC area under the curve, CAC coronary artery calcification, CI confidence interval, LDL low-density lipoprotein, SOx serum oxalate concentration. www.nature.com/scientificreports/ in only 10/30 (33.3%) patients with SOx lower than the median value; the risk of new-onset CVD events was greater in the patients with SOx greater than or equal to the median value than in those with SOx lower than the median value [hazard ratio (HR) 2.71, 95% CI 1.26–6.16, P = 0.008; Fig. 2a]. By restricting the analysis to patients with CAC score < 1000 and without history of CVD (n = 33), CVD events occurred in 10/13 (76.9%) patients with SOx greater than or equal to the median value and in 7/20 (35.0%) patients with SOx lower than the median value, respectively. The number of events was significantly higher in the patients with SOx greater than or equal to the median value than in the patients with lower than the median value (HR 2.94, 95% CI 1.10–7.85, P = 0.020; Fig. 2b). g We performed Cox proportional hazard analyses to examine the relationship between SOx and new-onset CVD events. In a univariate analysis, variables with a P value < 0.2 were SOx greater than or equal to the median value, age, male, history of CVD, alkaline phosphatase, HDL cholesterol, and hemoglobin (Table 4). In a multivari- able analysis using these variables after variable selection, SOx (HR 2.10, 95% CI 0.90–4.91, P = 0.086), history of CVD (HR 3.84, 95% CI 1.44–10.2, P = 0.007), and HDL cholesterol (HR 0.97, 95% CI 0.94–1.00, P = 0.062) were selected as factors associated with new-onset CVD events (Table 4). Discussion Association between SOx and major artery calcification volume by linear regression analysis. A linear regression analysis was performed for the association between SOx and major artery calcification volume. The explanatory variable was SOx, and the covariates were factors that are reported to be associated with vascular calcification8,25,32,33 or CVD29,34–36. A univariable analysis was performed first, and factors with a two-tailed P value < 0.2 were used for a multivariable analysis. For variable selection, we used Akaike’s information criteria with stepwise backward elimination. A two-tailed P value of < 0.05 was considered to indicate statistical significance. aCa albumin-adjusted calcium, AIC Akaike’s information criteria, ALP alkaline phosphatase, β2MG beta-2 microglobulin, BUN blood urea nitrogen, BMI body mass index, CaCO3 calcium carbonate, CI confidence interval, HD hemodialysis, HDL-C high-density lipoprotein cholesterol, iPTH intact parathyroid hormone, LDL-C low-density lipoprotein cholesterol, SOx serum oxalate concentration. Table 3. Association between SOx and major artery calcification volume by linear regression analysis. A linear regression analysis was performed for the association between SOx and major artery calcification volume. The explanatory variable was SOx, and the covariates were factors that are reported to be associated with vascular calcification8,25,32,33 or CVD29,34–36. A univariable analysis was performed first, and factors with a two-tailed P value < 0.2 were used for a multivariable analysis. For variable selection, we used Akaike’s information criteria with stepwise backward elimination. A two-tailed P value of < 0.05 was considered to indicate statistical significance. aCa albumin-adjusted calcium, AIC Akaike’s information criteria, ALP alkaline phosphatase, β2MG beta-2 microglobulin, BUN blood urea nitrogen, BMI body mass index, CaCO3 calcium carbonate, CI confidence interval, HD hemodialysis, HDL-C high-density lipoprotein cholesterol, iPTH intact parathyroid hormone, LDL-C low-density lipoprotein cholesterol, SOx serum oxalate concentration. example, mineral and bone disorder factors or LDL cholesterol. In addition, our study also revealed a relationship between SOx and CVD events, which is consistent with the findings from other study22. xample, mineral and bone disorder factors or LDL cholesterol. In addition, our study also revealed a relationship etween SOx and CVD events, which is consistent with the findings from other study22.i Oxi g y Although we found that higher SOx concentration was associated with vascular calcification and CVD events, it is important to note that many factors are involved in these outcomes. Discussion For variable selection, we used Akaike’s information criteria with stepwise backward elimination. A two-tailed P value of < 0.05 was considered to indicate statistical significance. aCa albumin-adjusted calcium, AIC Akaike’s information criteria, ALP alkaline phosphatase, β2MG beta-2 microglobulin, BUN blood urea nitrogen, BMI body mass index, CaCO3 calcium carbonate, CI confidence interval, HD hemodialysis, HDL-C high-density lipoprotein cholesterol, iPTH intact parathyroid hormone, LDL-C low-density lipoprotein cholesterol, SOx serum oxalate concentration. Discussion Univariable analysis Multivariable analysis Variable selection (AIC) β 95 %CI P β 95% CI P β 95% CI P SOx (µmol/L) 0.04 0.01 to 0.07 0.022 0.02 − 0.02 to 0.06 0.264 0.03 0.00–0.06 0.084 Age (years) 0.31 0.09 to 0.54 0.007 0.29 0.03 to 0.55 0.027 0.26 0.05–0.47 0.018 Male 4.73 –1.03 to 10.50 0.105 1.98 − 4.35 to 8.31 0.531 HD duration (months) 0.00 –0.03 to 0.03 0.972 Diabetes mellitus 1.66 –4.17 to 7.49 0.570 BMI (kg/m2) –0.29 –1.12 to 0.53 0.478 Ankle-brachial index 6.49 –15.71 to 28.70 0.559 aCa (mg/dL) 3.65 –1.75 to 9.05 0.180 3.26 − 2.35 to 8.87 0.248 Phosphate (mg/dL) –0.26 –3.47 to 2.95 0.871 iPTH (pg/dL) –0.01 –0.03 to 0.02 0.545 ALP (U/mL) –0.01 –0.04 to 0.01 0.352 Magnesium (mg/dL) –2.14 –11.94 to 7.67 0.663 Triglyceride (mg/dL) 0.00 –0.04 to 0.04 0.980 LDL-C (mg/dL) –0.01 –0.14 to 0.11 0.856 HDL-C (mg/dL) –0.16 –0.34 to 0.01 0.071 − 0.11 − 0.30 to 0.07 0.228 Uric acid (mg/dL) 0.72 –2.66 to 4.10 0.670 β2MG (µg/L) 0.74 0.17 to 1.30 0.013 0.42 − 0.23 to 1.06 0.200 0.55 0.00–1.09 0.048 BUN (mg/dL) –0.16 –0.44 to 0.12 0.267 Hemoglobin (g/dL) –0.07 –3.55 to 3.40 0.966 Serum albumin (g/dL) –7.36 –17.45 to 2.73 0.149 3.95 − 7.57 to 15.47 0.492 CaCO3 intake 6.53 –1.34 to 14.39 0.102 − 0.33 − 8.97 to 8.30 0.939 Vitamin D medication 2.20 –3.62 to 8.01 0.451 Statin intake –5.13 –13.08 to 2.82 0.201 Univariable analysis Multivariable analysis Variable selection (AIC) β 95 %CI P β 95% CI P β 95% CI P SOx (µmol/L) 0.04 0.01 to 0.07 0.022 0.02 − 0.02 to 0.06 0.264 0.03 0.00–0.06 0.084 Age (years) 0.31 0.09 to 0.54 0.007 0.29 0.03 to 0.55 0.027 0.26 0.05–0.47 0.018 Male 4.73 –1.03 to 10.50 0.105 1.98 − 4.35 to 8.31 0.531 HD duration (months) 0.00 –0.03 to 0.03 0.972 Diabetes mellitus 1.66 –4.17 to 7.49 0.570 BMI (kg/m2) –0.29 –1.12 to 0.53 0.478 Ankle-brachial index 6.49 –15.71 to 28.70 0.559 aCa (mg/dL) 3.65 –1.75 to 9.05 0.180 3.26 − 2.35 to 8.87 0.248 Phosphate (mg/dL) –0.26 –3.47 to 2.95 0.871 iPTH (pg/dL) –0.01 –0.03 to 0.02 0.545 ALP (U/mL) –0.01 –0.04 to 0.01 0.352 Magnesium (mg/dL) –2.14 –11.94 to 7.67 0.663 Triglyceride (mg/dL) 0.00 –0.04 to 0.04 0.980 LDL-C (mg/dL) –0.01 –0.14 to 0.11 0.856 HDL-C (mg/dL) –0.16 –0.34 to 0.01 0.071 − 0.11 − 0.30 to 0.07 0.228 Uric acid (mg/dL) 0.72 –2.66 to 4.10 0.670 β2MG (µg/L) 0.74 0.17 to 1.30 0.013 0.42 − 0.23 to 1.06 0.200 0.55 0.00–1.09 0.048 BUN (mg/dL) –0.16 –0.44 to 0.12 0.267 Hemoglobin (g/dL) –0.07 –3.55 to 3.40 0.966 Serum albumin (g/dL) –7.36 –17.45 to 2.73 0.149 3.95 − 7.57 to 15.47 0.492 CaCO3 intake 6.53 –1.34 to 14.39 0.102 − 0.33 − 8.97 to 8.30 0.939 Vitamin D medication 2.20 –3.62 to 8.01 0.451 Statin intake –5.13 –13.08 to 2.82 0.201 Table 3. Discussion In this study, higher SOx concentration was associated with vascular calcification in both the coronary artery and other major arteries, and with new-onset CVD events in Japanese dialysis patients. Several previous studies have concluded that CAC contains calcium oxalate12–14; however, no studies have examined SOx concentration and vascular calcification in dialysis patients. This was the first report revealed relationship between SOx and vascular calcification in dialysis patients as far as we know. Previous studies20,21 have revealed that excess SOx combines with calcium to form calcium oxalate crystals that are deposited in various tissues; for example, myocardium, renal tubules, and interstitium. In uremic atherosclerosis mice, excess SOx has been shown to alter intracellular calcium to increase in endothelial cells, promote oxidative stress, severely inhibit proliferation and migration of human endothelial cells, and induce endothelial injury23. Excess SOx is also correlated with aortic calcification containing a major oxalate component in the aortic wall in uremic mice24. Although the mechanism underlying how excess oxalate promotes vascular calcification is unknown, these previous findings support our present results. In this retrospective study, we found that SOx concentration was associated with CVD events in dialysis patients. Recently, Pfau et al.22 reported that SOx concentration was associated with CVD mortality in 1108 dialysis patients over a 2.5-year observation period. Our findings are similar to those of Pfau et al.; however, in our study we adjusted for factors for mineral and bone disorder factors as covariates, whereas Pfau et al. did not. We think it is important to adjust for mineral and bone disorder factors because they are currently considered some of the most powerful factors that promote vascular calcification in ESRD patients1. Our present findings are the first to reveal an association between SOx and CAC or vascular calcification. These findings are supported by our analysis selecting factors that are already known risk factors for vascular calcification or CVD events, for Scientific Reports \| (2023) 13:18558 \| https://doi.org/10.1038/s41598-023-45903-9 www.nature.com/scientificreports/ Table 3. Association between SOx and major artery calcification volume by linear regression analysis. A linear regression analysis was performed for the association between SOx and major artery calcification volume. The explanatory variable was SOx, and the covariates were factors that are reported to be associated with vascular calcification8,25,32,33 or CVD29,34–36. A univariable analysis was performed first, and factors with a two-tailed P value < 0.2 were used for a multivariable analysis. Discussion In other words, traditional factors, for example those related to mineral and bone disorder8 or elevated LDL-cholesterol25, are also important to prevent vascular calcification or CVD events in dialysis patients. Some gut microbes, for example, Oxalobacter formigenes, Lactobacillus, and Bifidobacterium produce specific enzymes that help in the degradation of oxalate salts; humans produce no enzymes for oxalate biotransformation26. The use of probiotics might be a logical treatment for lowering SOx. Indeed, Oxalobacter formigenes intervention lowered SOx concentrations in uremic atherosclerosis mice, however, it did not significantly improve vascular calcification24. This result indicated that vascular calcification in ESRD patients is the result of many interrelated factors, not only oxalate, multiple target therapies will likely be needed for optimal prevention for vascular calcification or CVD events.hhi i There are several limitations to the present study. The first is that we measured SOx concentrations retrospec- tively by using serum that had been in storage for around 10 years. As ascorbate converts nonenzymatically to oxalate at pH > 4, it is recommended that samples are immediately cooled and acidified to lower pH to halt this biochemical process27,28. However, a recent study has revealed that immediate freezing without acidic conditions and maintaining the samples at − 80 °C has been shown provide accurate and stable SOx assessments for up to 21 months28. The serum samples used in the present study were frozen immediately upon collected and stored at − 80 °C until analysis with strict thermal management. The second limitation is that this was a retrospective study in which confounding factors were not fully considered. For example, smoking and C-reactive protein are also associated with CVD events in ESRD patients29, but we were unable to add them as covariates because many of the patient records were missing these values. Moreover, the study’s sample size was relatively small. The final limitation is that this study might contain sampling bias. The study participants voluntarily underwent the initial atherosclerosis checkup, meaning they may comprise a group of particularly health-conscious individu- als. Although, the study population was similar to the whole Japanese dialysis population reported in 201230 https://doi.org/10.1038/s41598-023-45903-9 Scientific Reports \| (2023) 13:18558 \| www.nature.com/scientificreports/ Figure 2. Kaplan–Meier analysis of the relationship between SOx and new-onset cardiovascular disease events. Subjects were divided into two groups by median SOx concentration (SOx < 266.9 µmol/L and SOx ≥ 266.9 µmol/L). Discussion Kaplan–Meier analysis of the relationship between SOx and new-onset cardiovascular disease even Subjects were divided into two groups by median SOx concentration (SOx < 266.9 µmol/L and SOx ≥ 266.9 µmo New-onset cardiovascular events occurred in 29/60 (48.3%) patients: in 19/30 (63.3%) patients with SOx greater than or equal to the median value and in 10/30 (33.3%) patients with SOx less than the median value. The number of events was significantly higher in the patients with SOx ≥ 266.9 µmol/L than in those with SOx < 266.9 µmol/L (a), even after limiting the analysis only to patients with CAC score < 1000 and without history of cardiovascular disease (n = 33, b). CAC coronary artery calcification, CI confidence interval, HR hazard ratio, SOx serum oxalate. in terms of age, gender, dialysis duration, and diabetes mellitus, the estimated 5-year mortality of the present study population was 82.3% (95%CI, 72.9–92.9%), whereas that of the whole Japanese dialysis population was 60.0%31. It should be noted that the prognosis of even health-conscious ESRD patients is poorer than that of the general population, meaning that strategies to lower SOx concentrations may bring about desirable outcomes even in well-managed ESRD patients.i g p High SOx concentration was associated with Agatston CAC score ≥ 1000, major artery calcification volume, and new-onset CVD events in Japanese ESRD patients. A novel strategy for SOx control may bring a better prognosis to ESRD patients. Discussion New-onset cardiovascular events occurred in 29/60 (48.3%) patients: in 19/30 (63.3%) patients with SOx greater than or equal to the median value and in 10/30 (33.3%) patients with SOx less than the median value. The number of events was significantly higher in the patients with SOx ≥ 266.9 µmol/L than in those with SOx < 266.9 µmol/L (a), even after limiting the analysis only to patients with CAC score < 1000 and without history of cardiovascular disease (n = 33, b). CAC coronary artery calcification, CI confidence interval, HR hazard ratio, SOx serum oxalate. Figure 2. Kaplan–Meier analysis of the relationship between SOx and new-onset cardiovascular disease events. Subjects were divided into two groups by median SOx concentration (SOx < 266.9 µmol/L and SOx ≥ 266.9 µmol/L). New-onset cardiovascular events occurred in 29/60 (48.3%) patients: in 19/30 (63.3%) patients with SOx greater than or equal to the median value and in 10/30 (33.3%) patients with SOx less than the median value. The number of events was significantly higher in the patients with SOx ≥ 266.9 µmol/L than in those with SOx < 266.9 µmol/L (a), even after limiting the analysis only to patients with CAC score < 1000 and without history of cardiovascular disease (n = 33, b). CAC coronary artery calcification, CI confidence interval, HR hazard ratio, SOx serum oxalate. igure 2. Kaplan–Meier analysis of the relationship between SOx and new-onset cardiovascular disease events. Figure 2. Kaplan–Meier analysis of the relationship between SOx and new-onset cardiovascular disease events. Subjects were divided into two groups by median SOx concentration (SOx < 266.9 µmol/L and SOx ≥ 266.9 µmol/L). New-onset cardiovascular events occurred in 29/60 (48.3%) patients: in 19/30 (63.3%) patients with SOx greater than or equal to the median value and in 10/30 (33.3%) patients with SOx less than the median value. The number of events was significantly higher in the patients with SOx ≥ 266.9 µmol/L than in those with SOx < 266.9 µmol/L (a), even after limiting the analysis only to patients with CAC score < 1000 and without history of cardiovascular disease (n = 33, b). CAC coronary artery calcification, CI confidence interval, HR hazard ratio, SOx serum oxalate. Figure 2. Study design and population y g p p A total of 540 ESRD patients undergoing maintenance hemodialysis at a single hemodialysis center (Heisei Hidaka Clinic, Gunma, Japan) were enrolled in the study (Fig. 3). All subjects were receiving hemodialysis administered in 3.5–4.5 h sessions three times weekly using a polysulfone hollow-fiber dialyzer (APS-SA, Asahi Kasei Medical, Tokyo, Japan or NV-U, Toray Industries, Tokyo, Japan) and a membrane area of 0.8–2.5 m2. Blood and dialysate flows were 180–250 mL/min and 500 mL/min, respectively, with a constant ultrafiltration rate. The dialysate bath comprised 140 mmol/L sodium, 2.0 mmol/L potassium, 2.5 mmol/L calcium, 1.0 mmol/L magnesium, 8.0 mmol/L acetate, 25.0 mmol/L bicarbonate, and 150 mg/dL glucose (Kindaly 3D; Fuso, Osaka, Japan). From April 2011 to March 2012, 77 subjects voluntarily received an atherosclerosis checkup in which the patients were examined by multidetector spiral computed tomography (MDCT), and Agatston CAC score4 and major artery calcification volume were determined by a skilled radiologist; further details are provided in the section titled “MDCT and Measurement of CAC and Major Artery Calcification Volume”. We did not calculate the sample size. We included all the participants who voluntarily received an atherosclerosis checkup from April 2011 to March 2012 with consent (N = 77) to this study. Patients without consent were excluded. All of the study participants provided written informed consent at the time of the atherosclerosis checkup in 2011, and we afforded them the opportunity to opt-out from this secondary analysis in 2021. The data were collected by using the hospital chart from January 2021 to May 2021. The study complies with the Declaration of Helsinki, and the study was approved by the Ethics Committee on Human Research at Heisei Hidaka Clinic (Gunma, Japan; No. 46) and the Ethics Committee on Clinical Research at Teikyo University (Tokyo, Japan; No. 22-058). Confirms that all experiments were performed in accordance with relevant named guidelines and regulations. Cross‑sectional analysis of the association between SOx and vascular calcification C oss sect o a a a ys s o t e assoc at o bet ee SOx a d ascu a ca ci cat o In January 2021, SOx concentration was measured retrospectively by a laboratorian not directly involved in the planning of the study by using frozen serum collected around the atherosclerosis checkup date and stored at − 80 °C. Because our study targeted subjects with higher SOx concentrations, we excluded patients with SOx concentrations in the normal range (< 181 µmol/L) at the time of analysis. Detailed methods of SOx measurement were provided in the “Sample handling and measurement of SOx” section. https://doi.org/10.1038/s41598-023-45903-9 Scientific Reports \| (2023) 13:18558 \| www.nature.com/scientificreports/ Univariable analysis Multivariable analysis Variable selection (AIC) HR 95%CI P HR 95%CI P HR 95%CI P SOx ≥ 266.9 (µmol/L) 2.79 1.26–6.16 0.011 1.92 0.79–4.68 0.150 2.10 0.90–4.91 0.086 Age (years) 1.02 0.99–1.06 0.159 1.00 0.96–1.04 0.951 Male 2.07 0.87–4.89 0.099 1.34 0.53–3.39 0.531 HD duration (months) 1.00 0.99–1.00 0.403 History of CVD 5.88 2.28–15.15 < 0.001 3.23 1.06–9.85 0.039 3.84 1.44–10.2 0.007 Diabetes mellitus 1.41 0.65–3.07 0.390 BMI (kg/m2) 1.00 0.88–1.13 0.993 Ankle-brachial index 0.22 0.01–4.58 0.329 aCa (mg/dL) 0.58 0.24–1.39 0.221 Phosphate (mg/dL) 1.14 0.71–1.85 0.585 iPTH (pg/dL) 1.00 0.99–1.00 0.324 ALP (IU/L) 1.00 0.99–1.00 0.142 1.00 0.99–1.00 0.708 Magnesium (mg/dL) 0.44 0.13–1.55 0.201 Triglyceride (mg/dL) 1.00 1.00–1.01 0.306 LDL-C (mg/dL) 1.01 0.99–1.02 0.494 HDL-C (mg/dL) 0.96 0.93–1.00 0.030 0.97 0.94–1.00 0.091 0.97 0.94–1.00 0.062 Uric acid (mg/dL) 0.91 0.59–1.42 0.686 β2MG (µg/L) 0.99 0.93–1.06 0.837 BUN (mg/dL) 0.98 0.94–1.02 0.283 Hemoglobin (g/dL) 0.64 0.35–1.16 0.139 0.82 0.45–1.48 0.508 Serum albumin (g/dL) 0.53 0.13–2.11 0.367 CaCO3 intake 0.83 0.34–2.05 0.683 Vitamin D medication 1.33 0.63–2.81 0.451 Statin intake 1.03 0.39–2.73 0.952 Table 4. Relationship between SOx and new-onset cardiovascular disease events by Cox proportional hazard analysis. We divided the subjects into two groups by SOx value ≥ 266.9 μmol/L and conducted a Cox proportional hazard analysis. The explanatory variable was SOx, and the covariates were factors that are reported to be associated with vascular calcification8,25,32,33 or CVD29,34–36. A univariable analysis was performed first, and factors with a two-tailed P value < 0.2 were used for a multivariable analysis. For variable selection, we used Akaike’s information criteria with stepwise backward elimination. A two-tailed P value of < 0.05 was considered to indicate statistical significance. Table 4. Relationship between SOx and new-onset cardiovascular disease events by Cox proportional hazard analysis. We divided the subjects into two groups by SOx value ≥ 266.9 μmol/L and conducted a Cox proportional hazard analysis. The explanatory variable was SOx, and the covariates were factors that are reported to be associated with vascular calcification8,25,32,33 or CVD29,34–36. A univariable analysis was performed first, and factors with a two-tailed P value < 0.2 were used for a multivariable analysis. For variable selection, we used Akaike’s information criteria with stepwise backward elimination. A two-tailed P value of < 0.05 was considered to indicate statistical significance. aCa albumin-adjusted calcium, AIC Akaike’s information criteria, ALP alkaline phosphatase, β2MG beta-2 microglobulin, BUN blood urea nitrogen, BMI body mass index, CaCO3 calcium carbonate, CI confidence interval, CVD cardiovascular disease, HD hemodialysis, HDL-C high-density lipoprotein cholesterol, HR hazard ratio, iPTH intact parathyroid hormone, LDL-C low-density lipoprotein cholesterol, SOx serum oxalate concentration. Retrospective cohort analysis of the relationship between SOx and new‑onset CVD events p y p Ox To examine the relationship between SOx and new-onset CVD events, we conducted a retrospective cohort analysis. New-onset CVD events during the 10-year period from serum sample collection date to observation end date of May 15th, 2021 were recorded by medical staff blinded to the SOx results to avoid information bias. “New-onset CVD event” was defined as any admission due to non-fatal myocardial infarction, coronary artery disease, or heart failure during the observation period. We divided subjects into two groups by SOx median and compared new-onset CVD events between two groups. The explanatory variable was SOx lower or grater than median, and outcome was new-onset CVD event. Potential confounders were the same as listed on the “Cross- sectional Analysis of the Association Between SOx and Vascular Calcification” section. MDCT and measurement of CAC and major artery calcification volume j yi Multi-slice computed tomography (CT) scans were performed with an Aquilion TSX-101A (Toshiba, Tokyo, Japan). Slices of 1.25-mm thickness were acquired eight at a time under the following conditions: 120–140 kVp, 85–150 mA, 500 ms exposure, and 0.5 s gantry rotation time. The entire heart was covered in a single breath- hold (20–30 s). The CT images were transferred to a Ziosoft M900 QUADRA workstation (AMIN, Inc, Tokyo, Japan) and CAC score according to the algorithm suggested by Agatston et al.4 (area × cofactor; 1: 130–199H; 2: 200–299H; 3: 300–399H; 4: > 400H), along with the volume (area × slice increment), mass (area × slice incre- ment × mean CT density/250), and density (mass/volume) of the CAC were determined by a single radiologist. Using the electrocardiogram tracing, the workstation software automatically selected a reduced set of diastolic images from each cardiac cycle. All pixels with density > 130H were automatically highlighted on the images. The radiologist assigned one of four locations to each calcified plaque: left main, left anterior descending, circumflex, or right coronary artery. The score for each plaque equaled the plaque area × weighting factor × increment/slice width. The score for the entire specimen equaled the sum of the scores for each plaque. The mean intra-reader variability for CAC was 1.8%.iii y Quantification of major artery calcification volume was performed by three-dimensional calcified lesion reconstitution from multiple slices of the aorta from the top of the arch to the abdominal artery just before the bifurcation of the iliac artery along the longitudinal axis by using the Ziosoft M900 QUADRA workstation. Cross‑sectional analysis of the association between SOx and vascular calcification aCa albumin-adjusted calcium, AIC Akaike’s information criteria, ALP alkaline phosphatase, β2MG beta-2 microglobulin, BUN blood urea nitrogen, BMI body mass index, CaCO3 calcium carbonate, CI confidence interval, CVD cardiovascular disease, HD hemodialysis, HDL-C high-density lipoprotein cholesterol, HR hazard ratio, iPTH intact parathyroid hormone, LDL-C low-density lipoprotein cholesterol, SOx serum oxalate concentration. Figure 3. Study protocol. Figure 3. Study protocol. Figure 3. Study protocol. https://doi.org/10.1038/s41598-023-45903-9 Scientific Reports \| (2023) 13:18558 \| www.nature.com/scientificreports/ To examine the association between SOx and vascular calcification, we conducted a cross-sectional analysis using data collected at the atherosclerosis checkup (age, gender, hemodialysis duration, dialysis prescriptions, past medical history, medications, BMI, ankle-brachial index, CAC score, and major artery calcification volume). Laboratory data were also collected from medical records at the start of the dialysis session with the longest interdialytic interval around the atherosclerosis checkup date. We divided subjects into two groups: those with CAC score < 1000 and those with CAC score ≥ 1000 to define “very high” CAC5–7 group. The predictor was SOx, and outcome was Agatston CAC score ≥ 1000. A logistic regression analysis was performed for the association between SOx and CAC score ≥ 1000. We further analyzed the relationship between SOx and major artery calcifi- cation volume in addition to the relationship between SOx and CAC. Potential confounders8,25,29,32–36 were age, gender, hemodialysis duration, history of CVD, diabetes mellitus, BMI, ankle-brachial index, albumin-adjusted calcium, serum phosphate, intact parathyroid hormone, alkaline phosphatase, serum magnesium, triglyceride, LDL cholesterol, HDL cholesterol, uric acid, beta-2 microglobulin, blood urea nitrogen, hemoglobin, serum albumin, vitamin D medication, calcium carbonate intake, and statin intake. Sample handling and measurement of SOx Blood samples were collected at the start of the dialysis session with the longest interdialytic interval around the atherosclerosis checkup date. The collected samples were mixed immediately with 5 mg edetic acid, centrifuged at 3000 rpm for 5 min to separate the serum, and stored immediately at − 80 °C with strict thermal management until analysis. SOx concentration was measured in January 2021 by using an Oxalate Assay Kit (Colorimetric) (ab196990; Abcam, Cambridge, MA, USA). With this assay kit, the normal range of human SOx is < 181 µmol/L. All serum samples were analyzed in duplicate. Statistical analysis Continuous variables are reported as mean ± standard deviation for normally distributed data, or median (inter- quartile range) for non-normally distributed data. Discrete variables are expressed as numeral (percentage). Differences between subjects with CAC score ≥ 1000 and those with CAC score < 1000 were tested for statistical significance by using Welch’s t-test for normally distributed data, the Wilcoxon rank sum test for non-normally distributed data, and Fisher’s exact test for discrete variables. A two-tailed P value of < 0.05 was considered to indicate statistical significance. In univariate analyses, we did available-case analyses, whereas in multivariable analyses, we did complete-case analyses. No imputation methods for sensitivity analysis were used in our study. In the analysis of an association between SOx concentration and CAC score, we divided subjects into two groups: those with CAC score < 1000 and those with CAC score ≥ 1000. This cutoff value was selected from previous studies indicating that CAC score ≥ 1000 is associated with cumulative incidence of cardiovascular events5–7. A logistic regression analysis was performed for the association between SOx and CAC score ≥ 1000, and a linear regression analysis was performed for the association between SOx and major artery calcification volume. Moreover, we performed risk prediction modeling to predict CAC score ≥ 1000 for validation. We present a ROC curve for predicting CAC score ≥ 1000 by using variables selected from multivariable logistic regression with variable selection. In the analysis of an association between SOx and new-onset CVD events, we divided subjects into two groups by SOx median and performed a Kaplan–Meier analysis and log-rank test to compare the survival functions of the Scientific Reports \| (2023) 13:18558 \| https://doi.org/10.1038/s41598-023-45903-9 www.nature.com/scientificreports/ two groups and to determine whether SOx is associated with CVD events. We repeated the analysis, restricting it to patients with CAC score < 1000 and without history of CVD. We also conducted a Cox proportional hazard analysis using the SOx cut-off value to determine which factors were associated with CVD events. Statistical analysis y gf In each analysis, the explanatory variable was SOx and the covariates were factors that are reported to be associated with vascular calcification8,25,32,33 or CVD29,34–36; specifically, age, gender, hemodialysis duration, his- tory of CVD, diabetes mellitus, BMI, ankle-brachial index, albumin-adjusted calcium, serum phosphate, intact parathyroid hormone, alkaline phosphatase, serum magnesium, triglyceride, LDL cholesterol, HDL cholesterol, uric acid, beta-2 microglobulin, blood urea nitrogen, hemoglobin, and serum albumin, vitamin D medication, calcium carbonate intake, and statin intake were used as covariates. A univariable analysis was performed first, and factors with a two-tailed P value < 0.2 were used for a multivariable analysis. For variable selection, we used Akaike’s information criteria with stepwise backward elimination. All statistical analyses were performed using R software, version 4.1.3. References L., Runnells, D. E. & Le Geros, R. Z. 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Data availabilityh The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Received: 3 April 2023; Accepted: 25 October 2023 Received: 3 April 2023; Accepted: 25 October 2023 References 1. Cozzolino, M. et al. Cardiovascular disease in dialysis patients. Nephrol. Dial. Transplant 33, iii28–iii34. https://doi.org/10.1093/ ndt/gfy174 (2018).i 1. Cozzolino, M. et al. Cardiovascular disease in dialysis patients. Nephrol. Dial. Transplant 33, iii28–iii34. https://doi.org/10.1093/ ndt/gfy174 (2018).i g y ( ) 2. Raggi, P. et al. Cardiac calcification in adult hemodialysis patients. A link between end-stage renal disease and cardiovascular disease?. J. Am. Coll. Cardiol. 39, 695–701. https://doi.org/10.1016/s0735-1097(01)01781-8 (2002).i p g 3. Wang, X. R., Zhang, J. J., Xu, X. X. & Wu, Y. G. 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Acknowledgementsh g The authors thank all participating patients, medical staff of the dialysis center, and the laboratorian at the Heisei- Hidaka Clinic for collecting the medical records. g The authors thank all participating patients, medical staff of the dialysis center, and the laboratorian at the Heisei- Hidaka Clinic for collecting the medical records. References Calcium oxalate saturation in dialysis patients with and without primary hyperoxaluria. Urol. Res. 34, 12–16 https://doi.org/10.1007/s00240-005-0004-6 (2006). g 22. Pfau, A. et al. High oxalate concentrations correlate with increased risk for sudden cardiac death in dialysis patients. J. Am. Soc. Nephrol. 32, 2375–2385. https://doi.org/10.1681/ASN.2020121793 (2021). p p g 3. Recht, P. A. et al. Oxalic acid alters intracellular calcium in endothelial cells. Atherosclerosis 173, 321–328. https://doi.org/10.1016/j atherosclerosis.2003.11.023 (2004). 4. Sun, K. et al. Hyperoxalemia leads to oxidative stress in endothelial cells and mice with chronic kidney disease. Kidney Blood Press Res. 46, 377–386. https://doi.org/10.1159/000516013 (2021).i 25. McCullough, P. A., Sandberg, K. R., Dumler, F. & Yanez, J. E. Determinants of coronary vascular calcification in patients with chronic kidney disease and end-stage renal disease: A systematic review. J. Nephrol. 17, 205–215 (2004). 26. Sadaf, H., Raza, S. I. & Hassan, S. W. Role of gut microbiota against calcium oxalate. Microb. Pathog. 109, 287–291. https://doi.org/ 10.1016/j.micpath.2017.06.009 (2017). j p ( ) 7. Ladwig, P. M., Liedtke, R. R., Larson, T. S. & Lieske, J. C. Sensitive spectrophotometric assay for plasma oxalate. Clin. Chem. 51 2377–2380. https://doi.org/10.1373/clinchem.2005.054353 (2005). https://doi.org/10.1038/s41598-023-45903-9 Scientific Reports \| (2023) 13:18558 \| www.nature.com/scientificreports/ 28. Pfau, A. et al. Assessment of plasma oxalate concentration in patients with CKD. Kidney Int. Rep. 5, 2013–2020. https://doi.org/ 10.1016/j.ekir.2020.08.029 (2020). j 9. Ma, L. & Zhao, S. Risk factors for mortality in patients undergoing hemodialysis: A systematic review and meta-analysis. Int. J Cardiol. 238, 151–158. https://doi.org/10.1016/j.ijcard.2017.02.095 (2017).h p g j j 0. Nakai, S. et al. An overview of regular dialysis treatment in Japan (as of December 31, 2012). J. Jpn. Soc. Dial. Ther. 47, 1–56. https:// doi.org/10.4009/jsdt.47.1.[InJapanese] (2014).h g j p 1. Masakane, I. et al. An overview of regular dialysis treatment in Japan (as of December 31, 2012). J. Jpn. Soc. Dial. Ther. 50, 1–62 https://doi.org/10.4009/jsdt.50.1.[InJapanese] (2017).i p g j p 2. Shantouf, R. et al. Association of serum alkaline phosphatase with coronary artery calcification in maintenance hemodialysis patients. Clin. J. Am. Soc. Nephrol. 4, 1106–1114. https://doi.org/10.2215/CJN.06091108 (2009).i p p p g 33. Jono, S., Nishizawa, Y., Shioi, A. & Morii, H. 1,25-Dihydroxyvitamin D3 increases in vitro vascular calcification by modulating secretion of endogenous parathyroid hormone-related peptide. Circulation 98, 1302–1306. https://doi.org/10.1161/01.cir.98.13. 1302 (1998).f 34. Jamal, S. A. et al. Effect of calcium-based versus non-calcium-based phosphate binders on mortality in patients with chronic kidney disease: An updated systematic review and meta-analysis. Lancet 382, 1268–1277. Author contributions Y.N. planed the study; S.M. conducted statistical analyses; M.T., E.H., and K.O. drafted the manuscript; Y.H. and A.H. conducted the study; K.I. collected data; N.N. supervised the study; J.H. and T.O. reviewed and revised the manuscript. Competing interests h p g The authors declare no competing interests. © The Author(s) 2023 Additional information Correspondence and requests for materials should be addressed to Y.N. Correspondence and requests for materials should be addressed to Y.N. Reprints and permissions information is available at www.nature.com/reprints. 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 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. 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https://openalex.org/W4253374651	https://revistasinvestigacion.unmsm.edu.pe/index.php/letras/article/download/21124/17146	es		Julián M. Del Portillo. Amor y muerte. El hijo del crimen. Lima de aquí a cien años. Edición crítica y estudio preliminar de Christian Elguera. Lima: Ediciones MYL, 2021, 128 pp.	Escritura y pensamiento/Escritura & pensamiento	2,021	cc-by	2,150	Escritura y Pensamiento 20(41), 2021, 265-271 JULIÁN M. DEL PORTILLO. AMOR Y MUERTE. EL HIJO DEL CRIMEN. LIMA DE AQUÍ A CIEN AÑOS. EDICIÓN CRÍTICA Y ESTUDIO PRELIMINAR DE CHRISTIAN ELGUERA. LIMA: EDICIONES MYL, 2021, 128 PP. En el Bicentenario peruano, no debemos preguntarnos si hemos logrado constituir una nación, sino redirigir la pregunta hacia cómo nos hemos imaginado en ella, cuál fue el proyecto que se pensó para el país. En ese sentido, es importante celebrar el trabajo de Ediciones MYL, cuyo trabajo con la Colección Rumbo al Bicentenario nos permite redescubrir a autores de inicios de la República y, de esa manera, acceder a un modo específico en el que se buscó representar al país y sus habitantes. Precisamente uno de estos autores es Julián M. del Portillo, de quien la editorial ha tenido el acierto de publicar un volumen que reúne sus obras Amor y Muerte, El hijo del crimen y Lima de aquí a cien años. Se trata de un libro que agrupa estos tres títulos, con un extenso y documentado estudio del investigador Christian Elguera, titulado “Julián M. del Portillo, ideólogo del colonialismo liberal: espacio, jerarquías raciales y liberalismo en los inicios republicanos”. Se trata de un estudio que hace una revisión crítica acerca de las anteriores ediciones e interpretaciones que se le han dedicado a Julián M. del Portillo respecto de Lima de aquí a cien años y que se extiende hacia los otros dos títulos mencionados, en los que reconoce un continuo en el proyecto político. En ese sentido, Elguera deslinda de las exégesis que han optado por analizar a este autor desde la información biografista y propone explorar el contexto histórico del autor para entender su proyecto colonial. Reseñas Este estudio se instala así en el debate académico que gravita alrededor de este escritor decimonónico. Elguera refuta las interpretaciones que han intentado fijar su lectura ubicándola dentro de los géneros de la ciencia ficción, cuestionando a quienes únicamente han centrado su atención en las imágenes futuristas de la novela. La sugerencia de situarnos en el contexto del autor nos permite, en cambio, conocer sobre conflictos de los primeros años de la República en donde el sector liberal buscó proteger sus intereses particulares como clase social. Asimismo, en diálogo con una lectura decolonial, Elguera propone que la mirada de progreso de Portillo, ejemplificada en una Lima europeizada, no fue pensada para ni por sujetos indígenas o afrodescendientes. La primera novela es Amor y muerte, quizá la obra mejor lograda de un autor sin talentos. Es necesario hacer esta advertencia: estamos ante un escritor cuyas capacidades literarias se evidencian limitadas, incluso para la época en las que se escribieron (entre 1830 y 1840). El escenario es Estados Unidos durante la época de las tensiones políticas entre esclavistas y abolicionistas. En ella se relata la historia de dos amantes -Jenny Makensie y Jones Cokeril- quienes deben separarse por problemas económicos. La tensión de la trama se agrava cuando la salud del padre de Jenny se va deteriorando debido a las deudas que mantiene con Mr. Jackson, quien aprovecha esta oportunidad para satisfacer sus deseos sexuales. Mr. Jackson ofrece perdonar la deuda del padre a cambio de la joven Jenny. Enterado de la situación, el padre de la joven rechaza esta propuesta, aunque muere momentos después debido a su delicada salud. Jenny acude entonces a su tío, quien llega para asumir la deuda de su hermano y así salvar a su sobrina. Sin embargo, un nuevo giro -acierto de Portillo- brindará una nueva complicación a la trama, cuando Mr. Jackson revela a los acreedores que Jenny no es sólo la hija de Mr. Makensie, sino también parte de su propiedad, pues es la hija que tuvo con una esclava, motivo por el cual también adquiere esa misma 266 \| Escritura y Pensamiento 20(41), 2021 Reseñas condición. Ante esta revelación, Jenny acepta ser tomada como posesión de Mr. Jackson y así salvar lo que queda de la memoria de su padre. Una vez que la deuda es saldada, Jenny se lanza al río, protegiendo así su dignidad. Se trata, pues, de un melodrama con un trasfondo político: la esclavitud. Con el siguiente texto, El hijo del crimen, nos encontramos con un prólogo del mismo autor, el cual nos permite un mapeo del acontecer literario de los primeros años de la República. Es importante precisar que esta novela nunca ha sido publicada, ya sea porque los ejemplares se perdieron o porque Portillo nunca la escribió. El prólogo nos hace partícipes de lo que era y debía ser el país según Portillo. Es conveniente recordar que una de las características de la novela decimonónica era su espíritu pedagógico, antes que estético. Así entendido, sus producciones pueden y deben ser leídas como parte de un proyecto político más complejo, dentro del cual la literatura forma una parte anexada. Así, cuando Portillo comenta sobre el declive de nuestra literatura y la inestabilidad de nuestra República no solo está considerando que en algún momento del pasado colonial nuestra producción literaria gozó de un elevado nivel —aunque no profundiza sobré qué juicios parte para sostener esa consideración— sino que reduce y hasta invisibiliza años de violencia estructural cuando menciona las virtudes legadas por la corona sin que un proyecto político a la altura se haga presente para sucederla. Prestemos atención al siguiente pasaje: “Veinte años de desórdenes, de guerras fratricidas e incesantes, a la vez que escandalosas e injustas, todo lo aniquilaron, todo lo destruyeron. Nuestra literatura no salió libre de tan terribles luchas, y durante este periodo siguió con precipitados pasos la marcha decadente que la condujera hacia el abismo...” (Portillo, 2021, p. 145). Mención especial merece Lima de aquí a cien años. En esta edición, las cartas que estructuran el argumento se presentan en el orden cronológico en las que fueron publicadas originalEscritura y Pensamiento E-ISSN: 1609-9109 \| 267 Reseñas mente. Estamos ante un diálogo epistolar entre J.M de P. (quien luego se hará llamar Arthur) y su amigo cusqueño, llamado Carlos de A. La particularidad de esta historia es la ubicación del tiempo histórico del relato: 1943, siendo el texto publicado en 1843. Los personajes, pertenecientes a las primeras décadas de la República, aparecen de pronto cien años después sin que se brinde una explicación racional de este salto temporal. Lo importante para el narrador y para el autor es mostrarnos cómo sería Lima dentro de cien años. La segunda particularidad es cuando J.M de P. recibe la respuesta de otro autor, quien asume la personalidad del amigo cusqueño, y que firma como Carlos de A. De esta manera, estamos ante una obra escrita al alimón, en donde la interferencia del segundo autor termina redireccionando y dando forma a un proyecto político liberal. Para conocer las dimensiones de este proyecto político, quiero resaltar algunos puntos relevantes en el estudio crítico de Elguera. En este se resalta la necesidad de leer a Portillo dentro del contexto histórico al que perteneció. Precisamente Elguera se pregunta “¿Qué puede decirnos este texto en las postrimerías del Bicentenario de la República del Perú?” (Elguera, 2021, p. 13). Para el autor de este estudio es importante que tengamos en claro este carácter pedagógico o, en sus palabras, de “herramienta didáctica” de la novela, y que fue una característica propia de la literatura decimonónica del siglo XIX. Al respecto, recordemos los prólogos de las novelas decimonónicas, donde los propios autores expresan las intenciones de sus respectivos proyectos políticos. Citemos los casos de Mercedes Cabello en Blanca sol y Clorinda Matto de Turner con Aves sin nido, ambas autoras profundamente estudiadas por las profesoras Ana Peluffo y Rocío Ferreira, respectivamente. Sólo así entendido y partiendo de un análisis interdisciplinario, que dialogue con la historia y otras herramientas académicas, es posible leer con amplitud la obra de Portillo y no quedarnos solamente con un análisis formal de su estructura narrativa o alcances literarios. Elguera sugiere que este texto 268 \| Escritura y Pensamiento 20(41), 2021 Reseñas debe entenderse como un programa que buscaba “legitimar al colonialismo liberal del siglo XIX” (Elguera, 2021, p. 15). En ese sentido es importante prestar atención a lo que denomina contactología letrada; es decir a “ese intelectual zalamero, que busca siempre quedar bien, elogiar y defender a sus mecenas, a aquellos grupos de los que esperan un favor” (Elguera, 2021, p. 18). Así quedan expuestas las intenciones que significan la presencia de Domingo Elías en la novela de Portillo. Este fue uno de los hombres más ricos e influyentes durante las primeras décadas de la República del Perú y conspicuo representante del liberalismo, con amplia proyección política. La adulación a Elías se hace evidente en la referencia a su vino. Recordemos que este empresario fue un importante productor de vinos. Dicha escena permite inferir el olfato político de Portillo para reconocer y vaticinar el ascenso en la carrera pública de esta persona y así asegurarse una mejor posición dentro de un contexto de recuperación y posicionamiento de las elites liberales. Un aporte importante de Elguera es su idea de colonialismo liberal. Este concepto se refiere a los “discursos y las prácticas, tanto culturales y políticas, que han promovido la exterminación de grupos minoritarios, así como la imposición de jerarquías raciales y sociales en las primeras décadas republicanas” (Elguera, 2021, p. 15-16). Esto se comprueba en el diseño de la Lima de Portillo, que al fin ha alcanzado la civilización soñada. Estamos ante una Lima donde no se observan poblaciones indígenas o afrodescendientes. Lima, como centro del poder, es una ciudad europeizada, lo que se lee como un eurocentrismo que niega la valía de otras culturas que se consideran ajenas al ideal moderno de las clases liberales. Esta propensión hacia lo europeo y la eliminación de otras representaciones étnicas también se evidencia en Amor y Muerte y El hijo del crimen. Elguera observa que, la primera novela, puede leerse como una historia antiabolicionista, pero, al mismo tiempo, no se observa ninguna voz de quienes justamente son los oprimidos por la esclavitud. Ningún esclavo negro aparece con voz propia para Escritura y Pensamiento E-ISSN: 1609-9109 \| 269 Reseñas hablar de su situación y Jenny, quien se revela al final como esclava, es blanca y es precisamente por ello que su condición causa admiración. Respecto al prólogo de El hijo del crimen, nos encontramos frente al testimonio de una hispanofilia que, como sugiere el investigador, pudo haber significado uno de los motivos por el cual Portillo, siendo liberal, no fue reconocido ni recordado como otros liberales de su época: Ricardo Palma y Luis Benjamín Cisneros, por mencionar los casos más conocidos. Lima de aquí a cien años es la novela donde se hace más explícito lo que Elguera llama el colonialismo liberal del siglo XIX. Teniendo esto en consideración, su estudio va a contracorriente de investigadores que han propuesto leer esta novela como obra de ciencia ficción, prestando atención únicamente a su futurismo. Elguera sostiene que más allá de que esta historia se decanta en referencias futuristas, es necesario analizar el acontecer político dentro del cual se escribe: el fracaso de la confederación Perú-Boliviana y el resurgimiento de los liberales luego de la derrota del caudillismo. Fiel a una comprensión sociológica de la literatura, inspirada por Pierre Bourdieu, el investigador sostiene que estamos ante una novela histórica, reforzando la hipótesis sobre el colonialismo defendido en las obras de Portillo. La novela expresa una violencia colonial a través de la desaparición de poblaciones indígenas y afrodescendientes, a través de jerarquías de género que configuran a la mujer como un ángel del hogar, y a través de injusticias sociales que legitiman el poder de las clases dominantes limeñas. Los liberales negaban la realidad heterogénea del país y esta negación puede explicarnos los distintos fracasos de los proyectos nación, incapaces de pensar al país dentro de su pluridiversidad cultural y étnica. La Lima de Portillo hace ecos con el actual Perú rumbo al bicentenario. Lima sigue constituyéndose como un espacio de poder desde donde se valida o se subordina a sujetos que atentan contra la visión arcádica de la ciudad. Las actuales tensiones políticas, fruto de un contexto electoral y crisis económicas y 270 \| Escritura y Pensamiento 20(41), 2021 Reseñas de salud, han evidenciado la continuidad de nuestros fracasos debido a jerarquías coloniales. Como la Lima de 1943 de Portillo, en los planes de gobierno de los candidatos presidenciales las omisiones dicen más que lo propuesto. Así la ausencia de la población LGTBIQ dentro de las propuestas electorales puede leerse como un rezago de las intenciones liberales de desaparecer lo afro y lo indígena del país. Por estos motivos, la aparición de las obras de Portillo nos invita a una revisión de nuestro pasado y reconocer los vasos comunicantes que aún nos unen con el colonialismo liberal que no ha cerrado su ciclo en doscientos años de vida republicana. (Martín Carrasco Pena) Escritura y Pensamiento E-ISSN: 1609-9109 \| 271
https://openalex.org/W4380417268	https://zenodo.org/records/8031085/files/6.%20Bermudez%20et%20al.pdf	English	null	UTILIZATION OF A DUAL-AXIS SOLAR TRACKER TO INCREASE THE EFFECTIVENESS OF SOLAR PANELS IN THE STORAGE OF POWER IN LITHIUM-ION BATTERIES	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	5,277	Abstract Abstract This study was conducted to determine the effectiveness of utilizing the Dual-axis Solar Tracker in harnessing solar energy and storing it in lithium-ion batteries in terms of voltage and milliamps per hour gained. This study utilized two solar panel systems, the Static Solar Panel System and the Dual-Axis Solar Tracker and aimed to prove the significant difference generated by solar panel systems in terms of voltage and milliamps per hour. This study employed an Applied Experimental Research Design utilizing Arduino Uno, light-dependent resistors, micro servos, and solar panels to create the Dual-Axis Solar Tracker. The data gathered from the electric quantities during the seven-day sun exposure were tabulated, analyzed, and computed using mean tests, standard deviation, and T-tests to find the significant difference between the two solar panels. Based on the analyses of the data, it was revealed that the Dual-Axis Solar Tracker was superior in terms of voltage and milliamps per hour generated compared to the Static Solar Panel System. Moreover, computations using the T-test found that in terms of voltage and milliamps per hour generated, there was a significant difference between the Dual-Axis Solar Tracker and the Static Solar Panel System. Based on the findings of the study, it is concluded that the utilization of the Dual-Axis Solar Tracker statistically increases the effectiveness of solar panels in harnessing energy generation. Keywords: Dual-axis solar tracker, Effectiveness, Static Solar Panel System Keywords: Dual-axis solar tracker, Effectiveness, Static Solar Panel System Corresponding Email: bermudez.ge@southernchristiancollege.edu.ph Corresponding Email: bermudez.ge@southernchristiancollege.edu.ph Volume I Issue 2 (2023) DOI: 10.5281/zenodo.8031085 E-ISSN: 2984-7184 P-ISSN: 2984-7176 https://guildofeducatorsintesol.international/research/ Volume I Issue 2 (2023) DOI: 10.5281/zenodo.8031085 E-ISSN: 2984-7184 P-ISSN: 2984-7176 https://guildofeducatorsintesol.international/research/ Available Online: May 2023 Revised: April 2023 Accepted: February 2022 Received: January 2022 Available Online: May 2023 Revised: April 2023 Accepted: February 2022 Received: January 2022 Available Online: May 2023 Revised: April 2023 Accepted: February 2022 Received: January 2022 UTILIZATION OF A DUAL-AXIS SOLAR TRACKER TO INCREASE THE EFFECTIVENESS OF SOLAR PANELS IN THE STORAGE OF POWER IN LITHIUM-ION BATTERIES Geodizon Iman C. Bermudez1, Joanna Patricia U. Develleres2, Danniah Shamella N. Palao3, Kate Aileen M. Gromia4, Rainer Jade T. Daingan5, Raffy S. Virtucio, MEAL6 123456Southern Christian College, Midsayap, North Cotabato, Philippines Keywords: Dual-axis solar tracker, Effectiveness, Static Solar Panel System INTRODUCTION Students of today can be the ones to spearhead change and work toward universal access to clean and affordable energy for all. Studies in solar energy are relevant to address a significant problem in the modern world which is access to efficient renewable energy. Renewable energy impacts jobs, housing, food supplies, and climate change. The world could not function without energy, and access to inexpensive and clean energy is now more important than ever. Even with the advancements made by humanity in the development of sustainable energy sources like solar and wind power, energy still accounts for around 60% of the world's emissions of greenhouse gases, one of the primary factors in climate change (UNEP). Renewable energy is vital to the country's low-carbon sustainability emissions development strategy and is vital to addressing the challenges of energy security, climate change, and access to energy (Uy, 2022). The Philippines is subject to price changes and supply constraints due to its reliance on imported coal and oil (Brahim, 2014). The development and optimal use of the country's renewable energy resources are central to the Philippines' 87 sustainable energy agenda. Renewable energy is an essential part of the country's low-emissions development strategy. It is vital to address climate change challenges, energy security, and access to energy. This research used electronic components to fabricate a dual-axis solar tracker to increase the effectiveness of solar panels. In order to create a more effective way of harnessing solar energy using solar panels, the researchers applied robotics fundamentals to create the solar tracker, a system that positions a body at an angle relative to the sun. Solar trackers could generate more electricity than their stationary counterparts. This is due to its feature, which increases its direct exposure to solar rays (SPW, 2016). Solar trackers generate more electricity in approximately the same amount of space needed for the static panel system, making them ideal for optimizing land usage (McHale, 2015). If the trackers are installed in an environment with ideal conditions, they added value to the solar panel system and boost energy production. In terms of sustainability, the usage of solar trackers decreased reliance on the local grid and increase reliance on renewable energy as an alternative source for electricity generation (Gregus, 2021). Many studies have explored aspects of solar panels and the use of solar trackers (del Rosario, 2014). The present study explored creating a dual-axis solar tracker by utilizing micro servos instead of direct current (D.C.) motors for the solar tracker's movement, computer systems and microprocessors to control solar tracking systems to follow the sun's location, and introduces a power efficient method to minimize the power consumption of the dual-axis solar tracker. This study created a model of a dual-axis solar tracker that is not dependent on the electrical grid. Furthermore, it used batteries to store solar energy and use the energy to power up the dual-axis solar tracker. Thus, considering the foregoing claims, this study was conducted to utilize a dual-axis solar tracker to increase the effectiveness of solar panels. Background Electric power generation accounts for the second highest share of greenhouse gas emissions: carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O). The greenhouse gas emissions from electricity generation are attributed to the electricity sector. Approximately 60% of our electricity is generated by the combustion of fossil fuels like coal, oil, and natural gas, as stated by the U.S. Energy Information Administration (2019). Therefore, the Philippines' reliance on fossil fuels compromises its energy security (Lea, 2020). Renewable energy is in development. Denmark produced 43% of its energy from renewables, and it is working towards achieving 70% by 2020. At more than 25% now and 30% soon, Germany is going for 40% to 45% clean energy by 2025, 55% to 60% by 2035, and a possible 80% by 2050 (Ahuja, 2009). In the Philippines, renewable energy supplied 26.44% of total electricity in 2013 and 19,903 gigawatt-hours (GWh) of electrical energy out of a total demand of 75,266 gigawatt-hours (Membrere, 2013). The Philippine government has aimed to increase renewable energy contributions to 50% of its total electricity generating capacity (France-Presse, 2011) to 15.3 gigawatts (G.W.) by 2030 (Rhodium, n.d). The approach would enhance the country's objective of reducing carbon emissions by 70% by 2030. From the data given, it can be observed that in the Philippines, currently, the large-scale use of photovoltaic cells is not economically competitive in the market for electricity generation. Solar energy is the conversion of renewable energy from photons into electricity, either directly through photovoltaics, indirectly through concentrated solar power, or through a mix of the two. Several mechanisms have been put in place in order to derive maximum energy from solar power. An example of this is solar tracker which is a system that positions a body at an angle relative to the sun and is commonly used. More sunlight strikes the solar panel, and less light is reflected. Hence, more energy is collected by maintaining the panel perpendicular to the sun. Solar tracking employs complex devices to determine the sun's position in relation to the item being aligned. To track 88 Objectives The general objective of this study was to design and create a Dual-Axis Solar Tracker that can increase the effectiveness of the solar panels more than the static version of the system can harvest. Specifically, this project aimed to: 1. develop a dual-axis solar tracker that is self-sufficient having the characteristics of: a. an off-grid powered model that is not reliant on the electrical grid; and b. has the capacity to store power in lithium-ion batteries; 2. determine the amount of power that the solar panels can store in the lithium-ion batteries in terms of the batteries’: 2. determine the amount of power that the solar panels can store in the lithium-ion batteries in terms of the batteries’: a. voltage; and b. milliamps per hour harnessed; rmine the significant difference between the static and dual-axis systems in terms of the batteries’: etermine the significant difference between the static and dual-axis systems in terms of the batteries a. voltage; and b. milliamps per hour harnessed the sun, computers will be used to execute complex algorithms that allow the system to monitor the sun and sensors that provide information about the sun's location. A solar panel with a basic circuit board can also track the sun without the need for a computer (Encyclopedia Britannica, n.d.). Various solar tracking systems differ in their cost, functionality, and complexity (Jyoti, 2017). Solar tracking systems are typically classified into two categories, depending on the controlling mechanisms that drive the photovoltaic panels. These categories are active tracking systems and passive tracking systems. Both are considered essential methods for obtaining tracking efficiency. Active tracking systems utilize motors and gears to guide the panels in the sun's direction; thus, they must be provided with energy for their actuators to move. While passive tracking systems utilize compressed gas fluid with a low boiling point that boils with heat generated from the sun, these are the systems that do not need any external source of energy (Rooij, 2022). This study is centered on using the active tracking system, specifically the dual-axis tracking system. The dual-axis tracking device follows the sun to collect more solar energy. Using dual-axis trackers, maximum energy collection can be achieved due to its total freedom of movement (north-south and east-west). The tracker can face the sun's rays throughout the day (Chang, 2016). Instrumentation This study utilized a multimeter and the state of charge of the batteries for its capacity to measure voltage and milliamps per hour of the lithium-ion batteries. The voltmeter method (where the state of charge can be computed based on the outputted voltage of the batteries) was used in order to compute the state of charge of the batteries and their percentage. Population and Sampling The respondents of this study consisted of the members of this research paper. The respondents have the needed technical skills to acquire and record the necessary data for the study. This means that the respondents could measure the voltage and the milliamps per hour of the lithium-ion batteries. METHODS Research Design Applied Experimental Research was used as the research design of the study. This study relied on experiments and examine the electrical signals generated from the solar tracker system. Therefore, an applied experimental research design is the best way to examine the effectiveness of the application of solar trackers to the power output of solar panels. 89 89 Population and Sampling The respondents of this study consisted of the members of this research paper. The respondents have the needed technical skills to acquire and record the necessary data for the study. This means that the respondents could measure the voltage and the milliamps per hour of the lithium-ion batteries. Data Collection The researchers started the data collection by placing the solar panel systems for seven days directly under the sun where there was no shade. Then, the voltage and milliamps per hour of the lithium-ion batteries was measured. The milliamps per hour of the batteries were determined using the charge/discharge rate of the ICR3000 lithium-ion batteries. In addition, the data that was collected was the voltage and the milliamps per hour harnessed by the lithium- ion batteries of the dual-axis solar tracker and the static solar panel system in a span of 11 hours for 7 days. The system architecture shows the conceptual model that defines the structure, behavior, and views of the Dual-Axis Solar Tracker. It is a formal description and representation of a system, organized in a way that supports reasoning about the structures and behaviors of the system. The researchers divided the procedure into four steps to produce the system—the Flowchart, Code, Schematic, and Chassis. Figure 2 shows the system architecture flowchart of the Dual-Axis Solar Tracker. Figure 2. System Architecture flowchart Figure 2. System Architecture flowchart Figure 2. System Architecture flowchart Step 1: System Flow Diagram. Figure 3 in page 19 shows the flowchart to separate the processes and steps for tracking the position of the greatest light. The program will read the resistance value of the light-dependent resistors and store it in variables to be used for the next steps. There are two major tests that the data gathered must pass through before creating a movement in the hardware. Using the formulas: elevation = (average top - average bottom) and azimuth = (average right - average left), the program will compute the azimuth and elevation values of the system. Knowing that there is a portion of greater focus on sunlight, the process will move the system leftwards. The program starts by deciding whether the average top is greater than the average bottom. 90 If the first major decision of the system returns True, then the system proceeds to the minor decisions of the elevation movement. After this process, the system will repeat the loop starting from the first process, which is reading the analog values from the light-dependent sensors. The next decision tests whether the average left is less than the average right. If the boolean logic returns True, this defines that there is no significant amount of sunlight present between the left and right portions of the system. analog values from the light-dependent sensors. The next decision tests whether the average left is less than the average right. If the boolean logic returns True, this defines that there is no significant amount of sunlight present between the left and right portions of the system. analog values from the light-dependent sensors. The next decision tests whether the average left is less than the average right. If the boolean logic returns True, this defines that there is no significant amount of sunlight present between the left and right portions of the system. Figure 3. System flow diagram for tracking the position of greatest light. Step 2: Coding. Using the open-source Arduino Software (IDE), the codes were made to program the Arduino board and control the position of the dual-axis solar tracker. Step 3. Schematic. Figure 3 represents the elements of a system using a diagram of the components used in the electronic circuit. It also shows how the researchers wired the individual components in the system. Starting from the power supply, connect the 5v pin to the positive rail of the breadboard. Then, connect the ground pin to the negative power rail of the breadboard. The servo has three pins—the VCC, ground, and signal. Connect the ground pins to the negative rail of the breadboard. Then the VCC pins to the positive rail of the breadboard. For the horizontal servo, connect the signal pin to the digital pin number 9 of the Arduino, and for the vertical servo, connect the signal pin to the digital pin number 10. For the wirings of the light-dependent resistors, ground the 1kΩ resistors to the negative rail of the breadboard. Connect the VCC to one pin of the light-dependent resistors and connect the light-dependent resistors to the 1kΩ resistors. Connect the analog pins between the connection of the 1kΩ resistor and the light-dependent resistor. This voltage divider turns a large voltage into a smaller one making it possible to input an analog signal to the Arduino. From the code, the top left LDR is connected to the A0 analog pin, the top right LDR is connected to the A2 analog The servo has three pins—the VCC, ground, and signal. Connect the ground pins to the negative rail of the breadboard. Then the VCC pins to the positive rail of the breadboard. For the horizontal servo, connect the signal pin to the digital pin number 9 of the Arduino, and for the vertical servo, connect the signal pin to the digital pin number 10. The servo has three pins—the VCC, ground, and signal. Connect the ground pins to the negative rail of the breadboard. Then the VCC pins to the positive rail of the breadboard. For the horizontal servo, connect the signal pin to the digital pin number 9 of the Arduino, and for the vertical servo, connect the signal pin to the digital pin number 10. For the wirings of the light-dependent resistors, ground the 1kΩ resistors to the negative rail of the breadboard. Figure 4. Wirings of dual-axis solar tracker Figure 4. Wirings of dual-axis solar tracker Step 4: Chassis Fabrication. This study used Solidworks Premium 2021 to fabricate the 3d designs and Ultimaker Cura version 4.9.1 as the slicer for 3d files. Using the BIQU B1 3D printer, the pan-tilt servo motor, which includes the base, head, arm, support, and solar panel holder, which comprises of base and LDR separator, was fabricated. Using the BIQU B1 3D printer, the pan-tilt servo motor, which includes the base, head, arm, support, and solar panel holder, which comprises of base and LDR separator, was fabricated. Data Analysis After the data were gathered, the researchers tabulated, evaluated, and analyzed the state of charge of the lithium- ion batteries that were harvested by the dual-axis solar tracker and static solar panel system using mean and standard deviation. T-test was used to determine the significant difference in the effectivity between the Dual-Axis Solar Tracker and the Static Solar Panel System. Figure 3. System flow diagram for tracking the position of greatest light. Connect the VCC to one pin of the light dependent resistors and connect the light dependent resistors to the 1kΩ For the wirings of the light-dependent resistors, ground the 1kΩ resistors to the negative rail of the breadboard. Connect the VCC to one pin of the light-dependent resistors and connect the light-dependent resistors to the 1kΩ resistors. Connect the analog pins between the connection of the 1kΩ resistor and the light-dependent resistor. This voltage divider turns a large voltage into a smaller one making it possible to input an analog signal to the Arduino. From the code, the top left LDR is connected to the A0 analog pin, the top right LDR is connected to the A2 analog 91 pin, the bottom left LDR is connected to the A1 analog pin, and the bottom right LDR is connected to the A3 analog pin. pin, the bottom left LDR is connected to the A1 analog pin, and the bottom right LDR is connected to the A3 analog pin eft LDR is connected to the A1 analog pin, and the bottom right LDR is connected to the A3 analog pin, the bottom left LDR is connected to the A1 analog pin, and the bottom right LDR is connected to the A3 analog pin. Figure 4. Wirings of dual-axis solar tracker RESULTS and DISCUSSION Table I. Voltage output of the batteries that harvested solar power from the dual-axis solar panel system and the batteries that harvested solar power from the static solar panel system. The voltage output was measured using a multimeter. Table 1 Average voltage outputs of dual-axis solar tracker and static solar panel. Voltage Output Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Mean Static Solar Panel System 3.55 V 3.54 V 3.46 V 3.57 V 3.55 V 3.60 V 3.54 V 3.54V Dual-Axis Solar Tracker 3.61 V 3.60 V 3.54 V 3.68 V 3.62 V 3.65 V 3.61 V 3.62V Table 1 Average voltage outputs of dual-axis solar tracker and static solar panel. 92 Table 1 above indicates that in seven (7) days of data gathering, the voltage output of the batteries that harvested solar power from the Dual-Axis Solar Tracker generated an average of 3.62 volts, while the batteries that harvested solar power from the static solar panel system generated 3.54 volts. It further shows that the Dual-Axis Solar Tracker has generated a greater amount of voltage compared to the Static Solar Panel System with a difference of 0.08 volts and by a factor of 2.2%. The Dual-Axis Solar Tracker System outputs a greater voltage than the Static Solar Panel System. This indicates that more amperage (in milliamps) is stored in the batteries of the Dual-Axis Solar Panel System than in the Static Solar Panel System. The proportion between the voltage and amperage of an ICR3000 can be seen in figure 6 on page 24. Moreover, figure 6 shows that the graph between voltage and amperage is not linear. This could further explain how a difference of 0.08 could create about a greater number of milliamps per hour. Table II. Milliamps per hour generated by the dual-axis solar tracker system and the static solar panel system. The milliamp per hour is a measure of a battery’s capacity for supplying current. It was measured by measuring the voltage of the battery and solving the state of charge of the ICR 3600 lithium-ion battery. able 2. Average of current outputs of dual-axis solar tracker and static solar panel. Table 2. Average of current outputs of dual-axis solar tracker and static solar panel. Milliamps per hour Harnessed Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Mean Static Solar Panel System 2400 mAh 2400 mAh 1125 mAh 2850 mAh 2400 mAh 3000 mAh 2400 mAh 2367.86 mAh Dual-Axis Solar Tracker 3000 mAh 3000 mAh 2400 mAh 4425 mAh 3000 mAh 4050 mAh 3000 mAh 3267.86 mAh Table 2 above indicates that in seven (7) days of data gathering, the current output of the dual-axis solar tracker generated an average of 3267.86 milliamps per hour, while the static solar panel system generated 2367.86 milliamps per hour. This implies a 38% increase in the current output of the Dual-Axis Solar Tracker compared to the Static Solar Panel System. Table 2. Average of current outputs of dual-axis solar tracker and static solar panel. Table 2 above indicates that in seven (7) days of data gathering, the current output of the dual-axis solar tracker generated an average of 3267.86 milliamps per hour, while the static solar panel system generated 2367.86 milliamps per hour. This implies a 38% increase in the current output of the Dual-Axis Solar Tracker compared to the Static Solar Panel System. This result proved what Altenergy posited, that a dual-axis tracker can increase energy production by up to 40% because of the angle at which it enables the solar panel to face the sun. Hypothetically, if an appliance that uses 10 watts of power is connected to the battery of a static solar panel, it would take about 8.76 hours until the said appliance would stop functioning. Comparing the same conditions with the dual-axis solar tracker, it would take about 12.09 hours until the said appliance would stop functioning. Table III. T-test on the effectivity of the Dual-Axis Solar Tracker in terms of milliamps per hour. t-Test: Two Independent Means 93 Voltage Output Mean SD p-value t-value Result Decision Static Solar Panel System 3.54 0.0001 Significant Reject Ho2 Dual-Axis Solar Tracker 3.62 0.0001 0.004622 3.0969 Significant at a = 0.05 Significant at a = 0.05 The results revealed that the computed value of P is 0.004622, which is less than the 0.05 level of significance; therefore, Ho_1 which stated that “there is no significant difference in the effectivity of the Static Solar Panel System and the Dual-Axis Solar Tracker System in terms of the voltage stored in the lithium-ion batteries.” is rejected. This implies that there is a significant difference in the effectivity of the Static Solar Panel System and the Dual-Axis Solar Tracker System in terms of voltage output. Therefore, the utilization of a Dual-Axis Solar Tracker System is more effective in terms of voltage output than the Static Solar Panel System since there is a significant difference in the t- test done. With the T-test done, the significant difference shows that the voltage data that were gathered are not due to chance or sampling error. On a bright sunny day, the Dual-Axis Solar Tracker System will always generate more voltage than the Static Solar Panel System. Table IV. T-test on the effectivity of the Dual-Axis Solar Tracker in terms of milliamps per hour. t-Test: Two Independent Means Table 4 T-Test on the effectivity of the Dual-Axis Solar Tracker System and the Static Solar Panel System in terms of milliamps per hour output. N = 7 mAh Harnessed Mean SD p-value t-value Result Decision Static Solar Panel System 2367.86 mAh 602.89 Reject Ho2 Dual-Axis Solar Tracker 3267.86 mAh 706.03 0.012392 2.56476 Significant Significant at a = 0.05 Table IV. T-test on the effectivity of the Dual-Axis Solar Tracker in terms of milliamps per hour. t-Test: Two Independent Means The results revealed that the computed value of P is 0.012392. Since it is less than the 0.05 level of significance, Ho_2 which states that “There is no significant difference in the effectivity of the Static Solar Panel System and the Dual-Axis Solar Tracker System in terms of the milliamps per hour stored in the lithium-ion batteries.” is therefore rejected. This implies that there is a significant difference in the effectivity of the Static Solar Panel System and the Dual-Axis Solar Tracker System in terms of milliamps per hour output. Therefore, the Dual-Axis Solar Tracker System is more effective in terms of milliamps per hour output than the Static Solar Panel System since there is a significant difference in the t- test done. The results revealed that the computed value of P is 0.012392. Voltage Output Mean SD p-value t-value Result Decision Static Solar Panel System 3.54 0.0001 Significant Reject Ho2 Dual-Axis Solar Tracker 3.62 0.0001 0.004622 3.0969 Significant at a = 0.05 The study utilized an applied experimental research design. The study was conducted to develop a dual-axis solar tracker that is self-sufficient having the characteristics of an off-grid powered model that is not reliant on the electrical grid; and has the capacity to store power in lithium-ion batteries. Then, determine the amount of power that the solar panels can store in the lithium-ion batteries in terms of the batteries’: voltage; and milliamps per hour harnessed. And lastly, determine the significant difference between the static and dual-axis systems in terms of the batteries’ voltage; and milliamps per hour harnessed. The device was created and tested at Poblacion 1, Midsayap, North Cotabato. The Dual-Axis Solar Tracker was fabricated by first creating a working system flow diagram to track the sun's greatest light position. It was then followed by writing a code using Arduino sketches to utilize the micro servos as actuators of the system and, wiring the Arduino Uno board to the system's electrical parts. The researchers created a chassis for the Dual-Axis Solar Tracker. Additionally, the researchers connected the solar panels from the Dual-Axis Solar Tracker and the Static Solar Panel System to the lithium-ion batteries. The batteries then power the Dual-Axis Solar Tracker for about 3 minutes every hour. This function makes the Dual-Axis Solar Tracker independent from the power grid. After the seven-day data collection, for the voltage, the Dual-Axis Solar Tracker generated an average of 3.62 volts, while the static solar panel system generated an average of 3.54 volts. For the milliamps per hour, the dual-axis solar tracker generated an average of 3267.86 milliamps per hour, while the static solar panel system generated 2367.86 milliamps per hour. After the seven-day data collection, for the voltage, the Dual-Axis Solar Tracker generated an average of 3.62 volts, while the static solar panel system generated an average of 3.54 volts. For the milliamps per hour, the dual-axis solar tracker generated an average of 3267.86 milliamps per hour, while the static solar panel system generated 2367.86 milliamps per hour. **Significant at a = 0.05 Since it is less than the 0.05 level of significance, Ho_2 which states that “There is no significant difference in the effectivity of the Static Solar Panel System and the Dual-Axis Solar Tracker System in terms of the milliamps per hour stored in the lithium-ion batteries.” is therefore rejected. This The results revealed that the computed value of P is 0.012392. Since it is less than the 0.05 level of significance, Ho_2 which states that “There is no significant difference in the effectivity of the Static Solar Panel System and the Dual-Axis With the T-test done, the significant difference shows that the milliamps per hour data that were gathered are not due to chance or sampling error. On a bright sunny day, the Dual-Axis Solar Tracker System will always harness more milliamps per hour than the Static Solar Panel System. 94 94 Based on the given data, facts, and the computations using the T-test, it was found that in terms of voltage, there is a significant difference between the Dual-Axis Solar Tracker and the Static Solar Panel System; therefore, the first null hypothesis which states that “there is no significant difference in the effectivity of the Static Solar Panel System and the Dual-Axis Solar Tracker System in terms of the voltage stored in the lithium-ion batteries.” is rejected. Meanwhile, in terms of milliamps per hour, it was also found that there is a significant difference between the Dual-Axis Solar Tracker and the Static Solar Panel System; therefore, the second null hypothesis which states that “there is no significant difference in the effectivity of the Static Solar Panel System and the Dual-Axis Solar Tracker System in terms of the milliamps per hour stored in the lithium-ion batteries.” is rejected. CONCLUSIONS Based on the study's findings, it is concluded that the amount of voltage and milliamps per hour generated by the Dual-Axis Solar Tracker is greater compared to the static solar panel system. Moreover, this statement is supported by the t-test that was done. The t-test shows that there is a significant difference between the voltage and milliamps per hour. Thus, the results show that on a bright sunny day, the Dual-Axis Solar Tracker System will always generate more voltage and milliamps per hour than the Static Solar Panel System. 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https://openalex.org/W4289781030	https://www.nature.com/articles/s41598-022-17505-4.pdf	English	null	Forecasting of energy consumption by G20 countries using an adjacent accumulation grey model	Scientific reports	2,022	cc-by	14,035	Forecasting of energy consumption by G20 countries using an adjacent accumulation grey model OPEN Ijlal Raheem1, Nabisab Mujawar Mubarak2, Rama Rao Karri2, T. Manoj3, Sobhy M. Ibrahim4, Shaukat Ali Mazari5 & Sabzoi Nizamuddin6 This paper studies an adjacent accumulation discrete grey model to improve the prediction of the grey model and enhance the utilization of new data. The impact of COVID-19 on the global economy is also discussed. Two cases are discussed to prove the stability of the adjacent accumulation discrete grey model, which helped the studied model attain higher forecasting accuracy. Using the adjacent accumulation discrete grey model, non-renewable energy consumption in G20 countries from 2022 to 2026 is predicted based on their consumption data from 2011 to 2021. It is proven that the adjacent accumulation exhibits sufficient accuracy and precision. Forecasting results obtained in this paper show that energy consumption of all the non-renewable sources other than coal has an increasing trend during the forecasting period, with the USA, Russia, and China being the biggest consumers. Natural gas is the most consumed non-renewable energy source between 2022 and 2026, whereas hydroelectricity is the least consumed. The USA is the biggest consumer of Nuclear energy among the G20 countries, whereas Argentina consumed only 0.1 Exajoules of nuclear energy, placing it at the end of nuclear energy consumers. Abbreviations G20 Great 20 MAE Mean absolute error RMSE Root mean square error MAPE Mean absolute percentage error DAGM Discrete adjacent grey model EOGM Exponential optimization grey model ARX Autoregressive exogenous OECD Organization for Economic Co-operation and Development Global industrialization and urbanization have entered a crucial stage and are expected to play a role in global economic growth in the next 10 or 20 years. However, countries must consume high energy for rapid urbanization and industrialization to reach their financial targets1. Due to global warming threats and limited resources, it is not sustainable to keep consuming natural resources in high amounts in the long run. It is significant to control and reduce energy consumption growth globally, which is impossible until accurate energy consumption data of developed countries2. p Forecasting is a technique that uses small data to extract valuable insight and can be used for both short- term and long-term forecasts3. Energy consumption forecasting is a technique many countries use to predict the future prosperity of the energy consumption pattern4. Governments and businesses use energy consumption forecasting to determine their policies and strategies related to energy consumption for the upcoming years5. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Forecasting of energy con by G20 countries using an adjacent accumulation model Ijlal Raheem1, Nabisab Mujawar Mubarak2, Rama Rao Karri2, T. M Sobhy M. Ibrahim4, Shaukat Ali Mazari5 & Sabzoi Nizamuddin6 This paper studies an adjacent accumulation discrete grey model to improv grey model and enhance the utilization of new data. The impact of COVID- is also discussed. Two cases are discussed to prove the stability of the adjac grey model, which helped the studied model attain higher forecasting accu accumulation discrete grey model, non-renewable energy consumption in G 2026 is predicted based on their consumption data from 2011 to 2021. It is accumulation exhibits sufficient accuracy and precision. Forecasting results show that energy consumption of all the non-renewable sources other than trend during the forecasting period, with the USA, Russia, and China being Natural gas is the most consumed non-renewable energy source between 2 hydroelectricity is the least consumed. The USA is the biggest consumer of G20 countries, whereas Argentina consumed only 0.1 Exajoules of nuclear of nuclear energy consumers. Abbreviations G20 Great 20 MAE Mean absolute error RMSE Root mean square error MAPE Mean absolute percentage error DAGM Discrete adjacent grey model EOGM Exponential optimization grey model ARX Autoregressive exogenous OECD Organization for Economic Co-operation and Development Global industrialization and urbanization have entered a crucial stage and are ex economic growth in the next 10 or 20 years. However, countries must consume high and industrialization to reach their financial targets1. Due to global warming thre not sustainable to keep consuming natural resources in high amounts in the long and reduce energy consumption growth globally, which is impossible until accu of developed countries2. Forecasting is a technique that uses small data to extract valuable insight an term and long-term forecasts3. Energy consumption forecasting is a technique ma future prosperity of the energy consumption pattern4. Governments and busine forecasting to determine their policies and strategies related to energy consumpt PEN 1Department of Chemical Engineering, Faculty of Engineering and Science, Curtin U Malaysia. 2Petroleum and Chemical Engineering, Faculty of Engineering, Unive Seri Begawan 1410, Brunei Darussalam. 3Department of Physics and Materials Sc Institute of Information Technology, Noida 201309, India. 4Department of Biochem Saud University, P.O. Box: 2455, Riyadh 11451, Saudi Arabia. 5Department of C University of Engineering and Technology, Karachi 74800, Pakistan. 6School of M lb 3000 A t li * il b k @ il k iitd@ Forecasting of energy consumption by G20 countries using an adjacent accumulation grey model OPEN 1Department of Chemical Engineering, Faculty of Engineering and Science, Curtin University, 98009 Miri Sarawak, Malaysia. 2Petroleum and Chemical Engineering, Faculty of Engineering, Universiti Teknologi Brunei, Bandar Seri Begawan 1410, Brunei Darussalam. 3Department of Physics and Materials Science and Engineering, Jaypee Institute of Information Technology, Noida 201309, India. 4Department of Biochemistry, College of Science, King Saud University, P.O. Box: 2455, Riyadh 11451, Saudi Arabia. 5Department of Chemical Engineering, Dawood University of Engineering and Technology, Karachi 74800, Pakistan. 6School of Engineering, RMIT University, Melbourne 3000, Australia. *email: mubarak.yaseen@gmail.com; kramarao.iitd@gmail.com \| https://doi.org/10.1038/s41598-022-17505-4 Scientific Reports \| (2022) 12:13417 www.nature.com/scientificreports/ Figure 1. Population growth in G20 countries between 2021 and 2027 (in millions)13. Figure 1. Population growth in G20 countries between 2021 and 2027 (in millions)13. Grey models are widely used to forecast energy consumption by many countries. Previously, a grey prediction model with a very grey average weakening buffer operator was used to forecast China’s shale gas output. Apart from that, a new fractional accumulation grey model was used to forecast the nuclear energy consumption of China. Many forecast models are available, but the grey system theory, which Deng Julong proposed in the 1980s, has gained the attention of many researchers and scholars. Scholars have considered different aspects to improve the grey model4. For instance, the model proposed by Markov optimizes the background value and improves the central point weight function6. Bernoulli’s model proposed the nonlinear grey model that optimizes the initial condition with boundary value7. Nevertheless, the above-given models are only suitable for minor scale problems. Therefore, an optimized grey system theory was proposed for more big sample data with adjacent accumulation DAGM (1,1)8.i During the construction step, the grey model treats every model equally. Still, since the significance of each sample that contributes to the construction of the prediction model cannot be the same, it is feasible to estimate the weight of each piece independently to obtain better results9. In the discrete adjacent grey model, the sample size does not change or affect the forecasting model, making the percentage errorless and more stable10.hi gf g g g The discrete grey model has been previously used to predict the energy consumption of the Asia–Pacific Economic Cooperation10. Considering the size and magnitude of the G20, it is significant to predict their non- renewable energy consumption. G20 was formed in 1999; it consists of World’s largest economies. Literature review A Series of ecological and environmental issues, such as extreme weather, greenhouse effects and global warm- ing, affect human survival and development. High carbon emission issues have resulted from increased non- renewable energy consumption and rapid urbanization. Energy demand is expected to grow globally due to economic and population growth14. Short-term pollutants and greenhouse gases are mainly emitted from fossil fuel combustion. Human activities, especially GHG emissions, cause climate change in this industrial age. Energy consumption releases approximately 35 billion tons of carbon dioxide into the atmosphere each year. Therefore, it is significant to develop energy consumption plans by developed countries to reduce global carbon emissions15. gi p gy p p y p g Annually, due to ongoing urbanizing, a huge number of people have been moving from rural areas to urban areas worldwide. Over the past four decades, the urban population has increased from 39.1% in 1980 to 56.61 in 2021 worldwide16. Nevertheless, it is generally acceptable that economic development and quality of life can be improved by urbanization but it also causes other challenges such as energy demand and energy consumption by stimulating which makes urbanization another factor of staggering increase in CO2 emission level over the last four decades17. Relationship between energy consumption and economic factors in few developed countries are tabulated in Table 1. Impact of COVID‑19. Global economy and energy sector were majorly affected by COVID-19 pandemic. For a affect, it had caused worst damage than World War II as the global economy was shocked by it23. Uncertain- ties about long-lasting economic crisis were justified by the safety measures such as travel restrictions, border shutdowns and quarantine to make the pandemic curve linear24. Demand for many services and good were significantly declined by induced economic loss due to COVID-19. Consequently, demand for fossil fuel was drastically hindered all over the world25. According to IEA global energy review, energy demand was declined by 3.8% from January to March 2020 and further decline by 6% at the end of 202026. Decline in energy consumption from January 2020 to March 2020 also resulted in reduction of carbon dioxide emission in 202027.i y Additionally, economy dependent on non-renewable energy like oil, gas and coal was significantly decreased by 5%, 2% and 8%, respectively. It is predicted by the researchers that global energy economic crisis by the end 2030 can be greater than the energy economic crisis in 200826. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 1. Relationship between energy consumption and economic factors. Study region Method Variables Main findings References China and Taiwan Engle–Granger Energy consumption: coal, natural gas, electricity, GDP GDP → OC 18 India ARDL bounds testing approach GDP, high speed diesel HSD ↔ GDP GDP ↔ HDC 19 Portugal ARDL bounds test Primary energy consumption, GDP OC ↔ GDP 20 China Granger causality tests GDP, OC OC → GDP 21 South Korea Granger causality tests GDP, OC OC → GDP 22 Table 1. Relationship between energy consumption and economic factors. Forecasting of energy consumption by G20 countries using an adjacent accumulation grey model OPEN Every year, G20 discusses financial stability and international economic by bringing together the most important developing and industrialized economies11. G20 represents 80% of global trade, 60% of world’s population and 85% of global GDP. Its objective is to create financial mechanism, risk reduction, sustainable growth, energy policy and coordinate policy between its member countries12. Figure 1 shows the contribution of G20 in world population in millions. So, considering their non-renewable energy consumption, which results in an increase in carbon dioxide emission and global warming and is highly predicted to cause energy and fossil fuel shortfall in the future, it is critical to forecasting the energy consumption of G20 countries for the next 5 years. So, the relevant authorities can architect their energy policy for the future. gy p y In this paper, non-renewable energy consumption in G20 countries is predicted by using DAGM (1,1). Results are obtained for non-renewable energy consumption prediction in G20 countries. Hence, this paper makes the following contributions. • The background of the grey model is studied, and its comparison is reviewed to analyze the performance and accuracy of other grey models and DAGM (1,1). The background of the grey model is studied, and its comparison is reviewed to analyze the performance and accuracy of other grey models and DAGM (1,1). y g y ( ) • DAGM (1,1) weakens the constraints of GM (1,1) as it can be applied to exponential growth data and improves the development of the grey forecasting model.h g y g • The relationship between DAGM (1,1) and other forecasting grey models is also studied to estimate the accuracy of DAGM (1,1). The accuracy of model and coefficient development was analyzed according to actual data. • The model’s validity was analyzed by studying the non-renewable energy consumption in G20 countries. The obtained results show that the new model can be applied to other energy consumption forecasting and that it has predicted the non-renewable energy consumption in G20 countries to propose effective policy recommendations to the corresponding countries. https://doi.org/10.1038/s41598-022-17505-4 Scientific Reports \| (2022) 12:13417 \| www.nature.com/scientificreports/ Literature review • Topological forecasting. • Systematic forecasting. • Time series forecasting. • Calamity forecasting. • Seasonal calamity forecastin • Topological forecasting. • Systematic forecasting. • Time series forecasting. • Calamity forecasting. • Seasonal calamity forecasting. • Topological forecasting. • Systematic forecasting. GM (1,1) is main model of grey theory of prediction, i.e., single variable first order grey model which can provide high precision results by creating with few data (four or more). GM (!,1) model is one of the most used method in grey system35. Recently, it has exhibited satisfactory results after getting applied in many fields. Despite implementation of GM (1,1) model in many fields its performance can still be improved36. Many techniques to optimize the precision of model have been proposed over the years. Residual sequence amendable method was provided by Deng10. Method for optimum grey derivative’s whitening values were provided by Mu in which he proposed a method to estimate parameters by developing an unbiased GM (1,1). Model GM (1,1) was amended by providing the center approach in which adjusting grey model was derived37. The background values of struc- ture method were provided in model GM (1,1), which showed strong adaptability by reestablishing a simple calculating formula38. Optimum time response sequence was provided by utilizing the least square method in order to find the constant number c in basic equation of GM (1,1), eventually optimum time response sequence was created for model GM (1,1)39,40. Forecasting precision can be advanced by all these methods, apart from that it also reduces error partially but problem could not be solved completely. Many studies have been conducted to improve the application of Grey model, traditional GM (!,1) and other modelling technology are integrated in these improved Grey models but it does not include research Grey model’s accumulated generating operator7. Rolling mechanism was used to emphasize the principle of new information of small sample in Grey model. Rolling mechanism assisted Grey model in keeping equal series dimensions upon removing the older data and adding new one41.t Once the results are obtained, new data is added at the end of series after older data is deleted from system. It is recommended to use rolling mechanism Grey model for recent data, so the accuracy of forecasting could be increased for future prediction42. The future law of development for system with small data is represented by the new data. Literature review Hence, significance and principle of new information in grey model theory was put forward by researchers. To consider the principle of new information priority, a novel Grey model with fractional accumu- lation is used in this paper which indicates that used GM (1,1) has higher performance and precision not only for forecasting but also for model fitting43. Literature review Figure 2 shows the relationship Between Energy Consumption and GDP growth in 2020. Significance of forecasting. Forecasting is defined as a predicting future trend by taking into consid- eration past and present trends. Energy forecasting is very crucial as it can help global stakeholders to plan and implement their energy policies. G20 countries are major energy consumers in the World due to their rapid industrialization and economic growth. Therefore, accurate forecast of energy consumption can help these countries to assess the future energy demand and supply situation in their countries by making readjustments and optimization in energy structure, and diversify energy use. Based on forecasting and optimization, alloca- tion of social resource and healthy growth of economy can be focused. Forecasting methods. Most commonly used prediction methods are given below. orecasting methods. Most commonly used prediction methods are given below. Intelligent prediction methods and statistical method. A hybrid intelligent approach was developed by applying an adaptive algorithm to evaluate energy consumption, GDP, import and export data and taking population as inputs29. Artificial neutral network ARX (ANNARX) and conventional autoregressive exogenous (ARX) model were used to predict Malaysian oil data in 2020 on the basis of its per capita GDP, population and oil consump- tion data. Oil consumption in Canada, Japan and Germany were predicted by applying and adaptive neuro-fuzzy inference system which was optimized with a sine–cosine algorithm30. Apart from that, fuel consumption by marine engines were predicted by different machine learning models31. Malaysia’s oil demand was predicted by applying regression model whereas long term correlation between energy consumption and economic factors, labor productivity, oil prices and other influencing factors were predicted by using cointegration method32. Grey model. Various uncertainty theories are emerged due to limited and uncertain information obtained by people. For instance, Fuzzy mathematics and Rough sets theories were proposed but system structure could realize only small part due to the limited knowledge and information33. To resolve this issue, grey system theory was proposed by Deng in 198234. Many fields have successfully applied grey system theory. There are five main categories of grey prediction such as: Scientific Reports \| (2022) 12:13417 \| https://doi.org/10.1038/s41598-022-17505-4 ww.nature.com/scientificreports/ Figure 2. Relationship between energy consumption and GDP in 202028. www.nature.com/scientificreports/ Figure 2. Relationship between energy consumption and GDP in 202028. Figure 2. Relationship between energy consumption and GDP in 202028. • Time series forecasting. • Calamity forecasting. • Seasonal calamity forecasting. Performance evaluation of grey model. To analyze the accuracy and performance of studied discrete grey model, it is compared with other forecasting models by discussing two different cases. Performance evaluation of grey model. To analyze the accuracy and performance of studied discrete grey model, it is compared with other forecasting models by discussing two different cases. Case 1. In this case, four models were used to predict the added value of the tertiary industry in China using the data from 2005 to 2012 to forecast 2013 to 2014. The predicted values of the model are shown in Table 2. It can be seen that DAGM (1,1) possesses smaller MAPE values as compared to the other four models irrespective of the error of fitting result; hence it has been proven that DAGM (1,1) is very much effective44. Table 2 shows the forecasting results of different forecasting grey models. Case 2. This model was also applied in Jiangsu province as the conventional GM (1,1) only satisfies the fore- casting for small samples. Generally, Higher predictive accuracy can be obtained when the sample volume is small during the forecasting process. Hence, the accuracy of DAGM (1,1) is proven by applying it to smaller volume samples and then comparing it to GM (1,1). It is shown in Table that DAGM (1,1) possesses better results https://doi.org/10.1038/s41598-022-17505-4 Scientific Reports \| (2022) 12:13417 \| www.nature.com/scientificreports/ Table 2. Case 1: Forecasting results of different grey models44. Year Actual value GM (1,1) RPE % EOGM (1,1) RPE % DAGM (1,1)10 RPE % 2005 1590.7 1590.7 0 1590.7 0 1590.7 0 2006 1815.3 1896.7 4.484107 1847.3 1.762794 1825.6 0.567399 2007 2106.7 2084.3 1.06327 2077.8 1.37181 2073.4 1.58067 2008 2327.1 2291.1 1.54699 2302.6 1.05281 2320.5 0.28361 2009 2547.4 2518.5 1.13449 2536.2 0.43966 2568.1 0.812593 2010 2794.1 2768.5 0.91622 2786.9 0.25769 2813.1 0.680004 2011 3059.7 3043.2 0.53927 3056.4 0.10785 3058.5 0.03922 2012 3303.3 3345.3 1.271456 3333.8 0.923319 3303.3 0 MAPE – 38.65 – 21.15 – 15.85 – MAE – 31 – 17.2 – 11.26 – RMSE – 1.37 – 0.74 – 0.49 – 2013 3576 36,773 2.83277 3650.3 2.07774 3547.6 0.79418 2014 3856.6 4042.3 4.815122 3987.4 3.391588 3791.3 1.6932 MAPE – 149.58 – 106.37 – 50.35 – MAE – 143.5 – 102.55 – 46.85 – RMSE – 3.82 – 2.73 – 1.24 – Table 2. Case 1: Forecasting results of different grey models44. s it has a lesser influence on disturbance boundary as compared to GM (1,1)45. Table 3 shows the comparison f discrete grey model with other forecasting results. Table 3. Case 2: Forecasting results of different grey models45. f Methodology G d l46 y(0) = y(0)(1), y(0)(2), y(0)(3), y(0)(4) . . . y(0)(n) , where n represents the total number of periods and is greater or equal to 5. Th f ( ) b d b d h l d The precision of GM (1,1) can be increased by deriving the accumulated generating operator through original time series data. The accumulated generating data operator is derived. y(1)(k) = n m=1 y(0)(m), k = 2, 3 . . . , n. Obtained time series data can then be expressed as, Obtained time series data can then be expressed as, y(1) = y(1)(1), 2 m=1 y(0)(m), 3 m=1 y(0)(m), 4 m=1 y(0)(m), · · · n m=1 y(0)(m) , https://doi.org/10.1038/s41598-022-17505-4 https://doi.org/10.1038/s41598-022-17505-4 https://doi.org/10.1038/s41598-022-17505-4 Performance evaluation of grey model. To analyze the accuracy and performance of studied discrete grey model, it is compared with other forecasting models by discussing two different cases. Year Actual value GM6(1,1) RPE % GM4(1,1) RPE % DAGM (1,1)10 RPE % 2000 132.4 132.4 0 – – 132.4 0 2001 144.6 142 1.79806 – – 142.3 1.59059 2002 156.3 157.3 0.639795 156.3 0 155.8 0.3199 2003 1737 174.2 0.287853 172.1 0.92113 172.2 0.86356 2004 190.2 193 1.472135 192.5 1.209253 192.1 0.998948 2005 216.7 213.8 1.33826 215.3 0.64605 216.2 0.23073 RMSE – 1.12 – 0.93 – 0.66 – MAE – 1.63 – 1.33 – 1.12 – MAPE – 2.01 – 1.57 – 1.39 – 2006 249.4 236.8 5.05213 240.8 3.44828 245.4 1.60385 RMSE – 5.04 – 3.46 – 1.61 – MAE – 12.6 – 8.6 – 4 – MAPE – 12.6 – 8.6 – 4 – Table 3. Case 2: Forecasting results of different grey models45. Table 3. Case 2: Forecasting results of different grey models45. as it has a lesser influence on disturbance boundary as compared to GM (1,1)45. Table 3 shows the comparison of discrete grey model with other forecasting results. Methodology Grey Model46 https://doi.org/10.1038/s41598-022-17505-4 Scientific Reports \| (2022) 12:13417 \| www.nature.com/scientificreports/ Open Spreadsheet Make Time Data Available for Use In the First Column, Input the Data Including Duraon and Time In the Next Column, Input the Values for Forecasng Select Relevant Data Click Data then Forecast Group and Choose Forecast Sheet Choose the Line or Bar Graph as Required aer Accessing the Sheet In the Forecast Tab, Analyze end Data and Click Create Figure 3. Flowchart to estimate outcomes by time series model. Click Data then Forecast Group and Choose Forecast Sheet Bar Graph as Required aer Accessing the Sheet g Sheet In the Forecast Tab, Analyze end Data and Click Create In the Next Column, Input the Values for Forecasng Figure 3. Flowchart to estimate outcomes by time series model. y(1)(1), y(1)(2), y(1)(3) . . . . . . . . . y(0)(n)). Given below Fig. 3 shows the steps to follow to obtain time series model. Once the forecasting model is setup, formulate best estimation of future consumption by moving onto interpretation. d ff l h d h ( ) d l Given below Fig. 3 shows the steps to follow to obtain time series model. O h f d l f l b f f Once the forecasting model is setup, formulate best estimation of futu interpretation.f p Grey differential equation is then used to construct the GM (1,1) model. y(0)(k) + az(1)(k), = u, where a = Development coefficient, u = Grey Input, z = Background value. z(1)(k) = 1 2 × y(1)(k) + y(1)(k −1) , k = 2, 3 . . . .n, where y(1)(k) = y(0)(‘) −u a × e−a(k−1) + u a , where y(1)(k) = y(0)(‘) −u a × e−a(k−1) + u a , y(1)(k) = y(0)(‘) −u a × e−a(k−1) + u a , a u = (ATA)−1A TY, a u = (ATA)−1A TY, and A = −z(1) 2 1 −z(1) 3 1 −z(1) (n) 1 , Y = [y(0)(‘2), y(0)(‘2) . . . y(0)(n)]T. An inverse of accumulation grey model is employed to obtain the values for forecasting value, which gives the following forecasting equation3. y(0) n + p =y(1) n + p −y(1) n + p −1 , =(y(0)(1) −u a ) 1 −e0 e−a(n+p−1), p = 1, 2, 3. https://doi.org/10.1038/s41598-022-17505-4 y(0) n + p =y(1) n + p −y(1) n + p −1 , =(y(0)(1) −u a ) 1 −e0 e−a(n+p−1), p = 1, 2, 3. where y(0)(1) , y(0)(2) , y(0)(3)………y(0)(n) = GM (1,1). (0)( + 1) (0)( + 2) (0)( + 3) (0)( + P) F i l f h d l i h i d where y(0)(1) , y(0)(2) , y(0)(3)………y(0)(n) = GM (1,1). y(0)(n + 1), y(0)(n + 2), y(0)(n + 3) . . . . . . ..y(0)(n + P) = where y(0)(1) , y(0)(2) , y(0)(3)………y(0)(n) = GM (1,1). (0)( + 1) (0)( + 2) (0)( + 3) (0)( + P) F ti l f th d l i th i d where y(0)(1) , y(0)(2) , y(0)(3)………y(0)(n) = GM (1,1). y(0)(n + 1), y(0)(n + 2), y(0)(n + 3) . . . . . . ..y(0)(n + P) = Forecasting value of the model in the period, p where y (1) , y (2) , y (3)………y (n) = GM (1,1). y(0)(n + 1), y(0)(n + 2), y(0)(n + 3) . . . . . . ..y(0)(n + P) = Forecasting value of the model in the period, p y(0)(n + 1), y(0)(n + 2), y(0)(n + 3) . . . . . . ..y(0)(n + P) = Forecasting value of the model in the period, p https://doi.org/10.1038/s41598-022-17505-4 Discrete grey model and its stability10. Definition 1 X(0) = x(0)(1), x(0)(2), x(0)(3), x(0)(4) . . . . . . . . . x(0)(n) , where x(0) = orignal data, = adjacent accumulation parameter, x(1) = adjacent accumulation generating sequence, L[x(0)(k)] = perturbation bound of L[x(0)(k)]. x(0)(1) = (x(0)(1), x(0)(1) = (x(0)(1), Scientific Reports \| (2022) 12:13417 \| https://doi.org/10.1038/s41598-022-17505-4 www.nature.com/scientificreports/ x(1)(2) = x(0)(1) + x(0)(2), x(1)(3) = x(0)(2) + x(0)(3), x(1)(2) = x(0)(1) + x(0)(2), x (2) = x (1) + x (2), x(1)(3) = x(0)(2) + x(0)(3), x(1)(3) = x(0)(2) + x(0)(3), x(1)(n) = x(0)(n −1) + x(0)(n), x(1)(n) = x(0)(n −1) + x(0)(n), where = Adjacent accumulation parameter, X(1) = x(1)(1), x(1)(2), x(1)(3), x(1)(4) . . . x(1)(n) = Accumula- ion generating sequence pf X(0). where = Adjacent accumulation parameter, X(1) = x(1)(1), x(1)(2), x(1)(3), x(1)(4) . . . x(1)(n) = Accumula- tion generating sequence pf X(0). can be adjusted during the generation sequence of old and new data. Then the least-squares estimation parameter is satisfied by x(1)(k + 1) = β1x(1)(k) + β2, β = (BTB)−1BTY, x(1)(1) = x(0)(1), x(k + 1) = βk 1x(0)(1) + 1 −βk 1 1 −β2 .β2, k = 1, 2, 3, 4 . . . . . . n −1, x(0)(k + 1)x1(k + 1) −x(0)(k + 1). x(0)(k + 1)x1(k + 1) −x(0)(k + 1). x(0)(k + 1)x1(k + 1) −x(0)(k + 1). It is necessary to use the adaject accumulation method to accumulate the original data X(0) . The weight of old and new information is adjusted to introduce the adjacent accumulation parameter.hl j j p To perform the study and simulation, a Discrete adjacent grey model is used. The flowchart of the simula is shown in Fig. 4. The following lemma then tests the stability of the discrete adjacent grey model. x ≤K† γ† B2 B x+Y B +K† γ† B2 B K† γ† rx B = 1 + 2\|ε\|K† γ† 1 Bx + 1 B + K† γ† 1 B rx B . Lemma 1 Let A ∈Cm×n, b ∈Cm2 where At = Inverse of matrix A, B = A + E, C = b + k ∈Cm , Bx −c2 = max, Ax −b2 = min. Discrete grey model and its stability10. emma 1 Let A ∈Cm×n, b ∈Cm2 where At = Inverse of matrix A, B = A + E, C = b + k ∈Cm , Bx −c2 = max Ax −b2 = min. Then the equation becomes Then the equation becomes where Kt=A∗ 2A · γ∗= 1 −A∗ 2E2 · rx = b −Ax. h ≤Kt γt (E2 A x + k A + Kt γt E2 A rt A , h ≤Kt γt (E2 A x + k A + Kt γt E2 A rt A , where Kt=A∗ 2A · γ∗= 1 −A∗ 2E2 · rx = b −Ax. Theorem 110 Assume that X(0) = {x(0)(1), x(0)(2), x(0)(3), x(0)(4) . . . . . . . . . x(0)(n) is the original nonnegative sequence, B and Y are supposed to remain the same as mentioned earlier L[x(0)(k) = K† γ† B2 B x + Y B + K† γ† B2 B rx B , k = 1, 2, 3, 4 . . . ..n, where L[x(0)(k) = Perturbation Bound. x(0)(k) = x(0)(k)+ ∈andB†2B2 < 1, L[x(0)(k) Does not change with an increase in sample values. Hence, DAGM (1,1) solution is more suitable and stable. of x(0)(2) = x(0)(2)+ ∈ Proof of x(0)(2) = x(0)(2)+ ∈ Proof of x(0)(2) = x(0)(2)+ ∈ β = B + B =   x(1)(1) 1 x(1)(2) 1 x(1)(3) 1 x(1)(4) 1 . . . . .x(1)(n) 1   +   0 0 ∈ 0 0 . . . . .0 0   , β = B + B =   x(1)(1) 1 x(1)(2) 1 x(1)(3) 1 x(1)(4) 1 . . . . .x(1)(n) 1   +   0 0 ∈ 0 0 . . . . .0 0   , https://doi.org/10.1038/s41598-022-17505-4 Scientific Reports \| (2022) 12:13417 \| www.nature.com/scientificreports/ Step 1 Start Step 2 Data Processing Step 3 Estimating Parameters Step 4 Constructing the DAGM (1,1) model Step 5 Analyzing simulation performance of DAGM (1,1) Step 6 Analyzing the prediction performance of DAGM (1,1) Step 7 Minimize the Errors Stop Figure 4. Flow Chart to Design DAGM (1,1)10. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 4. Tools used for this study. Softwares Purpose MS Excel 2016 To analyze the results and get the values MATLAB To verify and analyze the results Microsoft Excel Solver To perform the Generalized Reduced Gradient (GRG) nonlinear method for optimization Origin Lab To plot the graphs and figures Table 4. Tools used for this study. Table 5. Oil consumption data of G20 countries47. All consumption in exajoules. Country 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 Argentina 1.22 1.30 1.38 1.37 1.39 1.35 1.34 1.27 1.17 1.07 1.23 Australia 1.97 2.02 2.08 2.07 2.05 2.06 2.16 2.20 2.18 1.88 1.93 Brazil 4.83 5.00 5.27 5.44 4.94 4.70 4.75 4.51 4.54 4.22 4.46 Canada 4.58 4.64 4.62 4.61 4.63 4.61 4.57 4.73 4.70 4.11 4.17 China 19.41 20.36 21.27 22.11 23.80 24.56 25.86 27.12 28.49 28.74 30.60 France 3.45 3.34 3.29 3.19 3.19 3.17 3.18 3.17 3.14 2.68 2.91 Germany 4.73 4.70 4.80 4.67 4.67 4.76 4.87 4.63 4.66 4.22 4.18 India 6.91 7.33 7.38 7.59 8.20 8.99 9.26 9.68 9.99 9.08 9.41 Indonesia 3.05 3.21 3.11 3.09 2.95 2.84 3.05 3.15 3.06 2.70 2.83 Italy 2.98 2.78 2.54 2.42 2.56 2.54 2.57 2.63 2.55 2.11 2.35 Japan 8.78 9.37 8.96 8.51 8.17 7.93 7.81 7.56 7.32 6.49 6.61 South Korea 4.63 4.80 4.81 4.79 5.02 5.47 5.42 5.40 5.35 5.06 5.39 Mexico 3.97 4.04 3.93 3.75 3.70 3.73 3.59 3.51 3.24 2.47 2.56 Russia 6.20 6.30 6.30 6.60 6.34 6.50 6.48 6.55 6.69 6.34 6.71 Saudi Arabia 6.03 6.34 6.36 7.02 7.29 7.36 7.14 6.90 6.78 6.54 6.59 South Africa 1.10 1.13 1.15 1.13 1.25 1.19 1.19 1.19 1.18 0.96 1.04 Turkey 1.34 1.41 1.51 1.55 1.85 1.98 2.07 2.00 2.01 1.84 1.89 United Kingdom 3.14 3.05 3.00 3.00 3.08 3.18 3.19 3.14 3.06 2.35 2.50 United States of America 34.90 34.10 34.66 34.90 35.61 35.86 36.21 37.08 37.02 32.52 35.33 European Union 24.03 23.05 22.47 22.02 22.38 22.81 23.20 23.25 23.19 20.24 21.32 Table 5. Oil consumption data of G20 countries47. All consumption in exajoules. L x(0)(n) = K† γ† \|∈\| B. It is to be noted here that L x(0)(n) It has no correlation with the sample size; hence it does not vary with the sample size. Usually L x(0)(n) Increases with an increase in sample size in conventional model GM (1,1) make DAGM (1,1) more stable. Discrete grey model and its stability10. Step 2 Data Processing Step 1 Start Step 3 Estimating Parameters Step 5 Analyzing simulation performance of DAGM (1,1) Step 6 Analyzing the prediction performance of DAGM (1,1) Step 4 Constructing the DAGM (1,1) model Step 7 Minimize the Errors Stop Figure 4. Flow Chart to Design DAGM (1,1)10. B vector is linearly independent Y = Y + Y =   x(1)(1) x(1)(2) . . . . . . x(1)(n)   +   0 ∈ ∈ . . . . .0   . B vector is linearly independent B2 = max BTB whereas the maximum value for BTB = 1 + 2 ǫ2. Thus Y2 = 1 + 2 ǫ2 = 1 + 2 ǫ2 = 1 + 2\| ∈\|, BTB = 0 0 0 1 + 2 ∈2 . B2 = max BTB whereas the maximum value for BTB = 1 + 2 ǫ2. Thus Y2 = 1 + 2 ǫ2 = 1 + 2 ǫ2 = 1 + 2\| ∈\|, BTB = 0 0 0 1 + 2 ∈2 . B2 = 1 + 2\|∈\|, Similarly, it has been proven that x(0)(r) = x(0)(r)+ ∈, So L x(0)(2) = 1 + 2\|ε\|K† γ† 1 Bx + 1 B + K† γ† 1 B rx B . L x(0)(r) = 1 + 2\|ε\|K† γ† 1 Bx + 1 B + K† γ† 1 B rx B . R = 3,4, …, n−2 When x(0)(n −1) = x(0)(n −1)+ ∈, x(0)(n −1) = x(0)(n −1)+ ∈, L x(0)(n −1) = \|ε\|K† γ† ( 1 Bx + √ 1 + 2 B + K† γ† 1 B rx B , L x(0)(n −1) = \|ε\|K† γ† ( 1 Bx + √ 1 + 2 B + K† γ† 1 B rx B , when x(0)(n) = x(0)(n)+ ∈, https://doi.org/10.1038/s41598-022-17505-4 Scientific Reports \| (2022) 12:13417 \| www.nature.com/scientificreports/ Figure 4 shows the steps that were followed in designing DAGM (1,1) whereas tools and softwares used are mentioned in Table 5. t Future prediction of natural resources in G20 countries. Natural resources always play a very sig- nificant role in developing any country. Demand for these resources is constantly increasing due to the rapid urbanization worldwide. The reserves of these resources are declining due to the rapid increase in consumption and the nonrenewable nature of these resources. The concerned authorities must start looking for alternatives to prevent the depletion of these resources. To perform the research, it was considered to select major industrial countries from all over the world; hence G20 countries are chosen to perform the forecast of these resources in G20 countries by analyzing the historical consumption of non-renewable resources. The recorded data is obtained from the 71th British Petroleum World Energy Statistical Year book 2022. Oil. Analyzing the current situation, the G20 countries include all the world’s primary consumers, such as China, USA, and Russia. Studying oil consumption in these countries is significant, so the future supply and demand can be predicted. Apart from that, the prediction can also help governments make future energy poli- cies. Table 5 includes all the primary oil consumers in the world from 2011 to 2021 to predict the oil consump- tion of G20 countries in the next 5 years. https://doi.org/10.1038/s41598-022-17505-4 Scientific Reports \| (2022) 12:13417 \| www.nature.com/scientificreports/ Table 6. Natural gas consumption data of G20 countries47. All consumption in exajoules. www.nature.com/scientificreports/ Country 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 Argentina 1.58 1.64 1.66 1.66 1.68 1.74 1.74 1.75 1.68 1.58 1.65 Australia 1.18 1.19 1.25 1.34 1.40 1.36 1.34 1.32 1.58 1.55 1.42 Brazil 0.99 1.17 1.38 1.46 1.55 1.34 1.35 1.29 1.29 1.13 1.46 Canada 3.62 3.58 3.80 3.95 3.97 3.78 3.96 4.16 4.22 4.08 4.29 China 4.87 5.43 6.19 6.78 7.01 7.54 8.69 10.22 11.10 12.12 13.63 France 1.55 1.60 1.63 1.36 1.47 1.60 1.61 1.54 1.57 1.46 1.55 Germany 2.91 2.92 3.06 2.66 2.77 3.06 3.16 3.09 3.21 3.14 3.26 India 2.17 2.01 1.76 1.75 1.72 1.83 1.93 2.09 2.13 2.18 2.24 Indonesia 1.54 1.55 1.60 1.59 1.65 1.61 1.56 1.60 1.58 1.35 1.33 Italy 2.67 2.57 2.40 2.12 2.32 2.43 2.58 2.49 2.55 2.43 2.61 Japan 4.03 4.44 4.45 4.49 4.27 4.19 4.21 4.17 3.89 3.75 3.73 South Korea 1.74 1.89 1.98 1.80 1.64 1.72 1.79 2.08 2.02 2.07 2.25 Mexico 2.55 2.65 2.80 2.84 2.91 2.99 3.10 3.15 3.17 3.01 3.18 Russia 15.68 15.43 15.30 15.20 14.71 15.14 15.52 16.36 16.00 15.25 17.09 Saudi Arabia 3.16 3.40 3.42 3.50 3.57 3.79 3.93 4.04 4.00 4.07 4.22 South Africa 0.15 0.16 0.15 0.15 0.16 0.13 0.14 0.16 0.15 0.14 0.14 Turkey 1.51 1.56 1.58 1.68 1.65 1.60 1.86 1.70 1.56 1.66 2.06 United King- dom 2.95 2.77 2.75 2.52 2.59 2.90 2.83 2.83 2.80 2.63 2.77 United States of America 23.70 24.77 25.45 26.00 26.77 26.97 26.64 29.58 30.62 29.95 29.76 European Union 14.01 13.76 13.48 11.93 12.48 13.25 13.87 13.61 14.11 13.69 14.28 Table 6. Natural gas consumption data of G20 countries47. All consumption in exajoules. Natural gas. Natural gas is one of the most significant non-renewable energy sources. Global influential con- sumers of natural gas are members of the G2048. It is critical to decide on the exploitation and utilization of natural gas carefully. For the reasonable utilization of natural gas, it is essential to know the future demand for natural gas so the policymakers can make the relevant decisions. Table 6 shows the natural gas consumption by the G20 countries from 2011 to 2021, which is used in this study to predict the natural gas consumption for the next five years by G20 countries. Nuclear energy. www.nature.com/scientificreports/ Nuclear energy is one of the most dangerous forms of energy as one mistake in handling nuclear power can cause loss of many lives and damage for centuries. Hence, it is a policy by all the United Nations members to reduce the use of nuclear energy. There is no doubt that the development of nuclear energy has a significant effect on coal, oil, and gas. Most G20 countries do not make their nuclear energy consumption data available due to the government policy, so predicted consumption is only available based on available data. Table 7 shows the nuclear energy consumption of G20 countries from 2011 to 2021 to predict the consumption of the next five years. Coal. Coal is the most reliable source as far as global energy consumption is considered; hence it is critical to analyze the coal consumption by the G20 countries closely. As the G20 countries include all the major coal consumers globally, it is easy to predict the global coal consumption by analyzing the coal consumption of G20 countries. To perform the forecasting, coal consumption data from 2011 to 2021 is used to indicate the consumption in the next five years by the G20 countries. Table 8 shows the coal consumption by G20 countries from 2011 to 2021. Hydroelectricity. Hydroelectricity is used to harness the power of water in motion, such as the flow of water over a waterfall. There are three types of hydroelectricity in which impoundment is the most common one. Hydroelectricity is not commonly used in most G20 countries; hence, a few countries’ consumption data is unavailable. Table 9 shows hydroelectric energy consumption from 2011 to 2021 to predict the consumption in the next 5 years by the G20 countries. Results and discussion Oil. Figure 5 shows the Forecasting of Oil Consumption by G20 countries in continent America. The United States is the biggest consumer of Oil in the American region, followed by Brazil and Canada due to their rapid industrialization49. Oil consumption declined in the United States between 2011 and 2014, and it is predicted to be increased from 2022 to 2026. Brazil’s oil consumption showed robust growth between 2011 and 2015 as their oil consumption was 4.83 exajoules in 2011, which increased to 4.94 exajoules in 2015. High consumption of oil forced Brazil to change its energy policy and reduce its oil consumption which they managed to do which can be https://doi.org/10.1038/s41598-022-17505-4 Scientific Reports \| (2022) 12:13417 \| www.nature.com/scientificreports/ Table 7. Nuclear energy consumption data of G20 countries47. All consumption in exajoules. Country 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 Argentina 0.06 0.06 0.06 0.05 0.07 0.08 0.06 0.06 0.08 0.10 0.10 Brazil 0.15 0.15 0.15 0.14 0.14 0.15 0.14 0.14 0.15 0.13 0.13 Canada 0.89 0.89 0.97 1.00 0.94 0.93 0.92 0.91 0.92 0.88 0.83 China 0.83 0.93 1.05 1.25 1.59 1.97 2.28 2.70 3.18 3.32 3.68 France 4.22 4.03 3.99 4.08 4.07 3.73 3.66 3.77 3.63 3.21 3.43 Germany 1.03 0.94 0.92 0.91 0.85 0.78 0.70 0.69 0.68 0.58 0.62 India 0.31 0.31 0.31 0.32 0.36 0.35 0.34 0.36 0.41 0.40 0.40 Japan 1.55 0.17 0.14 – 0.04 0.16 0.27 0.45 0.60 0.39 0.55 South Korea 1.47 1.42 1.31 1.46 1.53 1.50 1.36 1.22 1.33 1.45 1.43 Mexico 0.10 0.08 0.11 0.09 0.11 0.10 0.10 0.12 0.10 0.10 0.11 Russia 1.65 1.68 1.62 1.69 1.82 1.82 1.87 1.87 1.90 1.96 2.01 South Africa 0.12 0.12 0.13 0.13 0.11 0.14 0.13 0.11 0.12 0.13 0.09 United Kingdom 0.66 0.67 0.66 0.60 0.65 0.66 0.65 0.59 0.51 0.46 0.41 United States of America 7.93 7.67 7.82 7.85 7.81 7.84 7.79 7.76 7.76 7.54 7.40 European Union 7.99 7.69 7.59 7.61 7.32 7.10 6.98 6.97 6.97 6.20 6.62 Table 7. Nuclear energy consumption data of G20 countries47. All consumption in exajoules. Table 8. Coal consumption data of G20 countries47. All consumption in exajoules. Results and discussion Country 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 Argentina 0.05 0.05 0.05 0.06 0.06 0.04 0.05 0.05 0.03 0.04 0.07 Australia 2.13 2.00 1.89 1.88 1.95 1.94 1.88 1.83 1.75 1.69 1.63 Brazil 0.65 0.64 0.69 0.73 0.74 0.67 0.70 0.69 0.65 0.59 0.71 Canada 0.93 0.88 0.86 0.82 0.82 0.77 0.80 0.65 0.61 0.53 0.48 China 79.71 80.71 82.43 82.48 80.92 80.19 80.56 81.05 81.70 82.38 86.17 France 0.41 0.46 0.48 0.36 0.36 0.35 0.38 0.34 0.27 0.19 0.23 Germany 3.28 3.37 3.47 3.33 3.29 3.20 3.01 2.90 2.25 1.81 2.12 India 12.76 14.02 14.87 16.36 16.55 16.88 17.44 18.58 18.59 17.40 20.09 Indonesia 1.96 2.03 1.78 1.88 2.14 2.23 2.39 2.84 3.41 3.25 3.28 Italy 0.64 0.66 0.57 0.55 0.52 0.46 0.40 0.37 0.28 0.21 0.23 Japan 4.62 4.88 5.07 4.99 5.03 5.02 5.10 4.99 4.91 4.57 4.80 South Korea 3.50 3.38 3.41 3.53 3.58 3.41 3.61 3.63 3.44 3.02 3.04 Mexico 0.62 0.54 0.53 0.53 0.53 0.52 0.64 0.57 0.54 0.24 0.23 Russia 3.94 4.12 3.79 3.67 3.86 3.74 3.51 3.63 3.57 3.29 3.41 South Africa 3.79 3.70 3.70 3.75 3.52 3.78 3.72 3.53 3.76 3.56 3.53 Turkey 1.42 1.53 1.32 1.51 1.45 1.61 1.65 1.71 1.76 1.70 1.74 United Kingdom 1.32 1.63 1.55 1.25 0.97 0.46 0.38 0.32 0.22 0.20 0.21 United States of America 19.70 17.42 18.08 18.04 15.58 14.26 13.87 13.28 11.34 9.20 10.57 European Union 10.75 10.70 10.50 10.00 9.98 9.58 9.25 9.04 7.30 5.97 6.74 Table 8. Coal consumption data of G20 countries47. All consumption in exajoules. seen in Fig. 5 which shows that Brazil’s oil consumption reduced to 4.54 exajoules in 2019 However, the forecast period still shows growth as its oil consumption is predicted to be 5.14 exajoules at the end of the forecast period in 202650. Canada is the third-biggest consumer of oil in the American region. However, it still tries to reduce its dependence on oil by controlling oil consumption. Still, during the first three years of the forecast period from 2022 to 2024, oil consumption is predicted to significantly increase in Canada from 4.22 exajoules in 2022 to 4.79 exajoules in 2026. In contrast, the next two years of the forecast period show a decrease in oil consumption.f seen in Fig. Table 8. Coal consumption data of G20 countries47. All consumption in exajoules. Results and discussion 5 which shows that Brazil’s oil consumption reduced to 4.54 exajoules in 2019 However, the forecast period still shows growth as its oil consumption is predicted to be 5.14 exajoules at the end of the forecast period in 202650. Canada is the third-biggest consumer of oil in the American region. However, it still tries to reduce its dependence on oil by controlling oil consumption. Still, during the first three years of the forecast period from 2022 to 2024, oil consumption is predicted to significantly increase in Canada from 4.22 exajoules in 2022 to 4.79 exajoules in 2026. In contrast, the next two years of the forecast period show a decrease in oil consumption.f j y p p Oil consumption in Mexico is also predicted to be increased, but it offers still lesser consumption until 2026 than what was being consumed in 2012. Mexico controlled its oil consumption from 2012 to 2019, whereas it is predicted to slightly increase during the forecast period, as shown in Fig. 5. Argentina’s oil consumption shows a weak trend during the forecast period, increasing oil consumption from 1.33 exajoules to 2022 to 1.66 exajoules to 2026. j In Australia, oil consumption increased from 1.97 exajoules in 2011 to 2.18 exajoules in 2019, higher than in South Africa. Oil consumption growth showed a weak trend as it had an oil consumption of 1.11 exajoules in 2011, which then increased to 1.18 exajoules in 2019. During the forecast period, oil consumption is predicted to be increased from 2.02 exajoules to 2.34 and 1.11 exajoules to 2.01 exajoules in Australia and South Africa, Scientific Reports \| (2022) 12:13417 \| https://doi.org/10.1038/s41598-022-17505-4 www.nature.com/scientificreports/ Table 9. Hydroelectricity Consumption Data of G20 Countries47. All consumption in exajoules. Results and discussion Country 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 Argentina 0.31 0.29 0.32 0.32 0.31 0.29 0.30 0.31 0.26 0.22 0.18 Australia 0.18 0.16 0.18 0.13 0.13 0.16 0.12 0.16 0.13 0.14 0.15 Brazil 4.25 4.10 3.84 3.64 3.49 3.67 3.55 3.70 3.78 3.75 3.42 Canada 3.73 3.75 3.84 3.73 3.71 3.71 3.78 3.68 3.62 3.65 3.59 China 6.83 8.52 8.92 10.33 10.80 11.11 11.16 11.42 12.08 12.50 12.25 France 0.44 0.58 0.70 0.61 0.53 0.58 0.47 0.61 0.53 0.58 0.55 Germany 0.18 0.21 0.23 0.19 0.18 0.20 0.19 0.17 0.19 0.17 0.18 India 1.31 1.14 1.29 1.36 1.29 1.24 1.30 1.33 1.54 1.55 1.51 Indonesia 0.12 0.13 0.17 0.15 0.13 0.18 0.18 0.21 0.20 0.23 0.23 Italy 0.46 0.41 0.52 0.57 0.43 0.39 0.33 0.47 0.45 0.43 0.41 Japan 0.82 0.76 0.78 0.80 0.83 0.77 0.76 0.77 0.70 0.73 0.73 South Korea 0.05 0.04 0.04 0.03 0.02 0.03 0.03 0.03 0.03 0.04 0.03 Mexico 0.36 0.31 0.27 0.38 0.30 0.30 0.31 0.31 0.22 0.25 0.33 Russia 1.62 1.61 1.78 1.69 1.63 1.78 1.77 1.82 1.84 2.01 2.02 South Africa 0.03 0.01 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.01 0.01 Turkey 0.52 0.57 0.58 0.40 0.65 0.65 0.56 0.57 0.84 0.74 0.52 United Kingdom 0.06 0.05 0.05 0.06 0.06 0.05 0.06 0.05 0.06 0.06 0.05 United States of America 3.14 2.70 2.62 2.49 2.39 2.54 2.84 2.76 2.71 2.67 2.43 European Union 3.05 3.26 3.60 3.60 3.24 3.30 2.79 3.26 3.02 3.25 3.24 Table 9. Hydroelectricity Consumption Data of G20 Countries47. All consumption in exajoules. Table 9. Hydroelectricity Consumption Data of G20 Countries47. All consumption in exajoules. Figure 5. Oil consumption in American Region. Figure 5. Oil consumption in American Region. respectively, as shown in Fig. 6. Saudi Arabia is the biggest exporter of oil globally and predicted to have a sig- nificant and strong increase between 2022 and 2026 as far as oil consumption is concerned, as shown in Fig. 6. h h l h d b h T k d respectively, as shown in Fig. 6. Saudi Arabia is the biggest exporter of oil globally and predicted to have a sig- nificant and strong increase between 2022 and 2026 as far as oil consumption is concerned, as shown in Fig. 6. nificant and strong increase between 2022 and 2026 as far as oil consumption is concerned, as shown in Fig. 6. Results and discussion Turkey shows a very strong growth in its oil consumption as it consumed 1.34 exajoules in 2011 which increased to 1.89 exajoules in 2021 and is predicted to be increased to 3.01 exajoules in 2026. European Union’s oil consumption is predicted to show a weaker trend as its consumption is increase from 24.03 exajoules to 24.57 exajoules between 2011 and 2026. region. The United Kingdom had also reduced its oil consumption between 2011 and 2021 from 3.14 exajoules to 2.5 exajoules. Still, after the political situation due to Brexit, it increased to 3.06 exajoules in 2019, which is predicted to increase by healthy trend as Fig. 7 shows that it can grow to 4.49 exajoules in 2026. Turkey is the smallest oil consumers in the region. Turkey shows a very strong growth in its oil consumption as it consumed 1.34 exajoules in 2011 which increased to 1.89 exajoules in 2021 and is predicted to be increased to 3.01 exajoules in 2026. European Union’s oil consumption is predicted to show a weaker trend as its consumption is increase from 24.03 exajoules to 24.57 exajoules between 2011 and 2026. j j Asia has one of the biggest oil consumers among the G20 countries, with China leading the way. China’s oil consumption is the highest globally, increasing by huge numbers between 2011 and 2021, shown in Fig. 8. China was consuming 19.41 exajoules of oil in 2011, which increased by a massive number in the next 10 years, and in 2021 China’s total consumption of oil was 30.6 exajoules. China’s rapid industrialization and urbanization is the main reason behind the massive increase in its oil consumption53. During the forecast period, its consumption is predicted to be increased from 30.6 exajoules to 37.52 exajoules. Japan and India follow China in oil consumption in the region. Still, Japan had managed to reduce its oil consumption from 8.78 exajoules in 2011 to 6.61 exajoules in 2021, but in the forecast period, it shows just a slight increase in its oil consumption. p j g p On the contrary, India’s oil consumption has been increasing throughout the studied period as it consumed 6.91 exajoules in 2011, which is supposed to jump to 9.41 exajoules by 2021. Indonesia is the minor oil consumer in Asia, and its consumption is predicted to increase slightly between 2022 and 2026. Results and discussion Figure 7 shows the oil consumption in the European region and in its nearby countries such as Turkey and Russia. European Union and Germany are the biggest oil consumers in Europe, but Germany has the most opti- mistic stats regarding reducing oil consumption. Germany used 4.73 exajoules of oil in 2011, which decreased to 4.18 exajoules in 2021. Still, it controlled its consumption by relying more on renewable sources due to regional policies, showing that Germany’s oil consumption reduced between 2011 and 202151. During the forecast period, it shows robust growth in its oil consumption which is supposed to be increased from 4.37 exajoules to 5.07 exa- joules. France is the only country in the European region that worked on its oil consumption and had managed to consistently reduce it between 2011 and 2021 from 3.45 exajoules to 2.91 exajoules. Still, the forecast period shows linear growth from 3.14 exajoules in 2022 to 4.01 exajoules in 2026. This increase is perhaps due to France’s rise in its oil consumption between 2015 and 2019, as shown in Fig. 7. Italy had reduced its oil consumption between 2.98 exajoules to 2.35 exajoules from 2011 to 2021 by running its industry on renewable energy sources. Still, from 2014 and 2018, its consumption increased from 2.42 exajoules to 2.63 exajoules which slightly declined to 2.35 exajoules in 202152. During the forecast period from 2022 to 2026, the consumption is predicted from 2.46 exajoules in 2022 to 3.04 exajoules in 2026, which is still a weak trend compared to other countries in the https://doi.org/10.1038/s41598-022-17505-4 Scientific Reports \| (2022) 12:13417 \| www.nature.com/scientificreports/ Figure 6. Oil consumption in Australian, African and Middle Eastern Countries. Figure 6. Oil consumption in Australian, African and Middle Eastern Countries. Figure 6. Oil consumption in Australian, African and Middle Eastern Countries. Figure 7. Oil Consumption in European Region. Figure 7. Oil Consumption in European Region. region. The United Kingdom had also reduced its oil consumption between 2011 and 2021 from 3.14 exajoules to 2.5 exajoules. Still, after the political situation due to Brexit, it increased to 3.06 exajoules in 2019, which is predicted to increase by healthy trend as Fig. 7 shows that it can grow to 4.49 exajoules in 2026. Turkey is the smallest oil consumers in the region. Results and discussion Nuclear energy consumption in South Africa. Figure 11. Nuclear energy consumption in European Region. Figure 10. Nuclear energy consumption in South Africa. Figure 10. Nuclear energy consumption in South Africa. Figure 11. Nuclear energy consumption in European Region. Figure 11. Nuclear energy consumption in European Region. Figure 11. Nuclear energy consumption in European Region. Figure 11. Nuclear energy consumption in European Region. As shown in Fig. 12, China increased its nuclear energy consumption between 2011 and 2021. China’s nuclear energy consumption may increase from 3.68 exajoules in 2021 to 4.13 exajoules in 2026, whereas India’s con- sumption may increase from 0.4 exajoules in 2021 to 0.57 exajoules in 2026. Hydroelectricity. Figure 13 shows the Forecasting of Hydroelectricity Consumption by G20 Countries of continent America. Hydroelectricity consumption is not as typical as other non-renewable sources54. Canada is the biggest consumer of hydroelectricity in the region due to its rapid demand for dams and other water reserve plants55. Its consumption is predicted to increase from 3.62 exajoules in 2021 to 3.79 exajoules in 2026. Brazil and United States are the second and third biggest consumers of hydroelectricity in the studies region, respectively. Both countries show very strong growth in hydroelectricity consumption between 2021 and 2026, as shown in Fig. 13. Mexico and Argentina do not contribute much to hydroelectricity consumption, and they are not pre- dicted to significantly contribute to hydroelectricity consumption at the end of the forecast period. i Hydroelectricity consumption in Australia and South Africa is predicted to increase between 2022 and 2026 as the consumption in Australia is expected to grow from 0.15 exajoules to 0.18 exajoules, whereas in South Africa, from 0.014 exajoules in 2022 to 0.026 exajoules in 2026. Still, in the last 2 years of the forecast period, the hydroelectricity consumption in Australia is predicted to have a similar value as 2011, shown in Fig. 14. As shown in Fig. 15, the European union consumes more hydroelectricity than any other country. During the forecast period, consumption in European Union is expected to increase from 3.41 exajoules in 2022 to 3.69 exajoules in 2026. Russia is the second biggest consumer among the nearby counties of Europe as its consumption increased 1.62 exajoules in 2011 to 2.02 exajoules in 2022 and may further increase to 2.12 exajoules in 2026. Results and discussion On the other hand, South Korea has managed to reduce its dependence on oil and is expected to consistently follow the trend between 2022 and 2026. https://doi.org/10.1038/s41598-022-17505-4 Scientific Reports \| (2022) 12:13417 \| www.nature.com/scientificreports/ entificreports/ Figure 8. Oil consumption in Asian Region. Figure 9. Nuclear energy consumption in American Region. Figure 8. Oil consumption in Asian Region. Figure 8. Oil consumption in Asian Region. l Figure 9. Nuclear energy consumption in American Region. Nuclear energy. Figure 9 shows the Forecasting of Nuclear Energy Consumption by in G20 countries of continent America. The United States lead the way in nuclear energy consumption in the American region by consuming 7.93 exajoules in 2011 and is predicted to maintain its consumption during the forecast period. Canada and Brazil are the second and third most significant consumers of nuclear energy in the region, respec- tively. Brazil’s nuclear energy consumption is predicted to rise during the forecast period, as shown in Fig. 9. In contrast, Canada is expected to consume 1.21 exajoules of atomic energy in 2026 compared to 2021 when they used only 0.83 exajoules. Argentina and Mexico have minor contributions in nuclear energy consumption. In 2021 only 0.1 exajoules and 0.2 exajoules nuclear energy is used by Mexico and Argentina, respectively, which is not predicted to show any substantial growth in the next 5 years according to Fig. 9, which shows the Nuclear Energy Consumption in American Region. gy p g Figure 10 shows that Australia’s nuclear energy data is not available, while South Africa’s nuclear energy consumption shows a rising trend during the forecast period between 2022 and 2026, as shown in Fig. 10.t As shown in Fig. 11, France had reduced its consumption after 2014 and is expected to show a slight increase in its consumption during the forecast period. Russia is expected to have very slim growth over the next 5 years. Russia is also expected to significantly increase its nuclear energy consumption during the forecast period whereas Germany managed to reduce its nuclear energy consumption between 2011 and 2021 by almost half, as shown in Fig. 11, and are predicted to follow the same trend until the end of 2026. The United Kingdom has the lowest nuclear energy consumption in Europe and is expected to increase the consumption to 0.67 exajoules in 2026 from 0.41 exajoules in 2021. https://doi.org/10.1038/s41598-022-17505-4 Scientific Reports \| (2022) 12:13417 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 10. Results and discussion Other countries like Italy, Turkey, and Germany show an increase in hydroelectricity consumption between the Scientific Reports \| (2022) 12:13417 \| https://doi.org/10.1038/s41598-022-17505-4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 12. Nuclear energy consumption in Asian Region. Figure 13. Hydroelectricity consumption in American Region. Figure 12. Nuclear energy consumption in Asian Region. Figure 12. Nuclear energy consumption in Asian Region. Figure 12. Nuclear energy consumption in Asian Region. 16 2022) 12:13417 \| https://doi.org/10.1038/s41598-022-17505-4 Figure 12. Nuclear energy consumption in Asian Region. Figure 13. Hydroelectricity consumption in American Region. Figure 14. Hydroelectricity consumption in Australia and South Africa. Figure 12. Nuclear energy consumption in Asian Region. Figure 13. Hydroelectricity consumption in American Region. Figure 13 Hydroelectricity consumption in American Region Figure 13. Hydroelectricity consumption in American Region. Figure 14. Hydroelectricity consumption in Australia and South Africa. Figure 14. Hydroelectricity consumption in Australia and South Africa. Figure 14. Hydroelectricity consumption in Australia and South Africa. Scientific Reports \| (2022) 12:13417 \| https://doi.org/10.1038/s41598-022-17505-4 www.nature.com/scientificreports/ p / Figure 15. Hydroelectricity consumption in European Region. Figure 16. Hydroelectricity consumption in Asian Region. Figure 15. Hydroelectricity consumption in European Region. Figure 15. Hydroelectricity consumption in European Region. Figure 15. Hydroelectricity consumption in European Region. Figure 16. Hydroelectricity consumption in Asian Region. Figure 16. Hydroelectricity consumption in Asian Region. Figure 16. Hydroelectricity consumption in Asian Region. Figure 16. Hydroelectricity consumption in Asian Region. studied period. The United Kingdom is predicted to have an almost similar trend with no significant increase in its hydroelectricity consumption but may show a slight increase in its hydroelectricity consumption. studied period. The United Kingdom is predicted to have an almost similar trend with no significant increase in its hydroelectricity consumption but may show a slight increase in its hydroelectricity consumption. China consumes hydroelectricity almost equal to all the other countries in Asia. China’s hydroelectricity consumption is expected to keep increasing until 2026. China was consuming 6.83 exajoules in 2011 and is expected to reach the consumption of 12.83 exajoules by 2026. India and Japan are the second and third biggest consumers of hydroelectricity, respectively in the studied region. They are expected to maintain their positions with a minor increase in their hydroelectricity consumption, as shown in Fig. 16. Indonesia is also expected to increase its consumption during 2011 to 2026 whereas South Korea is expected to hold its position throughout the studied period. Natural gas. Results and discussion Figure 17 shows the forecasting of Natural Gas Consumption by G20 Countries in continent America. There is a massive gap between the countries regarding natural gas consumption56. The United States consumes the highest amount of natural gas in the American region. United States natural gas consumption had increased from 23.7 exajoules in 2011 to 29.76 exajoules in 2021, which is predicted to show the same linear growth by 2026 with a consumption of 33.14 exajoules. Canada and Mexico are second and third-biggest natural gas consumers, respectively with rapid growth in their consumption between 2011 and 2021. Canada’s rapid growth in natural gas consumption is caused by the increase in its population, which is considered to remain the same by the end of the forecast period in 202657. Brazil and Argentina are predicted to show minor increase in their natural gas consumption until 202658. Figure 17 shows the Natural Gas Consumption in American Region. It can be observed that natural gas consumption in all the countries dropped down between 2019 and 2021 which may have caused due to COVID-19 as industry had to be shut down for few months as a safety measure. https://doi.org/10.1038/s41598-022-17505-4 Scientific Reports \| (2022) 12:13417 \| www.nature.com/scientificreports/ scientificreports/ Figure 17. Natural gas consumption in American Region. Figure 18. Natural gas consumption in Australia and South Africa. Figure 17. Natural gas consumption in American Region. Figure 17. Natural gas consumption in American Region. Figure 17. Natural gas consumption in American Region. Figure 18. Natural gas consumption in Australia and South Africa. Figure 18. Natural gas consumption in Australia and South Africa. Figure 18. Natural gas consumption in Australia and South Africa. Australia’s natural gas consumption had shown growth between 2011 and 2026 as it rose to 1.18 exajoules from 1.73 exajoules which is shown in Fig. 18. In contrast, consumption in South Africa has almost similar numbers between the same period, as shown in Fig. 18. A middle eastern country like Saudi Arabia also consumes a high amount of natural gas as they consumed 4.22 exajoules in 2021. It is predicted that the consumption can reach 4.73 exajoules by the end of 2026. j y Russia led in gas consumption in the region with consumption of 15.68 exajoules in 2011 to 17.09 exajoules in 2021 and is predicted to maintain its position with slight increase in its consumption until the end of 2026. Results and discussion Figure 22. Coal consumption in Australia and South Africa. Figure 23. Coal consumption in European Region. Figure 23. Coal consumption in European Region. 2011 and 2021 as consumption fell from 19.7 exajoules in 2011 to 10.57 exajoules in 2021, which is predicted to follow the same trend during the forecast period with consumption maintaining its position of 2011, to 13.06 exajoules at the end of 2026 according to Fig. 21. Argentina and Brazil are the only countries in the American region that show minor fluctuation in coal consumption, which remains the same in the next five years, as shown in Fig. 21. Canada and Mexico declined their coal consumption between 2011 and 2021, but Mexico decreased its consumption to half between 2011 and 2021. 2011 and 2021 as consumption fell from 19.7 exajoules in 2011 to 10.57 exajoules in 2021, which is predicted to follow the same trend during the forecast period with consumption maintaining its position of 2011, to 13.06 exajoules at the end of 2026 according to Fig. 21. Argentina and Brazil are the only countries in the American region that show minor fluctuation in coal consumption, which remains the same in the next five years, as shown in Fig. 21. Canada and Mexico declined their coal consumption between 2011 and 2021, but Mexico decreased its consumption to half between 2011 and 2021. p Australia’s coal consumption fell from 2.13 exajoules in 2011 to 1.63 exajoules in 2021, and it is predicted to rise its coal consumption slightly to 1.97 exajoules by the end of 2026. On the other hand, South Africa had almost similar coal consumption from 2011 to 2021, which is predicted to remain the same during the forecast period, as shown in Fig. 22.i p g Europe do not have a significant consumer of coal compared to other parts of the world. European Union is the biggest consumer of coal in Europe with a consumption of 10.75 exajoules in 2011 and is expected to consume 8.73 exajoules at the end of 2026 according to the forecasting result shown in Fig. 23. All the countries except Turkey show a decline in their coal consumption between 2011 and 2021. Turkey was consuming 1.42 exajoules of coal in 2011, which reached 1.74 exajoules in 2021 and is predicted to remain the same until the end of the forecast period in 2026, as shown in Fig. 23. Results and discussion As mentioned earlier, natural gas significantly impacts countries’ progress. Figure 19 shows that in Europe, all the countries are expected to increase their natural gas consumption, with the European Union leading the way from 14.01 exajoules in 2011 to 16.04 exajoules in 2026. In contrast, France and Turkey are the minor consumers of natural gas in the region and are expected to maintain their position until 2026. g g p p Rapid growth in natural gas consumption is shown by China, where the consumption was 4.87 exajoules in 2011, and it jumped to 13.63 exajoules in 2021, which is forecasted to reach 16.73 exajoules at the end of 2026, which can be seen in Fig. 20. Other countries like Japan, India, Indonesia, and South Korea also have significant contributions to natural gas consumption. They are expected to remain in the picture with almost same contri- bution until the end of 2026, with substantial consumption shown by Japan and India. Coal. Figure 21 shows the Forecasting of Coal Consumption by G20 Countries in continent America. Coal consumption keeps falling due to it being replaced by natural gas and renewables; hence, coal’s contribution in the energy mix fell to 27%, reaching its lowest level in the last 18 years59. Like natural gas, the United States coal is the biggest consumer of coal in the American region. Still, it had shown a decline in coal consumption between Scientific Reports \| (2022) 12:13417 \| https://doi.org/10.1038/s41598-022-17505-4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ cientificreports/ Figure 19. Natural gas consumption in European Region. Figure 20. Natural gas consumption in Asian Region. Figure 19. Natural gas consumption in European Region. Figure 19. Natural gas consumption in European Region. 19 022) 12:13417 \| https://doi.org/10.1038/s41598-022-17505-4 Figure 19. Natural gas consumption in European Region. Figure 20. Natural gas consumption in Asian Region. Figure 21. Coal consumption in American Region. Figure 19. Natural gas consumption in European Region. Figure 20. Natural gas consumption in Asian Region. Figure 20. Natural gas consumption in Asian Region. Figure 20. Natural gas consumption in Asian Region. Figure 21. Coal consumption in American Region. Figure 21. Coal consumption in American Region. Figure 21. Coal consumption in American Region. Figure 21. Coal consumption in American Region. Scientific Reports \| (2022) 12:13417 \| https://doi.org/10.1038/s41598-022-17505-4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 22. Coal consumption in Australia and South Africa. Figure 22. Coal consumption in Australia and South Africa. Figure 22. Coal consumption in Australia and South Africa. Conclusion and policy implications p y p In conclusion, the adjacent accumulation generation operator can be defined by introducing the parameters based on available data. The stability of the studied model is proven by discussing two confirmed cases, and the results were then compared with other forecasting grey models. It is found that more stability was shown exhibited by the disturbance bound of the least squares’ solution. The obtained results predicted that prediction accuracy in many cases could be improved. To analyze the model’s suitability for forecasting, it was interpreted by first predicting the already given data, and the prediction error was then calculated. Hence, DAGM (1,1) can be applied for forecasting as it has an excellent theoretical contribution and shows low error. Furthermore, the practical significance of DAGM (1,1) was proven by applying it to predict the non-renewable energy consump- tion in G20 countries. In this paper, the non-renewable energy consumption of G20 countries was analyzed, and the future con- sumption until 2026 was predicted where DAGM (1,1) was found to be effective. This paper indicates that oil consumption has an increasing trend in all the G20 countries, with the USA leading by China until 2026. Many G20 countries do not make their nuclear energy consumption data available. Based on available data, it can be predicted that China, France, and Russia are expected to consume the most nuclear energy until 2026, with Argentina being the least. Coal is predicted to show a downfall in its consumption by G20 countries except for South Africa and France. Argentina is expected to consume a minuscule amount of coal, with a consumption of 0.09 until 2026. Hydroelectricity is the least consumed form of nonrenewable energy and is predicted to main- tain its position until 2026. China, the USA, and European Union are expected to maintain their position as the biggest consumers of hydroelectricity among the G20 countries. Natural gas consumption is predicted to show a massive rise in G20 countries, especially in Russia, China, and the USA, justifiable considering their economic progress. The results can help authorities make the decision more easily as a proven forecasting method is used in this study. It may have policy implications especially related to natural gas and Oil as the obtained data demands reduction in these two non-renewable energy consumption to not only reduce the carbon dioxide emission but also prevent the energy shortfall in the future. Results and discussion p g China consumes the most amount coal than any other country globally. Coal is the most consumed nonre- newable resource59. China’s consumption was 79.71 exajoules in 2011, increasing to 86.17 exajoules in 2021, it is expected to keep fluctuating and may increase during the forecast period and end at a consumption of 96.13 exajoules at the end of 2026, as shown in Fig. 24. Unlike other countries, coal consumption in India and Indonesia Scientific Reports \| (2022) 12:13417 \| https://doi.org/10.1038/s41598-022-17505-4 www.nature.com/scientificreports/ ntificreports/ Figure 24. Coal consumption in Asian Region. Figure 24. Coal consumption in Asian Region. Figure 24. Coal consumption in Asian Region. is expected to grow significantly during the next 5 years. On the contrary, Japan, and South Korea are expected to decline their coal consumption which can be seen in Fig. 24. Conclusion and policy implications p gy As a new forecasting model, the studied model DAGM (1,1) can be combined with other models to obtain better and optimized results. 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https://openalex.org/W4200302101	https://zenodo.org/records/7767532/files/SupInf_Vasileiadis_et_al_PDA_final.pdf	English	null	Fast Light-Driven Motion of Polydopamine Nanomembranes	Nano letters	2,021	cc-by	3,152	Supplementary Information: Fast light-driven motion of polydopamine nanomembranes Thomas Vasileiadis1,2, Tommaso Marchesi D’Alvise2, Clara-Magdalena Saak2,3, Mikołaj Pochylski1, Sean Harvey2, Christopher V. Synatschke2, Jacek Gapiński1, George Fytas2, Ellen H.G. Backus2,3, Tanja Weil2,, and Bartlomiej Graczykowski1,2, Thomas Vasileiadis1,2, Tommaso Marchesi D’Alvise2, Clara-Magdalena Saak2,3, Mikołaj Pochylski1, Sean Harvey2, Christopher V. Synatschke2, Jacek Gapiński1, George Fytas2, Ellen H.G. Backus2,3, Tanja Weil2,, and Bartlomiej Graczykowski1,2, y y , y, y g 2, 61-614 Poznan, Poland 2. Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany. 2, 61-614 Poznan, Poland 2. Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany. 2. Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany. 3. Department of Physical Chemistry, University of Vienna, Währinger Strasse 42, 1090 Vienna * weil@mpip-mainz.mpg.de * bartlomiej.graczykowski@amu.edu.pl 3. Department of Physical Chemistry, University of Vienna, Währinger Strasse 42, 1090 Vienna * weil@mpip-mainz.mpg.de * bartlomiej.graczykowski@amu.edu.pl Supplementary Information: Fast light-driven motion of polydopamine nanomembranes Thomas Vasileiadis1,2, Tommaso Marchesi D’Alvise2, Clara-Magdalena Saak2,3, Mikołaj Pochylski1, Sean Harvey2, Christopher V. Synatschke2, Jacek Gapiński1, George Fytas2, Ellen H.G. Backus2,3, Tanja Weil2,, and Bartlomiej Graczykowski1,2, 1. Faculty of Physics, Adam Mickiewicz University, Uniwersytetu Poznanskiego 2, 61-614 Poznan, Poland 2. Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany. 3. Department of Physical Chemistry, University of Vienna, Währinger Strasse 42, 1090 Vienna * weil@mpip-mainz.mpg.de * bartlomiej.graczykowski@amu.edu.pl Supplementary Information: Fast light-driven motion of polydopamine nanomembranes Thomas Vasileiadis1,2, Tommaso Marchesi D’Alvise2, Clara-Magdalena Saak2,3, Mikołaj Pochylski1, Sean Harvey2, Christopher V. Synatschke2, Jacek Gapiński1, George Fytas2, Ellen H.G. Backus2,3, Tanja Weil2,, and Bartlomiej Graczykowski1,2, 1. Faculty of Physics, Adam Mickiewicz University, Uniwersytetu Poznanskiego 2, 61-614 Poznan, Poland 2. Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany. 3. Department of Physical Chemistry, University of Vienna, Währinger Strasse 42, 1090 Vienna * weil@mpip-mainz.mpg.de * bartlomiej.graczykowski@amu.edu.pl Supplementary Information: Fast light-driven motion of polydopamine nanomembranes Thomas Vasileiadis1,2, Tommaso Marchesi D’Alvise2, Clara-Magdalena Saak2,3, Mikołaj Pochylski1, Sean Harvey2, Christopher V. Synatschke2, Jacek Gapiński1, George Fytas2, Ellen H.G. Backus2,3, Tanja Weil2,, and Bartlomiej Graczykowski1,2, Method Before the SFG experiments, the PDA film already transferred on Si3N4 was incubated in the oven at 70 ºC for 30 min. Briefly, PDA films were first incubating in carbonate buffer at pH 10 for 30 min before applying a removal cycle by sweeping the potential between -0.8 and +1.2 V for 3 cycles with a scan rate of 20 mV/s to desorb it from the gold surface. The film was then covered with a polyvinyl alcohol (PVA) 80% hydrolyzed solution, 10% mass in water, and once dried for ca 25 min in the oven at 40 ºC, it was stripped off the surface and transferred onto the respective substrates. The PVA layer was removed by washing with water. Before the SFG experiments, the PDA film already transferred on Si3N4 was incubated in the oven at 70 ºC for 30 min. 1. Preparation and Characterization of PDA membranes. Polydopamine (PDA) films were polymerized on a gold electrode surface using cyclic voltammetry (see Fig. S1 and section 1.1). Grazing incidence angle FTIR was applied to characterize the prepared films before and after transfer from the gold electrode surface (section 1.2). Before and after transfer, both films revealed similar thickness of 16 to 14 nm, respectively, as determined by AFM (see section 1.3). Figure S1: Cyclic voltammogram for polydopamine film formation. Cyclic voltammogram of PDA film in phosphate buffer pH 7 where the potential was swept between +0.5 and –0.5 V for 5 cycles starting and ending at 0 V vs Ag/AgCl reference electrode with a scan rate of 2 mV/s. The first oxidation peak for the reaction from catechol to quinone form of dopamine molecule can be distinguished at 0.3 V. Other oxidation and reduction peak are barely present due to the slow scan rate. Figure S1: Cyclic voltammogram for polydopamine film formation. Cyclic voltammogram of PDA film in phosphate buffer pH 7 where the potential was swept between +0.5 and –0.5 V for 5 cycles starting and ending at 0 V vs Ag/AgCl reference electrode with a scan rate of 2 mV/s. The first oxidation peak for the reaction from catechol to quinone form of dopamine molecule can be distinguished at 0.3 V. Other oxidation and reduction peak are barely present due to the slow scan rate. 1 Materials Gold-coated (1000 Å) microscope slide (Sigma-Aldrich), were cut using a diamond tip. Phosphate buffer pH 7, 100 mM was prepared using sodium phosphate dibasic anhydrous (99%) and sodium phosphate monobasic (99%) (Sigma-Aldrich) in Milli-Q water. Carbonate buffer pH 10, 100 mM was prepared using sodium bicarbonate (>99.7%) and sodium carbonate (>99.8%) from Sigma-Aldrich, in Milli-Q water. Polyvinyl alcohol 80% hydrolyzed 9–10 kDa Mw (Sigma-Aldrich) was used to prepare the 10 wt% solution in Milli-Q water. Custom-made silicon nitride substrates were purchased from Silson and Norcada. Method Electropolymerization was performed using a Metrohm Autolab N series potentiostat (AUTOLAB PGSTAT 204) and a three-electrode configuration. A gold-covered glass microscope slide was used as the working electrode, a Ag/AgCl with 3M KCl as the reference electrode, and a gold wire as the counter electrode. All reactions were performed in a 30 mL electrochemical cell (Metrohm) filled with 25 mL of solution in air atmosphere and at room temperature. The working electrode was pre-treated for 10 minutes with Ar plasma cleaning at a pressure of 6 mbar. The gold substrate was immersed in a 1 mg/mL solution of dopamine hydrochloride dissolved in 100 mM phosphate buffer at pH 7.0 previously bubbled with Nitrogen to remove oxygen from the solution and prevent dopamine self-polymerization. The film was then transferred either to silicon support for AFM measurements or to Si3Ni4 substrates for actuation and SFG experiments through a previously established transfer method1. Electropolymerization was performed using a Metrohm Autolab N series potentiostat (AUTOLAB PGSTAT 204) and a three-electrode configuration. A gold-covered glass microscope slide was used as the working electrode, a Ag/AgCl with 3M KCl as the reference electrode, and a gold wire as the counter electrode. All reactions were performed in a 30 mL electrochemical cell (Metrohm) filled with 25 mL of solution in air atmosphere and at room temperature. The working electrode was pre-treated for 10 minutes with Ar plasma cleaning at a pressure of 6 mbar. The gold substrate was immersed in a 1 mg/mL solution of dopamine hydrochloride dissolved in 100 mM phosphate buffer at pH 7.0 previously bubbled with Nitrogen to remove oxygen from the solution and prevent dopamine self-polymerization. The film was then transferred either to silicon support for AFM measurements or to Si3Ni4 substrates for actuation and SFG experiments through a previously established transfer method1. Briefly, PDA films were first incubating in carbonate buffer at pH 10 for 30 min before applying a removal cycle by sweeping the potential between -0.8 and +1.2 V for 3 cycles with a scan rate of 20 mV/s to desorb it from the gold surface. The film was then covered with a polyvinyl alcohol (PVA) 80% hydrolyzed solution, 10% mass in water, and once dried for ca 25 min in the oven at 40 ºC, it was stripped off the surface and transferred onto the respective substrates. The PVA layer was removed by washing with water. 1.2 Grazing incidence angle FTIR. The infrared spectrum of the film on gold obtained by grazing-angle reflectance FTIR (Vertex 70, Bruker) after purging the sample with dry air for 15 minutes and recording 4 spectra at 3000 scans with an interval of 1 minute between each one. 2 2 Figure S2: Grazing incidence angle FTIR. Spectra of polydopamine film 5 cycles before (orange) and after (blue) the carbonate buffer incubation and the removal procedure was performed. 1.3 Atomic force microscopy. Atomic force microscopy was used to characterize the film, measuring the morphology, thickness of the polymer films on gold by scratching it with a plastic tip after it was freshly prepared and after the transfer on Si support. The profile was then recorded by AFM (Park NX20 and Bruker Dimension ICON) with a cantilever 70 KHz resonance frequency and an elastic constant of 2 N/m in tapping mode. The film freshly prepared on gold shows a mean surface roughness of 2.84 nm while the one transferred on silicon substrate of 2.7 nm. The profile and the roughness were measured by analyzing the images using the Gwyddion software on a region of ca 1.5 x 3.0 μm. 3 3 Figure S3: AFM images and height profiles. Atomic force micrograph of the film on gold (left) and transferred on Si substrate (right) and the corresponding profiles (down). Figure S3: AFM images and height profiles. Atomic force micrograph of the film on gold (left) and transferred on Si substrate (right) and the corresponding profiles (down). 2. Optical absorption of PDA membranes The absorption, thickness (𝜏) and penetration depth (𝛿) are connected with the formula 𝐴(%) = 100 ∙[1 −𝑒−𝜏𝛿 ⁄ ] (Beer–Lambert law) from which we derive 𝛿= 0.33 𝜇𝑚 at 660 nm. These measurements are in good agreement with recent spectroscopic ellipsometry studies of various PDA thin films2, which give 𝑘= 0.1 at λ=660 nm and 𝛿= 𝜆4𝜋𝑘 ⁄ = 0.53 𝜇𝑚. 1.4 Wrinkle formation. The nanoscopic wrinkles that are present on the film are formed during the transfer procedure. Such wrinkles arise already when the removal procedure is applied on gold even before removing the film from the surface, and are probably due to the partial desorption of the film from the substrate (Fig. S4). Wrinkles are mostly noticeable when the sample is transferred on a continuous substrate and not as much on free-standing membranes (Fig. S5). The film is also showing stripes, which are folds that can occur once the membrane is transferred to a substrate, due to the manual transfer procedure, such folds are twice thicker than the membrane (see AFM Figs. S4 and S5) confirming that the film is folding on itself. Finally, macroscopically visible wrinkles are induced during temperature treatment to trigger the phenomena, which are then disappearing upon photoactuation. Interestingly the higher magnification AFM of the film surface shows a porous structure (Figure S5D). 4 4 Figure S4: Wrinkle formation and visualization with AFM. PDA film on gold after removal procedure was applied.; wrinkles with circular pattern are appearing due to the desorption from the substrate. Figure S4: Wrinkle formation and visualization with AFM. PDA film on gold after removal procedure was applied.; wrinkles with circular pattern are appearing due to the desorption from the substrate. Figure S5: AFM microscopy of free-standing membranes. A. Micrograph of the free-standing membrane over a hole on the perforated Si3N4 film; B. Micrograph of the free-standing part where small wrinkles and folds are present;. C. micrograph of a fold present at the free standing film. D. Surface of the free standing film. A B C D A B D C C Figure S5: AFM microscopy of free-standing membranes. A. Micrograph of the free-standing membrane over a hole on the perforated Si3N4 film; B. Micrograph of the free-standing part where small wrinkles and folds are present;. C. micrograph of a fold present at the free standing film. D. Surface of the free standing film. 5 3. Laser power damage threshold 3. Laser power damage threshold Figure S6: Optical microscopy images showing rack formation for laser powers larger than 90 mW. Figure S6: Optical microscopy images showing rack formation for laser powers larger than 90 mW. 4. Fitting of time-constants To quantify how fast the response of the PDA membranes to laser light is, we have fitted the time-resolved reflectivity data with exponential decay functions of the form: 𝑓(𝑡) = 𝛩(𝑡−𝑡𝑜) ∙𝐴∙(1 −exp [−𝑡−𝑡𝑜 𝜏 ]) where 𝑡𝑜 is the time that the laser turns on or off, 𝛩(𝑡−𝑡𝑜) is the Heaviside step function, 𝐴 is the amplitude of the change, and 𝜏 is the time-constant. For fitting the experimental data, the 𝑓(𝑡) is convoluted with a Gaussian function, the width of which represents the time-resolution of the instrument, which is estimated to be 100 μs. 6 6 Figure S7: Bi-exponential fitting of relaxation (up) after the laser is switched off and residuals of the fit (down) showing additional oscillatory dynamics. Figure S7: Bi-exponential fitting of relaxation (up) after the laser is switched off and residuals of the fit (down) showing additional oscillatory dynamics. The time-constant for contraction and flattening is found to be τc=(140 ± 10) μs, which approaches the instrumental time-resolution. For this process the bioexponential fitting is adequately reproducing the data – see Fig. 3c of the main manuscript. The reverse processes of swelling and wrinkling can be represented with a bi-exponential function with time-constants τ1=(1.1 ± 0.1) ms and τ2=(15 ± 2) ms. For the relaxation (wrinkling and swelling) the signal contains oscillations in addition to the exponential dynamics (residuals in Fig. S7), which can originate from mechanical instabilities such as buckling and wrinkling. Methods Laser light was produced by a Ti:Sapphire-based amplifier (Coherent Libra), yielding 40 fs pulses at 800 nm with a 1 kHz repetition rate and approximately 5 mJ in energy. Part of the laser output was passed through an OPA (TOPAS Prime) to generate infrared light (IR) at 3200 cm-1. Another part passed through an etalon which narrowed the spectral width to ~20 cm-1. The 800 nm and the IR pulses were subsequently overlapped on the sample in space and time to generate light at their sum frequency (SFG). The generated SFG signal was focused onto a spectrometer (Shamrock 303i) and detected on a CCD camera (Newton EMCCD Andor). The SFG data shown in this work was collected in SSP polarization. This experimental setup has previously been described by Schlegel et al.3 As previously mentioned SFG involves the generation of light at the sum-frequency of two incident light beams. This nonlinear process is dependent on the second-order nonlinear susceptibility of the material in which the two beams are overlapped. Since the second-order nonlinear susceptibility becomes zero in centrosymmetric media, SFG is only allowed in the non-centrosymmetric parts of the investigated systems. In the case of the studied PDA film this means that the SFG 7 process is inherently surface sensitive. A more detailed summary of the SFG process can be found in the article of Lambert et al.4 In order to change the temperature of the polymer film, the sample is placed onto a copper block through which water is circulated at a defined temperature. The heating block is in turn placed on a 3D translational stage to bring the sample into alignment with the SFG pulse pair. The temperature of the circulating water is heated/cooled and circulated by a commercially available chiller. The set temperature of the chiller and the measured temperature on the surface of the copper block was 20/20 °C and 80/72°C, respectively. In total 5 set points, corresponding to two temperature cycles were measured (20 °C - 80 °C - 20 °C - 80 °C - 20 °C) to investigate the reversibility of any spectral changes due to temperature. Data Treatment Each SFG spectrum was background subtracted by a spectrum measured with the IR beam blocked and normalized to an SFG spectrum of quartz, which was obtained under identical conditions. Quartz spectra measured at the beginning and end of the temperature cycle confirmed the stability of the setup. At each temperature point, 3 spectra were accumulated for 5 minutes each. These spectra were individually fitted. Since the Si3N4 substrate contributes a pronounced non-resonant background to the spectra, which is reproducibly affected by the sample temperature, a nonlinear function was used to describe the background signal. The observed OH and CH stretch bands were fitted using one Lorentzian function each. A similar fitting procedure has previously been used by, e.g., Schlegel et al.2, with the only difference being the nonlinear frequency dependence of the amplitude used to fit the non-resonant background signal. The area of the Lorentzian function corresponding to the OH stretch band was then extracted and averaged at each temperature set point during the cycle to yield the values summarised in Table 1. The OH stretch mode of the PDA film was found at 3371(4) cm-1 with a line width of 125(8) cm-1. Correspondingly, the CH stretch mode was found at 2938(5) cm-1 with a width of approximately 32(17) cm-1. 8 Table 1: Summary of fitted OH stretch area Set/Sample Temperature Cycle Nr. Spectrum Nr. Fitted Area Average area [°C] [#] [#] [a.u.] [a.u.] 20 / 20 1 20_1 18.34 16.53 20_2 16.95 20_3 14.29 80 / 72 2 80_1 12.91 11.59 80_2 10.64 80_3 11.23 20 / 20 3 20_4 16.68 15.29 20_5 15.00 20_6 14.19 80 / 72 4 80_5 12.23 11.32 80_6 10.41 20 / 20 5 20_7 14.71 14.00 20_8 14.61 20_9 12.69 Table 1: Summary of fitted OH stretch area 6. Response of larger membranes The reflectivity experiment shown in Figure 3 of the main manuscript have been repeated for a PDA membrane of 80 μm diameter. The larger PDA membranes show the same behaviour albeit the dynamics for initial contraction are slower, in the millisecond timescale – see Figure S8. 9 9 Figure S8: The light-driven motion of a wider (80 μm diameter) PDA membrane measured with time-resolved reflectivity (532 nm). (a) Periodic contraction and flattening of the irradiated PDA membrane with the driving laser (660 nm) on for 0.25 s and with a repetition rate of 2 Hz. (b) The initial contraction of the wider membrane is completed in less than 5 ms. (c) The relaxation (expansion) of the wider membrane takes place within approximately 10-20 ms. Figure S8: The light-driven motion of a wider (80 μm diameter) PDA membrane measured with time-resolved reflectivity (532 nm). (a) Periodic contraction and flattening of the irradiated PDA membrane with the driving laser (660 nm) on for 0.25 s and with a repetition rate of 2 Hz. (b) The initial contraction of the wider membrane is completed in less than 5 ms. (c) The relaxation (expansion) of the wider membrane takes place within approximately 10-20 ms. 3. Schlegel, S. J. et al. How water flips at charged titanium dioxide: an SFG-study on the water–TiO2 interface. Phys. Chem. Chem. Phys. 21, 8956–8964 (2019). 4. Lambert, A. G., Davies, P. B. & Neivandt, D. J. Implementing the Theory of Sum Frequency Generation Vibrational Spectroscopy: A Tutorial Review. Appl. Spectrosc. Rev. 40, 103–145 (2005). References 1. Marchesi D’Alvise, T. et al. Ultrathin Polydopamine Films with Phospholipid Nanodiscs Containing a Glycophorin A Domain. Adv. Funct. Mater. 30, 2000378 (2020). 2. Qie, R., Zajforoushan Moghaddam, S. & Thormann, E. Parameterization of the optical constants of polydopamine films for spectroscopic ellipsometry studies. Phys. Chem. Chem. Phys. 23, 5516-5526 (2021). 10 4. Lambert, A. G., Davies, P. B. & Neivandt, D. J. Implementing the Theory of Sum Frequency Generation Vibrational Spectroscopy: A Tutorial Review. Appl. Spectrosc. Rev. 40, 103–145 (2005). 4. Lambert, A. G., Davies, P. B. & Neivandt, D. J. Implementing the Theory of Sum Frequency Generation Vibrational Spectroscopy: A Tutorial Review. Appl. Spectrosc. Rev. 40, 103–145 (2005). 11 11
https://openalex.org/W2799781838	https://digital.library.adelaide.edu.au/dspace/bitstream/2440/113787/2/hdl_113787.pdf	English	null	Efficiency Improvement Using Molybdenum Disulphide Interlayers in Single-Wall Carbon Nanotube/Silicon Solar Cells	Materials	2,018	cc-by	6,921	Received: 27 March 2018; Accepted: 12 April 2018; Published: 21 April 2018 Abstract: Molybdenum disulphide (MoS2) is one of the most studied and widely applied nanomaterials from the layered transition-metal dichalcogenides (TMDs) semiconductor family. MoS2 has a large carrier diffusion length and a high carrier mobility. Combining a layered structure of single-wall carbon nanotube (SWCNT) and MoS2 with n-type silicon (n-Si) provided novel SWCNT/n-Si photovoltaic devices. The solar cell has a layered structure with Si covered ﬁrst by a thin layer of MoS2 ﬂakes and then a SWCNT ﬁlm. The ﬁlms were examined using scanning electron microscopy, atomic force microscopy and Raman spectroscopy. The MoS2 ﬂake thickness ranged from 5 to 90 nm while the nanosheet’s lateral dimensions size ranged up to 1 µm2. This insertion of MoS2 improved the photoconversion efﬁciency (PCE) of the SWCNT/n-Si solar cells by approximately a factor of 2. ywords: molybdenum disulphide (MoS2); single-wall carbon nanotubes (SWCNTs); solar cells materials materials Materials 2018, 11, 639; doi:10.3390/ma11040639 Efﬁciency Improvement Using Molybdenum Disulphide Interlayers in Single-Wall Carbon Nanotube/Silicon Solar Cells Shaykha Alzahly 1, LePing Yu 1, Cameron J. Shearer 1,2 ID , Christopher T. Gibson 1 and Joseph G. Shapter 1,3,* ID 1 Flinders Centre for Nanoscale Science and Technology, College of Science and Engineering, Flinders University, Bedford Park, Adelaide, SA 5042, Australia; alza0112@uni.ﬂinders.edu.au (S.A.); yu0252@uni.ﬂinders.edu.au (L.Y.); cameron.shearer@ﬂinders.edu.au (C.J.S.); christopher.gibson@ﬂinders.edu.au (C.T.G.) 1 Flinders Centre for Nanoscale Science and Technology, College of Science and Engineering, Flinders University, Bedford Park, Adelaide, SA 5042, Australia; alza0112@uni.ﬂinders.edu.au (S.A.); yu0252@uni.ﬂinders.edu.au (L.Y.); cameron.shearer@ﬂinders.edu.au (C.J.S.); christopher.gibson@ﬂinders.edu.au (C.T.G.) 2 Department of Chemistry, The University of Adelaide, Adelaide, SA 5005, Australia 3 Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St. Lucia, QLD 4072, Australia y y 3 Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St. Lucia, QLD 4072, Australia * Correspondence: joe.shapter@ﬂinders.edu.au; Tel.: +61-07-3343-1165 * Correspondence: joe.shapter@ﬂinders.edu.au; Tel.: +61-07-3343-1165 1. Introduction However, there are several important issues remaining that hinder the performance of CNT/Si-solar cells which need to be solved to allow commercial development. One issue that remains is the improvement of the Schottky barrier, which could be accomplished by using an effective carrier transfer layerto enable the transport of a high current without losses due to carrier recombination [9]. The CNT ﬁlms typically used in CNT/Si solar cells consist of a mat of CNTs, in contact with one another, to form a conductive grid. The CNT ﬁlm must be transparent so the ﬁlm is very thin (<10 nm) or sparse. The resistance of thin ﬁlms made with SWCNTs is largely due to the hopping of charge carriers from one tube to another. There is also a resistance transferring charge carriers across the CNT/Si interface. Two-dimensional (2D) materials like molybdenum disulphide (MoS2) can be successfully applied in this case because they will extend charge carrier lifetimes due to the long carrier diffusion length (200–500 cm2/Vs) [12] in the 2D material. Thus, MoS2 is capable of lowering the charge carrier resistance by both helping transfer across the interface and decreasing the number transfers between CNTs. This reduction in resistance means that the insertion of MoS2 could enhance efﬁciency [13]. MoS2 is made of stacked monolayers that are bonded together by weak van der Waals forces. The monolayers consist of S-Mo-S units that are hexagonally packed [14]. MoS2 is one of the most studied and widely applied nanoelectronic transition metal dichalcogenides (TMDs). Like other TMDs, the material in its bulk state is an indirect band gap semiconductor with a band gap of 1.2 eV [15,16]. When the material is formed into a monolayer, the band gap becomes a direct band gap of 1.9 eV [16]. Due to this effect, bulk MoS2 and its monolayer have been studied extensively [17], with considerable research conducted on the potential applications of monolayer MoS2 in 2D devices. The existence of the large band gap in the monolayer of MoS2 has found applications in areas such as ﬁeld effect transistors with an on/off ratio as high as 108 [18], integrated circuits [19], sensors [20] and logic operations [21]. There are two exfoliation methods to produce 2D MoS2: micromechanical exfoliation and liquid phase exfoliation [16]. Both methods begin with bulk MoS2 to produce ﬂakes of MoS2. 1. Introduction Global energy demand has increased dramatically in recent years due to the rapid increase in world population, use of modern technologies and improved standards of living. According to the U.S Energy department, by 2040, the global energy demand will increase by 28% [1]. With the increasing global energy consumption and the commitment to reduce the amount of CO2 and other greenhouse gases emitted into the atmosphere due to the burning of fossil fuels, there is a great need to improve the current photovoltaic systems and develop other sources of renewable energy such as wind power [2], fuel cells [3], biofuels [4] and solar cell technologies [5]. These alternative energy technologies have the capability to meet the world’s energy demand if well developed. However, the challenge is that the production from these resources is small in relation to the required energy supply and are geographically limited to areas where the resource is in abundance and consistent. It is only solar energy that can be harnessed almost everywhere in the world, providing a possible solution to the current energy demand [6]. There are many solar photovoltaics of next generation including carbon nanotube-silicon (CNT/Si) heterojunction solar cells [7]. Since nanotube-silicon heterojunction solar cells were reported by Wei et al. in 2007, they have been widely studied because of their potential to replace the expensive p-type emitter layer in crystalline Materials 2018, 11, 639; doi:10.3390/ma11040639 www.mdpi.com/journal/materials 2 of 11 Materials 2018, 11, 639 silicon solar panels that are in use today [8]. CNT/Si solar cells possess a high solar-to-electricity conversion efﬁciency and can be manufactured using simple, inexpensive materials using an easy fabrication procedure. Studies have reported 17% efﬁciency with the use of metal oxide layers for both efﬁcient carrier transport and as an antireﬂection layer [9]. These ﬁndings present solid evidence that CNT/Si devices will possibly replace silicon solar cells [10]. Typically, the CNT/Si cell is similar to the conventional n-type silicon cell but in CNT/Si devices, a highly transparent ﬁlm of CNT replaces the p-type silicon layer and the front metallization. In air, CNTs adsorb O2 and hence are p-type. When deposited on n-type Si, a depletion region is established and the built-in potential in this heterojunction can separate charge carriers to yield a current. CNT/Si cells are potentially cheap, semi-transparent, ﬂexible, have excellent conductivity and efﬁcient even under low light [11]. 2.2. Molybdenum Disulphide Dispersion Molybdenum disulphide aqueous dispersion (FlexeGRAPH, Australian National University, Canberra, Australia) was bath sonicated (S 30H, Elmasonic) for three minutes to make a homogeneous suspension. 12.5 mL of the suspension was diluted with 37.5 mL Milli-Q water (Millipore Corporation, Burlington, MA, USA) (in order to keep the concentration of MoS2 suspension at 25 v/v%). The suspension was then centrifuged (Beckman Coulter Allegra X-22 Centrifuge (Brea, CA, USA)) at 500 g for 10 min, whereby afterwards the upper two thirds of the supernatants was collected and then centrifuged again in the same manner as previously, with the bottom residue being discarded. The upper two thirds of the supernatants from this second centrifuge cycle were then collected to yield the stock solution, with the bottom residue being discarded. 2.1. Single-Wall Carbon Nanotube Dispersion A single-wall carbon nanotube stock solution was prepared by dispersing 5 mg arc-discharge powder (P3-SWNT, Carbon Solutions Inc., Riverside, CA, USA) in 50 mL of 1% (v/v) aqueous Triton X-100 (Sigma-Aldrich, Sydney, Australia) by bath sonication (≈50 WRMS (root mean squared Watts), Elmasonic S 30H (Elma Schmidbauer GmbH, Singen, Germany) for 3 × 1 h intervals at room temperature. The resulting SWCNT suspension was centrifuged for 1 h, at 17,500 g (Beckman Coulter Allegra X-22 Centrifuge (Brea, CA, USA)). Then, the upper two thirds of the supernatants were carefully collected and then centrifuged again in the similar manner as previously, with the bottom residue being discarded. The upper two thirds of the supernatants from this second centrifuge cycle were then collected and combined to yield the stock solution. The remaining third of unsuspended carbon was discarded [26]. 1. Introduction In micromechanical exfoliation, the ﬂakes are produced by manual removal and transfer using adhesive tape. Liquid phase exfoliation involves mechanical means such as shearing, sonication, stirring, bubbling and grinding [22], or via atomic intercalation through solution chemistry [23]. MoS2 exhibits robust mechanical properties. It has good photo-responsivity when used as a monolayer, allowing use in innovative solar cell devices [24]. When MoS2 and CNTs are combined, they can provide novel photovoltaic devices with excellent performance. The performance of SWCNT/n-Si hybrid solar cells also depends on the thickness of the CNT ﬁlm, particularly for ﬁlms with an average transmittance above 70% [11]. Other research studies done in this ﬁeld have shown that the addition of a conducting polymer, such as polyaniline, into the CNT ﬁlm enhances electrical conductivity and therefore, improves the performance of the solar cells [25]. There is a need to increase the energy conversion efﬁciency of photovoltaic systems and given CNTs and MoS2 have unique physical properties and it is possible to use simple solution processes for their deposition and application, these materials present an obvious avenue for increasing PCE and achieving high-energy efﬁciency compared to current solar panels. In this report, the performance of SWCNT/n-Si solar cells will be enhanced with the addition of a MoS2 interlayer (see Figure 1) 3 of 11 Materials 2018, 11, 639 between the CNT layer and the Si substrate (making a SWCNT/MoS2/n-Si cell) to help with charge carrier transport. Figure 1. Schematic structure for (a) single-wall carbon nanotube (SWCNT)/n-Si solar cells and (b) SWCNT/ Molybdenum disulphide (MoS)2/n-Si layered solar cells. Figure 1. Schematic structure for (a) single-wall carbon nanotube (SWCNT)/n-Si solar cells and (b) SWCNT/ Molybdenum disulphide (MoS)2/n-Si layered solar cells. 2.4. Device Fabrication Nanotube ﬁlms were prepared using vacuum ﬁltration. This was completed by initially mixing an appropriate amount of SWCNT suspension with milli-Q water (Kansas City, MO, USA) to make a solution of 250 mL which when ﬁltered would yield a ﬁlm with a transmittance of about 80%. The suspension was vacuum ﬁltered with the aid of a water aspirator through two membranes. The ﬁlter paper on the bottom (VSWP Millipore, 0.025 µm pore size, nitrocellulose) was patterned with four holes similar to the size of the desired SWCNT ﬁlms. The top ﬁlter paper (HAWP Millipore Burlington, MA, USA, 0.45 µm pore size, mixed cellulose ester (MCE)) remained unpatterned. The difference in the rate of ﬂow through the ﬁlter papers causes preferential ﬂow of solution through the top ﬁlm where the bottom ﬁlm is patterned. Thus, the CNTs are stacked by the top ﬁlm in a similar shape as that of the template ﬁlm. After the solution passed through both ﬁlms, it was passed through the ﬁltration media two more times to allow enough CNTs to be retained on the ﬁlm. After this, Milli-Q water was passed through the CNTs again to remove the surfactants. The template used in these experiments produces four identical 0.5 cm2 ﬁlms in each ﬁltration. One ﬁlm was deposited on a microscope slide for measurement of sheet resistance and optical transmittance, while the others were attached to solar cells substrates for measurement of cell efﬁciency. For ﬁlm deposition, the ﬁlms were cut from the MCE membrane and placed (CNT side down) on the substrate. Wetting was done using a small drop of water and the SWCNT/MCE layer sandwiched between a piece of Teﬂon (on top of MCE paper) and substrate was clamped by two pieces of glass slides. The substrate was then heated at 80 ◦C for about 15 min and then cooled in darkness for 30 min. The substrates were then washed in acetone three times (30 min each) to dissolve the MCE membrane; second and third washes with stirring facilitate the removal of the MCE membrane. To complete the preparation of cells, the oxide on the reverse side of all Si pieces was manually removed by scratching. A gallium indium eutectic layer (eGaIn, Sigma-Aldrich, Saint Louis, MO, USA) was then painted on the back surface of Si before attaching a piece of stainless steel as the back contact of the device (see Figure 1). 2.3. Preparation of Si Wafer-Photolithography An n-Si wafer doped with phosphorous was rinsed using acetone and dried under a stream of nitrogen. The resistivity of the 525 µm thick wafer was 1–5 Ωm, with 100 nm thermal oxide layer, (ABC GmbH, Munich, Germany). In a clean room (Class 1000), the Au grid structure with an active area of 0.087 cm2 was deﬁned by photolithography [27]. By using spin coating at 3000 rpm for 30 s, a positive photoresist (AZ1518, Micro resist technology GmbH, Munich, Germany) was placed on the Si wafer 4 of 11 Materials 2018, 11, 639 and then softbaked on a hot plate (AREC heating magnetic stirrer from Rowe Scientiﬁc, Lonsdale, SA, Australia) at 100 ◦C for 50 s. The coated wafer was cooled to room temperature before deﬁning the grid patterns using a mask aligner—EVG 610 (EV Group, Braunau am Inn, Austria). The wafer was then immersed in a developer solution—(AZ 726 MIF, AZ Electronic Materials, GmbH, Munich, Germany) for 15 s to develop photoresist. The wafer was then rinsed with water and dried under a stream of nitrogen gas. The Si wafer post-baking process was done on a hot plate with the pattern deﬁned at 115 ◦C for 50 s. A Quorumtech Q300T-D sputter coater (Quorumtech, East Sussex, UK), equipped with a quartz crystal microbalance, was used to deposit gold and chromium layers (Au/Cr 145/5 nm) on a silicon wafer to form the metal electrode. The substrate was then immersed in acetone for about 90 min followed by a mild rub with a cotton stick in order to dissolve the photoresist. Solar cell substrates were then prepared by cutting pieces of Si sized 1.5 cm2. One drop of buffered oxide etch (6:1 of 40% NH4F and 49% hydroﬂuoric acid (HF), Sigma-Aldrich, Saint Louis, MO, USA) was applied on the active area to remove the SiO2 layer on the surface (The SiO2 layer was considered removed when the surface expelled the aqueous droplet) [27]. 2.7. Film Characterization A series of (SWCNT, MoS2 and SWCNT over MoS2) ﬁlms were fabricated from each sample using the SWCNT and MoS2 solutions and deposited on a glass slide (deposited similarly to the previously described ﬁlm deposition on silicon). Sheet resistance (Rsheet) measurements were completed on SWCNT, MoS2 and SWCNT on MoS2 ﬁlms using a four-point probe linked to Keithlink Film Resistivity Measurement Tool 1.0 (KeithLink Technology, New Taipei City, Taiwan), four readings on each ﬁlm at various locations were taken and then results were averaged. Optical absorption spectroscopy (UV-Vis spectroscopy, (Cary 60, Agilent, Santa Clara, CA, USA) was used to determine the transmittance of the ﬁlms as this affects the amount of light passing through the ﬁlms and subsequently, the amount of energy produced by the cell. Scanning Electron Microscopy (SEM) was used to characterize the surface structure of the nanostructures after fabrication. Dispersions were deposited on Si for examination using an FEI Inspect F50 SEM (Hillsboro, OR, USA). Atomic Force Microscopy (AFM) was used to investigate the topographical structure of the nanostructures formed. This imaging technique was speciﬁcally applied in order to determine the size and structure of SWCNTs, ﬂakes of MoS2 and MoS2/SWCNT composites. AFM data was acquired under ambient conditions in tapping mode using a Bruker Multimode8 AFM ( Bruker, Santa Barbara, CA, USA) with Nanoscope V controller. The cantilevers used were Silicon (HQNSC15 Mikromasch) with a fundamental resonance frequency of 200–500 kHz and nominal spring constant of 40 N/m. AFM imaging parameters, including set-point, scan rate and feedback gains, were adjusted to optimize image quality and ensure accurate measurement of ﬂake thickness [28,29]. The scanner was calibrated in x, y and z directions using a Si calibration grid (Bruker model number VGRP: 10 µm pitch, 180 nm depth). All analysis of AFM images was performed using Nanoscope analysis software version 1.4 (Bruker, Santa Barbara, CA, USA). Raman spectra was acquired with a Witec alpha300R Raman microscope (Witec, Ulm, Germany) at an excitation wavelength of 532 nm using a ×40 objective with a numerical aperture of 0.60. The gratings used for measurements was either the 1800 grooves/mm or the 600 grooves/mm. Integration times for single Raman spectra ranged between 30 s and 60 s for between 2 and 3 accumulations. Confocal Raman images were also acquired with integration times ranging from 5 s to 10 s per pixel, with each pixel being a Raman spectrum. 2.5. Layered SWCNT/ MoS2/n-Si Solar Cells The dispersions of SWCNTs and MoS2 were sonicated for 5 min and 1 h respectively. Then, using vacuum ﬁltration, the SWCNT dispersion was ﬁltered to give a constant thickness (250 µL) CNT ﬁlm. The MoS2 ﬁlm was then formed on the CNT ﬁlm using different volumes (100–1000 µL) of the MoS2 dispersion ﬁltered through the CNT ﬁlm already in place on the ﬁlter paper. The cells were made by turning the ﬁltered ﬁlms over such that the MoS2 layer was in contact with the Si and then dissolving the ﬁlter paper. 2.6. Solar Cell Characterization At each stage of preparation, the solar cells produced were tested by applying voltage to the electrodes under a solar simulator in the absence of natural light. An AM 1.5G ﬁlter (obtained from Irvine, CA, USA) was used to ﬁlter the light. A silicon reference cell (PV Measurements, from the National Institute of Standards and Technology, Gaithersburg, MD, USA) was used to calibrate the irradiance to be 100 mW cm−2 on the surface of the sample. A Keithley 2400 SourceMeter (from Newport Corporation, Solon, OH, USA) was used to acquire data that was captured and sent to a computer with LabVIEW 8.2 (National Instruments, Austin, TX, USA). 2.4. Device Fabrication The cells were then tested 3 times and further subjected to different post-fabrication treatment procedures. First, a drop of 2% HF was applied on the active area to etch off the SiO2 formed between the nanotube ﬁlm and the Si during the attachment step of the ﬁlms. This was then followed by treating the SWCNT ﬁlm with two drops of thionyl chloride (SOCl2) which was left to evaporate to increase conductivity. Before testing, the residue was washed with ethanol and dried under a stream of nitrogen. In the last step, the devices were again treated with 2% HF in the same manner as previously described which signiﬁcantly improved performance [26]. 5 of 11 Materials 2018, 11, 639 3. Results and Discussion The MoS2 ﬁlms used in this work were characterized with representative examples of the results shown in Figure 2. Figure 2. (a) Raman spectrum (collected using the 1800 grooves/mm grating); (b) Scanning electron microscopy (SEM) image and (c) Atomic Force Microscopy (AFM) image of MoS2 ﬁlm deposited on a Si substrate and the corresponding line scan of the MoS2-nanosheet ﬁlm. Figure 2. (a) Raman spectrum (collected using the 1800 grooves/mm grating); (b) Scanning electron microscopy (SEM) image and (c) Atomic Force Microscopy (AFM) image of MoS2 ﬁlm deposited on a Si substrate and the corresponding line scan of the MoS2-nanosheet ﬁlm. Raman spectra give a qualitative characterization of MoS2 nanosheets. MoS2 was deposited on an Si substrate and Raman spectra for the exfoliated MoS2 are shown in Figure 2. The expected E 1 2g peak at 384 cm−1 originates from the Mo−S in-plane vibration mode while, the A1g peak is observed near 408 cm−1 from vibrations of out-of-plane which yields a peak difference between E 1 2g and A1g of ~25 cm−1. This is consistent with previously determined values of bulk MoS2 [16] where the frequency difference is about 25 cm−1 for bulk and 19 cm−1 for monolayer [16,30]. Figure 2b displays a typical SEM image of exfoliated MoS2 ﬂakes with crystalline straight edges and evidence of partial exfoliation through the semitransparent layers (other representative SEM images are provided in Figure S1). AFM imaging (Figure 2c) shows that the MoS2 has a thickness on the order of 25 nm conﬁrming the ﬂakes are many layers thick given the interlayer spacing is ~0.65 nm [16]. The range of lateral size was 100–1000 nm2. Raman, SEM and AFM combine to show the exfoliated MoS2 is multilayered but remains highly two dimensional. Similar characterization of the SWCNT ﬁlm is provided in the supplemental material (Figure S2). 2.7. Film Characterization To generate Raman images, the intensity of a given region in the Raman spectrum, corresponding to the material of interest, is plotted against the X-Y position of the laser during a surface scan. 6 of 11 Materials 2018, 11, 639 3.1. Characterization of Control Solar Cells In order to investigate the effect of molybdenum disulphide on SWCNT/n-Si solar cells, separate SWCNTs and MoS2 aqueous dispersions were prepared and used to fabricate solar cells (SWCNT/n-Si and MoS2/n-Si). This experiment was carried out to understand the properties of each material 7 of 11 Materials 2018, 11, 639 individually for solar cells. Figure 3 illustrates the photocurrent-voltage (J-V) characteristics of the best-performing SWCNT/n-Si and MoS2/n-Si solar cells and their detailed photovoltaic performance are summarized in Table 1. It can be seen that SWCNTs only based solar cells exhibit a higher efﬁciency compared to the MoS2 based solar cells. The electrodes made with SWCNTs have a lower sheet resistance than those made with MoS2 and hence the SWCNT-based cells exhibit better performance. MoS2 can be either an n-type or p-type semiconductor depending on the level of impurities [31]. The MoS2 dispersions used in this work are very dilute which lead to very low coverages on the Si substrates. The intimate contact of MoS2 and Si in few areas will lead to very few depletion regions with the n-Si being established leading to very low efﬁciencies. Figure 3. J-V curves of (a) SWCNT/n-Si; (b) MoS2/n-Si and (c) SWCNT/MoS2/n-Si solar cells. Inset in (b) show an expanded view of the J-V curves for MoS2/n-Si cells. Figure 3. J-V curves of (a) SWCNT/n-Si; (b) MoS2/n-Si and (c) SWCNT/MoS2/n-Si solar cells. Inset in (b) show an expanded view of the J-V curves for MoS2/n-Si cells. 8 of 11 Materials 2018, 11, 639 Table 1. Selected solar cell and diode properties for SWCNTs, MoS2 and SWCNT/MoS2. Data shown for champion cells (bold typeface) and average properties with standard deviation (regular typeface). Three devices of such type are included in the analysis. Jsc (mA cm−2) Voc (V) T% Rsheet (Ωsq−1) FF Eff (%) SWCNT 25.17 25.4 ± 1 0.529 0.532 ± 0.009 74 531 ± 74 0.53 0.49 ± 0.05 7.04 6.6 ± 0.4 MoS2 0.025 0.021 ± 0.004 0.236 0.208 ± 0.027 96 1621 ± 236.6 0.18 0.2 ± 0.005 1.06 × 10−3 7.99 × 10-4 ± 2.67 × 10-4 SWCNT@600 µL MoS2 35.46 32.1 ± 3.1 0.557 0.557 ± 0.002 59 410 ± 90 0.61 0.6 ± 0.01 12.04 11.2 ± 0.8 In order to adjust the thickness of the SWCNT ﬁlms, the volume of the ﬁltered solutions was varied. 3.1. Characterization of Control Solar Cells Previous work has shown the best CNT ﬁlm transmittance in CNT/Si solar cells is 75% [24]. A sheet resistance of 531 Ωsq−1 is in good agreement with the previously reported value in the literature [11]. On the basis of that, with the current dispersion, 250 µL is an optimal value and its solar cell exhibited an average power conversion efﬁciency (PCE) of ~6.6% (Table 1). In an effort to improve performance, a very thin layer of MoS2 was placed between the n-Si and the SWCNT. Various coverages of MoS2 were used to ﬁnd the optimal conditions. 3.2. Layered SWCNT/MoS2/n-Si Solar Cells The J-V curves for the best cells in Figure 3 show that the addition of the MoS2 interlayer leads to signiﬁcant changes in the short circuit current density (Jsc) and the ﬁll factor (FF) with a negligible change in open circuit voltage (Voc), which leads to a near doubling of the PCE. In order to determine the optimal coverage of the MoS2 ﬁlms, a set of ﬁlms were prepared by ﬁrst ﬁltering a constant volume of SWCNT dispersion (250 µL) followed by ﬁltration with different volumes (100–1000 µL) of the MoS2 dispersion to give a layered ﬁlm. Figure 4 shows that the transmittance decreased by ~10% with increasing MoS2 volume (from 100 to 1000 µL) as expected as adding more material should increase the ﬁlm mass and increase the number of scattering elements (leading to more light absorption or scattering). The sheet resistance stayed reasonably constant (Figure 4). Variation in the volume of MoS2 dispersion shows a clear maximum in photovoltaic performance when 600 µL of MoS2 was used in the ﬁltration to fabricate the layered design. Figure 5 shows that the addition of the MoS2 layer leads to small changes in the average Jsc while the average Voc is largely unchanged (also see Table 1). The largest changes are observed for the average FF. MoS2 has a very long charge carrier diffusion length [12] which should reduce opportunity for charge recombination at junctions such as those between two SWCNTs. The increase in average FF shows that the layers with more MoS2 are more effective at extracting the charge carriers and this leads to improve performance. Figure S3 shows that there are no obvious trends in the diode properties (reverse saturation current density (Jsat) or Ideality) with the values relatively constant for all devices. Figure 4. Sheet resistance (left) and optical transmittance @550 nm (right) of varying layered SWCNTs@ MoS2 ﬁlms thickness after all three chemical treatments. Figure 4. Sheet resistance (left) and optical transmittance @550 nm (right) of varying layered SWCNTs@ MoS2 ﬁlms thickness after all three chemical treatments. 9 of 11 Materials 2018, 11, 639 Figure 5. Layered SWCNT/MoS2/n-Si solar cells parameters ((a) Jsc; (b) Voc; (c) Fill factor (FF) and (d) Photoconversion efficiency (PCE) extracted after all three chemical treatments. The close association of the p-type SWCNTs with the n-type Si substrate is critical for the unctioning of these cells as this produces the required depletion region. 3.2. Layered SWCNT/MoS2/n-Si Solar Cells The addition of an n-type MoS2 layer clearly improves the performance (average PCE increase from 6.6% to 11.2%). The oncentration of the MoS2 is quite low, so at all volumes used the coverage is certainly not complete. This means there are always SWCNTS in contact with the Si to give a working cell. The MoS2 flakes are excellent hole conductors and will help improve carrier lifetimes and this will improve performance. The carrier MoS2 layer efficiently decreases the recombination loss of carriers which esults in an improvement of FF. Once the number of MoS2 flakes increases to give reasonably high overages, the number of nanotubes in close contact with the Si will decrease and performance will decrease again. There is a lot of work showing that MoS2 density of states shift up and down depending on how Materials 2018, 11, x FOR PEER REVIEW 9 of 11 Figure 5. Layered SWCNT/MoS2/n-Si solar cells parameters (a) Jsc; (b) Voc; (c) Fill factor (FF) and (d) Photoconversion efﬁciency (PCE) extracted after all three chemical treatments. Materials 2018, 11, x FOR PEER REVIEW Materials 2018, 11, x FOR PEER REVIEW 9 of 11 meters ((a) Jsc; (b) Voc; (c) Fill factor (FF) and (d) hree chemical treatments. with the n-type Si substrate is critical for the ed depletion region. The addition of an n-type rage PCE increase from 6.6% to 11.2%). The mes used the coverage is certainly not complete. h the Si to give a working cell. The MoS2 flakes rove carrier lifetimes and this will improve eases the recombination loss of carriers which f MoS flakes increases to give reasonably high 9 of 11 verages, the number of nanotubes in close contact with the Si will decrease and performance will crease again. There is a lot of work showing that MoS2 density of states shift up and down depending on how Figure 5. Layered SWCNT/MoS2/n-Si solar cells parameters (a) Jsc; (b) Voc; (c) Fill factor (FF) and (d) Photoconversion efﬁciency (PCE) extracted after all three chemical treatments. it interacts with other materials [32]. This may mean the shift in the band gap of the MoS2 provides the opportunity for the MoS2 to act as an intermediate facilitating transfer of charge carriers. 4. Conclusions Molybdenum disulphide (MoS2) has been successfully used to improve the performance of CNT/Si solar photovoltaics. 3.2. Layered SWCNT/MoS2/n-Si Solar Cells A number of techniques such as AFM, SEM and Raman were used to characterize the MoS2 and CNTs in order to examine their structure, thickness and/or lateral size. The average performance achieved from the CNT/Si-solar cells only was ~6.6% whereas, the highest PCE after the insertion of MoS2 layer between SWCNTs and silicon was 11.2%. Addition of the interlayer of molybdenum disulphide led to an improved fill factor which was largely responsible for the Figure 5. Layered SWCNT/MoS2/n-Si solar cells parameters ((a) Jsc; (b) Voc; (c) Fill factor (FF) and (d) Photoconversion efficiency (PCE) extracted after all three chemical treatments. The close association of the p-type SWCNTs with the n-type Si substrate is critical for the functioning of these cells as this produces the required depletion region. The addition of an n-type MoS2 layer clearly improves the performance (average PCE increase from 6.6% to 11.2%). The concentration of the MoS2 is quite low, so at all volumes used the coverage is certainly not complete. This means there are always SWCNTS in contact with the Si to give a working cell. The MoS2 ﬂakes are excellent hole conductors and will help improve carrier lifetimes and this will improve performance. The carrier MoS2 layer efﬁciently decreases the recombination loss of carriers which results in an improvement of FF. Once the number of MoS2 ﬂakes increases to give reasonably high coverages, the number of nanotubes in close contact with the Si will decrease and performance will decrease again. y p p g y p improved performance. Supplementary Materials: The following are available online at www.mdpi.com/link, Figure S1: SEM images of a iou MoS flake o Si Fi u e S2 (A) Ra a e t u (a ui ed u i the 600 oo e / ati a d The close association of the p-type SWCNTs with the n-type Si substrate is critical for the functioning of these cells as this produces the required depletion region. The addition of an n-type MoS2 layer clearly improves the performance (average PCE increase from 6.6% to 11.2%). The There is a lot of work showing that MoS2 density of states shift up and down depending on how it interacts with other materials [32]. This may mean the shift in the band gap of the MoS2 provides the opportunity for the MoS2 to act as an intermediate facilitating transfer of charge carriers. 4. Conclusions Molybdenum disulphide (MoS2) has been successfully used to improve the performance of CNT/Si solar photovoltaics A number of techniques such as AFM SEM and Raman were used to Supplementary Materials: The following are available online at http://www.mdpi.com/1996-1944/11/4/639/s1, Figure S1: SEM images of various MoS2 ﬂakes on Si, Figure S2: (A) Raman spectrum (acquired using the 600 grooves/mm grating and (B) Raman optical image, (C,D) SEM images and (E,F) AFM images of SWCNTs ﬁlm that were deposited on Si substrates, Figure S3: Diode properties of the devices as a function of the volume of MoS2 used. References 1. International Energy Outlook 2017. Available online: https://www.eia.gov/outlooks/ieo/pdf/0484% 282017%29.pdf (accessed on 19 April 2018). 1. International Energy Outlook 2017. Available online: https://www.eia.gov/outlooks/ieo/pdf/0484% 282017%29.pdf (accessed on 19 April 2018). 2. Díaz-González, F.; Sumper, A.; Gomis-Bellmunt, O.; Villafáﬁla-Robles, R. A Review of energy storage technologies for wind power applications. Renew. Sustain. Energy Rev. 2012, 16, 2154–2171. [CrossRef] 3. Hawkes, A.; Staffell, I.; Brett, D.; Brandon, N. Fuel cells for micro-combined heat and power generation. 2. Díaz-González, F.; Sumper, A.; Gomis-Bellmunt, O.; Villafáﬁla-Robles, R. A Review of energy storage technologies for wind power applications. Renew. Sustain. Energy Rev. 2012, 16, 2154–2171. [CrossRef] 2. Díaz-González, F.; Sumper, A.; Gomis-Bellmunt, O.; Villafáﬁla-Robles, R. A Review of energy storage technologies for wind power applications. Renew. Sustain. Energy Rev. 2012, 16, 2154–2171. [CrossRef] p gy g technologies for wind power applications. Renew. Sustain. Energy Rev. 2012, 16, 2154–2171. [CrossRef] 3. Hawkes, A.; Staffell, I.; Brett, D.; Brandon, N. Fuel cells for micro-combined heat and power generation. Energy Environ. Sci. 2009, 2, 729–744. [CrossRef] 3. Hawkes, A.; Staffell, I.; Brett, D.; Brandon, N. Fuel cells for micro-combined heat and power generation. Energy Environ. Sci. 2009, 2, 729–744. [CrossRef] 3. Hawkes, A.; Staffell, I.; Brett, D.; Brandon, N. Fuel cells for micro-combined heat and power generation. Energy Environ. Sci. 2009, 2, 729–744. [CrossRef] gy 4. Brennan, L.; Owende, P. Biofuels from microalgae—A review of technologies for production, processing and extractions of biofuels and co-products. Renew. Sustain. Energy Rev. 2010, 14, 557–577. [CrossRef] 4. Brennan, L.; Owende, P. Biofuels from microalgae—A review of technologies for production, processing and extractions of biofuels and co-products. Renew. Sustain. Energy Rev. 2010, 14, 557–577. [CrossRef] h l l ll h l l [ f] 5. Wenham, S.; Green, M. Silicon solar cells. Prog. Photovolt. Res. Appl. 1996, 4, 3–33. [CrossRef] Rinkesh. What Are Alternative Energy Sources? Available online: http://www.conserve-energy-future com/alternativeenergysources.php (accessed on 26 March 2018). 7. Tune, D.D.; Flavel, B.S. Advances in carbon nanotube–silicon heterojunction solar cells. Adv. Energy Mater. 2018. [CrossRef] 7. Tune, D.D.; Flavel, B.S. Advances in carbon nanotube–silicon heterojunction solar cells. Adv. Energy Mater. 2018. [CrossRef] 8. Jia, Y.; Cao, A.; Kang, F.; Li, P.; Gui, X.; Zhang, L.; Shi, E.; Wei, J.; Wang, K.; Zhu, H. Strong and reversible modulation of carbon nanotube–silicon heterojunction solar cells by an interfacial oxide layer. Phys. Chem. Chem. Phys. 2012, 14, 8391–8396. [CrossRef] [PubMed] 9. (B) Raman optica b t t Fi concentration o This means ther 4. Conclusions wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest. (B) Raman optica b t t Fi concentration o This means ther 4. Conclusions substrates, Figure S3: Diode properties of the devices as a function of the volume of MoS2 used. y g g are excellent hole conductors and will help improve carrier lifetimes and this will improve performance. The carrier MoS2 layer efficiently decreases the recombination loss of carriers which results in an improvement of FF. Once the number of MoS2 flakes increases to give reasonably high coverages, the number of nanotubes in close contact with the Si will decrease and performance will decrease again. There is a lot of work showing that MoS2 density of states shift up and down depending on how it interacts with other materials [32]. This may mean the shift in the band gap of the MoS2 provides Molybdenum disulphide (MoS2) has been successfully used to improve the performance of CNT/Si solar photovoltaics. A number of techniques such as AFM, SEM and Raman were used to characterize the MoS2 and CNTs in order to examine their structure, thickness and/or lateral size. The average performance achieved from the CNT/Si-solar cells only was ~6.6% whereas, the highest PCE after the insertion of MoS2 layer between SWCNTs and silicon was 11.2%. Addition of the interlayer of molybdenum disulphide led to an improved ﬁll factor which was largely responsible for the improved performance. pp y g g 4. Conclusions Molybdenum disulphide (MoS2) has been successfully used to improve the performance of CNT/Si solar photovoltaics. A number of techniques such as AFM, SEM and Raman were used to Supplementary Materials: The following are available online at http://www.mdpi.com/1996-1944/11/4/639/s1, Figure S1: SEM images of various MoS2 ﬂakes on Si, Figure S2: (A) Raman spectrum (acquired using the 600 grooves/mm grating and (B) Raman optical image, (C,D) SEM images and (E,F) AFM images of SWCNTs ﬁlm that were deposited on Si substrates, Figure S3: Diode properties of the devices as a function of the volume of MoS2 used. 10 of 11 10 of 11 Materials 2018, 11, 639 Acknowledgments: We acknowledge the use of South Australian nodes of the Australian Microscopy & Microanalysis Research Facility (AMMRF) and Australian National Fabrication Facility (ANFF) at Flinders University and the University of Adelaide. The support of the Australian Research Council Discovery Program (DP150101354 and DP160101301) is gratefully acknowledged. 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https://openalex.org/W4323363020	https://phsreda.com/e-articles/10471/Action10471-105358.pdf	Russian	null	Features of professional self-determination of high school students from dysfunctional families	null	2,023	cc-by	2,274	Content is licensed under the Creative Commons Attribution 4.0 license (CC-BY 4.0) Publishing house "Sreda" g Аксенова Мария Анатольевна бакалавр, магистрант ФГБОУ ВО «Московский государственный психолого-педагогический университет» г. Москва DOI 10.31483/r-105358 ОСОБЕННОСТИ ПРОФЕССИОНАЛЬНОГО САМООПРЕДЕЛЕНИЯ СТАРШЕКЛАССНИКОВ ИЗ НЕБЛАГОПОЛУЧНЫХ СЕМЕЙ Аннотация: в статье представлены особенности профессионального са- моопределения обучающихся в старших классах и влияния неблагополучия семьи на характер протекания данного процесса. Неблагополучие семьи может при- водить к возникновению различных деформаций в процессе профессионального самоопределения старшеклассников. Результаты исследования показали, что старшеклассники из неблагополучных семей чаще выбирают профессии, связан- ные со взаимодействием с людьми, сферой обслуживания, а также профессии, связанные творчеством. При этом полученные данные свидетельствуют о том, что их уровень готовности к выбору профессии намного ниже уровня са- моопределения старшеклассников из благополучных семей, что обусловлено их меньшей осведомленностью о существующих профессиях. Ключевые слова: профессиональное самоопределение, старшеклассники, Аксенова Мария Анатольевна бакалавр, магистрант ФГБОУ ВО «Московский государственный психолого-педагогический университет» г. Москва Аксенова Мария Анатольевна бакалавр, магистрант ФГБОУ ВО «Московский государственный психолого-педагогический университет» г. Москва р р ф моопределения обучающихся в старших классах и влияния неблагополучия семьи на характер протекания данного процесса. Неблагополучие семьи может при- водить к возникновению различных деформаций в процессе профессионального самоопределения старшеклассников. Результаты исследования показали, что старшеклассники из неблагополучных семей чаще выбирают профессии, связан- ные со взаимодействием с людьми, сферой обслуживания, а также профессии, связанные творчеством. При этом полученные данные свидетельствуют о том, что их уровень готовности к выбору профессии намного ниже уровня са- моопределения старшеклассников из благополучных семей, что обусловлено их меньшей осведомленностью о существующих профессиях. Ключевые слова: профессиональное самоопределение, старшеклассники, неблагополучная семья, профессиональные деформации. Профессиональное самоопределение является важной и неотъемлемой ча- стью социализации учеников старших классов, а также необходимым условием их успешности в дальнейшей жизни за стенами школы. Вступая в самостоятель- ную жизнь, выпускникам предстоит определиться с будущей профессией. Дан- ный выбор является весьма сложным, т. к. здесь приходится учитывать множе- ство внешних и внутренних факторов современных социальных и экономиче- ских реалий, а его последствия будут влиять на все стороны жизни и ее качество. Одним из важнейших факторов, влияющих на выбор профессии является мнение, помощь и поддержка семьи. Семейное неблагополучие, в той или иной 1 Издательский дом «Среда» степени, безусловно влияет на профессиональное самоопределение выпускни- ков, что мало отражено в современных исследованиях. В связи с этим возникает необходимость в изучении специфики профессионального самоопределения старшеклассников из неблагополучных семей. степени, безусловно влияет на профессиональное самоопределение выпускни- ков, что мало отражено в современных исследованиях. В связи с этим возникает необходимость в изучении специфики профессионального самоопределения старшеклассников из неблагополучных семей. Е.А. Климов (основоположник отечественной дифференциальной психофи- зиологии и разработчик основ теории индивидуального стиля деятельности) определяет профессиональное самоопределение как важнейшую составляющую психического развития человека и как процесс формирования полноценной лич- ности, участника общественных отношений [1]. Самоопределение старшеклассника в профессиональной сфере является до- статочно сложным и длительным процессом в его жизни, касающегося всех зна- чимых аспектов его личностного развития в целом. Данный этап проходит каж- дый старшеклассник в своей жизни. После школы одной из основных задач яв- ляется выбор профессионального пути и учебного заведения. Здесь как никогда важна помощь и поддержка со стороны родителей ученика [4]. Неблагополучие семьи оказывает сильное негативное влияние на процесс социализации подростка в целом и, как следствие, может приводить к различным трудностям в выборе профессии у старшеклассников. По определению В.М. ttps://phsreda.com Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) 2 https://phsreda.com Аксенова Мария Анатольевна бакалавр, магистрант ФГБОУ ВО «Московский государственный психолого-педагогический университет» г. Москва Целуйко, «неблагополучная семья – это семья, в ко- торой нарушена структура, обесцениваются или игнорируются основные семей- ные функции, имеются явные или скрытые дефекты воспитания, в результате чего дети, воспитывающиеся в ней, попадают в категорию “трудных”» [3]. Профессиональное самоопределение старшеклассников из неблагополуч- ных семей может претерпевать различного рода деформации, и такие ученики сталкиваются с серьезными проблемами, требующими помощи со стороны спе- циалистов. Деформации профессионального самоопределения представляют со- бой ложный выбор профессии, не соответствующий реальным способностям и интересам ребенка [2]. Среди таких деформаций выделяют: разногласия между образцом профессии и самооценкой; различие между возможностями и требова- ниями определённой профессии; ошибочный выбор специальности; 2 https://phsreda.com Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) Publishing house "Sreda" противоречия между осознанными и неосознанными сторонами высокопрофес- сионального сознания. Целью нашего исследования являлось изучение особенностей профессио- нального самоопределения старшеклассников из неблагополучных семей. Нами было выдвинуто предположение, что для старшеклассников из неблагополучных семей характерен низкий уровень готовности к выбору профессии, который вы- ражен в низкой осведомленности о многообразии профессий. Исследование проводилось среди учеников параллели 10-х классов. Всего испытуемых 20 (10 из благополучных семей и 10 из неблагополучных семей). В качестве диагностического материала для выявления особенностей про- фессионального самоопределения старшеклассников были проведены следую- щие методики: методика «Дифференциально-диагностический опросник» (ДДО Е.А. Климов); опросник «Готовность учащихся к выбору профессии» (В.Б. Успенский); для дополнительного определения благополучия / неблагопо- лучия семьи испытуемого (помимо характеристики на каждого ученика от школьного педагога-психолога), а также выявления профессионального само- определения старшеклассников была создана и проведена авторская анкета «Ан- кета-опросник для выявления профессионального самоопределения». В ходе исследования особенностей профессионального самоопределения старшеклассников из благополучных и неблагополучных семей были получены следующие результаты. Соотношение средних показателей склонности к тому или иному типу про- фессии у обучающихся из благополучных и неблагополучных семей представ- лено на рисунке 1. Content is licensed under the Creative Commons Attribution 4.0 license (CC-BY 4.0) 3 Content is licensed under the Creative Commons Attribution 4.0 license (CC-BY 4.0) Издательский дом «Среда» Издательский дом «Среда» Рис. 1. Соотношение средних значений показателей профессиональных предпочтений старшеклассников из неблагополучных и благополучных семей Из рисунка 1 видно, что наибольшие различия между старшеклассниками из неблагополучных и благополучных семей заметны по таким сферам профес- сиональной деятельности, как «человек ‒ человек» и «человек ‒ художественный образ», т. е. старшеклассники из неблагополучных семей более склонны выби- рать профессии, связанные со сферой обслуживания или творческими специаль- ностями. Аксенова Мария Анатольевна бакалавр, магистрант ФГБОУ ВО «Московский государственный психолого-педагогический университет» г. Москва Обучающиеся из благополучных семей в среднем больше выбирают техни- ческие профессии и профессии, связанные с цифровым расчётом, а также про- фессии, связанные с животноводством, растениеводством и лесным хозяйством. Для выявления статистически значимых различий профессиональной направленности между группой обучающихся из благополучных семей и груп- пой из неблагополучных семей мы использовали U-критерий Манна – Уитни. Ре- зультаты расчета представлены в таблице 1. ttps://phsreda.com Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) Content is licensed under the Creative Commons Attribution 4.0 license (CC-BY 4.0) Таблица 1 Таблица 1 Таблица 1 Результаты выявления статистически значимых различий профессионального интереса и склонности обучающихся из благополучных и неблагополучных семей (методика ДДО Е.А. Климов: U-критерий Манна – Уитни) Тип Коэф. Манна ‒ Уитни (Uэмп) Уровень значимости (р)* Средний ранг Благополучные Неблагополучные «Человек – природа» 35 27 120 90 «Человек – техника» 35,5 119,5 90,5 «Человек – человек» 16,5 71,5 138,5 «Человек – знак» 41,5 113,5 96,5 «Человек – художественный образ» 41 96 114 Различия статически достоверны при p≤0,05. ц Результаты выявления статистически значимых различий профессионального интереса и склонности обучающихся из благополучных и неблагополучных семей (методика ДДО Е.А. Климов: U-критерий Манна – Уитни) Результаты выявления статистически значимых различий профессионального интереса и склонности обучающихся из благополучных б й ( ДДО Е А К U-критерий Манна – Уитни) Тип Коэф. Манна ‒ Уитни (Uэмп) Уровень значимости (р) Средний ранг Благополучные Неблагополучные «Человек – природа» 35 27 120 90 «Человек – техника» 35,5 119,5 90,5 «Человек – человек» 16,5 71,5 138,5 «Человек – знак» 41,5 113,5 96,5 «Человек – художественный образ» 41 96 114 Различия статически достоверны при p≤0,05. 4 https://phsreda.com Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) Publishing house "Sreda" Анализ результатов с помощью U-критерия Манна – Уитни показал, что статистически значимыми различиями является данные по типу «Человек ‒ че- ловек» (16,5). Следовательно, наше предположение о том, что обучающиеся из неблагополучных семей выбирают чаще профессии, связанные с частым взаимо- действием с людьми, подтверждается и является статистически значимым отли- чием от обучающихся из благополучных семей. Результаты изучения готовности обучающихся из благополучных и небла- гополучных семей к выбору профессии, а также уровня их самоопределения (опросник «Готовность учащихся к выбору профессии» В.Б. Успенский) пред- ставлены на рисунке 2. 0 2 4 6 8 10 Неготовность Средняя готовность Благополучные Проблемные 10 Рис. 2. Соотношение средних показателей по уровням выраженности готовности старшеклассников из благополучных и неблагополучных семей к выбору профессии Из рисунка 2 видно, что у старшеклассников из неблагополучных семей в большинстве случаев выражена низкая готовность к выбору профессии. Также, ни один испытуемый из данной группы не показал высокую готовность. У старшеклассников из благополучных семей чаще отмечается средняя го- товность к выбору профессии, т. е. у обучающихся из данной группы выше уро- вень понимания того, кем они хотят быть в будущем. Также, у них отмечается отсутствие показателей по шкале «неготовность». 6 https://phsreda.com Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) 6 https://phsreda.com ttps://phsreda.com Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) Таблица 1 Результаты, полученные в ходе проведения авторской анкеты, позволили выявить следующие особенности: 1) старшеклассники из обоих типов семей показали, что все задумывались о выборе профессии (100%); 2) старшеклассники из благополучных семей лучше знают о многообразии профессий (90%), чем старшеклассники из неблагополучных семей (40%); 6 https://phsreda.com Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) Publishing house "Sreda" 3) для старшеклассников из благополучных семей на первом месте стоит та- кой фактор, влияющий на их профессиональный выбор, как заработок (80%), на втором месте – личный интерес (30%), а у старшеклассников из неблагополуч- ных семей – наоборот (40% и 60%, соответственно); 3) для старшеклассников из благополучных семей на первом месте стоит та- кой фактор, влияющий на их профессиональный выбор, как заработок (80%), на втором месте – личный интерес (30%), а у старшеклассников из неблагополуч- ных семей – наоборот (40% и 60%, соответственно); 4) абсолютно все старшеклассники из неблагополучных семей (100%) хо- тели бы или уже посоветовались с кем-либо о выборе профессии; 5) среди учеников из благополучных семей преобладают ответы, связанные с описанием личностных качеств своей будущей личности (40%). Также у них чаще (20%), чем у старшеклассников из неблагополучных семей (10%), звучали ответы, касающиеся больших заработков; 6) на вопрос «Кем вы видите себя в будущем?» ученики из неблагополуч- ных семей в 50% случаев продемонстрировали затруднения в ответе на данный вопрос, при этом 40% обучающихся отметили примерные профессиональные предпочтения («кем-то из киноиндустрии», «государственное и муниципальное управление», «ИП», «финансист»). 6) на вопрос «Кем вы видите себя в будущем?» ученики из неблагополуч- ных семей в 50% случаев продемонстрировали затруднения в ответе на данный вопрос, при этом 40% обучающихся отметили примерные профессиональные предпочтения («кем-то из киноиндустрии», «государственное и муниципальное управление», «ИП», «финансист»). Таким образом, подводя итог всему вышесказанному, можно сделать вывод: для старшеклассников из неблагополучных семей характерен низкий уровень го- товности к выбору профессии, который выражен в низкой осведомленности о многообразии профессии. Таблица 1 Для выявления статистически значимых различий по уровню готовности к профессиональному выбору у старшеклассников из неблагополучных и 5 Content is licensed under the Creative Commons Attribution 4.0 license (CC-BY 4.0) Издательский дом «Среда» благополучных семей мы использовали U-критерий Манна – Уитни. Результаты расчета представлены в таблице 2. Таблица 2 Результаты выявления статистически значимых различий по уровню выраженности готовности к профессиональному выбору обучающихся из благополучных и неблагополучных семей (опросник «Готовность учащихся к выбору профессии» В.Б. Успенский: U-критерий Манна – Уитни) Уровни готовности Коэф. Манна ‒ Уитни (Uэмп) Уровень значимости (р) Средний ранг Благополучные Неблагополучные 1. Неготовность 45 27 100 110 2. Низкая готовность 18,5 73,5 136,5 3. Средняя готовность 29,5 125,5 84,5 4. Высокая готовность 40 115 95 * Различия статически достоверны при p≤0,05. Результаты выявления статистически значимых различий по уровню выраженности готовности к профессиональному выбору обучающихся из благополучных и неблагополучных семей (опросник «Готовность учащихся к выбору профессии» В.Б. Успенский: U-критерий Манна – Уитни) Уровни готовности Коэф. Манна ‒ Уитни (Uэмп) Уровень значимости (р)* Средний ранг Благополучные Неблагополучные 1. Неготовность 45 27 100 110 2. Низкая готовность 18,5 73,5 136,5 3. Средняя готовность 29,5 125,5 84,5 4. Высокая готовность 40 115 95 * Различия статически достоверны при p≤0,05. * Различия статически достоверны при p≤0,05. * Различия статически достоверны при p≤0,05. Анализ результатов исследования уровня готовности к выбору профессии обучающихся из неблагополучных и благополучных семей показал, что стати- стически достоверными различиями являются данные по шкале «низкая готов- ность» (18,5), т. е. можно сделать вывод, что старшеклассники из неблагополуч- ных семей имеют более низкую готовность к выбору профессии. Данный резуль- тат является статистически значимым отличием от результатов старшеклассни- ков из благополучных семей. Content is licensed under the Creative Commons Attribution 4.0 license (CC-BY 4.0) 4. Широкова Е.Ю. Влияние семьи на профессиональное самоопределение подростков / Е.Ю. Широкова // Молодой ученый. – 2020. – №50 (340). – С. 448– 449. 8 https://phsreda.com Содержимое доступно по лицензии Creative Commons Attribution 4.0 license (CC-BY 4.0) Издательский дом «Среда» Список литературы Список литературы 1. Климов Е.А. Психология профессионального самоопределения / Е.А. Климов. – 3-е изд., стер. – М.: Академия, 2007. – 304 с. 1. Климов Е.А. Психология профессионального самоопределения / Е.А. Климов. – 3-е изд., стер. – М.: Академия, 2007. – 304 с. 2. Поняев А.Н. Социальные последствия деформаций профессионального самоопределения старшеклассников из неблагополучных семей / А.Н. Поняев // 2. Поняев А.Н. Социальные последствия деформаций профессионального самоопределения старшеклассников из неблагополучных семей / А.Н. Поняев // Педагогика в теории и на практике: актуальные вопросы и современные аспекты: 2. Поняев А.Н. Социальные последствия деформаций профессионального самоопределения старшеклассников из неблагополучных семей / А.Н. Поняев // Педагогика в теории и на практике: актуальные вопросы и современные аспекты: сб. ст. VIII Междунар. науч.-практич. конф. (Пенза, 15 марта 2021 года). – Пенза: Наука и Просвещение, 2021. – С. 103–106. Педагогика в теории и на практике: актуальные вопросы и современные аспекты: сб. ст. VIII Междунар. науч.-практич. конф. (Пенза, 15 марта 2021 года). – Пенза: Наука и Просвещение, 2021. – С. 103–106. 3. Целуйко В.М. Психология неблагополучной семьи: книга для педагогов и родителей / В.М. Целуйко. – М.: Владос-пресс, 2004. 7 4. Широкова Е.Ю. Влияние семьи на профессиональное самоопределение подростков / Е.Ю. Широкова // Молодой ученый. – 2020. – №50 (340). – С. 448– 449.
https://openalex.org/W2581677259	https://hal.science/hal-01758952/document	English	null	PSHA after a strong earthquake: hints for the recovery	Annals of geophysics	2,016	cc-by	5,838	To cite this version: L. Peruzza, R. Gee, B. Pace, Gregory Roberts, O. Scotti, et al.. PSHA after a strong earthquake: hints for the recovery. Annals of Geophysics, 2016, 59 (5), 10.4401/ag-7257. hal-01758952 L. Peruzza, R. Gee, B. Pace, Gregory Roberts, O. Scotti, et al.. PSHA after a strong earthquake: hints for the recovery. Annals of Geophysics, 2016, 59 (5), 10.4401/ag-7257. hal-01758952 Distributed under a Creative Commons Attribution 4.0 International License PSHA after a strong earthquake: hints for the recovery L. Peruzza, R. Gee, B. Pace, Gregory Roberts, O. Scotti, F. Visini, L Benedetti, M. Pagani To cite this version: L. Peruzza, R. Gee, B. Pace, Gregory Roberts, O. Scotti, et al.. PSHA after a strong earthquake: hints for the recovery. Annals of Geophysics, 2016, 59 (5), 10.4401/ag-7257. hal-01758952 PSHA after a strong earthquake: hints for the recovery L. Peruzza, R. Gee, B. Pace, Gregory Roberts, O. Scotti, F. Visini, L Benedetti, M. Pagani HAL Id: hal-01758952 https://hal.science/hal-01758952v1 Submitted on 25 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 ANNALS OF GEOPHYSICS, 59, FAST TRACK 5, 2016; DOI: 10.4401/ag-7257 L. PERUZZA1, R. GEE1,2, B. PACE3, G. ROBERTS4, O. SCOTTI5, F. VISINI6 , L. BENEDETTI7, M. PAGANI2 (1) Istituto Nazionale di Oceanografia e di Geofisica Sperimentale - OGS, Trieste, Italy (2) Hazard Team, GEM Foundation, Pavia, Italy (3) DiSPUTer, Università “G. d’Annunzio” Chieti- Pescara, Chieti Scalo, Italy (4) Department of Earth and Planetary Sciences, Birkbeck, University of London, UK (5) Institut de Radioprotection et de Surete Nucleaire IRSN, Fontenay-aux-Roses, France (6) Istituto Nazionale di Geofisica e Vulcanologia, Sezione di Pisa, Italy (7) Aix-Marseille Université, CEREGE CNRS-IRD UMR 34, Aix en Provence, France lperuzza@inogs.it * The WG has been formally approved during the XXXV ESC General Assembly: to join the WG fill the form at https://sites.google.com/site/linkingfaultpsha/ fault2sha-esc-wg Abstract We perform aftershock probabilistic seismic hazard analysis (APSHA) of the ongoing aftershock sequence following the Amatrice August 24th, 2016 Central Italy earthquake. APSHA is a time-dependent PSHA calculation where earthquake occurrence rates decrease after the occurrence of a mainshock following an Omori-type decay. In this paper we propose a fault source model based on preliminary evidence of the complex fault geometry associated with the mainshock. We then explore the possibility that the aftershock seismicity is distributed either uniformly or non-uniformly across the fault source. The hazard results are then computed for short-intermediate exposure periods (1-3 months, 1 year). They are compared to the background hazard and intended to be useful for post-earthquake safety evaluation. PSHA after a strong earthquake: hints for the recovery L. PERUZZA1, R. GEE1,2, B. PACE3, G. ROBERTS4, O. SCOTTI5, F. VISINI6 , L. BENEDETTI7, M. PAGAN (1) Istituto Nazionale di Oceanografia e di Geofisica Sperimentale - OGS, Trieste, Italy (2) Hazard Team, GEM Foundation, Pavia, Italy (3) DiSPUTer, Università “G. d’Annunzio” Chieti- Pescara, Chieti Scalo, Italy (4) Department of Earth and Planetary Sciences, Birkbeck, University of London, UK (5) Institut de Radioprotection et de Surete Nucleaire IRSN, Fontenay-aux-Roses, France (6) Istituto Nazionale di Geofisica e Vulcanologia, Sezione di Pisa, Italy ANNALS OF GEOPHYSICS, 59, FAST TRACK 5, 2016; DOI: 10.4401/ag-7257 Figure 1: faults and earthquakes overlain on the E-W component of INSAR data [Marinkovic and Larsen, 2016], blue means eastwards; white pins for M>4 before Sep, 28th, 2016 (cnt.rm.ingv.it), dotted for M>5; A=Mt. Vettore Fault, B=Laga Fault, C=Sibillini Thrust. Although fault segmentation, expected magni- tude, recurrence time and associated historical earthquakes differ somewhat among authors, there is a clear agreement concerning the lack of surface faulting between the southern ter- mination of Mt. Vettore fault and the north- ernmost limit of Mt. Gorzano fault (hereinafter Laga fault) where the main shock of the 2016 Amatrice earthquake is located and most of the INSAR deformation is concentrated (Fig. 1). Figure 2: surface effects after the 24 Aug earthquake: a) location map of ruptures by EMERGEO WG [2016], Piccardi et al. [2016], Pace et al. [2016]; b) rupture detail at Mt. Vettore. Figure 2: surface effects after the 24 Aug earthquake: a) location map of ruptures by EMERGEO WG [2016], Piccardi et al. [2016], Pace et al. [2016]; b) rupture detail at Mt. Vettore. g f ff f g q location map of ruptures by EMERGEO WG [2016], Piccardi et al. [2016], Pace et al. [2016]; b) rupture detail at Mt. Vettore. a) b) After August 24th, researchers that went into the field documented coseismic ground rup- tures with maximum throw up to 25 cm, de- tectable along segments 3-9 km long (Fig. 2). Most of these surface deformations can be as- cribed to coseismic exhumation (rejuvenation) a) In this paper we gathered pieces of infor- mation for a first formulation of aftershock probabilistic seismic hazard assessment (APSHA, as defined by Yeo and Cornell [2009]) in the damaged area. The topography of the area, 3D geometry of the faults and time- dependent estimates of seismicity rates enter in our computations. The results should super- sede the standard PSHA practice for the in- termediate-term (months to year). Thus, these estimates may be suitable for the impending activities of microzonation, retrofitting and re- building. We hope this work will form a basis for gathering more detailed information and to support activities aimed at post-earthquake recovery. a) b) I. INTRODUCTION 0826-index-e.html; www.eqclearinghouse.org /2016-08-24-italy/]. Since August 30th, some participants of the in- formal Fault2SHA working group* opened a forum [http://earthquake2016.prophpbb.com] to circulate ideas, publish material, draft pa- pers and news within the scientific communi- ty. This attempt to debate scientific issues in real-time helped to focus investigations in the field, and motivated and boosted this work. 0826-index-e.html; www.eqclearinghouse.org /2016-08-24-italy/]. he event of August 24th, 2016 in Central Italy caused devastating damage and 298 deaths, in spite of the well-known high seismic hazard of the region. The earthquake provoked not only a prompt mobilization of rescue teams but also an unprecedented mobi- lization of national and international scientific teams. Preliminary scientific data have been progressively provided, complemented and updated [e.g. Gruppo di Lavoro INGV, 2016a, b; Marinkovic and Larsen, 2016; ReLUIS-INGV Workgroup, 2016; GL IREA-CNR and INGV, 2016;ran.protezionecivile.it/IT/dettaglio_evid. php?evid=340867;www.gsi.go.jp/cais/topic16 T T Since August 30th, some participants of the in- formal Fault2SHA working group* opened a forum [http://earthquake2016.prophpbb.com] to circulate ideas, publish material, draft pa- pers and news within the scientific communi- ty. This attempt to debate scientific issues in real-time helped to focus investigations in the field, and motivated and boosted this work. 1 1 II. METHODS Within the Fault2SHA scientific forum, we faced the parameterization of the causative seismogenic source, based on very preliminary and uncer- tain data, with the aim to discern between the activation of one or more seismogenic sources. As the aftershock sequence evolved, the poten- tial link at depth of Mt. Vettore and Laga faults became more evident: this interpretation is ac- tually the most widely accepted one after the quake (see e.g. Lavecchia et al. [2016]). Adopting surface traces based on our mapping activities that acknowledge earlier work on ex- isting published maps, focal mechanisms re- leased from various seismological agencies, and the hypothesis of one unique source, we modeled a SW dipping plane representing a single fault at depth that splits into separate branches towards the surface as the Mt. Vet- tore and Laga Faults: it represents the prelimi- nary fault geometry for the purpose of our cal- culations. This complex fault geometry is one way to ex- plain the two distinct patches of slip distribu- tion obtained by INSAR data inversion located on 50 degree dipping planes [GL IREA-CNR and INGV, 2016]. It is coherent with outcrop- ping observations of two steeper dipping planes (60-70 degrees) separated by about 5 km, in the area between the localities of Ar- quata del Tronto and Accumuli, where negli- gible surface expressions have been observed in the field. The hypothesized fault geometry is compatible with the preliminary aftershock locations, but we do expect refinement and a better resolu- tion when high-quality hypocentral locations will be released. Conversely, this geometry does not account for the alternate stripes of east-westwards movement detected by INSAR (see Fig. 1) for which two distinct conjugate faults may be needed. As these patterns sug- gesting east-dipping planes are controversial, we decided not to model such a plane in this study. Figure 3a is the 3D sketch of the fault source, created as input for the OpenQuake- engine software [Pagani et al., 2014; OQ, www.globalquakemodel.org/openquake/]. Due to its complex geometry, the surface is represented by a set of point sources (Fig. 3b) on which the global seismicity rate of the fault system is partitioned. The red rectangle rough- ly corresponds to the rupture area as modeled by GdL INGV [2016a]. II. METHODS Active normal faulting in the Central Apen- nines has been recognized for many decades; several authors [e.g. Barchi et al., 1999; Galadini and Galli, 2003; Boncio et al., 2004a, b; Roberts and Michetti, 2004; Benedetti et al., 2013] have described potential seismic sources in the region, which has been recently and his- torically hit by deadly earthquake sequences. After August 24th, researchers that went into the field documented coseismic ground rup- tures with maximum throw up to 25 cm, de- tectable along segments 3-9 km long (Fig. 2). Most of these surface deformations can be as- cribed to coseismic exhumation (rejuvenation) After August 24th, researchers that went into the field documented coseismic ground rup- tures with maximum throw up to 25 cm, de- tectable along segments 3-9 km long (Fig. 2). Most of these surface deformations can be as- cribed to coseismic exhumation (rejuvenation) 2 ANNALS OF GEOPHYSICS, 59, FAST TRACK 5, 2016; DOI: 10.4401/ag-7257 of existing SW dipping normal faults outcrop- ping along the western flank of Mt. Vettore; minor features were reported also along the Laga fault. The expected magnitude predicted by these displacements and surface rupture length, using existing empirical magnitude vs size relations, is compatible with that of the mainshock, but quite different values (Mw 6.0- 6.2, downdip length 4-8 km or more) can be expected due to the uncertainty in the scaling relationship and field measurements. Within the Fault2SHA scientific forum, we faced the parameterization of the causative seismogenic source, based on very preliminary and uncer- tain data, with the aim to discern between the activation of one or more seismogenic sources. As the aftershock sequence evolved, the poten- tial link at depth of Mt. Vettore and Laga faults became more evident: this interpretation is ac- tually the most widely accepted one after the quake (see e.g. Lavecchia et al. [2016]). of existing SW dipping normal faults outcrop- ping along the western flank of Mt. Vettore; minor features were reported also along the Laga fault. The expected magnitude predicted by these displacements and surface rupture length, using existing empirical magnitude vs size relations, is compatible with that of the mainshock, but quite different values (Mw 6.0- 6.2, downdip length 4-8 km or more) can be expected due to the uncertainty in the scaling relationship and field measurements. ANNALS OF GEOPHYSICS, 59, FAST TRACK 5, 2016; DOI: 10.4401/ag-7257 weeks, as shown in Fig. 4b, whilst a too rapid decay (black line) is predicted by 10-day learn- ing period only. Finally, we integrate eq.(1) to derive the cumulative number of events pre- dicted by the theoretical curve in 30 days, 3 months, and 1 year, starting on October 1 st. In Table 1, the number of events and correspond- ing a-values of a G-R distribution are given, as- suming b=1. weeks, as shown in Fig. 4b, whilst a too rapid decay (black line) is predicted by 10-day learn- ing period only. Finally, we integrate eq.(1) to derive the cumulative number of events pre- dicted by the theoretical curve in 30 days, 3 months, and 1 year, starting on October 1 st. In Table 1, the number of events and correspond- ing a-values of a G-R distribution are given, as- suming b=1. although the surface itself may extend down to depths of 18 km [e.g. Boncio et al., 2004b]. The basic principle for an APSHA is to treat the earthquake sequence with time-dependent seismicity rates and superimpose it on the tra- ditional Poisson estimate. The traditional PSHA we use as reference is the one stated by law [http://zonesismiche.mi.ingv.it/] since 2004, namely MPS04. For the earthquake se- quence, we assume an Omori-Utsu decay of earthquakes with time after the main event. This simple model can be assumed as a realis- tic one provided that: Figure 4: Omori-Utsu decay for the 2016 sequence: a) fit on the first 20 days of INGV bulletin data with M ≥ 1.8; b) observed versus predicted n(t) from Aug 23 until Sep 16 (24th day), Model 1 of Fig. 4a by light green curve. p • the seismicity belongs to a unique source, with no triggering of nearby cascade events; • the coefficients are properly calibrated. p • the seismicity belongs to a unique source, with no triggering of nearby cascade events; • the coefficients are properly calibrated. p • the seismicity belongs to a unique source, with no triggering of nearby cascade events; p • the seismicity belongs to a unique source, with no triggering of nearby cascade events; f a) b) Note that despite the a-value increases with time, the seismicity decay is preserved if the prediction time in PSHA is set equal to the ob- servation time of the Omori-Utsu modelling. a) gg g y • the coefficients are properly calibrated. II. METHODS Considering the prelim- inary hypocentral distribution of aftershocks, we limit the fault source to a depth of 12 km, The hypothesized fault geometry is compatible with the preliminary aftershock locations, but we do expect refinement and a better resolu- tion when high-quality hypocentral locations will be released. Conversely, this geometry does not account for the alternate stripes of east-westwards movement detected by INSAR (see Fig. 1) for which two distinct conjugate faults may be needed. As these patterns sug- gesting east-dipping planes are controversial, we decided not to model such a plane in this study. Figure 3a is the 3D sketch of the fault source, created as input for the OpenQuake- engine software [Pagani et al., 2014; OQ, www.globalquakemodel.org/openquake/]. Figure 3: geometry of the fault source proposed for the 2016 earthquake sequence: a) 3D model implemented in OQ; b) surface projection of point sources (black dots) on earthquake locations, fault traces and rupture model; c) fault surface projection on the topography. a) b) c) a) Due to its complex geometry, the surface is represented by a set of point sources (Fig. 3b) on which the global seismicity rate of the fault system is partitioned. The red rectangle rough- ly corresponds to the rupture area as modeled by GdL INGV [2016a]. Considering the prelim- inary hypocentral distribution of aftershocks, we limit the fault source to a depth of 12 km, b) c) 3 ANNALS OF GEOPHYSICS, 59, FAST TRACK 5, 2016; DOI: 10.4401/ag-7257 ANNALS OF GEOPHYSICS, 59, FAST TRACK 5, 2016; DOI: 10.4401/ag-7257 p p y We therefore downloaded the INGV bulletin [http://cnt.rm.ingv.it/, data accessed on Sep 17, 2016], for deriving the coefficients of the theoretical model that forecasts the number of events (n) versus time (t) as: n(t) = k/(c+t) p (1) n(t) = k/(c+t) p (1) III. RESULTS We briefly describe here the results in terms of Peak Ground Acceleration (PGA is defined as Spectral Acceleration SA at 0s) and SA(0.3s) for rock sites (Vs30= 800m/s) obtained with the fault aftershock models only (Fig. 5), thus comparing the proxy hazard curve for the 3 observation times with the ones given by MPS04 (Fig. 6). Table 1: APSHA, maximum PGA/SA at 10% probabil- ity of exceedance in different observation times since Oct, 1st, 2016; values obtained with uniform and non-uniform partitioning of aftershock seismicity rates on the fault. Fault Model PGA /SA(0.3s) (g) Time Oct, 1 Num M≥1.8 a-value (a-year) uniform non- uniform 30 days 745 4.672 (5.751) 0.13/0.22 0.13/0.23 90 days 1597 5.003 (5.605) 0.18/0.34 0.19/0.50 1 year 3236 5.310 (5.310) 0.24/0.47 0.25/0.50 Figure 5: APSHA maps showing: a) PGA for rock sites (CY14) at 10% in 1 year from October 1, 2016, using uniform and non-uniform rates with topography; b) the same but SA (0.3s) in the next 3 months. a) a) a) b) Note the effects of the inclusion of a proper fault geometry, distance metrics and of topog- raphy (Fig. 5 and ESM) in the hazard calcula- tions: for the town of Amatrice, the aftershock hazard is slightly lower when the non-uniform Similarly to Yeo and Cornell [2009], we set uni- form and non-uniform partitioning of the seismicity rates: in the second case the a-value decreases as a function of distance away from the patches with highest coseismic slip, as it has been observed that aftershocks are often clustered at the ends of faults [e.g. Das and Henry, 2003]. These hypotheses are speculative and uncertainties can be handled as branches of a logic tree. Finally, by applying the new OQ features specifically developed for volcan- ic areas [Gee et al., 2016], we introduce topog- raphy in the computation by defining sites in terms of their 3D location, via a DEM (1km horizontal resolution, Fig. 3c) that results in some minor changes in rupture-to-site distanc- es (Rrup). The ground motion prediction equa- tion (GMPE) of Chiou and Youngs [2014] (CY14) is used, because it is defined in terms of Rrup; it has been derived using earthquakes from active shallow crustal regions, and is ap- a) a) b) b) Note the effects of the inclusion of a proper fault geometry, distance metrics and of topog- raphy (Fig. (1) where k, c, and p are empirical constants relat- ed to a particular aftershock sequence. The fit- ting was performed by ZMAP code, at www.seismo.ethz.ch/prod/software/zm ap/. a) b) A preliminary Gutenberg-Richter (G-R) analy- sis of this dataset suggests that b-value is close to 1, with completeness magnitude at about M2. We fit different sub-samples, by varying the minimum magnitude threshold and learn- ing period: the best candidate by checking the total number events within the longest period available at the time of the analysis, is obtained with Mmin≥ 1.8 and 20 days of learning period, represented in Fig. 4a. We are conscious that during the first hours or days small events have probably gone undetected, merged in the coda of bigger events; similarly the complete- ness and location quality has increased with the deployment of temporary stations. None- theless, the coefficients representing Model 1 in Fig. 4a, are suitable to represent the global seismic activity detected during the first b) Note that despite the a-value increases with time, the seismicity decay is preserved if the prediction time in PSHA is set equal to the ob- servation time of the Omori-Utsu modelling. 4 ANNALS OF GEOPHYSICS, 59, FAST TRACK 5, 2016; DOI: 10.4401/ag-7257 With this fundamental limitation (i.e. no ex- trapolation to other observation times), there is no need to generalize the seismicity rates into non-poissonian earthquake probabilities. Thus in OQ, G-R distributions truncated at Mmin=3.5 and Mmax=5.5 (based on magnitude of the largest aftershock) and a-values given in Tab. 1 are partitioned on the point sources rep- resenting the fault plane. plicable down to Mw 3.5. Some examples showing the impact of different ingredients in the APSHA (fault geometry, distance metric, topography, seismicity rate distribution on- fault) are given as Electronic Supplement. IV. DISCUSSION IV. DISCUSSION distribution of a-values is considered, com- pared to the assumption of uniform seismicity, due to the proximity of the city to the epicenter of the mainshock. We see the opposite trend, approximately same amount, for cities located at the tip ends of the fault, e.g. the town of Visso. Note also that the values obtained at 10% p.e. in 1-year since October 1, 2016 (ap- proximately the time in which the theoretical decay is flattening, at a rate of about 2 events per day with M ≥ 1.8) are higher than the val- ues expected in MPS04 in 50 years. Hypotheses of a more rapid decay, e.g. stated by the black curve in Fig. 4b, lead to lower values of about 20-40%. We set up a model for APSHA, in Central Ita- ly, after the devastating event of August, 24. The theoretical approach is not novel [Yeo and Cornell, 2009; Iervolino et al., 2014], but this is the first time it has been applied in a real case in Italy, modeling aftershocks with a fault plane, within an ongoing seismic sequence. In the aim of supporting the impending recovery and rebuilding actions, we set up investigation times of 1 month/1 year since October 1 st, 2016, as this is a reasonable time frame for the se- quence extinction, if no triggering of nearby faults happen. pp The 3D fault geometry is essentially derived from data available before the sequence, some hypothesized details have to be refined, or re- shaped with a community consensus, after that more accurate data will be available. However, the introduction of a realistic fault surface and proper computation of distances from ruptures via topography leads to a hazard map that captures complexities that are as detailed as those obtained via full ground wave propaga- tion modeling. Through a comparison of ob- servations with event-based scenarios we are planning further analyses, in order to check to what extent this hazard map representation is realistic, and whether it can also be adopted in microzonation studies. The simple assump- tions here adopted (Omori-Utsu decay of the earthquake number with time, non-uniform partitioning of seismicity rates outside the rup- ture area) cannot at present predict more com- plex fault interactions which, however, are strongly speculative: this will be an interesting aim of future work. III. RESULTS 5 and ESM) in the hazard calcula- tions: for the town of Amatrice, the aftershock hazard is slightly lower when the non-uniform 5 5 IV. DISCUSSION [2014] and references there- in) suggested an increase of the probability of occurrences of earthquakes in the northern Laga fault that ruptured in 2016, as well as southwards. The coupling of earthquakes on different fault segments is the main limitation of this study that we hope to overcome by this new coupling of knowledge from PSHA and structural geology. We hope this work may be an important new avenue for re-insurance companies to appreciate losses in real-time af- ter a major earthquake as well as for research to improve disaster response. patible with the aftershock G-R and Mmax adopted by this study, and with the non- uniform distribution of earthquakes as given in Fig. 5. But 2 hours later, another “main” event (Mw 6.1) broke the northernmost patch of Mt. Vettore fault, followed on Oct 30 th at 6:40 UTC by the actual biggest earthquake of the Amatrice - Mt. Vettore sequence (Mw 6.6). Thus, one basic assumption of this study, that no triggered and cascading events occur, does not hold anymore, as pinpointed by the re- viewer, and the results provided since Oct 1 st are no longer valid. g In light of the recent events, considered that the failure of a test hypothesis is itself a result, we recomputed our results for 30 days starting on Sep, 20 th. This exposure period is prior to the Mw 6.1 earthquake of Oct 26 th and posterior to the learning period of the O-U calibration. The number of M>1.8 events increases (see Table 2) as the limits for the integration of Omori-Utsu decay curve shifts left; maximum PGA/SA values rise as well but the pattern remains the same, with maxima nearby Amatrice assuming a uniform rate distribution, and northeast of Visso in the non-uniform case. These are the results we plan to analyze under rigorous test- ing against real observations, in future work. IV. DISCUSSION The time-dependent seis- mic hazard in PGA and SA suggests that the region may experience acceleration values in the next year since October 1 st that are compa- rable or higher to the ones stated by the Italian law in 50 years. These results cannot be ex- trapolated to different periods than the ones in the Omori-Utsu forecast. % Fig. 6 shows that the hazard curves computed in this study (APSHA) and those considered in the Italian law (MPS04) for the town of Ama- trice, for both PGA and SA at 0.3s, cannot be reconciled, at least if we do not consider epis- temic uncertainties of the whole logic tree. Prospective work should aim at merging APSHA with MPS04, or with updated release of the Italian reference hazard map; future ef- forts should include also the new information about site-effects that are being currently ac- quired by several institutions [www.centro microzonazionesismica.it/it/attivita/41-il- centroms-per-il-terremoto-italia-centrale-2016]. Figure 6: hazard curves in Amatrice. Figure 6: hazard curves in Amatrice. Figure 6: hazard curves in Amatrice. This is the first time that forecasts have been made for aftershocks based on information 6 6 ANNALS OF GEOPHYSICS, 59, FAST TRACK 5, 2016; DOI: 10.4401/ag-7257 gathered rapidly in the wake of the mainshock. Previous knowledge of fault geometries was crucial to define the fault plane that hosted the earthquake rupture. We believe the inclusion of such information improves the forecast rela- tive to those that do not include such infor- mation. It is particularly interesting because this unusual earthquake ruptured two separate faults as mapped at the surface. Our approach allows us to deal with this complex geometry by using realistic fault geometries that are well known in the structural geology literature where en echelon faults at surface merge downwards into a single fault at depth [Walsh et al., 1999]. A similar style of faulting may ap- ply to other earthquakes such as the 2009 L'Aquila earthquake [Wilkinson et al., 2015], or to less known faults not as recently activated. It is worth remembering that Central Italy is often subjected to earthquake clusters, and that several faults are indicted of high time- dependent earthquake occurrence probabilities [Peruzza et al., 2011]. They motivated post L’Aquila earthquake temporary seismometric monitoring [Romano et al., 2013] in the south- ern Middle Aterno Valley, still silent today. In addition, the modelling of static stress varia- tion (see Pace et al. IV. DISCUSSION g g For the purpose of this special issue, we decid- ed to keep the manuscript as it was at its first submission, with the exception of this last chapter, for the following reasons: 1) the source we designed in September, largely based on field work existing before the se- quence, is still coherent with the most recent events and observations; 2) the many novelties introduced in space and time characterization of the source depict a new hazard picture, much closer to deterministic modeling than ever; 3) the time-dependency introduced for a “simplistic” decay of seismicity with no trig- gering of similar-sized ruptures demonstrates that the survived buildings and temporary in- stallations after a major earthquake are statisti- cally exposed to similar/higher shaking than those expected for long return periods. We be- ACKNOWLEDGEMENTS EMERGEO Working Group (2016). The 24 August 2016 Amatrice Earthquake: Coseismic Effects. doi: 10.5281/zenodo.61568 For their work in the background, a special mention is due to G. Weatherill, J. Faure- Walker, S. Baize, H. Jomard. Thanks to the personell from various institutions that in labs and in the field guarantee new data acquisi- tions. We simpathize with the victims families and population, hoping a prompt and endur- ing recovery. Galadini F., P. Galli (2003). Paleoseismology of silent faults in the Central Apennines (Italy): the Mt. Vettore and Laga Mts.Faults. Ann. Ge- ophys., .46:815-36. Gee R., L. Peruzza, B. Pace, R. Azzaro, S. D’Amico, and M. Pagani (2016). When proba- bilistic seismic hazard climbs volcanoes: how 3D topography and scaling relationships influ- ence hazard estimates. An example from Mt. Etna (Italy) http://meetingorganizer. coperni- cus.org/ESC2016/ESC2016-545-1.pdf V. EPILOGUE V. 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https://openalex.org/W3198318582	https://www.research-collection.ethz.ch/bitstream/20.500.11850/572568/3/journal.pcbi.1009393.pdf	English	null	Signal neutrality, scalar property, and collapsing boundaries as consequences of a learned multi-timescale strategy	bioRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	19,019	ETH Library ETH Library RESEARCH ARTICLE Signal neutrality, scalar property, and collapsing boundaries as consequences of a learned multi-timescale strategy Luca ManneschiID1☯, Guido GiganteID2,3☯, Eleni VasilakiID1,4, Paolo Del Giudice2,3† 1 Department of Computer Science, University of Sheffield, Sheffield, United Kingdom, 2 Istituto Superiore di Sanità, Rome, Italy, 3 INFN, Sezione di Roma, Rome, Italy, 4 Institute of Neuroinformatics, University of Zurich and ETH Zurich, Switzerland Luca ManneschiID1☯, Guido GiganteID2,3☯, Eleni VasilakiID1,4, Paolo Del Giudice2,3† 1 Department of Computer Science, University of Sheffield, Sheffield, United Kingdom, 2 Istituto Superiore di Sanità, Rome, Italy, 3 INFN, Sezione di Roma, Rome, Italy, 4 Institute of Neuroinformatics, University of Zurich and ETH Zurich, Switzerland 1 Department of Computer Science, University of Sheffield, Sheffield, United Kingdom, 2 Istituto Superiore di Sanità, Rome, Italy, 3 INFN, Sezione di Roma, Rome, Italy, 4 Institute of Neuroinformatics, University of Zurich and ETH Zurich, Switzerland ☯These authors contributed equally to this work. † Deceased * l.manneschi@sheffield.ac.uk a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Editor: Stefano Palminteri, Ecole Normale Superieure, FRANCE Received: August 10, 2021 Accepted: June 8, 2022 Published: August 5, 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.pcbi.1009393 Abstract We postulate that three fundamental elements underlie a decision making process: percep- tion of time passing, information processing in multiple timescales and reward maximisation. We build a simple reinforcement learning agent upon these principles that we train on a ran- dom dot-like task. Our results, similar to the experimental data, demonstrate three emerging signatures. (1) signal neutrality: insensitivity to the signal coherence in the interval preceding the decision. (2) Scalar property: the mean of the response times varies widely for different signal coherences, yet the shape of the distributions stays almost unchanged. (3) Collapsing boundaries: the “effective” decision-making boundary changes over time in a manner remi- niscent of the theoretical optimal. Removing the perception of time or the multiple timescales from the model does not preserve the distinguishing signatures. Our results suggest an alternative explanation for signal neutrality. We propose that it is not part of motor planning. It is part of the decision-making process and emerges from information processing on multi- ple timescales. OPEN ACCESS Citation: Manneschi L, Gigante G, Vasilaki E, Del Giudice P (2022) Signal neutrality, scalar property, and collapsing boundaries as consequences of a learned multi-timescale strategy. PLoS Comput Biol 18(8): e1009393. https://doi.org/10.1371/ journal.pcbi.1009393 Author(s): Originally published in: PLoS Computational Biology 18(8), https://doi.org/10.1371/journal.pcbi.1009393 This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. PLOS COMPUTATIONAL BIOLOGY 1 Introduction Perceptual decision-making is one of the most fundamental interactions of a biological agent with its environment. Perceptual decision-making processes have been long studied in the context of operant conditioning [1]. In these scenarios, an animal learns to associate choices and consequences by trial and error. Sub-optimal performance is considered a consequence of imperfect learning or the reflex of the learning strategy itself [2]. Outside this context, the research on perceptual decision-making has mainly focused on tasks where uncertainty (typically in the form of noisy signals) and time (e.g., duration of the observation and response delays) play a pivotal role [3–7]. In such scenarios, the errors made by the subject at the end of a training phase, as well as the relevant performance metrics (e.g. accuracy or speed of response), are deemed informative of the cognitive mechanisms involved [8–11]. There have been numerous attempts to compare the behaviour of animal subjects to the performance of different algorithms and determine how optimal the displayed behaviour is [8, 12–16]. Competing interests: The authors have declared that no competing interests exist. One of the key ideas in perceptual decision-making is accumulating evidence over time [6, 8, 17–20]. The drift-diffusion model (also known as the ‘bounded evidence accumulation’ model) consists of two or more competing traces. These traces accumulate sensory evidence for different choices; the first trace to hit a threshold makes the associated option the final deci- sion [21]. The drift-diffusion model is a continuous-time variant of the sequential probability ratio test [22, 23]. In the case of two-alternative forced choices, it is optimal in selecting between two hypotheses. Despite its simplicity, this model accounts for many psychophysical and neural observations. Examples are the distribution of response times and performance when varying sensory coherence [22, 23]. Notwithstanding its success, there are several alternatives to the standard drift-diffusion model [8, 24, 25] to account for unexplained phenomena such as primacy and recency effects, asymptotic accuracy, and “fast errors” [26–28]. Of notable importance is the Ornstein–Uhlen- beck model, which modifies the standard drift-diffusion model by including a decay term in the dynamics of the accumulation. Although the Ornstein–Uhlenbeck model can account for many experimental observations, including neurophysiological ones [24, 28], it introduces a characteristic timescale over which the model ‘forgets’ the past sensory information. Author summary Humans and animals integrate sensory information before making a decision. The inte- gration rate varies depending on the task. While driving could require quick reactions, evaluating the authenticity of a painting typically requires long observations. Conse- quently, the concept of representations created over multiple timescales appears neces- sary. Nevertheless, there is a lack of theoretical research that exploits multiple timescales, despite experimental evidence for the variety of integration rates. We, therefore, devel- oped a decision-making model based on simple integrators with multiple characteristic times. We analysed its behaviour on a highly volatile, biologically relevant task. Through reward maximisation based on trial and error, the model discovers an effective strategy that is surprisingly different and more robust than the “classical” single timescale approach. This learned strategy exhibits a remarkable agreement with experimental Copyright: © 2022 Manneschi 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. Data Availability Statement: The source code and data used to produce the results and analyses presented in this manuscript are available at 1 / 29 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries https://github.com/GuidoGigante/Signal-neutrality- scalar-property-and-collapsing-boundaries---Code. https://github.com/GuidoGigante/Signal-neutrality- scalar-property-and-collapsing-boundaries---Code. findings, suggesting a fundamental role of multiple timescales for decision-making. Our abstract model achieves a degree of biological realism while performing robustly in differ- ent environments. findings, suggesting a fundamental role of multiple timescales for decision-making. Our abstract model achieves a degree of biological realism while performing robustly in differ- ent environments. Funding: EV acknowledges the support from a Google Deepmind Award. EV was funded by the Engineering and Physical Sciences Research Council (Grant Nos. EP/V055720/1, EP/V006339/1, EP/S030964/1, and EP/P006094/1). LM acknowledges the support from the Engineering and Physical Sciences Research Council (Grant No. EP/V006339/1). PD and GG were partially funded by the European Union Horizon 2020 Research and Innovation program under the FET Flagship Human Brain Project (SGA2 Grant agreement No. 785907 and SGA3 Grant agreement No. 945539). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: EV acknowledges the support from a Google Deepmind Award. EV was funded by the Engineering and Physical Sciences Research Council (Grant Nos. EP/V055720/1, EP/V006339/1, EP/S030964/1, and EP/P006094/1). LM PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 1 Introduction A com- mon approach in the literature is to treat the timescale of the accumulation as a free parameter that is optimised to match experimental data [28, 29]. Here we take a different approach. We study a decision-making problem within the context of reinforcement learning. The task is is intended to mimic a typical perceptual decision mak- ing setup [30]: an actor-critic agent has the task of determining whether a noisy signal has a positive or negative mean value. This agent can also decide when to decide, i.e., it can choose to wait instead of making a decision. We, thereby, postulate that the concept of reward maxi- misation is inherent in such problems. Whilst not theoretically impossible, it is not straightforward to devise a biologically plausi- ble mechanism to tune a single timescale parameter to the statistics of a task. To circumvent this issue, we propose a more biologically plausible process. The agent receives the signal from multiple integrators, each with a different time constant. Via reinforcement learning, the agent PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 2 / 29 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries learns how to weigh them appropriately to maximise the collected reward. We hypothesise that multiple timescales lead to robust performance across different tasks since it is unrealistic to expect one time constant to fit any problem. In the context of our model, we will explore robustness when varying the task difficulty, i.e. the signal to noise ratio, and contrast it with models of one time constant. Beyond the computational advantage, such approach is consistent with the ample evidence of the coexistence of many timescales in brain functionality [31–35], even at the single neuron level [36–38], and for reward memory in reinforcement learning [39]. Another fundamental element of our model is that the agent perceives the passage of time. The agent has a “clock” available, several integrators with various time constants that increase by a fixed amount at each time step. In our model, we pair the clock’s time constants with the time constants of the signal integrators. We do this to facilitate our mathematical analysis. However, we expect multiple time constants in the clock to implement a scalable population code for time, akin to what experimentally observed [40]. And, more specifically, to allow for more complex decision-making boundaries. 1 Introduction We contrast an agent without any clock mechanism, an agent with a “single time constant” clock, and an agent with a multiple timescales clock. Our results highlight the performance advantages that a multiple timescales clock brings in. We evaluate our agent concerning three properties observed in experimental data or theo- retical analyses of decision-making processes. (1) Signal neutrality. We use this term as a short- hand to denote the observation that, for several hundred of milliseconds before the decision, the neurons in the lateral intraparietal cortex that correlate with the decision show the same response to different signal-to-noise ratios, with a time course of the firing activity that is indis- tinguishable in the different cases [5, 41]. One prior explanation is that the signals in that stage prepare the motor action. Here we evaluate this behaviour as part of the decision making pro- cess. (2) Scalar property or Weber’s law [42]. The coefficient of variation (CV, the ratio of the standard deviation to the mean) remains constant as the task difficulty varies. (3) Collapsing boundaries. In the beginning, the agent should wait to integrate information to make an informed decision. However, the decision time is not unlimited; as time passes, the decision boundaries decrease to force the agent to act. Our setup has similarities to a Partially Observable Markov Decision Process [43] with opportunity costs. The agent cannot access the real state of the world (in our case, the mean of the noisy signal and the time elapsed from the beginning of the trial). Instead, it has access to several observations at each time step. These observations are continuous variables that inte- grate noisy information about the state in terms of signal information and the time passed. These observations progressively correlate with the world’s true state as the integration filters out the noise. The option to defer this decision in case of insufficient evidence complements the desirable action to find the sign of the stimulus. Yet, the presence of a time limit effectively imposes a cost on deferring the decision to accumulate more evidence. 2.1 Task definition Inspired by classical random dots experiments [30], we model a two-alternative forced-choice task as a decision over the sign of the mean value of a noisy signal s(t) (see Fig 1). The signal (black line) consists of independent samples from a Gaussian distribution of mean μ and stan- dard deviation σ, each drawn every time step Δt = 10 ms. The agent is not required to decide at a prescribed time, it has the option to wait and then see another sample, or to perform one of two actions, ‘left’ and ‘right’, respectively associated with the decision μ < 0 and μ > 0 at each step. When an action is made, the episode ends, and 3 / 29 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries Fig 1. Task and model schematic. The environment corresponds to the random movement of a group of dots on a screen, which is represented as a uni-dimensional noisy signal s(t) (black line), sampled at discrete time steps Δt = 10 ms from a Gaussian distribution of mean μ and variance σ2. The task requires the subject to guess the sign of μ, by moving a lever to the right (positive sign) or to the left (negative sign); the subject can ‘choose when to choose’, within a maximum episode duration Tmax. The learning agent integrates the signal over different timescales τ (xs tðtÞs, blue lines); the agent integrates a constant input (depicted in red as a constant from the start of the episode) over the same timescales (xc tðtÞs, yellow-red lines) to simulate an internal clock mechanism estimating the passage of time. In both cases, the darker the colour the longer the corresponding timescale. At each time instance, the weighted sums of the integrators (far right) are fed into a decision layer (the actor) that computes the probability of choosing ‘left’ and ‘right’, thus terminating the episode, or to ‘wait’ to see another sample of s(t). If the subject gives the correct answer (the guessed sign coincides with the actual sign of μ) within the time limit, a reward is delivered; otherwise, nothing happens. In any case, a new episode starts. The agent learns by observing the consequences (obtained rewards) of its actions, adapting the weights assigned to the xs tðtÞs and xc tðtÞs. 2.1 Task definition Task and model schematic. The environment corresponds to the random movement of a group of dots on a screen, which is represented as a uni-dimensional noisy signal s(t) (black line), sampled at discrete time steps Δt = 10 ms from a Gaussian distribution of mean μ and variance σ2. The task requires the subject to guess the sign of μ, by moving a lever to the right (positive sign) or to the left (negative sign); the subject can ‘choose when to choose’, within a maximum episode duration Tmax. The learning agent integrates the signal over different timescales τ (xs tðtÞs, blue lines); the agent integrates a constant input (depicted in red as a constant from the start of the episode) over the same timescales (xc tðtÞs, yellow-red lines) to simulate an internal clock mechanism estimating the passage of time. In both cases, the darker the colour the longer the corresponding timescale. At each time instance, the weighted sums of the integrators (far right) are fed into a decision layer (the actor) that computes the probability of choosing ‘left’ and ‘right’, thus terminating the episode, or to ‘wait’ to see another sample of s(t). If the subject gives the correct answer (the guessed sign coincides with the actual sign of μ) within the time limit, a reward is delivered; otherwise, nothing happens. In any case, a new episode starts. The agent learns by observing the consequences (obtained rewards) of its actions, adapting the weights assigned to the xs tðtÞs and xc tðtÞs. During learning, the model estimates at each step t the total future expected reward V(t) (the critic) for the current episode as a linear summation of the integrators. Learning of the parameters is accomplished through a standard actor-critic reinforcement learning model, where the reward delivered by the environment is used to update the V value function, which is then used to update the actor’s parameters (see S1 Text for more details) https://doi.org/10.1371/journal.pcbi.1009393.g001 a reward is delivered only if the agent correctly guessed the sign of μ; otherwise, the agent receives nothing. Each episode has a maximum duration Tmax. When Tmax is reached, another ‘wait’ from the agent leads to the end of the episode and no reward is delivered. Whilst σ is constant, the value of μ is instead re-sampled at the beginning of each episode from a Gaussian distribution p(μ) of zero mean and variance σμ. 2.1 Task definition During learning, the model estimates at each step t the total future expected reward V(t) (the critic) for the current episode as a linear summation of the integrators. Learning of the parameters is accomplished through a standard actor-critic reinforcement learning model, where the reward delivered by the environment is used to update the V value function, which is then used to update the actor’s parameters (see S1 Text for more details) https://doi.org/10.1371/journal.pcbi.1009393.g001 Fig 1. Task and model schematic. The environment corresponds to the random movement of a group of dots on a screen, which is represented as a uni-dimensional noisy signal s(t) (black line), sampled at discrete time steps Δt = 10 ms from a Gaussian distribution of mean μ and variance σ2. The task requires the subject to guess the sign of μ, by moving a lever to the right (positive sign) or to the left (negative sign); the subject can ‘choose when to choose’, within a maximum episode duration Tmax. The learning agent integrates the signal over different timescales τ (xs tðtÞs, blue lines); the agent integrates a constant input (depicted in red as a constant from the start of the episode) over the same timescales (xc tðtÞs, yellow-red lines) to simulate an internal clock mechanism estimating the passage of time. In both cases, the darker the colour the longer the corresponding timescale. At each time instance, the weighted sums of the integrators (far right) are fed into a decision layer (the actor) that computes the probability of choosing ‘left’ and ‘right’, thus terminating the episode, or to ‘wait’ to see another sample of s(t). If the subject gives the correct answer (the guessed sign coincides with the actual sign of μ) within the time limit, a reward is delivered; otherwise, nothing happens. In any case, a new episode starts. The agent learns by observing the consequences (obtained rewards) of its actions, adapting the weights assigned to the xs tðtÞs and xc tðtÞs. During learning, the model estimates at each step t the total future expected reward V(t) (the critic) for the current episode as a linear summation of the integrators. Learning of the parameters is accomplished through a standard actor-critic reinforcement learning model, where the reward delivered by the environment is used to update the V value function, which is then used to update the actor’s parameters (see S1 Text for more details) Fig 1. https://doi.org/10.1371/journal.pcbi.1009393.g001 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 2.1 Task definition This second-order a reward is delivered only if the agent correctly guessed the sign of μ; otherwise, the agent receives nothing. Each episode has a maximum duration Tmax. When Tmax is reached, another ‘wait’ from the agent leads to the end of the episode and no reward is delivered. Whilst σ is constant, the value of μ is instead re-sampled at the beginning of each episode from a Gaussian distribution p(μ) of zero mean and variance σμ. This second-order PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 4 / 29 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries uncertainty makes the agent experience a wide range of values of μ, putting severely to the test its ability to generalise to episodes of varying signal-to-noise ratios. 2.2 Relationship between μ and random dots coherence In random dots experiments, usually a number of dots moves randomly on a screen, with a fraction of them moving instead coherently in one direction (either left or right in different episodes). The percentage of coherently moving dots (‘coherence’) is a measure of how diffi- cult an episode is, not unlike \|μ\| in the model (with sign of μ corresponding to a coherent movement towards left or towards right respectively). To make the parallel between the pres- ent task and the experimental settings more evident, in the following we will show results using either \|μ\| or the coherence of the signal, the two measures being related by: jmj ¼ 0:216 coherence ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 100 coherence p : ð1Þ ð1Þ fififififififififififififififififififififififififififififififififi In fact, in [5], every three frames on the screen, a fraction c (‘coherence’) of dots are moved fififififififififififififififififififififififififififififififififi In fact, in [5], every three frames on the screen, a fraction c (‘coherence’) of dots are moved coherently in the chosen direction by dx, while the other 1 −c dots are randomly displaced. We assume that each of the randomly moving dots is subjected to a change Δx in their position following a probability distribution, with hdxi = 0 and Var½dx ¼ s2 x. Imagining that neurons with different receptive fields help to estimate the average movement of the dots at each time step, we end up with a signal s of mean: m hsi ¼ c dx ð2Þ ð2Þ m hsi ¼ c dx and variance: s2 Var½s ¼ ð1 cÞ s2 x ð3Þ ð3Þ Then, we have the relationship: Then, we have the relationship: m s ¼ c ffiffiffiffiffiffiffiffiffiffiffi 1 c p dx sx ð4Þ ð4Þ or: or: m / coherence ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 100 coherence p ; ð5Þ ð5Þ fififififififififififififififififififififififififififififififififi where we have expressed the coherence as a percentage. Eq 1 is a special case of this one, with a proportionality constant chosen to match experimental ranges. 2.3 An agent over multiple timescales The section is dedicated to the definition of the proposed model. In contrast to previous research works on the decision making process, the agent makes decisions thanks to a reser- voir of multiple timescales of integration and an estimate of the passage of time. The agent comprises nτ = 10 leaky integrators xs t (dark blue to cyan lines in Fig 1) that independently integrate the noisy signal s(t) over different timescales τ: _xs t ¼ xs t sðtÞ t ; ð6Þ ð6Þ and correspondingly nτ leaky integrators xc t (yellow to red lines in Fig 1) that integrate a con- stant input (a ‘time signal’, here valued 1), to account for the possible effects of an internal PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 5 / 29 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries ‘clock’: ‘clock’: _xc t ¼ xc t 1 t : ð7Þ ð7Þ Both the xs t and the xc t are reset to 0 at the beginning of each episode (note, therefore, that xc tðtÞ ¼ 1 et t 0 for all t). Moreover, we added noise to the values of the integrators at a given time (Eqs 6 and 7) redefining: Both the xs t and the xc t are reset to 0 at the beginning of each episode (note, therefore, that xc tðtÞ ¼ 1 et t 0 for all t). Moreover, we added noise to the values of the integrators at a given time (Eqs 6 and 7) redefining: xs tðtÞ xs tðtÞ þ xs t ð8Þ xc tðtÞ xc tðtÞ þ xc t ð9Þ xs tðtÞ xs tðtÞ þ xs t ð8Þ ð8Þ ð9Þ xs tðtÞ and xc tðtÞ are drawn independently for each t and each τ from a Gaussian distribution with zero mean and standard deviation σI. The xs tðtÞs and xc tðtÞs are introduced to model the intrinsic noise implied in any plausible biological implementation of the integration process, such as fluctuations in the instantaneous firing rate of a network of neurons. The τs are chosen on a logarithmic scale (i.e., τi = α τi−1, with α a suitable constant), with τ1 = τmin = 100 ms and tnt ¼ tmax ¼ 10 s, so as to allow the agent to accumulate information over a wide range of different timescales. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 2.3 An agent over multiple timescales The specific choice of the distribution of timescales is not critical to the following results, assuming that the values of τs are densely spread over a wide range (see Results and S1 Text). At each time step t, the agent computes six weighted sums, three for the signal xs tðtÞ and three for the clock xc tðtÞ. The first four of these weighted sums are related to the two possible actions: Ss rightðtÞ X t ys right;t xs tðtÞ ð10Þ Ss rightðtÞ X t ys right;t xs tðtÞ ð10Þ Sc rightðtÞ X t yc right;t xc tðtÞ þ bright ð11Þ Ss leftðtÞ X t ys left;t xtðtÞ ð12Þ Sc leftðtÞ X t yc left;t xc tðtÞ þ bleft ð13Þ ð10Þ Sc rightðtÞ X t yc right;t xc tðtÞ þ bright ð11Þ ð11Þ ð12Þ Sc leftðtÞ X t yc left;t xc tðtÞ þ bleft ð13Þ ð13Þ where bright and bleft are constants and can be described as the propensity of the agent to make the corresponding actions before the beginning of an episode. The Sss and the Sc carry infor- mation, respectively, on the signal and the time elapsed since the beginning of each episode. Even though the xc t increase with time, the Scs can be non-monotonic, something that will play an important role in implementing an effective ‘moving threshold’ for the decision mechanism. The other two sums are instead related to the ‘wait’ option: Ss waitðtÞ X t ys wait;t jxs tðtÞj ð14Þ Ss waitðtÞ X t ys wait;t jxs tðtÞj ð14Þ Sc waitðtÞ X t yc wait;t xc tðtÞ þ bwait; ð15Þ Ss waitðtÞ X t ys wait;t jxs tðtÞj ð14Þ ð14Þ Sc waitðtÞ X t yc wait;t xc tðtÞ þ bwait; ð15Þ ð15Þ 6 / 29 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries where the absolute value in Eq 14 is taken to account for the intuition that a signal and its neg- ative mirror should equally affect the agent’s propensity to defer a decision. The constant bwait has similar meaning to the biases bright and bleft, but related to the ‘wait’ action. 2.3 An agent over multiple timescales By setting: Sx Ss x þ Sc x ð16Þ ð16Þ (with x 2 {left, right, wait}), the six sums are then non-linearly combined through a softmax function (the circles corresponding to the actor on the right of Fig 1) to define a probability distribution over the possible actions: prightðtÞ ¼ eSrightðtÞ eSleftðtÞ þ eSwaitðtÞ þ eSrightðtÞ ð17Þ ð17Þ and analogous expressions for ‘left’ and ‘wait’. By definition, pleft(t) + pwait(t) + pright(t) = 1 for every t. The agent then randomly chooses an option according to the three probabilities. The agent is thus completely determined by the choice of the six sets of nτ weights: ys left;t, ys wait;t, ys right;t, yc left;t, yc wait;t, yc right;t, and three constant offsets bleft, bwait, and bright. We note how this set of parameters is redundant, because of the way they enter Eq 17. For example, we could make the substitution bright bright −bwait, bleft bleft −bwait, and bwait = 0 and the resulting agent would be mathematically equivalent to the original one. We use such redun- dant definition in order to simplify the description of the model, making it the most symmetric for ‘left’, ‘right’, and ‘wait’. These weights and offsets are learned by trial-and-error through a reinforcement learning procedure aiming to maximise reward. All the results shown, if not otherwise stated, are obtained using the same set of weights, at the end of the training proce- dure, with Tmax = 2 s, s ¼ 0:18 s1 2, σμ = 0.25, and σI = 0.02. Training of the parameters of the model is achieved through a standard actor-critic reinforcement learning algorithm [43], which is described in S1 Text. During learning, the model estimates at each step t the total future expected reward V(t) for the current episode. Such estimate is computed by a linear summation of the integrators (Fig 1, bottom-right) and is used to establish a moving baseline to modulate the changes in the model’s weights during training. The parameters of the actor and the critic are then updated thanks to the utilisation of eligibility traces [43]. 2.4 Comparative models To understand the role of multiple timescales and of the internal clock in the results, we com- pare the performance of the proposed agent with other decision making models. 1. Single integrator with optimised threshold. This refers to the Ornstein-Uhlenback decision process [24, 28], which is a generalisation of the standard drift diffusion model [21]. The model is composed by an integrator with one timescale and a threshold. The dynamic of the integrator is given by Eqs 6 and 8. A decision is triggered when the latter activity reaches ± a threshold value Θτ. In our case, the action ‘right’ is made when xs tðtÞ Yt, while the agent performs the ‘left’ action when xs tðtÞ Yt. Considering the presence of a single timescale of integration, we will consider multiple versions of the process, each with a different value of τ. For each model with a specific τ, the threshold Θτ will be optimised through grid search by maximising the accuracy on the considered task. In this way, we are certain that the process will exhibit the highest possible performance on the considered task, or performance that are negligibly distant to its theoretical optimal. 2. Agent with a single timescale. The model refers to a reinforcement learning agent similar to the proposed one, but with only one timescale of integration. Practically, the agent defini- tion is again based on Eqs 10–14, but every summation over τ reduces to a single term. The 2. Agent with a single timescale. The model refers to a reinforcement learning agent similar to the proposed one, but with only one timescale of integration. Practically, the agent defini- tion is again based on Eqs 10–14, but every summation over τ reduces to a single term. The PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 7 / 29 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries total number of parameters in this case is thus nine (ys left, ys wait, ys right, yc left, yc wait, yc right, bleft, bwait, and bright). As for the single integrator with optimised threshold, we will simulate mul- tiple versions of the model to vary the timescale of integration τ. We note how, in contrast to the previous comparative model, this process has an estimate of the passage of time over one single time constant. 2.4 Comparative models For this feature, the process departs from the other decision mak- ing models in the literature. This agent will help us to understand the role of multiple time- scales further, providing a baseline where a basic knowledge of the internal clock is present, but where integration occurs over a single τ. 3. Agent with multiple signal integrators, but without internal clock. The model is again defined by Eqs 10–14, but without temporal information, that is yc left ¼ yc wait ¼ yc right 0. The model will constitute an additional comparison to separate the roles of the availability of multiple timescales on the signal and on the internal clock mechanism. Because of the presence of multiple integrators, the proposed agent effectively lowers the total noise by summing up nτ integrators xs t affected by independent sources of noise xs t (Eq 8). Thus, when comparing the proposed agent with one of the above models that exploits a single time constant, we rescaled the amount of noise σI affecting the single integrator by a factor αI, defined as aI ¼ 1 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi P ty2 ;t=max t ðy2 ;tÞ q 1 ð18Þ ð18Þ where y;t refers to the optimal weights θright,τ found after training of the proposed model (we could equivalently use the optimal yleft;t, since after training yleft;t ’ yright;t, as it should be considering the symmetry of the task considered). Thus, αI = 1 when just one of the y;t is dif- ferent from 0, i.e. when the agent utilises just one integrator. On the other hand, the maximum aI ¼ 1ffiffiffi nt p is attained when the agent weights equally all the integrators. In this way, the total amount of noise in the single timescale model is effectively equivalent to the one present in the multiple timescales agent. 3 Results First, we analyse how the behaviour of the optimised agent is different from the standard drift- diffusion model by exploiting integration over a variety of timescales. Fig 2 shows the evolu- tion of pright(t) (blue line) and pleft(t) (red) during an episode where the correct action is ‘right’ (that is, μ > 0). As expected, pright(t) is for the most part greater than pleft(t) (although this is unnoticeable in the plot where the probabilities are very small), signalling that the agent favours the action associated with the correct decision. Nevertheless, both probabilities are very low most of the time, implying that pwait(t) is often close to one (not shown). Thus, the agent appears to select a strategy in which decisions are made within short ‘active’ windows of time during which fleeting bursts of pleft(t) or pright(t) make an action possible. Such strategy is not trivially associated with the intuitive picture of a process accumulating information over time until some threshold is met (for instance, see model 1. in section 2.4). In fact, the agent exploits the information carried by the different integrators by waiting for their consensus, akin to a majority vote. A short-lived fluctuation in the fastest integrators would not be enough for a decision. Yet, in conjunction with a longer-lived fluctuation of the slower integrators, a burst in one of the actions is triggered. Being the consensus fleeting, such probability bursts are usually quite low (they often stay below a probability of 0.1) and Fig 2. Learned decision strategy. Evolution of pright(t) (blue line) and pleft(t) (red) during an episode (signal s(t) in dashed grey) where the correct action is ‘right’ (that is, μ> 0). Decisions are made within short ‘active’ windows of time during which fleeting bursts of pleft(t) or pright(t), corresponding to the alignment of many integrators, make an action possible. The coloured circles correspond to the values of a subset of 5 of the 10 integrators (slow to fast associated timescales from top to bottom). The colours (blue to red) represent the ‘tendency’ of an integrator toward a decision. Blues correspond to positive (toward the ‘right’ action) values, while reds to negative values (toward the ‘left’ action). These tendencies are computed using the average behaviour of the specific integrator as a reference value. 2.5 signal neutrality and scalar property measures To measure signal neutrality, we take the average ΔSright(t) (see Eq 21), aligned to decision time, for six different coherences (0%, 3.2%, 6.4%, 12.8%, 25.6%, 51.2%); each curve is consid- ered for an interval between 0 and 600 ms before the decision is taken; if the number of points to average for a given coherence drops below 100 before the 600 ms, the interval of definition of that curve is shrunk accordingly. We then rescale all the curves to fit inside the range 0–1, so that the minimum of the minimum values attained by each curve is 0; and the maximum of the maxima is 1. Then we compute, for each time, the maximum distance between any pairs of rescaled curves (this distance is of course always 1 thanks to the rescaling). Finally we take the average of such maximum distance, and take the inverse: this is the operative measure of signal neutrality used throughout the paper. To give a measure of scalar property, we compute the coefficient of variation CV for the dis- tribution of response times corresponding to six values of coherence (0%, 3.2%, 6.4%, 12.8%, 25.6%, 51.2%). We then take the inverse of the difference between the maximum and the mini- mum value of CV: this is the reported measure of the scalar property. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 8 / 29 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries 3 Results In other words, if the circle is blue, it means that the value assumed by the integrator was higher than usual at that specific time. Uniformly positive (negative) values for the integrators are associated with bursts of pright (pleft, see times denoted with 1 and 2 in the plot). Outside bursts (point 3) or when a burst withers (point 4), not all the integrators assume low absolute values. https://doi.org/10.1371/journal.pcbi.1009393.g002 Fig 2. Learned decision strategy. Evolution of pright(t) (blue line) and pleft(t) (red) during an episode (signal s(t) in dashed grey) where the correct action is ‘right’ (that is, μ> 0). Decisions are made within short ‘active’ windows of time during which fleeting bursts of pleft(t) or pright(t), corresponding to the alignment of many integrators, make an action possible. The coloured circles correspond to the values of a subset of 5 of the 10 integrators (slow to fast associated timescales from top to bottom). The colours (blue to red) represent the ‘tendency’ of an integrator toward a decision. Blues correspond to positive (toward the ‘right’ action) values, while reds to negative values (toward the ‘left’ action). These tendencies are computed using the average behaviour of the specific integrator as a reference value. In other words, if the circle is blue, it means that the value assumed by the integrator was higher than usual at that specific time. Uniformly positive (negative) values for the integrators are associated with bursts of pright (pleft, see times denoted with 1 and 2 in the plot). Outside bursts (point 3) or when a burst withers (point 4), not all the integrators assume low absolute values. https://doi.org/10.1371/journal.pcbi.1009393.g002 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 9 / 29 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries therefore function as ‘open windows’ paving the way to a decision, more than as ‘funnels’ forc- ing it. Decisions therefore happen when the different timescales stay in agreement for an extended period (roughly 100 ms). This is illustrated in Fig 2 with coloured circles, each row representing the evolution of one integrator (for a subset of 5 of the 10 integrators, with slow to fast timescales from top to bot- tom). As expected, inside a burst of pright(t) almost all the integrators present large positive val- ues (dark blue, see for example temporal instance number 1 in Fig 2). 3 Results On the other hand, integrators typically assume negative values (light to dark red) in correspondence of bursts of pleft(t), as it is shown in the temporal instance number 2. The converse is not true: in absence of probability bursts, not all the integrators assume low absolute values (see, for example, col- oured circles corresponding to number 3). This is due to the fact that the integrators, though correlated, detect fluctuations in the signal over different timescales. Moreover, the non-linear nature of the probability function (Eq 17) dampens integrators’ fluctuations falling below a given range of values. When a burst fades away (see for example points between 2 and 4) not all the integrators go down together. Initially the faster integrators become neutral or even slightly change sign. Afterwards the slower integrators follow suit. Of course, the process is not completely linear, and intermediate integrators can assume (see instance number 4 and neigh- bouring points) higher values, while the slowest (fastest) ones are still decreasing (fluctuating rapidly). A more detailed analysis of the behaviour of the agent can be found in S1 Text and S1 Fig. 3.1 Model’s performance Fig 3A shows the fraction of correct choices as a function of the decision time, both for the agent at the end of training (black line) and for the optimal fixed-t observer (blue line) that, at each time t, simply chooses according to the sign of the sum of the signal up to time t. The lat- ter’s performance can be derived analytically: Fraction CorrectðtÞ ¼ 1 2 þ 1 p arctan ffiffiffiffiffiffiffi s2 m t s2 s ð19Þ ð19Þ If the task were to decide exactly at time t, no other decision maker could outperform it; for this reason it is deemed optimal. The comparison with the fixed-t observer sheds light on the agent’s strategy and the underlying trade-offs. The agent is free to“choose when to choose”, thus it is not surprising that its performance is higher than the optimal fixed-t observer for shorter decision times (the inset of Fig 3A shows the distribution of decision times for the agent). We see that the two performances cross slightly above the average decision time for the agent. Beyond this point, the fixed-t observer dominates. Indeed, the agent can make the easy decisions early on and wait to see how the sig- nal evolves when the choice appears more uncertain. In contrast, the fixed-t observer is bound to decide at time t, no matter how clear or ambiguous the observed signal was up to that point. The steep rise of the agent’s performance for very short decision times is mainly a reflection of its ability to tell apart the easy episodes from the hard ones. The fixed-t observer catches up for longer times, where the agent is left with only the most difficult decisions and its performance consequently declines. For the fixed-t observer, instead, larger ts always mean more informa- tion and therefore its performance monotonically increases. We notice how at the crossing point, the agent has already made the large part of its decisions, as it is apparent from the dis- tribution of decision times. Fig 3B shows how the agent (horizontal line) outperforms all the single integrators with optimised thresholds (circles, see section 2.4 for the model definition). The performance of the Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 10 / 29 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries Fig 3. Performance after training. 3.1 Model’s performance A: Fraction of correct choices as a function of the decision time, both for the agent at end of training (black line) and for optimal fixed-t observer (blue line) that simply chooses according to the sign of the accumulated signal up to time t(see text). The agent clearly outperforms the fixed-t observer for shorter decision times, thanks to its freedom to ‘choose when to choose’. The steep rise of the agent’s performance for very short decision times is mainly a reflection of its ability to tell apart the easy episodes from the hard ones. Inset: response time histograms for correct (grey) and wrong (green) decisions B: the agent (horizontal line) outperforms, considering the fraction of correct choices on a sample of episodes, all the single-timescale integrators with optimised decision threshold (dots; the continuous line is a second-degree polynomial fit for illustration purposes). The performance of the single-timescale integrator peaks for intermediate values of the associated timescale τ, though it always stays below the performance attained by the agent. The grey strip around the agent’s line marks the 25%-75% of the values obtained for the performance upon 100 repetitions of the training procedure (see Fig 5 for further details). C and D: Accuracy and mean response times for different values of coherence (dots). C: The accuracy curve for the agent is in very good agreement with experimental findings: the black line is the result of a fit on experimental data ([5]; see text for more details). D: As accuracy increases, responses become faster, as found in experiments (black line: fit with a sigmoid-like function). https://doi.org/10.1371/journal.pcbi.1009393.g003 Fig 3. Performance after training. A: Fraction of correct choices as a function of the decision time, both for the agent at end of training (black line) and for optimal fixed-t observer (blue line) that simply chooses according to the sign of the accumulated signal up to time t(see text). The agent clearly outperforms the fixed-t observer for shorter decision times, thanks to its freedom to ‘choose when to choose’. The steep rise of the agent’s performance for very short decision times is mainly a reflection of its ability to tell apart the easy episodes from the hard ones. https://doi.org/10.1371/journal.pcbi.1009393.g003 3.1 Model’s performance Inset: response time histograms for correct (grey) and wrong (green) decisions B: the agent (horizontal line) outperforms, considering the fraction of correct choices on a sample of episodes, all the single-timescale integrators with optimised decision threshold (dots; the continuous line is a second-degree polynomial fit for illustration purposes). The performance of the single-timescale integrator peaks for intermediate values of the associated timescale τ, though it always stays below the performance attained by the agent. The grey strip around the agent’s line marks the 25%-75% of the values obtained for the performance upon 100 repetitions of the training procedure (see Fig 5 for further details). C and D: Accuracy and mean response times for different values of coherence (dots). C: The accuracy curve for the agent is in very good agreement with experimental findings: the black line is the result of a fit on experimental data ([5]; see text for more details). D: As accuracy increases, responses become faster, as found in experiments (black line: fit with a sigmoid-like function). https://doi.org/10.1371/journal.pcbi.1009393.g003 single-timescale integrator peaks for intermediate values of the associated timescale τ, though it always stays well below the performance attained by the agent. The agent, therefore, is able to leverage the information on multiple timescales from the signal and the internal clock to gain a clear performance advantage with respect to the drift-diffusion model on the whole single-timescale integrator peaks for intermediate values of the associated timescale τ, though it always stays well below the performance attained by the agent. The agent, therefore, is able to leverage the information on multiple timescales from the signal and the internal clock to gain a clear performance advantage with respect to the drift-diffusion model on the whole PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 11 / 29 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries spectrum of τs. A more detailed comparison between the performance of the different models considered will be given in section 3.5. Fig 3C and 3D show the accuracy and the mean response time of the agent as the coherence of the signal varies (Eq 1). The black line in panel Fig 3C is computed as: Fraction CorrectðcoherenceÞ ¼ 1 1 2 exp coherence 7:97 1:62 " # ð20Þ ð20Þ as in Fig 3 of [5], where the parameters of the curve were fitted to experimental data. 3.2 Signal neutrality A more microscopic look at the decision process surprisingly uncovers shared features between the internal dynamics of the artificial agent and the activity observed in neurons in the lateral intraparietal cortex (LIP) during a random dots task [5, 41]. We now define a key observable of the model that will be central in the following (see Eqs 16, 10 and 11): ð21Þ DSrightðtÞ SrightðtÞ SwaitðtÞ ð21Þ and its ‘left’ counterpart ΔSleft(t) Sleft(t) −Swait(t). Eq 21 (ΔSleft) provides a direct measure of the propensity of the agent to make a ‘right’ (‘left’) decision at time t. Fig 4A shows the evolution of ΔSright, averaged over many episodes in which the agent has made the correct decision ‘right’. The traces are grouped by signal coherence. The left part of Fig 4A shows the evolution of the average ΔSright, with traces aligned to the beginning of the episode (onset of the external signal). ΔSright shows a marked sensitivity to the coherence of the signal. Moreover, the traces do not saturate over several hundreds of milliseconds, highlighting how the agent is making use of its slower integrators. Ramp-like changes in the discharge of LIP neurons have been repeatedly observed, with steeper rise in spike rate for higher stimulus coherence (see, e.g., Figure 7 in [5]). Such ramps, originating in the extrastriate visual cortex in the case of LIP neurons, have been interpreted as a signature of the accumulation of evidence for or against a specific behavioural response [10, 17]. This interpretation is fully compatible with what is seen in the agent. However, when the averages of the ΔSright traces (or of the activity of LIP neurons) are per- formed by aligning the episodes to the time of the decision, a clear signature of signal neutral- ity emerges. The sensitivity to the stimulus’s coherence is lost and all the lines surprisingly collapse on the same curve for several hundreds of milliseconds (Fig 4A, right). We emphasise that such collapse over an extended period of time is key to recognise signal neutrality: any decision model with a deterministic threshold, for example, would display a collapse at deci- sion time (exactly at the threshold), but not necessarily at previous times; in this case, accord- ing to our definition, the model would not display signal neutrality. 3.1 Model’s performance The match between the experimental fit and the result of the agent is striking. In Fig 3D, instead, the black line is a generic sigmoidal function plotted for illustration purposes. As found in the experiments, the agent’s responses become faster as the task becomes easier (larger coherences). as in Fig 3 of [5], where the parameters of the curve were fitted to experimental data. The 3.2 Signal neutrality The similarities with what is found in the discharge of LIP neurons during a motion-discrimination task are striking (see, e.g., Figure 7 in [5]). B: Time course of xs t for a single-timescale integrator with τ = 2s and optimised Fig 4. signal neutrality. ΔSright(t) (see Eq 21) provides a direct measure of the propensity of the agent to make a ‘right’ decision at time t. A Evolution of ΔSright, averaged over many successful episodes with the same signal coherence. On the left, the episodes are aligned to the beginning of the episode and ΔSright shows a marked sensitivity to the coherence of the signal. When the average is performed by aligning all the episodes to the time of the decision (right), signal neutrality clearly appears: the sensitivity to the signal strength is completely lost and all the lines collapse on the same curve for several hundreds of milliseconds. Inset: the same analysis on wrong episodes. The similarities with what is found in the discharge of LIP neurons during a motion-discrimination task are striking (see, e.g., Figure 7 in [5]). B: Time course of xs t for a single-timescale integrator with τ = 2s and optimised decision threshold (xs t, for an integrator with threshold, plays the role that ΔSright has in the agent). C: Time course of ΔSright (see Eq 21 for an agent optimised with a single timescale τ = 2s). In both B and C the collapse of the curves for different signal coherences is imperfect (rightmost part of the plots). But this cannot hold for the agent, where instead signal neutrality arises precisely from the presence of multiple timescales. Fig 4B and 4C show the time course of the equivalent of ΔSright for the models with a single timescale (see Section 2.4). For both these models, we dis- play the results obtained from an example time constants of τ = 2.0 s. In the single integrator with optimised threshold, xs t plays the role that ΔSright has in the agent. In the latter, the collapse of the curves for different signal coherences is not as evident (Fig 4B and 4C, rightmost part). To make this statement more systematic, we introduce an opera- tive measure of signal neutrality. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 3.2 Signal neutrality For the experimental data, a reasonable explanation for such collapse is that the neuronal circuitry is engaged in stereotyped dynamics, independent from the signal, just after a decision is made and before it is manifested with a physical action, perhaps as the result of a feedback from downstream areas. Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 12 / 29 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries Fig 4. signal neutrality. ΔSright(t) (see Eq 21) provides a direct measure of the propensity of the agent to make a ‘right’ decision at time t. A Evolution of ΔSright, averaged over many successful episodes with the same signal coherence. On the left, the episodes are aligned to the beginning of the episode and ΔSright shows a marked sensitivity to the coherence of the signal. When the average is performed by aligning all the episodes to the time of the decision (right), signal neutrality clearly appears: the sensitivity to the signal strength is completely lost and all the lines collapse on the same curve for several hundreds of milliseconds. Inset: the same analysis on wrong episodes. The similarities with what is found in the discharge of LIP neurons during a motion-discrimination task are striking (see, e.g., Figure 7 in [5]). B: Time course of xs t for a single-timescale integrator with τ = 2s and optimised decision threshold (xs t, for an integrator with threshold, plays the role that ΔSright has in the agent). C: Time course of ΔSright (see Eq 21 for an agent optimised with a single timescale τ = 2s). In both B and C the collapse of the curves for different signal coherences is imperfect (rightmost part of the plots). Fig 4. signal neutrality. ΔSright(t) (see Eq 21) provides a direct measure of the propensity of the agent to make a ‘right’ decision at time t. A Evolution of ΔSright, averaged over many successful episodes with the same signal coherence. On the left, the episodes are aligned to the beginning of the episode and ΔSright shows a marked sensitivity to the coherence of the signal. When the average is performed by aligning all the episodes to the time of the decision (right), signal neutrality clearly appears: the sensitivity to the signal strength is completely lost and all the lines collapse on the same curve for several hundreds of milliseconds. Inset: the same analysis on wrong episodes. 3.2 Signal neutrality We computed the inverse of the maximum distance between the curves for different coherences averaged over an interval of up to 600 ms prior to the deci- sion (see Methods). In Fig 5A we report this measure for the agent (horizontal line) and the models with a single timecale (coloured upper bars). The comparative models report lower val- ues in terms of signal neutrality and accuracy (Fig 5B). The propensity of the agent ΔSleft to make the erroneous ‘left’ decision does not display sig- nal neutrality. The same holds true for its experimental counterpart, that is the activity of LIP 13 / 29 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries Fig 5. Comparison of signal neutrality (A) and performance (B) for the single-τ agent and the single-timescale integrator as τ varies. The proposed model (black horizontal lines) shows better accuracy while exhibiting the experimentally observed collapse of the time course of neuronal activity aligned at the decision time. The grey area marks the 25%-75% of the values obtained for each of the two observables upon 100 repetitions of the training procedure; more specifically, each independent training has been halted where signal neutrality peaked, conditioned to having already reached a performance of 0.81 or above; this translates to an average training length of about 73000 episodes (10%-90% range: 39000–110000). https://doi.org/10.1371/journal.pcbi.1009393.g005 Fig 5. Comparison of signal neutrality (A) and performance (B) for the single-τ agent and the single-timescale integrator as τ varies. The proposed model (black horizontal lines) shows better accuracy while exhibiting the experimentally observed collapse of the time course of neuronal activity aligned at the decision time. The grey area marks the 25%-75% of the values obtained for each of the two observables upon 100 repetitions of the training procedure; more specifically, each independent training has been halted where signal neutrality peaked, conditioned to having already reached a performance of 0.81 or above; this translates to an average training length of about 73000 episodes (10%-90% range: 39000–110000). https://doi.org/10.1371/journal.pcbi.1009393.g005 https://doi.org/10.1371/journal.pcbi.1009393.g005 neurons when the random dot motion is away from their receptive field (see Figure 7 in [5], dashed lines). Finally, the comparison between the models in Fig 4 emphasises how in our sim- ulations the signal neutrality is a consequence of the availability of multiple timescales. 3.3 The scalar property The agent’s behaviour conforms to one of the hallmarks of temporal cognition: the scalar property or Weber’s law for interval timing [42]. This is illustrated in Fig 6A, where the distri- butions of response times of the agent are shown for three different values of coherence. As the coherence increases, the average response time of the agent decreases from 4.6 s to 370 ms. Simply stated, the scalar property—as observed for example in interval timing [42], and multistable perception [44]—implies that higher moments of the intervals’ distribution scale as appropriate powers of the mean. This implies a constant coefficient of variation. In other words, the shape of the distribution does not change when its mean varies even over wide ranges. The agent’s behaviour conforms to one of the hallmarks of temporal cognition: the scalar property or Weber’s law for interval timing [42]. This is illustrated in Fig 6A, where the distri- butions of response times of the agent are shown for three different values of coherence. As the coherence increases, the average response time of the agent decreases from 4.6 s to 370 ms. Simply stated, the scalar property—as observed for example in interval timing [42], and multistable perception [44]—implies that higher moments of the intervals’ distribution scale as appropriate powers of the mean. This implies a constant coefficient of variation. In other words, the shape of the distribution does not change when its mean varies even over wide ranges. Notwithstanding a mean value that varies by more than one order of magnitude, the coeffi- cient of variation of the agent moves in a very narrow range which is compatible with the experimental findings [42, 44]. The invariance of the shape of the distribution is made imme- diately evident in the inset of Fig 6A. Here the fitted Gamma distributions (black lines in the main plot) are rescaled to have mean equal to 1. The similarity of the three curves is striking. Fig 6B shows the coefficient of variation CV as the coherence varies for the proposed agent (black) and the comparative models (blue and red colours, see Section 2.4 for more details). The coefficient of variation has an approximately constant value for the proposed agent only. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 3.3 The scalar property We remark that an agent with a single integrator has information regarding the passage of time over a single time constant, and that the model depicted in blue has multiple integrators PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 14 / 29 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries Fig 6. Scalar property. A: The average response time of the agent decreases as the signal coherence increases; still the coefficient of variation of the response times varies in a very narrow range (see legend). The black lines are the best fit of the simulation histograms with a Gamma distribution. Inset: the fitted Gamma distributions are rescaled to have mean equal to 1, making immediately evident how the shape of the distribution stays almost unchanged as its average moves over almost one order of magnitude (colours consistent with the histograms in the main plot). Note how the highest value of coherence is very unlikely under the distribution used for training the agent (corresponding to a value of μ five times the standard deviation σμ of the distribution of μ). The ‘invariant shape’ property of the response time distribution therefore holds well beyond the typical range of functioning of the agent. B: Coefficient of variation (CV) of the different models as the coherence increases. The scalar property is satisfied exclusively by the proposed agent (black line). The single timescale models are reported with two different values of τ. Other choices of τ give comparable results. https://doi.org/10.1371/journal.pcbi.1009393.g006 Fig 6. Scalar property. A: The average response time of the agent decreases as the signal coherence increases; still the coefficient of variation of the response times varies in a very narrow range (see legend). The black lines are the best fit of the simulation histograms with a Gamma distribution. Inset: the fitted Gamma distributions are rescaled to have mean equal to 1, making immediately evident how the shape of the distribution stays almost unchanged as its average moves over almost one order of magnitude (colours consistent with the histograms in the main plot). Note how the highest value of coherence is very unlikely under the distribution used for training the agent (corresponding to a value of μ five times the standard deviation σμ of the distribution of μ). The ‘invariant shape’ property of the response time distribution therefore holds well beyond the typical range of functioning of the agent. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 3.3 The scalar property B: Coefficient of variation (CV) of the different models as the coherence increases. The scalar property is satisfied exclusively by the proposed agent (black line). The single timescale models are reported with two different values of τ. Other choices of τ give comparable results. https://doi.org/10.1371/journal.pcbi.1009393.g006 https://doi.org/10.1371/journal.pcbi.1009393.g006 but lacks any explicit temporal information. Thus, the key ingredient for the scalar property is again the availability of multiple timescales, in particular on the estimate of the passage of time. On the other hand, it is not surprising that the single integrator with optimised threshold is unable to display the scalar property. In fact, for the pure drift-diffusion model (τ = 1), the coefficient of variation as a function of the coherence c can be computed analytically [45] (see also Eq 1): CV ¼ 100 c c2 1=4 ; ð22Þ ð22Þ and it is clearly not constant. and it is clearly not constant. Lastly, we note how the highest values of coherence reported in the plots are very unlikely under the distribution used during the training phase. A coherence of 50% roughly corre- sponds to a value of μ that is five times the standard deviation σμ of the distribution of μ. Thus, the scalar property appears to be a very robust property of the learned decision strategy of the proposed agent, holding well beyond the range of functioning to which the agent has been accustomed during training. In view of the above considerations, signal neutrality and the scalar property share a similar origin. Further evidence of this can be found in the evolution of the two measures during the training phase. Fig 7 shows the average evolution of signal neutrality (black line; the same measure reported in Fig 4D), scalar property (blue line; see Methods for the definition of the metric), and 15 / 29 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries Fig 7. Signal neutrality and scalar property during training. Evolution of signal neutrality (black line), scalar property (blue line), and accuracy (dashed red line, scale on the right) as the training progresses. signal neutrality attains a broad maximum where the performance has almost plateaued. Thus signal neutrality can be interpreted as the signature of a ‘satisficing’ strategy, rather than of an optimal one. The scalar property, on the other hand, keeps growing even for very long training. 3.3 The scalar property Yet, the evolution of signal neutrality and the scalar property are highly correlated, suggesting a common origin for the two (see text for discussion). Fig 7. Signal neutrality and scalar property during training. Evolution of signal neutrality (black line), scalar property (blue line), and accuracy (dashed red line, scale on the right) as the training progresses. signal neutrality attains a broad maximum where the performance has almost plateaued. Thus signal neutrality can be interpreted as the signature of a ‘satisficing’ strategy, rather than of an optimal one. The scalar property, on the other hand, keeps growing even for very long training. Yet, the evolution of signal neutrality and the scalar property are highly correlated, suggesting a common origin for the two (see text for discussion). Fig 7. Signal neutrality and scalar property during training. Evolution of signal neutrality (black line), scalar property (blue line), and accuracy (dashed red line, scale on the right) as the training progresses. signal neutrality attains a broad maximum where the performance has almost plateaued. Thus signal neutrality can be interpreted as the signature of a ‘satisficing’ strategy, rather than of an optimal one. The scalar property, on the other hand, keeps growing even for very long training. Yet, the evolution of signal neutrality and the scalar property are highly correlated, suggesting a common origin for the two (see text for discussion). https://doi.org/10.1371/journal.pcbi.1009393.g007 accuracy (dashed red line, scale on the right y-axis) during training. All the lines are computed by averaging the results of 100 different realisations of the training. The evolution of signal neutrality and the scalar property are highly correlated for much of the training phase, with an initial fast increase that continues up to about 104 −105 episodes, where the accuracy has almost plateaued (the region used for the results of Figs 4A and 6; note how, after the first 105 episodes, the following 9 105 lead to a modest performance gain of ’ 1%). Such correlated progress naturally hints to a common origin for the two measures, and makes us advance the hypothesis that a behavioural policy displaying these two properties could represent an ‘optimal’ information-extraction strategy for dealing with a decision task in a volatile environment. It wouldn’t be by chance that the agent robustly finds such a strategy by tuning its parameters in a ecologically plausible way. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 https://doi.org/10.1371/journal.pcbi.1009393.g007 3.3 The scalar property Yet, after about 105 training episodes, and therefore probably far beyond the experimental training duration, the behaviour of the two curves in Fig 7 starts to diverge. Whilst the scalar property keeps improving, signal neutrality attains a broad peak, after which it gradually breaks down in the face of very modest performance gains. Therefore, the scalar property seems to be more fundamental than signal neutrality, at least for what concerns the strategy asymptotically discovered by the learning agent. In this sense, signal neutrality cannot be viewed per se as signature of an optimal strategy for the agent, but rather of a ‘satisficing’ one [46]. Faced with a wide distribution of coherences, 16 / 29 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries the agent pretty quickly finds a robust strategy that, at around decision time, disregards coher- ence by relying on fluctuations to make decisions, and still ensures a very good performance. Nevertheless, the agent can do slightly better, given enough training time, by giving more weight to the ‘drift’ component and less to the ‘diffusion’ component: this is what happens on the far right of the plot. In this region, we postulate, the learning enters an ‘overfitting’ phase, meaning that the agents become finely attuned to the exact statistics of the task: any slight changes, for example, in the shape of p(μ) would require many training episodes to revert to a good performance. In this sense, the signal neutral strategy generalises better to novel situa- tions. This is something we plan to study elsewhere. Finally, it is tempting to hypothesise that animal subjects, during perceptual decision experiments, display signal neutrality as a reflex of adopting such a satisficing strategy, given also the high number of training episodes the model needs to refine its strategy beyond signal neutrality. 3.4 Collapsing boundaries It is known that in the presence of a distribution of signal-to-noise ratios and limited decision time, as in the task at hand, the drift-diffusion model is not optimal anymore [15]. More spe- cifically, one ingredient that allows to re-establish optimality is a time-varying threshold. As it has been observed in [9] [11], the optimal decision threshold is not constant when the agent has a finite amount of time to make decisions, but is characterised by a non-monotonic trend across time. This optimal moving threshold is defined as collapsing boundaries. In this sense, the hypothesised optimality of the agent’s strategy finds indirect support in the behaviour dis- played by the component of ΔS that depends only on the passage of time and not on the signal. As we will show, this perception of the passage of time, defined in the model as integration of a constant input over multiple timescales, permits the agent to discover the collapsing bound- aries. We rewrite Eq 21 as (see Eqs 10–16): DSright ¼ DSs c DSc ð23Þ ð23Þ where: where: where: DSs right Ss right Ss wait ð24Þ ð24Þ is a term that provides information on the signal only. And: DSc Sc wait Sc right ð25Þ ð25Þ carries information on the passage of time only. We note that on the r.h.s. of Eq 25 we could insert Sc left in place of Sc right with no notable numerical difference in the result. This is because the right and left choices are a priori equivalent in the present task, and therefore the inferred yc right;t and yc left;t are in fact very similar. For this reason ΔSc does not carry a ‘right’ label. ΔSc(t) measures the propensity of the agent at time t to wait for another input instead of making a (either right or left) decision, independently from the signal. Looking back at Eq 23, ΔSc effectively acts as a time-dependent bias term that, in the context of a drift-diffusion model, could be easily interpreted as a time-dependent threshold. Despite the lack of an explicit threshold mechanism for the proposed agent, it is reasonable to expect that the range of values attained by Ss right at decision time shifts in accordance with the time-dependent bias. This is indeed the case. Fig 8A shows (black thick line) the evolution of ΔSc(t) from 0 to Tmax = 2 s for the proposed agent. In addition, three sample trajectories of ΔSs(t) (coloured lines) are shown from t = 0 to decision time (marked by the big coloured circles). The shaded grey area marks the region of 17 / 29 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries Fig 8. Collapsing boundaries. ΔSright (see Eqs 21 and 23) can be decomposed in a signal-dependent part (DSs right) and a time-dependent part (ΔSc; see Eq 25), that measures the propensity of the agent at each time to wait for another input instead of making a decision. In all panels, DSs right (coloured lines) is depicted for three sample episodes, alongside ΔSc (thick black line). The big coloured circles correspond to the decision times. A: The behaviour of the proposed agent. ΔSc acts as a time-dependent threshold: most of the decisions fall inside a strip running parallel to it (the grey area is where 80% of the decisions are made). The resulting boundaries collapse for longer response times. where: Until about 200 ms, a rise of the effective threshold discourages early decisions. This trend is analogous to the theoretically optimal decision threshold when the trial has a maximum allowed time to make a decision [9, 11]. B: The behaviour of an agent with a single integrator (see Section 2.4). Thanks to the internal clock over a single timescale, the agent can implement a suboptimal, monotonically decreasing threshold. C: Behaviour of the agent without internal clock, but with multiple signal integrators. The model is unable to exhibit the collapsing boundaries. Fig 8. Collapsing boundaries. ΔSright (see Eqs 21 and 23) can be decomposed in a signal-dependent part (DSs right) and a time-dependent part (ΔSc; see Eq 25), that measures the propensity of the agent at each time to wait for another input instead of making a decision. In all panels, DSs right (coloured lines) is depicted for three sample episodes, alongside ΔSc (thick black line). The big coloured circles correspond to the decision times. A: The behaviour of the proposed agent. ΔSc acts as a time-dependent threshold: most of the decisions fall inside a strip running parallel to it (the grey area is where 80% of the decisions are made). The resulting boundaries collapse for longer response times. Until about 200 ms, a rise of the effective threshold discourages early decisions. This trend is analogous to the theoretically optimal decision threshold when the trial has a maximum allowed time to make a decision [9, 11]. B: The behaviour of an agent with a single integrator (see Section 2.4). Thanks to the internal clock over a single timescale, the agent can implement a suboptimal, monotonically decreasing threshold. C: Behaviour of the agent without internal clock, but with multiple signal integrators. The model is unable to exhibit the collapsing boundaries. https://doi.org/10.1371/journal.pcbi.1009393.g008 values assumed by Ss right where 80% of the (correct) decisions are made. As expected, this region mostly run parallel to ΔSc(t), demonstrating how the latter observable can be inter- preted as a soft threshold for the decision that arises from the time integrators. Such threshold drops at longer times, a behaviour that finds normative support in the study of perceptual deci- sion making [20, 47]. 3.5 Robustness The utilisation of a wide range of timescales makes the performance of the agent robust to vari- ation of the task and to the intrinsic noise. This is shown in Fig 9A and 9B. We varied Tmax (the maximum duration of an episode) and σI (the standard deviation of the intrinsic noise, xs ts and xc ts in Eqs 10–15) systematically and, for each value, run the learning process from scratch. The results of the agent are then compared to the models of Section 2.4. While Fig 9 reports the comparison with the single integrators with optimised thresholds, the results for the agent with a single timescale can be found in S1 Text and S1 Fig. In Fig 9A, as Tmax increases (and σI stays at its reference point of 0.02), the fraction of cor- rect responses rises monotonically for all models, with the performance of the agent staying superior on the whole range of Tmax explored. Two features are noteworthy. First, the lines for the single integrators (τ = 0.1 s and τ = 10 s respectively) cross at intermediate values of Tmax, with the longer τ surpassing the shorter ones for higher episode durations. Second, the advan- tage of the proposed agent shrinks in comparison to the longer τ for longer Tmax. These fea- tures have a common origin. From Eq 6, a signal s(t) of mean μ will asymptotically lead all the integrators to the same (statistically) stationary value of μ, but with different levels of noise. Integrators with longer τs will have a smaller variance and thus will be more reliable in detect- ing whether μ > 0 or μ < 0. On the other hand, the time needed to reach the stationary state will be longer for larger τs. Slower integrators will still be integrating the signal for shorter Tmax and, as a consequence, their value will carry less information on the μ. Hence, the smaller τs will dominate for shorter Tmax, the larger τ for longer Tmax. In Fig 9B, the level σI of intrinsic noise is varied, with Tmax kept constant at 2 s. The perfor- mance of the agent (black line) is always substantially higher than that of the single integrators (coloured lines). where: Conversely, looking at Eq 23, one can view −ΔSc as an ‘urgency’ signal that pushes for a decision as the episode time elapses, not unlike what has been observed exper- imentally in the lateral intraparietal area [48]. Fig 8B and 8C report the same analysis for an agent with a single timescale (panel B) and an agent with multiple timescales on the signal but without the internal clock (panel C). It is evi- dent how the agent in Fig 8B exploits the unique timescale available for the internal clock to implement a monotonically decaying threshold. In contrast, the agent without internal clock is unable to create such mechanism, considering that the large majority of the decisions occur in PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 18 / 29 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries an area that is parallel to the constant bias bwait −bright. The agent of panel C is unable to clearly infer the passage of time from the multiple timescale of the signal. If this limited behaviour can be surprising at first, it can be understood by considering that the present task is highly vola- tile, with a broad range of signal to noise ratios. Since specific values of the signal integrators xs t can be reached rapidly (slowly) for episodes with high (low) coherences of the signal, such fea- tures do not constitute a reliable estimate of the passage of time. Indeed, the model in panel C fails in the implementation of any form of urgency signal. In this respect we want to point out how the soft threshold ΔSc of the proposed model (panel A) does not simply behave as an urgency signal. In fact the decision is made more and more likely as the time passes only after about 200 ms (when ΔSc reaches a peak). Initially, ear- lier decisions are discouraged by a rise of the threshold. Interestingly, such a non-monotonic trend of the moving threshold has been demonstrated to be theoretically optimal in [9] (see Fig 2B therein; see also [11]). Even if the models in the references and in the present paper are not structurally equivalent, it is nonetheless striking that the agent can approximate such optimal behaviour by trial-and- error. We note how the monotonically decreasing ΔSc shown in panel B is consequently sub- optimal. where: Thus, the results of Fig 8 demonstrate the necessity of multiple timescales also for an efficient implementation of the collapsing boundaries. 3.5 Robustness As expected, performance deteriorates as σI increases from 0 to 0.2; yet the decrease is only surprisingly slight, considering that the maximum value attained by σI is com- parable with the typical dynamical range of the integrators xτ. Such range is determined by the distribution p(μ) (here, a Gaussian of standard deviation σμ = 0.25). It is then clear that the highest levels of intrinsic noise really affect the typical value of the integrators. This is even more true taking into account that the slowest integrators operate far from the asymptotic PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 19 / 29 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries Fig 9. The wide range of timescales makes the agent’s performance robust to variations of the task and to the intrinsic noise. A: as Tmax increases, the fraction of correct responses rises monotonically both for the agent (dashed black line) and for all the single integrators, with the performance of the agent staying superior on the whole range of Tmax explored. B: varying the level σI of intrinsic noise, the performance of the agent (dashed black line) stays always substantially higher than that of the single integrators, notably for stronger noise. As expected, the performance does deteriorate, but the decrease is surprisingly slight, considering that the maximum value attained by σI is comparable with the typical dynamical range of the integrators xτ. The performance of the agent without internal clock (dashed blue in panels A and B) are close (or superior to) the best single integrators reported. However, this agent is more robust than the single integrators over the range of parameters’ values considered. Thus, the results show how the model (dashed blue) is able to select the appropriate timescale for different situations (see text for more details). C: evolution of the ‘moving threshold’ ΔSc (Eq 25) for three values of Tmax. For higher values of Tmax (see also Fig 8), the moving threshold presents a peak whose position shifts with Tmax. D: ys right;t after training (Eq 10) for different values of intrinsic noise σI (continuous lines are fourth degree polynomial fits for illustrative purposes). The peak of the lines, corresponding to the most exploited timescale, shifts towards lower τ values as σI increases. Fig 9. The wide range of timescales makes the agent’s performance robust to variations of the task and to the intrinsic noise. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 3.5 Robustness A: as Tmax increases, the fraction of correct responses rises monotonically both for the agent (dashed black line) and for all the single integrators, with the performance of the agent staying superior on the whole range of Tmax explored. B: varying the level σI of intrinsic noise, the performance of the agent (dashed black line) stays always substantially higher than that of the single integrators, notably for stronger noise. As expected, the performance does deteriorate, but the decrease is surprisingly slight, considering that the maximum value attained by σI is comparable with the typical dynamical range of the integrators xτ. The performance of the agent without internal clock (dashed blue in panels A and B) are close (or superior to) the best single integrators reported. However, this agent is more robust than the single integrators over the range of parameters’ values considered. Thus, the results show how the model (dashed blue) is able to select the appropriate timescale for different situations (see text for more details). C: evolution of the ‘moving threshold’ ΔSc (Eq 25) for three values of Tmax. For higher values of Tmax (see also Fig 8), the moving threshold presents a peak whose position shifts with Tmax. D: ys right;t after training (Eq 10) for different values of intrinsic noise σI (continuous lines are fourth degree polynomial fits for illustrative purposes). The peak of the lines, corresponding to the most exploited timescale, shifts towards lower τ values as σI increases. https://doi.org/10.1371/journal.pcbi.1009393.g009 value, given the limited integration time. This consideration is clearly reflected in the behav- iour of the single integrators. The fast integrators (τ = 0.1 s and τ = 2.1 s) indeed are scarcely affected by the increase in noise. On the other hand, the slowest integrator (τ = 10 s) shows good accuracy for very low levels of noise, but then becomes rapidly ineffective for higher val- ues of σI. The agent without the internal clock reports robust performance as Tmax and σI vary, dem- onstrating its capability to select the appropriate signal integrator for different conditions. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 20 / 29 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries However, this agent can also report lower performance in comparison to the best single inte- grators. This phenomenon is a consequence of the reduction of the noise performed on the model with optimised threshold. 3.5 Robustness Indeed, integration of the signal over a single timescale can carry the large majority of the relevant information for a specific simulation. In a specific parameters’ setting and in terms of accuracy only, the performance of this agent can be conse- quently inferior to the model with a single integrator with an optimal value of τ. However, it is clear how the agent depicted in blue is more robust over the range of values shown in the fig- ure, having the possibility to choose the appropriate integration time. For the sake of accuracy optimisation, the role of the reservoir of integrators is to select the appropriate timescale for the considered situation. Instead, the advantage of an internal clock parameterised by multiple timescales is to implement the optimal shape of collapsing boundaries of Fig 8. The latter state- ment is emphasised by the improved performance of the proposed model also over the agent with a single timescale (S3 Fig), which has information about the passage of time limited over a single τ. Fig 9C shows the evolution of the ‘moving threshold’ ΔSc (Eq 25, proposed agent) for three values of Tmax. For very low Tmax (black line) the threshold only decays, always pushing for a decision. For higher values of Tmax, instead, as we have already seen in Fig 8, the moving threshold initially rises; it reaches a peak and then decays afterwards, making a decision ever more likely. Such peak shifts with Tmax and so does, even more clearly, the time at which the threshold reaches back its initial value (around 1 s for Tmax = 2.0 s, and around 5 seconds for Tmax = 10 s). Fig 9D shows ys right;t after training (ys right;t ’ ys left;t for the symmetry of the problem after optimisation, as it is shown in Fig 10D) for different values of intrinsic noise σI (continuous lines are fourth degree polynomial fits for illustrative purposes). Coherently with what we have seen in Fig 9B, the peak of the lines, corresponding to the most exploited timescale, shifts towards lower τ values as σI increases. 3.6 Evolution during training The initial descending trend (around 100 episodes) of the response times common to all coherences is due to the initial ignorance of the agent about the nature of the task, on the tendency to avoid late decisions and to prefer immediate rewards. C: Probability of not making a decision before the end of the episode, i.e. after Tmax. D-E-F: Evolution of different parameters and of the collapsing boundaries for the training instances corresponding to the vertical grey lines of the top panels. D: Evolution of the weights corresponding to the integrators of the signal. The weights are positive (negative) for the ‘right’ (‘left’) action. E-F: Values of the parameters (E) defining the contribution of the internal clock to the decision making process and relative collapsing boundaries (F). Fig 10. Learning is characterised by a non-monotonic adaptation of the average response time that is consequent to the necessity of finding a fine balance between integrating information and the cost of waiting to make decisions. A: Accuracy of the model for signals with different coherences across learning. B: Average response times. Trials with increasing level of coherences correspond to greater response times and greater probabilities of ‘late’ responses. The initial descending trend (around 100 episodes) of the response times common to all coherences is due to the initial ignorance of the agent about the nature of the task, on the tendency to avoid late decisions and to prefer immediate rewards. C: Probability of not making a decision before the end of the episode, i.e. after Tmax. D-E-F: Evolution of different parameters and of the collapsing boundaries for the training instances corresponding to the vertical grey lines of the top panels. D: Evolution of the weights corresponding to the integrators of the signal. The weights are positive (negative) for the ‘right’ (‘left’) action. E-F: Values of the parameters (E) defining the contribution of the internal clock to the decision making process and relative collapsing boundaries (F). https://doi.org/10.1371/journal.pcbi.1009393.g010 Fig 10D and 10E show the evolution of ys right;t, ys left;t, and of yc wait;t yc right;t respectively (the shape of yc wait;t yc left;t is similar). The different colours (grey scale) and line styles correspond to the training instances highlighted with the vertical lines of the above panels. Each set of weights is separately rescaled, for each instance, by its absolute maximum value. 3.6 Evolution during training Fig 10 illustrates how the behaviour of the agent evolves as it encounters new episodes during learning. Fig 10A shows the performance attained on average for four different values of signal coherence at different times during the training phase. The performance is of course always higher for higher values of coherence (‘easier’ episodes), and tends to increase monotonically for all the values of coherence during training. This monotonic trend is not preserved, instead, looking at the average response time (Fig 10B). The response time drops at the beginning of training with values that are very close for every value of coherence. The reason for such behaviour is related to how the agent is initia- lised. At the beginning, the agent is ignorant about the rules of the task and pre-programmed to make a random choice after having waited for a finite random length of time. Without such random initialisation, the learning would not proceed, since the agent needs to perform actions to learn the relative consequences. While the agent is unable to tell apart signals with different coherences, the response time then decreases. In fact, longer average response times are detrimental due to late responses (no decision before the maximum time allowed Tmax) that are not rewarded. This is made clear in Fig 10B, that shows how the fraction of late responses quickly drops to almost zero, and it stays there. Afterwards, the model starts to statistically differentiate between signals with different coherences (the four lines diverge in Fig 10B) and the response time begins to rise. In this regime, waiting means accumulating more information and helps to improve the performance. 21 / 29 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries Fig 10. Learning is characterised by a non-monotonic adaptation of the average response time that is consequent to the necessity of finding a fine balance between integrating information and the cost of waiting to make decisions. A: Accuracy of the model for signals with different coherences across learning. B: Average response times. Trials with increasing level of coherences correspond to greater response times and greater probabilities of ‘late’ responses. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 4 Discussion Decision making and reinforcement learning are fields with overlapping contributions that attempt to demystify how humans and animals make decisions. Our work unifies the two approaches by using a reinforcement learning agent to solve a task reminiscent of a classical perceptual decision making setup, similar to [10, 49–51]. The reinforcement learning agent receives sensory information and information from vari- ous “clocks” integrated on multiple timescales. Timescales have been implicit in the reinforce- ment learning framework, in the context of propagating information about the success (or failure) of the task in cases where the reward is not immediate, see eligibility traces [43, 52]. However, this is not the same as the concept of timescales in this model, where the emphasis is on acquiring and retaining sensory information from the environment, not unlike what hap- pens in the field of Reservoir Computing [53]. We argue that reward maximisation, multiple time constants and perception of time are the fundamental ingredients for faithfully reproduc- ing (i) an optimal decision-making boundary, (ii) the scalar property, and (iii) signal neutrality. Indeed, the agent learns to solve the task in a relatively small number of episodes, perform- ing better than any single-timescale drift-diffusion integrator while fitting well the psycho- physical data. The agent’s policy is markedly different from the drift-diffusion model, where a decision happens when one of the integrating processes reach the decision threshold. The rein- forcement learning agent makes decisions within short ‘active’ time windows when fleeting bursts in the probability of choosing an action make that action possible. These “bursts” result from the broad agreement on the decision of many integrators with different timescales, akin to the concept of majority voting. The behaviour of our agent is compatible with the analysis performed in [54] on single-neuron single-trial spike trains in LIP area to uncover sudden activity jumps and their informativeness about choice. The multiple clock time constants lead to a decision boundary with a shape similar to the theoretical optimal for decisions with bounded time. We demonstrate in simulations that such a complex boundary is not learnable with a single timescale in the clock or without using a clock. The initial increment and then collapse of the decision boundary happen due to the interplay of clock integrators with multiple time constants. Another direct consequence of the clock with multiple timescales is the scalar property [42, 44], i.e. 3.6 Evolution during training This has been done to emphasise the relative importance among the parameters rather than their magnitude. At the beginning of training (light grey lines) and despite the rescaling, the weights are close to zero because we initialised the biases of the model at higher absolute values. We chose this initialisation so that the model could exhibit reasonable starting response times without weighting the contribution of the different timescales a priori. As the simulation progresses, the weights for the signal integration toward the ‘right’ and ‘left’ actions become stronger while maintaining an approximately symmetric trend with respect to zero (panel D). Also the parameters reflecting the internal clock in panel E grow across training, but fast (slow) timescales become positively (negatively) weighted. Thus, the PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 22 / 29 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries weights at a given instance compose an overall descending trend. To understand this, we need to make two simple observations. First, time integrators receive a constant positive signal of one as input. Thus, the sign of the contribution of a time integrator to the decision process cor- responds to the sign of its relative weight. Second, such weights are the ones responsible for the implementation of the collapsing boundaries (Fig 10F shows the boundaries corresponding to the considered training instances). By giving positive importance to the fast integrators, which are dominant at the beginning of an episode, the agent is implementing the initial rise of the effective, moving threshold (panel F). In contrast, the negative contribution from the slow τs is responsible for the collapsing trends reported in panel F. PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 4 Discussion the ratio of the standard deviation over the mean of the response times remains con- stant. The removal of the clock results in a fixed boundary over time and cannot exhibit the scalar property. Even a single timescale clock, which leads to a non-optimal decaying decision boundary, fails to capture the scalar property. Our results suggest that an optimal decision- making boundary may lead to the scalar property. As a side note, and contrary to [42, 44], the experimental results reported in [55] seem to support a linear relationship between response means and standard deviations (y = ax + b) Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 23 / 29 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries rather than an exact scalar property (y = ax). Yet the very low coefficients of variation for fast responses could result from ignoring the effect of non-decision times, which can be assumed to have low variability [20]. In principle, the drift-diffusion model is capable of exhibiting a lin- ear relationship between the mean response time and its standard deviation [55]. Yet, it seems difficult to display the scalar property (see Eq 22). The multiple timescale signal integrators offer a way to “learn” the integration time con- stant from the data instead of treating it as a free parameter. Their presence makes the agent robust, as it can perform well with various task difficulties (expressed as a signal to noise ratio or signal coherence). There are optimal integration time constants for specific task difficulties. Varying much the signal to noise ratio would inevitably reduce the performance of an integra- tor with a single time constant. A consequence of signal integration with multiple time constants is signal neutrality, the stereotypical collapse of the decision-making signal just before the agent decides. This charac- teristic is noticeable in the activity of neurons in LIP area during a motion-discrimination task [5, 41]. We can intuitively understand this characteristic in the following way. The agent has to find a policy that works across various coherences. A strategy independent of the specific coherences, if achievable, is an appropriate solution to the problem. The simulations suggest the agent discovers such a policy. To some extent, also the single integrator agents find such a policy. However, if we vary the coherence much, signal neutrality progressively fails. Fluctuations play a significant role in signal neutrality. 4 Discussion And indeed, as far as we can discern, the observation of the phenomenon in [17], where no multiple timescales are present, is rooted in the presence of large fluctuations in the activity traces being averaged. These fluctuations are smoothed out by a first-order filter and a random post-decision time. Nonetheless, they contribute significantly to the observed collapse, as testified by peak values well above the deci- sion threshold. In summary, our agent learns solely by maximising its reward. There is no strategy a priori prescribed, similar to a biological agent during a perceptual decision-making experiment. And yet, our model provides little information about the corresponding mechanisms at the circuit level. Nevertheless, it offers insights into complex processes. We argue that it is a good trade- off between complexity and simplicity [56]: the agent learns when to take actions in an “opti- mal” way. The learning process suggests that the optimal decision boundary is a consequence of time perception in multiple timescales. We underline how the proposed agent could be extended to tackle different perceptual decision tasks. For example, being probabilistic, the agent inherently computes an ongoing estimate of the confidence related to each of the possible options. Thus the agent could be pre- sented with the possibility to opt-out from a trial when the choice appears too uncertain. Con- fidence has moreover been related, in the perceptual decision making literature, to optimal learning [47, 57]. It is interesting to note, in this respect, that the learning rate for the proposed agent is indeed strongly modulated by confidence: an easy correct decision would trigger little learning; on the other hand, a confident but wrong decision would engender large changes in the model’s parameters. In [47], moreover, parameters’ fluctuations due to the ongoing learn- ing have been shown to account for differences in psychometric curves in an identification task, not unlike the one examined here, versus a categorisation task, to which the multi-τ agent could be adapted with minor modifications. Since we have largely focused the attention on the post-learning phase, the role of such fluctuations in the agent remains an open, and stimulat- ing, issue. The building blocks of the present model, i.e. the signal accumulators, may have biological counterparts [58, 59]. It is possible to use pools of noisy attractors to implement integrators with wildly different timescales, as required by a multi-scale system. 4 Discussion Attractor dynamics has PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 24 / 29 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries been long one of the main staples of theoretical neuroscience [60]. Several winner-take-all spiking networks capable of implementing a probabilistic classification of the noisy signal have been described in the literature [61, 62]. Therefore we see no conceptual barriers to a more detailed, spiking model mimicking the workings of the agent. Beyond specific interpretations in this work, we would like to advocate the consideration of multiple timescales in models handling non-stationary and noisy information. There is increasing evidence that performance improves or becomes more robust to changes in the environment when various elements are performing nearly the same task. Adapting to differ- ent conditions becomes possible by selectively choosing among those. We notice this general strategy, known as “degeneracy”, is present in many biological systems [63–66]. Degeneracy permits rapid adaptation to novel conditions leading to robust performance, adaptability and survivability. S2 Fig. Surface of accuracy as the number of integrators (x-axis) and maximum timescale (y-axis) vary. For this specific result, the intrinsic noise has not been rescaled for the different models. S2 Fig. Surface of accuracy as the number of integrators (x-axis) and maximum timescale (y-axis) vary. For this specific result, the intrinsic noise has not been rescaled for the different models. S3 Fig. Robustness of the proposed model to different parameters’ settings and compari- son with an agent that exploits a different distribution of characteristic times and an agent with a single integrator (see Section 2.4, Main Text). A-B: The model with a linear distribu- tion of timescales (red, dashed line) reports comparable performance to the one proposed (black, exponential distribution). This demonstrates that the performance of the proposed agent is robust with respect to changes in the distribution of timescales, assuming that the cho- sen distribution has time constants over different orders of magnitudes and that is enough dense to cover the range considered. The performance of the agents with single integrators shows similar trends to the one reported in Fig 9 in the Main Text for the integrators with opti- mised thresholds. Thus, we refer to Fig 9 in the Main Text (Panels A and B) for more detail. (TIF) Supporting information S1 Text. Supplementary information containing three sections. In the first, the actor-critic learning model is described. In the second, we analyse the interpretation of the strategy learned by the agent as a majority vote. In the last, we show how the model maintains high perfor- mance despite changes in the distribution of timescales. (PDF) S1 Fig. The agent waits for an alignment of the different integrators before making a deci- sion. The measures are computed for the episodes where the agent correctly selects the ‘right’ action. A: Fraction of integrators that are positively contributing to the ‘right’ action. The mea- sure is aligned with the decision time (extreme right at zero). When a decision is made, more than nine (out of ten) integrators have a positive contribution to the decision on average. B: Probability of the ‘right’ action as the fraction of positively contributing integrators changes. The probability of making a decision is considerably different than zero when the majority of the integrators align. (TIF) Author Contributions Conceptualization: Luca Manneschi, Guido Gigante, Eleni Vasilaki, Paolo Del Giudice. 25 / 29 PLOS Computational Biology \| https://doi.org/10.1371/journal.pcbi.1009393 August 5, 2022 PLOS COMPUTATIONAL BIOLOGY Signal neutrality, scalar property, and collapsing boundaries Data curation: Luca Manneschi. Data curation: Luca Manneschi. Data curation: Luca Manneschi. Formal analysis: Luca Manneschi, Guido Gigante, Paolo Del Giudice. Funding acquisition: Guido Gigante, Eleni Vasilaki, Paolo Del Giudice. Investigation: Luca Manneschi, Guido Gigante, Eleni Vasilaki, Paolo Del Giudice. Methodology: Luca Manneschi, Guido Gigante, Paolo Del Giudice. Software: Luca Manneschi, Guido Gigante. Supervision: Guido Gigante, Eleni Vasilaki, Paolo Del Giudice. Validation: Luca Manneschi, Guido Gigante. Visualization: Luca Manneschi, Guido Gigante. Writing – original draft: Luca Manneschi, Guido Gigante, Paolo Del Giudice. Writing – review & editing: Luca Manneschi, Guido Gigante, Eleni Vasilaki, Paolo Del Giudice. Formal analysis: Luca Manneschi, Guido Gigante, Paolo Del Giudice. Funding acquisition: Guido Gigante, Eleni Vasilaki, Paolo Del Giudice. Investigation: Luca Manneschi, Guido Gigante, Eleni Vasilaki, Paolo Del Giudice. Methodology: Luca Manneschi, Guido Gigante, Paolo Del Giudice. Software: Luca Manneschi, Guido Gigante. Supervision: Guido Gigante, Eleni Vasilaki, Paolo Del Giudice. Validation: Luca Manneschi, Guido Gigante. Visualization: Luca Manneschi, Guido Gigante. Writing – original draft: Luca Manneschi, Guido Gigante, Paolo Del Giudice. Writing – review & editing: Luca Manneschi, Guido Gigante, Eleni Vasilaki, Paolo Del Giudice. References Mazurek ME, Roitman JD, Ditterich J, Shadlen MN. A role for neural integrators in perceptual decision making. 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https://openalex.org/W3000404596	https://europepmc.org/articles/pmc6945658?pdf=render	English	null	AJCC 8th edition prognostic staging provides no better discriminatory ability in prognosis than anatomical staging in triple negative breast cancer	BMC cancer	2,020	cc-by	6,853	© 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. Abstract Background: We retrospectively compared the prognostic value between the AJCC 8th edition anatomic (AS) and prognostic staging (PS) system for triple negative breast cancer (TNBC) in a cohort from two involved institutions and a large population database. Methods: Clinicopathological data of TNBCs were identified in two involved institutions (SYSUCC-PWH cohort). Data from SEER database during 2010–2015 was also accessed. We restaged all cases into AS and PS group according to the AJCC 8th staging system. Results: A total of 611 and 31,941 TNBCs were identified in two cohorts, with a median follow-up of 53.5 and 27 months respectively. PS upstaged 46.1% of patients in SYSUCC-PWH cohort, and 62.4% in SEER cohort. No significant difference was observed in C index between AS and PS models for disease-specific survival (DSS), progression-free survival (PFS) or overall survival (OS) in either cohort. χ2 statistic and Hazard Ratio for PFS, DSS and OS showed better discrimination between IA and IB, IIB and IIIA, IIIA and IIIB in AS model than PS model. Besides, patients with IIIC unchanged stage showed worse PFS compared to those with AS IIIA or IIIB upstaged to PS IIIC in both cohorts(p = 0.049, p < 0.001). Conclusions: Our findings demonstrated that prognostic staging system did not provide better discriminatory ability in predicting TNBCs prognosis than anatomic staging system. Keywords: Triple negative breast cancer, AJCC 8th, Prognostic stage, Anatomic stage AJCC 8th edition prognostic staging provides no better discriminatory ability in prognosis than anatomical staging in triple negative breast cancer Jiehua He1,2,3†, Julia Y. Tsang4†, Xiaodan Xu5†, Jibin Li2,3,6, Mei Li1,2,3, Xue Chao1,2,3, Yuanyuan X Rongzhen Luo1,2,3, Gary M. Tse4 and Peng Sun1,2,3* Jiehua He1,2,3†, Julia Y. Tsang4†, Xiaodan Xu5†, Jibin Li2,3,6, Mei Li1,2,3, Xue Chao1,2,3, Yuanyuan Xu5, Rongzhen Luo1,2,3, Gary M. Tse4 and Peng Sun1,2,3* He et al. BMC Cancer (2020) 20:18 https://doi.org/10.1186/s12885-019-6494-3 He et al. BMC Cancer (2020) 20:18 https://doi.org/10.1186/s12885-019-6494-3 Open Access Background and extent of tumors (T), Lymph node involvement (N) and the presence or absence of distant metastasis (M). Once the T, N, and M are determined, they are com- bined for assigning the overall anatomic stage (AS) of 0, I, II, III, IV. However, a significant limitation of AS for BCs is that it does not include the biologic factors and may not represent the BC biologic behavior sufficiently. Cancer staging helps clinicians to determine prognosis and design treatment plans for individual patient. Since 1977, the American Joint Committee on Cancer (AJCC) staging system for breast cancer (BC) has assigned stage based on anatomical parameters, including tumor size Identification of BCs molecular subtypes and signifi- cant progress in genomics studies have led to a better understanding of BCs biologic behavior, which provides valuable information for the individualized treatment of BCs. Therefore, AJCC 8th Edition Cancer Staging * Correspondence: sunpeng1@sysucc.org.cn †Jiehua He, Julia Y Tsang and Xiaodan Xu contributed equally to this paper as co-first authors. 1Department of Pathology, Sun Yat-sen University Cancer Center, Guangzhou, China 2State Key Laboratory of Oncology in South China, Guangzhou, China Full list of author information is available at the end of the article © 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. He et al. BMC Cancer (2020) 20:18 He et al. BMC Cancer (2020) 20:18 Page 2 of 9 Page 2 of 9 Manual has updated with a prognostic stage (PS) group for breast cancer by incorporating traditional TNM ana- tomic parameters and additional biologic factors, includ- ing estrogen receptor (ER) and progesterone receptor (PR) status, HER2 status, tumor grade and Recurrence Scores (Oncotype Dx), which are known to have predict- ive and prognostic value [1, 2]. Previous validation stud- ies have demonstrated that AJCC 8th edition PS provided more accurate prognostic information than AS in HR positive and HER2 positive breast cancer [3–6]. However, for triple negative breast cancer (TNBC) pa- tients that lack expression of ER, PR and HER2, whether PS exhibits a better prognostic value than AS in these patients is still debatable [7, 8]. this cohort [11]. Since the HER2 status was not available for cases before 2010, the cases from SEER database was limited to the duration during 2010–2015. “Breast” was selected in side recode ICD-O-3/WHO 2008 category and “Triple Negative” was selected in Breast Subtype (2010+) category. Clinicopathologic data including age at diagnosis, sex, year of diagnosis, primary site, histo- logic type, histologic grade, AJCC 7th edition stage group (T, N, M), survival months, cause-specific death classification, treatment information, such as surgery, chemotherapy and radiotherapy, were collected. Cases with stage IV disease and those with incomplete or un- known clinicopathologic data were excluded. Statistical analysis In this study, we retrospectively compared the prog- nostic value between the AJCC 8th edition AS and PS for TNBCs in a large local cohort (Sun Yat-sen University Cancer Center and Prince of Wales Hospital) and performed a SEER population-based analysis. We aim to examine whether this novel PS system provide more accurate risk stratification in overall survival, disease-specific survival, progression- free survival than the AS system. PS and AS were determined for cases in both SYSUCC- PWH cohort and SEER cohort according to the AJCC 8th edition staging manual. Cases with histologic grade of grade III (poorly differentiated) and IV (Undifferenti- ated; anaplastic) in SEER cohort were both classified as G3 in PS system. Distributions of AS and PS were com- pared in each cohort. We evaluated the number of pa- tients with alterative stage by the PS system. PFS was define as the duration between date of diagnosis to date of disease progression or last contact. DSS was define as the duration between date of diagnosis to date of death because of breast cancer progression or last contact. OS was define as the duration between date of diagnosis to date of death or last contact. PFS and DSS according to AS and PS were compared for SYSUCC-PWH cohort. OS and DSS between the stages were compared for SEER cohort. Identification of SYSUCC-PWH cohort Identification of SYSUCC-PWH cohort Clinicopathologic data, including tumor histologic grade, pathologic T and N categories, and ER, PR, and HER2 status, for patients who underwent surgery as the initial intervention with pathological confirmed invasive breast cancer in Sun Yat-sen University Cancer Center (SYSUCC) during 2005–2013 and Prince of Wales Hospital (PWH) during 2002–2008 were recorded. Patients receiving neoadjuvant therapy, and those with stage IV disease, re-operation for local recurrence,or incomplete or unknown clinicopatho- logic data were excluded. The log-rank test was used to compare differences of survival rates between groups. The Harrell con- cordance index (C index) was calculated using SAS (Version 9.4) to measure the model’s predictive per- formance for the AS and PS models. A higher C index indicates a better predictive performance. Sig- nificance between the C index of AS and PS models was determined using t test. The χ2 statistic of the log-rank test was used to further calculate the dis- crimination between groups. A larger χ2 statistic indi- cates further distance between survival curves, and its P value reflects the statistical significance of this dis- tance. The relationship of AS and PS with DSS, PFS or OS was modeled using a Cox proportional hazards regression model. The results were expressed in haz- ard ratios (95% CIs). Two-tailed p value < 0.05 was considered statistically significant, and IBM SPSS (Version 21.0) was used for analysis. All pathologic specimens were reviewed by experi- enced pathologists to confirm the clinicopathologic characteristics including the tumor size, axillary nodal status, resection margin. ER, PR, and HER2 status were determined according to immunohistochemical (IHC) staining. For patients before 2010, ER and PR status was reclassified as negative using a cut-off of 1% according to the American Society of Clinical On- cology/College of American Pathologists (ASCO/CAP) guidelines [9]. HER2 status was define as negative with 0, 1+ on IHC as well as 2+ on IHC without HER2 gene amplification on fluorescence in situ hybridization (FISH) [10]. Results Identification of SEER cohort The SEER 18 Registry Research Data was accessed through the SEERstat (Version 8.3.5) to extract cases in A total of 611 patients with stage I to IIIC TNBC were identified in the SYSUCC-PWH cohort, of which 427 He et al. BMC Cancer (2020) 20:18 Page 3 of 9 and 184 patients were from Sun Yat-sen University Can- cer Center (SYSUCC) and Prince of Wales Hospital (PWH) respectively. All patients were assigned a PS. Compared to AS, PS upstaged 282 patients (46.1%) and, unchanged stage was in 329 patients (53.9%). No patient showed downstaged PS in this cohort. For those with upstaged PS, 232 patients (232/282, 82.3%) showed one stage up (including IA to IB, IIB to IIIA, IIIA to IIIB and IIIB to IIIC) and 50 patients (50/282, 17.7%) showed two stages up (IIIA to IIIC). accurate model for either DSS (p = 0.943) or PFS (p = 0.887) than AS model. Similar outcomes were observed in SEER cohort, the C index for AS was 0.86 (95% CI 0.81–0.89) in DSS and 0.90 (95% CI 0.87–0.92) in OS. The C index for PS was 0.85 (95% CI 0.81–0.89) in DSS and 0.90 (95% CI 0.87–0.92) in OS. No significant difference of C index between AS and PS models again in either DSS (p = 0.95) or OS (p = 0.98). and 184 patients were from Sun Yat-sen University Can- cer Center (SYSUCC) and Prince of Wales Hospital (PWH) respectively. All patients were assigned a PS. Compared to AS, PS upstaged 282 patients (46.1%) and, unchanged stage was in 329 patients (53.9%). No patient showed downstaged PS in this cohort. For those with upstaged PS, 232 patients (232/282, 82.3%) showed one stage up (including IA to IB, IIB to IIIA, IIIA to IIIB and IIIB to IIIC) and 50 patients (50/282, 17.7%) showed two stages up (IIIA to IIIC). The χ2 statistic was applied to evaluate the discrimin- atory power between different stages among AS and PS in both cohorts. In the SYSUCC-PWH cohort, χ2 on PFS between IIA and IIB for AS was 10.36 (p = 0.0013) and that for PS was similar, showing a χ2 of 10.05 (p = 0.0015). Comparing between IIB and IIIA, χ2 on PFS was 5.765 (P = 0.0163) for AS while that for PS was 0.6587 (P = 0.417). Results Similarly, between IIIA and IIIB, AS model also displayed a larger χ2 statistic than PS on DSS (χ2=4.204, P = 0.0403 vs χ2=1.239, P = 0.2656). χ2 statistic and P value on pair-wise stage comparison of the cohort are shown in Table 3. A total of 31,941 patients with stage I to IIIC TNBC were identified in the SEER cohort. Compared to AS, PS upstaged 19,924 patients (62.4%), and unchanged stage was in 12,009 patients (37.6%). Eight patient showed downstaged PS (IIIC to IIIB) in this cohort. For those with upstaged PS, 17771 out of 19,924 patients (89.2%) showed one stage up (including IA to IB, IIB to IIIA, IIIA to IIIB and IIIB to IIIC) and 2153 patients (10.8%) showed two stages up (IIIA to IIIC). Clinicopathologic characteristics and staging alterations for patients in both cohorts are listed in Additional file 3: Tables S1 and S2. In the SEER cohort, compared to PS model, χ2 stat- istic on DSS of AS model showed a larger and statis- tically significant difference between IA and IB (24.94, P < 0.0001 vs 1.272, P = 0.2593), IIA and IIB (137.6, P < 0.0001 vs 23.93, P < 0.0001), IIB and IIIA (98.42, P < 0.0001 vs 0.249, P = 0.6178) as well as IIIA and IIIB (97.38 P < 0.0001 vs 9.91, P = 0.0016). However, PS model displayed larger χ2 statistic on DSS than AS model between IB and IIA (0.0045, P = 0.9461 vs 220, P < 0.0001) as well as IIIB and IIIC (0.68, P = 0.4088 vs 21.1, P < 0.0001). Similar results were also observed on the discrimination between stages for OS. χ2 stat- istic and P value on pair-wise stage comparison of the cohort are shown in Table 4. Survival analyses were conducted in both cohorts and survival curves are shown in Fig. 1. The median follow- up was 53.5 months for the SYSUCC-PWH cohort and 27 months for the SEER cohort. Five-year DSS and PFS for SYSUCC-PWH cohort by AS and PS are summa- rized in Table 1. Five-year DSS and OS for SEER cohort by AS and PS are summarized in Table 2. In SYSUCC- PWH cohort, the C index for AS was 0.83 (95% CI 0.63–0.98) in DSS and 0.80 (95% CI 0.59–0.96) in PFS. Results at risk 5-year DSS (95% CI) p-value AS I(A) 100 86.5(76.2,92.5) < 0.001 100 92.9(83.4,97.0) 0.001 II 371 82.0(77.0,86.0) 0.001# 371 90.6(86.3,93.6) 0.080# IIA 240 87.9(82.4,91.9) 240 92.7(87.7,95.7) IIB 131 71.0(60.9,79.0) 131 86.2(76.8,92.0) III 140 50.9(41.1,59.8) 0.045△ 140 74.1(64.2,81.7) 0.099△ IIIA 78 56.6(43.7,67.6) 78 80.4(67.8,88.5) IIIB 29 55.2(28.6,75.4) 29 51.4(23.1,73.9) IIIC 33 34.9(18.3,52.2) 33 70.4(46.9,85.0) PS I 100 86.5(76.2,92.5) < 0.001 100 92.9(83.4,97.0) < 0.001 IA 3 NA NA 3 NA NA IB 97 86.0(75.4,92.3) 97 92.7(88.5,97.0) II 291 83.9(78.3,88.2) 0.002# 291 91.9(87.2,94.9) 0.209# IIA 240 87.9(82.4,91.9) 240 92.7(89.7,95.7) IIB 51 64.1(46.1,77.5) 51 87.3(79.4,95.1) III 220 60.1(52.3,66.9) < 0.001△ 220 78.2(70.7,83.9) 0.023△ IIIA 83 74.7(62.2,83.6) 83 85.5(78.7,92.3) IIIB 26 73.7(50.4,87.3) 26 83.5(72.5,94.5) IIIC 111 44.9(34.0,55.1) 111 70.7(61.8,79.7) *Log-rank test comparing proportions among all stage; #Log-rank test comparing proportions among Stage II; △Log-rank test comparing proportions among Stage III; No case was classified as anatomic stage IB in this cohort TNBC triple negative breast cancer, AS anatomic stage, PS prognosis stage, PFS progression-free survival, DSS disease specific survival, CI confidence interval Table 1 5-year Progression-Free Survival and Disease-Specific Survival by Anatomic and Prognosis Stage of TNBCs included in Table 1 5-year Progression-Free Survival and Disease-Specific Survival by Anatomic and Prognosis Stage of TNBCs included in SYSUCC-PWH cohort (N = 611) Table 1 5-year Progression-Free Survival and Disease-Specific Survival by Anatomic and Prognosis Stage of TNBCs included in *Log-rank test comparing proportions among all stage; #Log-rank test comparing proportions among Stage II; △Log-rank test comparing proportions among Stage III; No case was classified as anatomic stage IB in this cohort TNBC triple negative breast cancer, AS anatomic stage, PS prognosis stage, PFS progression-free survival, DSS disease specific survival, CI confidence interval Furthermore, we also compare survival data between patients with unchanged stage and patients with upstaged PS. In SYSUCC-PWH cohort, patients with IIIC unchanged stage showed worse survival outcome than those patients with AS IIIA or IIIB upstaged to PS IIIC in PFS (p = 0.049, Fig. 2b), but not in DSS (p = 0.515, Fig. 2a). Similarly, in SEER cohort, patients with IIIC unchanged stage showed worse survival outcome in both OS and DSS when compared to patients with AS IIIA or IIIB upstaged to PS IIIC (p < 0.001, Fig. 2e-f). Besides, patients with IIA un- changed stage also showed worse OS than patients with AS IA or IB upstaged to PS IIA (p = 0.0083, Fig. 2c). Results On the other hand, we found no significant difference in either DSS, PFS or OS between patients with IIB unchanged stage and those with AS IIB upstaged to PS IIIA in both cohorts. Survival curve and p value are shown in Additional file 1: Figure S1 and Additional file 2: Figure S2. and HER2 status, as well as genomic assays, which are generally accepted as important predictive and prognos- tic factors in clinical practice [9–14]. This mean that prognostic stage is more likely to reflect individual prog- nosis when patients receive the recommended treatment strategies [1, 2, 6]. Previous studies have demonstrated that the PS model provided a better prognosis value than the AS model in individuals with breast cancer [3, 4, 6, 15], including the subgroup analysis for ER positive [5] and HER2 positive BCs [16]. This may due to the se- lection of genomic low risk patients and the improved effectiveness of endocrine therapy and anti-HER2 therapy. When looking into patients with TNBC, who predominantly receive chemotherapy, are more likely to have mid-high nuclear grade and worse prognosis for overall survival and disease-free survival [17, 18]. It is not surprising that, 46.1 and 62.4% TNBC pa- tients were upstaged from AS to PS model in our two study cohorts. The upstaged rate in TNBC is greater than that reported for all breast cancer (6.8– 34.7% approximately) [3, 4, 6, 15]. On the other hand, only 8 out of 31,941 patients with TNBC in SEER co- hort were downstaged from AS IIIC to PS IIIB be- cause of the low nuclear grade (grade 1). Compared to the reported downstaged rate of 23.4–35.6% ap- proximately in overall breast cancer [3, 4, 6, 15], A Cox proportional hazards regression model was then used to look into the Hazard Ratio (HR) for DSS, PFS or OS by stage in both cohorts. The hazard ratios for anatomic and prognostic stages in both cohorts are summarized in Additional file 3: Tables S2, S3 and S4. Results The C index for PS was 0.84 (95% CI 0.63–0.98) in DSS and 0.82 (95% CI 0.62–0.97) in PFS (Fig. 1). No signifi- cant difference of C index between AS and PS models, reflecting that PS model may not be a more predictive Fig. 1 Kaplan-Meier curves of DSS and PFS for the SYSUCC-PWH cohort in anatomical staging system (a-b) and prognostic staging system (c-d). Kaplan-Meier curves of OS and DSS for the SEER cohort in anatomical staging system (e-f) and prognostic staging system (g-h) Fig. 1 Kaplan-Meier curves of DSS and PFS for the SYSUCC-PWH cohort in anatomical staging system (a-b) and prognostic staging system (c-d). Kaplan-Meier curves of OS and DSS for the SEER cohort in anatomical staging system (e-f) and prognostic staging system (g-h) He et al. BMC Cancer (2020) 20:18 Page 4 of 9 Table 1 5-year Progression-Free Survival and Disease-Specific Survival by Anatomic and Prognosis Stage of TNBCs included in SYSUCC-PWH cohort (N = 611) Stage No. at risk 5-year PFS (95% CI) p-value No. Discussion The greatest change of AJCC 8th edition cancer staging system is the incorporation of tumor grade, and ER, PR, Page 5 of 9 He et al. BMC Cancer (2020) 20:18 Table 2 5-year Overall Survival and Disease-Specific Survival by Anatomic and Prognosis Stage of TNBCs included in SEER cohort (N = 31,941) Stage No. at risk 5-year OS (95% CI) p-value No. at risk 5-year DSS (95% CI) p-value AS I 12,700 87.2(86.2,88.0) < 0.001 12,700 93.5(82.8,94.2) < 0.001 IA 12,293 87.4(86.5,88.3) 0.004* 12,293 93.8(93.1,94.4) 0.001* IB 407 80.4(73.9,85.5) 407 86.2(80.6,90.2) II 14,292 75.3(74.2,76.4) < 0.001# 14,292 82.7(81.8,83.7) < 0.001# IIA 9713 78.5(77.2,79.7) 9713 86.0(84.9,87.0) IIB 4579 68.3(66.1,70.4) 4579 75.5(73.5,77.4) III 4949 46.8(44.8,48.7) < 0.001△ 4949 54.0(52.0,56.0) < 0.001△ IIIA 2515 57.1(54.3,59.7) 2515 63.6(60.8,66.2) IIIB 1164 38.5(34.5,42.5) 1164 47.0(42.6,51.2) IIIC 1270 34.3(30.5,38.2) 1270 41.4(37.3,45.2) PS I 12,293 87.4(86.5,88.3) < 0.001 12,293 93.8(93.1,94.4) < 0.001 IA 407 89.4(83.6,93.2) 0.212* 407 94.6(89.2,97.3) 0.259* IB 11,886 87.4(86.5,88.3) 11,886 93.8(93.1,94.4) II 10,726 77.9(76.7,79.1) < 0.001# 10,726 85.5(84.4,86.5) < 0.001# IIA 10,120 78.6(77.3,79.8) 10,120 86.0(85.0,87.0) IIB 606 67.4(61.2,72.9) 606 75.4(69.3,80.4) III 8922 56.3(54.8,57.8) < 0.001△ 8922 63.5(62.0,65.0) < 0.001△ IIIA 3986 68.7(66.4,70.8) 3986 75.6(73.4,77.6) IIIB 365 56.3(47.8,64.0) 365 62.1(53.0,70.0) IIIC 4571 45.9(43.9,48.0) 4571 53.3(51.2,55.4) *Log-rank test comparing proportions among all stage;Log-rank test comparing proportions among Stage I; #Log-rank test comparing proportions among Stage II; △Log-rank test comparing proportions among Stage III TNBC triple negative breast cancer, AS anatomic stage, PS prognosis stage, OS overall survival, DSS disease specific survival, CI confidence interval Table 2 5-year Overall Survival and Disease-Specific Survival by Anatomic and Prognosis Stage of TNBCs included in SEER cohort (N = 31,941) Table 2 5-year Overall Survival and Disease-Specific Survival by Anatomic and Prognosis Stage of TNBCs included in SEER cohort rarely patients with TNBC were downstaged in PS model. the AJCC 8th Edition Updates and Corrections, that N1mi disease in patients with T2, T3 and T4 cancers in- cludes N1mi. In the present study, all TNBCs were per- fectly assigned to a proper prognostic stage according to the criterion. However, the results of present study in both TNBC cohorts do not show better prognostic value for PS model compared to the traditional AS model. PS model showed better discrimination for both OS and Weiss, et al. g TNBC triple negative breast cancer, AS anatomic stage, PS prognosis stage aNo case was classified as anatomic stage IB in this cohort BC triple negative breast cancer, AS anatomic stage, PS prognosis stage o case was classified as anatomic stage IB in this cohort Discussion [6] found that 13.6% BC patients could not be assigned to a prognostic stage due to the pres- ence of N1mi disease in patientsnwith tumors larger than T1 or uncategorized combinations of T and N cat- egories with grade and HR and HER2 status. Some sub- sequent changes had been made and demonstraded in Table 3 The χ2 statistic on Disease-Specific Survival and Progression-Free Survival for Anatomic and Prognostic Stage of TNBCs included in SYSUCC-PWH cohort (N = 611) Stage Disease-Specific Survival Progression-Free Survival AS PS AS PS χ2 p-value χ2 p-value χ2 p-value χ2 p-value Ia vs IIA 0.5876 0.4433 0.5876 0.4433 0.006753 0.9345 0.0068 0.9345 IIA vs IIB 3.214 0.073 1.572 0.2099 10.36 0.0013 10.05 0.0015 IIB vs IIIA 2.16 0.1417 0.0016 0.9681 5.765 0.0163 0.6587 0.417 IIIA vs IIIB 4.204 0.0403 1.239 0.2656 0.5332 0.4653 0.7811 0.3768 IIIB vs IIIC 0.2007 0.6541 0.7913 0.3737 1.922 0.1656 2.294 0.1299 aNo case was classified as anatomic stage IB in this cohort TNBC triple negative breast cancer, AS anatomic stage, PS prognosis stage isease-Specific Survival and Progression-Free Survival for Anatomic and Prognostic Stage of TNBCs hort (N = 611) Page 6 of 9 He et al. Discussion As a retrospective study, we enrolled TNBCs diagnosed at the time during 2005–2015 (SYSUCC, 2005–2013; PWH, 2002–2008; SEER, 2010–2015), when PARPi and immunotherapy were not widely ap- proved for clinical practice and the predominant treatment of TNBC remains to be chemotherapy and radiotherapy, especially in Asian countries. However, subsequent clinical trials, including EMBRACA [21] Trial, OlympiAD [22] Trial, PrECOG 0105 Trial [22] and IMpassion130 Trial [26], suggested the significant effects of novel treatment modalities especially for ad- vanced TNBCs. Another limitation of the present study is DSS between IB and IIA as well as IIIB and IIIC. How- ever, worse discrimination between IA and IB as well as IIB and IIIA were found in PS compared to AS model. Those upstaged from AS (IIIA/IIIB to IIIIC) showed a significant worse survival than those with unchanged PS (IIIC). Similarly, those upstaged to a higher PS (e.g. IIB to IIIA) did not show significant worse survival than PS stage equivalent to their original AS (IIB unchanged). Our find- ings demonstrated that the new prognostic staging system do not provide better discriminatory ability in predicting TNBCs prognosis than anatomic staging system. Upstaged TNBCs by PS in the present study are mostly from AS IA, IIB, IIIA and IIIB, however, we no- ticed that those upstaged cases do not demonstrate the relevant worse survival outcomes. These findings are consistent with previous study by Liu, et al. [8]. On the contrary, contradictory studies by Li, et al. [7] and Luo, et al. [19] indicated that the PS system displayed a more optimistic prognostic stratification and predictability than traditional AS system. However, Li, et al. [7] ap- plied a earlier version of AJCC 8th criterion without subsequent corrections in a small sample cohort includ- ing stage IV disease. They also excluded special types of invasive breast cancer, and no relevant statistical methods had been applied to further assess and compare prognostic ability of the two staging systems. Luo, et al. [19] used the goodness-of-fit test, included statistics as −2likeli- hood, AIC, and BIC, to describe the prediction capability of the two competing staging systems in TNBCs and found that new version of AJCC staging system were higher than before. However, they also mentioned that statistics such as AIC and BIC are not convertible to a clinical meaningful relevance. Discussion BMC Cancer (2020) 20:18 Table 4 The χ2 statistic on Disease-Specific Survival and Overall Survival for Anatomic and Prognostic Stage of TNBCs included in Table 4 The χ2 statistic on Disease-Specific Survival and Overall Survival for Anatomic and Prognostic Stage of TNBCs included in SEER h (N 31 941) Table 4 The χ2 statistic on Disease-Specific Survival and Overall Survival for Anatomic and Prognostic Stage of TNBCs included in SEER cohort (N = 31,941) Table 4 The χ2 statistic on Disease-Specific Survival and Overall Survival for Anatomic and Prognostic Stage of TNBCs included in SEER cohort (N = 31,941) Stage Disease-Specific Survival Overall Survival AS PS AS PS χ2 p-value χ2 p-value χ2 p-value χ2 p-value IA vs IB 24.94 < 0.0001 1.272 0.2593 8.254 0.0041 1.561 0.2115 IB vs IIA 0.0045 0.9461 220 < 0.0001 1.247 0.2641 183.3 < 0.0001 IIA vsIIB 137.6 < 0.0001 23.93 < 0.0001 109.5 < 0.0001 22.64 < 0.0001 IIB vs IIIA 98.42 < 0.0001 0.249 0.6178 92.07 < 0.0001 0.005236 0.9423 IIIA vs IIIB 97.38 < 0.0001 9.91 0.0016 128.1 < 0.0001 11 0.0009 IIIB vs IIIC 0.6823 0.4088 21.1 < 0.0001 0.0601 0.8063 21.44 < 0.0001 TNBC triple negative breast cancer, AS anatomic stage, PS prognosis stage about the overtreatments for TNBCs. However, PS could not accurately stratify risk in TNBC, thus fur- ther updates should be required. Additional morpho- logical and genomic information with prognostic significance in TNBCs have been demonstrated. Up- coming studies should consider the incorporation of biologic factors that closely related to the develop- ment of novel clinical therapies in TNBCs, for ex- ample, Poly-ADP-ribose polymerase inhibitors (PARPi) including Olaparib or Talazoparib and plat- inum therapy may provided a significant benefit over standard chemotherapy with respect to progression- free survival for metastatic TNBCs with germline BRCA1/2 mutation [21–23]; Presence and increasing percentage of stromal tumor infiltrating lymphocytes (sTILs) [24, 25] are associated with better response to anthracycline-based neoadjuvant chemotherapy and improved long-term survival in TNBCs; Atezolizumab plus nab-paclitaxel prolonged progression-free survival among patients with metastatic triple-negative breast cancer in PD-L1-positive subgroup [26]. Moreover, activation PI3K/AKT signaling [27], AR expression [28], histopathological and molecular subtypes [29, 30] should also be considered for the modification of the prognostic staging system in TNBCs. One limita- tion of the present study is that we cannot acquire data regarding administration of PARPi and immuno- therapy. Discussion The calculation of the C-index at different time points did not show significant differences in the two com- peting stage systems, which is in line with our observations. The mainstay treatment of TNBC remains to be chemotherapy. Despite the upstages in PS, the clinical treatment decision will unlikely be changed for the time being [6, 20]. So it is unnecessary to worry Page 7 of 9 He et al. BMC Cancer He et al. BMC Cancer (2020) 20:18 (2020) 20:18 Fig. 2 Kaplan-Meier curves of DSS (a) and PFS (b) for the SYSUCC-PWH cohort between patients with IIIC unchanged and those with anatomical stage IIIA/IIIB upstaged to prognostic stage IIIC. Kaplan-Meier curves of OS (c) and DSS (d) for the SEER cohort between patients with IIA unchanged and those with anatomical stage IA/IB upstaged to prognostic stage IIA; Kaplan-Meier curves of OS (e) and DSS (f) for the SEER cohort between patients with IIIC unchanged and those with anatomical stage IIIA/IIIB upstaged to prognostic stage IIIC Fig. 2 Kaplan-Meier curves of DSS (a) and PFS (b) for the SYSUCC-PWH cohort between patients with IIIC unchanged and those with anatomical stage IIIA/IIIB upstaged to prognostic stage IIIC. Kaplan-Meier curves of OS (c) and DSS (d) for the SEER cohort between patients with IIA unchanged and those with anatomical stage IA/IB upstaged to prognostic stage IIA; Kaplan-Meier curves of OS (e) and DSS (f) for the SEER cohort between patients with IIIC unchanged and those with anatomical stage IIIA/IIIB upstaged to prognostic stage IIIC the unchanged PS IB following the AJCC 8th criteria, which may not affect outcomes of the comparative study. the unchanged PS IB following the AJCC 8th criteria, which may not affect outcomes of the comparative study. that no case was classified as anatomic stage IB in SYSUCC-PWH cohort. In SEER cohort, we found that few cases of TNBC (1.3%) were classified as AS IB (T0NmiM0 and T1N1miM0). So it can be happened that no AS IB case was found in the SYSUCC-PWH cohort with a smaller sample size. This may also due to the reluc- tance for pathologists to make the diagnosis of Nmi at the time of initial diagnosis. However, all the AS IB cases, re- gardless of the variety in histologic grade, are classified as Supplementary information Guangzhou, China. 4Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China. 5Department of Physiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China. 6Department of Clinical Research, Sun Yat-sen University Cancer Center, Guangzhou, China. Supplementary information accompanies this paper at https://doi.org/10. 1186/s12885-019-6494-3. Additional file 1: Figure S1. Kaplan-Meier curves of DSS (A) and PFS (B) for the SYSUCC-PWH cohort between patients with IIB unchanged and those with anatomical stage IIB upstaged to prognostic stage IIIA. Funding Th d 10. Wolff AC, Hammond MEH, Allison KH, Harvey BE, Mangu PB, et al. Human epidermal growth factor receptor 2 testing in breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Focused Update. J Clin Oncol. 2018;36(20):2105–22. This study was funded by the Guangdong Medical Research Foundation (A2018241) and National Natural Science Foundation of China (81902679) in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. 11. Surveillance, epidemiology and end results program. about the SEER program. http://seer.cancer.gov/about. Accessed 10 Oct 2016. program. http://seer.cancer.gov/about. Accessed 10 Oct Acknowledgements 8. Liu YY, Yu TJ, Liu GY. The predictive value of the prognostic staging system in the 8th edition of the American joint committee on Cancer for triple- negative breast cancer: a SEER population-based analysis. Future Oncol. 2019;15(4):391–400. Received: 9 October 2019 Accepted: 23 December 2019 Received: 9 October 2019 Accepted: 23 December 2019 Additional file 2: Figure S2. Kaplan-Meier curves of OS (A) and DSS (B) for the SEER cohort between patients with IA unchanged and those with anatomical stage IA upstaged to prognostic stage IB; Kaplan-Meier curves of OS (C) and DSS (D) for the SEER cohort between patients with IIB un- changed and those with anatomical stage IIB upstaged to prognostic stage IIIA. Availability of data and materials 12. National Comprehensive Cancer Network. Clinical Practice Guidelines in Oncology: breast. Clinical Practice Guidelines in Oncology: breast. https:// www.nccn.org/professionals/physician_gls/pdf/breast.pdf. Published 2019. Accessed 5 Apr 2019. The SEER-database is publicly available. The datasets generated and/or ana- lysed during the current study are also available from the corresponding au- thor on reasonable request. 13. Curigliano G, Burstein HJ, Winer EP, Gnant M, Dubsky P, et al. De-escalating and escalating treatments for early-stage breast cancer: the St. Gallen International Expert Consensus Conference on the Primary Therapy of Early Breast Cancer 2017. Ann Oncol. 2018;29(10):2153. Competing interests 16. Zhou B, Xu L, Ye J, Xin L, Duan X, et al. The prognostic value of the 8th edition of the American joint committee on Cancer (AJCC) staging system in HER2-enriched subtype breast Cancer, a retrospective analysis. Anticancer Res. 2017;37(8):4615–21. The authors declare that they have no competing interests. Author details 1Department of Pathology, Sun Yat-sen University Cancer Center, Guangzhou, China. 2State Key Laboratory of Oncology in South China, Guangzhou, China. 3Collaborative Innovation Center for Cancer Medicine, Authors’ contributions Conceptualisation: PS, JH, JYT, GMT; Data acquisition: XX, ML, XC, YX, RL; Methodology: PS, JH, JYT, JL,XX, GMT; Data analysis: PS, JYT, XX, JL,ML; Writing original draft and editing: JH, PS, JYT, XX, GMT, JH, JL; Data curation: PS, JYT, XX, GMT, JH; Project administration: PS, GMT, JH; Funding acquisition: PS. All authors read and approved the final manuscript. 9. Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, et al. American Society of Clinical Oncology/college of American pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Clin Oncol. 2010;28(16):2784–95. References 1. Hortobagyi GN, Connolly JL, Edge SB, et al. Breast. In: Amin MB, Edge S, Greene F, et al., editors. AJCC Cancer staging manual. 8th ed. New York: Springer International Publishing; 2016. 1. Hortobagyi GN, Connolly JL, Edge SB, et al. Breast. In: Amin MB, Edge S, Greene F, et al., editors. AJCC Cancer staging manual. 8th ed. New York: Springer International Publishing; 2016. 2. Hortobagyi GN, Edge SB, Giuliano A. New and important changes in the TNM staging system for breast Cancer. Am Soc Clin Oncol Educ Book. 2018; 38:457–67. Additional file 3: Table S1. Clinicopathologic characteristics of TNBCs included in SYSUCC-PWH cohort (N = 611) and SEER cohort (N = 31,941). Table S2. Alteration from Anatomic to Prognostic Stage of TNBCs in- cluded in SYSUCC-PWH cohort (N = 611) and SEER cohort (N = 31,941). Table S3. Hazard Ratio for Disease-Specific Survival and Progression-Free Survival by Stage of TNBCs included in the SYSUCC-PWH cohort (N = 611). Table S4. Hazard Ratio for Disease-Specific Survival and Overall Sur- vival by Stage of TNBCs included in the SEER cohort (N = 31,941). 3. Abdel-Rahman O. Validation of the 8th AJCC prognostic staging system for breast cancer in a population-based setting. Breast Cancer Res Treat. 2018; 168(1):269–75. 3. Abdel-Rahman O. Validation of the 8th AJCC prognostic staging system for breast cancer in a population-based setting. Breast Cancer Res Treat. 2018; 168(1):269–75. 4. Shao N, Xie C, Shi Y, Ye R, Long J, et al. Comparison of the 7th and 8th edition of American joint committee on Cancer (AJCC) staging systems for breast cancer patients: a surveillance, epidemiology and end results (SEER) analysis. Cancer Manag Res. 2019;11:1433–42. 5. Wang M, Wu K, Zhang P, Zhang M, Ding A, et al. The prognostic significance of the Oncotype DX recurrence score in T1-2N1M0 estrogen receptor-positive HER2-negative breast Cancer based on the prognostic stage in the updated AJCC 8th edition. Ann Surg Oncol. 2019;26(5):1227–35 Conclusions Our findings demonstrated that prognostic staging system did not provide better discriminatory ability in predicting TNBCs prognosis than anatomic staging system. The prog- nosis staging system should be further modified following the development of novel clinical therapies in TNBCs. Page 8 of 9 Page 8 of 9 Page 8 of 9 Page 8 of 9 He et al. BMC Cancer He et al. BMC Cancer (2020) 20:18 Ethics approval and consent to participate All procedures performed in studies involving human participants were in accordance with the ethical standards of the Sun Yat-sen University Cancer Center (SYSUCC) research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The formal writ- ten informed consent was obtained from all individual participants included in the study. 14. Krop I, Ismaila N, Andre F, Bast RC, Barlow W, et al. Use of biomarkers to guide decisions on adjuvant systemic therapy for women with early-stage invasive breast Cancer: American Society of Clinical Oncology clinical practice guideline focused update. J Clin Oncol. 2017;35(24):2838–47. 15. Wang M, Chen H, Wu K, Ding A, Zhang M, et al. Evaluation of the prognostic stage in the 8th edition of the American joint committee on Cancer in locally advanced breast cancer: an analysis based on SEER 18 database. Breast. 2018;37:56–63. Abbreviations AJCC Th A AJCC: The American Joint Committee on Cancer; AS: Anatomic stage; BC: Breast cancer; DSS: Disease-specific survival; OS: Overall survival; PFS: Progression-free survival; PS: Prognostic stage; PWH: Prince of Wales Hospital; SEER database: Surveillance, epidemiology, and end results database; SYSUCC: Sun Yat-sen University Cancer Center; TNBC: Triple negative breast cancer 6. Weiss A, Chavez-MacGregor M, Lichtensztajn DY, Yi M, Tadros A, et al. 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Activated PI3K/AKT and MAPK pathways are potential good prognostic markers in node-positive, triple-negative breast cancer. Ann Oncol. 2014;25(10):1973–9. 28. Ogawa Y, Hai E, Matsumoto K, Ikeda K, Tokunaga S, et al. Androgen receptor expression in breast cancer: relationship with clinicopathological factors and biomarkers. Int J Clin Oncol. 2008;13:431–5. 28. Ogawa Y, Hai E, Matsumoto K, Ikeda K, Tokunaga S, et al. Androgen receptor expression in breast cancer: relationship with clinicopathological factors and biomarkers. Int J Clin Oncol. 2008;13:431–5. 29. Jiang YZ, Ma D, Suo C, Shi J, Xue M, et al. Genomic and Transcriptomic landscape of triple-negative breast cancers: subtypes and treatment strategies. Cancer Cell. 2019;35(3):428–40. 29. Jiang YZ, Ma D, Suo C, Shi J, Xue M, et al. Genomic and Transcriptomic landscape of triple-negative breast cancers: subtypes and treatment strategies. Cancer Cell. 2019;35(3):428–40. 30. 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https://openalex.org/W2981019890	https://www.matec-conferences.org/articles/matecconf/pdf/2019/44/matecconf_suslille2019_03002.pdf	English	null	Investigation of rock bolting for tunnels based on an efficient CATIA-ABAQUS model	MATEC web of conferences	2,019	cc-by	5,263	Investigation of rock bolting for tunnels based on an efficient CATIA-ABAQUS model Hui Lee1,2, Wei-zhong Chen 1,3,, Xian-jun Tan 1, Qiao-chu Yang 4, and Xiao-gang Wang 1 1State Key Laboratory of Geomechanics and Geotechnical Engineering, Institute of Rock and Soil Mechanics, Chinese Academy of Sciences, Wuhan 430071, China y 2University of Chinese Academy of Sciences, Beijing 100049, China y y , j g , 3Geotechnical and Structural Engineering Research Center, Shandong University, Jinan, Shandong 250061, China 4BIM Research Center, Sichuan Highway Planning, Survey, Design and Research Institute Ltd, Ch d Si h 610000 Chi 3Geotechnical and Structural Engineering Research Center, Shandong University, Jinan, Shandong 250061, China 4BIM Research Center, Sichuan Highway Planning, Survey, Design and Research Institute Ltd, Chengdu, Sichuan 610000, China Abstract. As a widely used reinforcement method in tunnel engineering, the accuracy of models in numerical analysis has a vital influence on the reliability of the analysis results. To solve the problem of hard modeling for complicated models, this paper proposes to build models of different characteristics automatically by the means of Enterprise Knowledge Language (EKL) of CATIA, combining knowledge engineering template with parameterization. Furthermore, the stability of surrounding rocks and the distribution features of plastic areas were studied in different cases by CATIA-ABAQUS model. Besides, the effects and contributions of several parameters, including the rock-bolt diameter and interval between rock- bolts have been studied. It was found that: (1) Rock-bolt has a notable effect on the stability of the surrounding rocks. Increasing the diameter of rock-bolts contributes to significantly reduce the plastic area and the vertical displacement. With the increase of the quantity and the diameter of anchors, the degree of influence gradually weakens, showing the logarithmic relationship; (2) When the quantity and diameter of rock-bolts are constant, in the case in which the intervals between rock-bolts are various, the plastic area of surrounding rocks and the axial stress of rock- bolts are larger than that of the same distance, while the vertical displacement are uniform. (3) The excavation process has a great influence on the axial stress of rock-bolts and the redistribution of stresses in the surrounding rock. Keyword: BIM, Anchoring system, Rock-bolts Corresponding author : wzchen@whrsm.ac.cn. , 0 (2019) MATEC Web of Conferences 295 SUS - Lille 2019 3002 , 0 (2019) MATEC Web of Conferences 295 SUS - Lille 2019 3002 https://doi.org/10.1051/matecconf /201929503002 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 1 Introduction The equilibrium state of stress in the surrounding rock will be destroyed owing to excavation. For the surrounding rock of the affect region, a large amount of energy is going to be released by coordinate deformation as well as stress redistribution. If the redistribution stress exceeds the surrounding rock’s strength, a lot of new cracks will occur * Corresponding author : wzchen@whrsm.ac.cn. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). , 0 (2019) MATEC Web of Conferences 295 SUS - Lille 2019 3002 , 0 (2019) MATEC Web of Conferences 295 SUS - Lille 2019 3002 https://doi.org/10.1051/matecconf /201929503002 and at the same time, the existed cracks will extend. Finally, all of them affect each other leading to instability and destruction. and at the same time, the existed cracks will extend. Finally, all of them affect each other leading to instability and destruction. Anchoring system can transfer and enhance the strength and self-stability of the rock body by being embedded into it. Scholars did a plenty of works on the anchoring mechanism and optimal design of the anchors and concluded the anchor mechanism of suspension theory, supporting theory, composite theory, enhancement theory and pin theory [1,2]. However, the rock bolting design is still empirical or semi-empirical in practical engineering, due to the lack of understanding of bolt reinforcement mechanism [3,4], and the effects and contributions of several parameters are not very clear[5]. Ideally, large-scale physical experiments should be carried out to study the effects and contributions of these parameters. Moreover, (1) it seems to be difficult to get a consistent result, owing to the limitation of the laboratory experimental conditions. For example, the size of the test body can’t be cast large enough to satisfy the requirement of testing or to simulate the practical underground geological conditions[6,7]; (2) it is not very convenient for the test bodies to accommodate so many rock-bolts; (3) for larger diameter rock-bolt tests, there was always a lack of sufficient confinement to prevent the split of the test bodies under loading[8]. While, numerical simulations have the advantages of that (1) it is conceivable to satisfy the actual geological conditions as accurate as we need; (2) it can provide further insights and several significant improvements to these models including consistent testing environment which avoids issues of unbalanced forces and insufficient confinement; (3) it is reliable to examine every influence factors whilst obtaining reasonable results[8]; (4) it has the characteristics of repeatability, adjustability, convenience, accuracy, low cost and strong operability[9]. So numerical simulation is a good way to study the optimization of anchoring parameters. * Corresponding author : wzchen@whrsm.ac.cn. On the other hand, whether to generate numerical model accurately is an important factor affecting the accuracy of analysis results, and it seems to be difficult to simulate anchors of different design parameters quickly, accurately and parametrically. CATIA provides a way to build models by parameterizations automatically to reach the purpose of accurate and quick modelling. In this paper, the simulation of excavation and rock-bolt reinforcement in a large scale of rock body has been performed by parameterized modeling in CATIA and analyzing in ABAQUS. It aims to further understanding of the influence of relevant parameters, the distribution of stress and stability of surrounding rocks for the optimization of anchors. 2.1 Model building The parameters of the practical project including intervals and diameters of each anchor are easy to adjust for various research purpose in order to optimize the anchoring system by the means of parameterized design module of CATIA[10,11], so as to build the design model (Fig.1). The model is imported into ABAQUS for the CATIA-ABAQUS simulation model to analyze the influence of excavation on the stability and stress redistribution of surrounding rock, and investigate the factors and contributions of the influence factors on the stability of surrounding rock to realize the fusion of numerical simulation and BIM. The simulation process is shown in Fig.2. For the sake of simplification, the geological-terrain model was simplified as a cube in this research. 2 2 https://doi.org/10.1051/matecconf /201929503002 , 0 (2019) MATEC Web of Conferences 295 SUS - Lille 2019 3002 , 0 (2019) MATEC Web of Conferences 295 SUS - Lille 2019 3002 Fig. 1. The layout of the model Fig. 2. Simulation process Fig. 1. The layout of the model Fig. 2. Simulation process 2.2 Cases study To avoid steel wasting and to shorten the period of construction, it’s important to optimize the design of rock-bolts, including the length and the diameter of rock-bolts, rock-bolt inclination, interval between rock-bolts, and so on. So numerical simulation was carried out to investigate the influences and contributions of various parameters on the stability of rock body, stress distribution of rock body and rock-bolts, and the convergence of surrounding rock. Rock-bolts with different setting formations and diameters, 18, 22, 25, 28, and 32mm, were embedded in rock body which was with the size of 260m×20m×154m. The length of rock-bolts was 4m, whole of which was embedded into the rock body. The boundary conditions applied in the model were that the bottom side of the model was displacement limited at 3 directions, and the side face was limited to normal displacement constraint. Every two layers of rock-bolts were set as rectangular (Fig.1). In this paper, we considered the length and the inclination of rock-bolt as invariants, and the variables investigated are listed in Table 3. Besides, this paper considered the effect of excavation process[12]by dividing the tunnel into three sections for full section excavation. Table 3. The variables and invariants investigated in this paper Variables Invariants Intervals between rock-bolts 1.6m×1.6m 1.2m×1.2m 0.8m×0.8m 0.6m×0.6m 0.4m×0.4m 90º, 4000 mm length, 260MPa pretension, rock body strength Rock-bolt diameter 18mm, 22mm, 25mm, 28mm, 32mm Table 3. The variables and invariants investigated in this paper Variables Invariants Intervals between rock-bolts 1.6m×1.6m 1.2m×1.2m 0.8m×0.8m 0.6m×0.6m 0.4m×0.4m 90º, 4000 mm length, 260MPa pretension, rock body strength Rock-bolt diameter 18mm, 22mm, 25mm, 28mm, 32mm Table 3. The variables and invariants investigated in this paper 3 Numerical analysis and results As the quantity of output results were so large, only the main results were presented here. We considered the convergence to be one of the crucial criterion for the stability of the tunnel. So this paper discussed how the quantity (interval between rock-bolts longitudinal and tangent) and the diameter of rock-bolts affect the vertical displacement of the tunnel. Additionally, the characteristics of plastic zone, stress field and the axial stress of rock-bolts were studied. Fig. 2. Simulation process The study model consisted of two types of materials: rock body and steel (rock-bolt). In the numerical simulation, the rock body was modelled as the Mohr-Coulomb criterion, elasto-plastically and the rock-bolt was modelled as truss of linear elastic model. The interface between rock-bolts and rock mass was simulated by embedded elements which means the nodes of rock-bolt elements are inserted in the rock mass element by interpolation method. Reasonable material property is crucial to obtain correct simulation results. Table 1 and 2 show the material properties that were used in the numerical simulation. Table 1. Material properties of the rock body Density/(kg﹒ m-3) Elastic modulus/MPa Poisson’s ratio Cohesion/MPa Friction angle/(°) 2500 1200 0.35 0.2 25 Table 2. Material properties of rock-bolt Density /(kg﹒ Elastic modulus Poisson’s Coefficient of expa Pretension/M 3 , 0 (2019) MATEC Web of Conferences 295 SUS - Lille 2019 3002 https://doi.org/10.1051/matecconf /201929503002 3.1 Plastic zone analysis Only part of typical characteristics of the plastic zones were discussed in which no rock- bolt existed or the distance between rock-bolts longitudinal and tangent are both 1.6m, 0.8m, 0.4m, respectively. Fig.3 shows that the plastic zone without rock-bolt is mainly concentrated on the arch hance and arch spandrel, like a couple of wing. While the features changed when rock-bolt existed and different characteristics could be seen as the distance between rock-bolts differs. The area of plastic zone reduces as the setting density becoming large and focuses on the bottom of the arch which means rock-bolt is useful for inhibiting the extension of stress and strengthening the surrounding rock to improve its own strength. In fact, what rock-bolts do to improve the stability of the surrounding rock is called anchorage effect. Excavation of tunnel leading to unload of rock and stress redistribution is the key factor to rock deformation. To avoid exceeding displacement, anchoring system is 4 , 0 (2019) MATEC Web of Conferences 295 SUS - Lille 2019 3002 https://doi.org/10.1051/matecconf /201929503002 needed. As long as the surrounding rock deforms, the rock-bolts embedded in the rock body will lead to additional radial force and the stress distribution of the rock translates from two-directional stress state to three-directional stress state[13]. The tunnel whose section is circle forms a supporting ring and the tunnel with arch section forms a supporting arch, both of which avoid the deterioration of the property of rock body. Additionally, the physical and mechanical properties of the medium composed of steel and rock are ideally much higher than rock. In this paper, we improved the properties Elastic module, Poisson’s ratio, Cohesion by 10%[14]. needed. As long as the surrounding rock deforms, the rock-bolts embedded in the rock body will lead to additional radial force and the stress distribution of the rock translates from two-directional stress state to three-directional stress state[13]. The tunnel whose section is circle forms a supporting ring and the tunnel with arch section forms a supporting arch, both of which avoid the deterioration of the property of rock body. Additionally, the physical and mechanical properties of the medium composed of steel and rock are ideally much higher than rock. In this paper, we improved the properties Elastic module, Poisson’s ratio, Cohesion by 10%[14]. at o, Co es o by 0%[ ]. （a）without rock-bolt （b）1.6m×1.6m （c）0.8m×0.8m （d）0.4m×0.4m Fig. 3. 3.1 Plastic zone analysis The plastic zone distribution with setting density increasing , y [ ] （a）without rock-bolt （b）1.6m×1.6m （d）0.4m×0.4m （d）0.4m×0.4m （c）0.8m×0.8m Fig. 3. The plastic zone distribution with setting density increasing 3.2 Stress field analysis Take the stress distribution of the tunnel vault (called “line” below) with various rock-bolt’s setting formations for example. Take the stress distribution of the tunnel vault (called “line” below) with various rock-bolt’s setting formations for example. Fig.4(a) shows that the regular of the radial stress distribution without rock-bolt is different from that with rock-bolt. If no rock-bolt is embedded, with an increase of the distance from the tunnel wall, the radial stress of “line” increases gradually, then it’s going to be stable. When rock-bolts exist, the radial stress of “line” increases to a maximum and then decreases gradually in the anchoring area. Beyond the anchoring area, the radial stress increases to a stable value. Take the case in which the distance between longitudinal and tangent rock-bolt are both 0.6m for example, the radial stress of the first observation point on the tunnel wall is 0.6MPa, while it increases with the distance from the tunnel wall, and reaches the maximum at the fourth observation point. Then, it decreases to 0.28MPa at the fifth observation point. At last, out of the anchoring area, the radial stress raises as the distance from the tunnel wall. What’s more, the more the rock-bolts are, the larger the radial stress on the tunnel wall is, and the maximum of the radial stress increases as well. When the distance of longitudinal and tangent rock-bolts are both 1.6m, 1.2m, 0.8m, 0.6m, and 0.4m, the radial stress on the tunnel wall is 0.167MPa, 0.207MPa, 0.398MPa, 0.604MPa, 1.006MPa, respectively; the maximum stress of this line is 0.247MPa, 0.314MPa, 0.483MPa, 0.659MPa, 1.062MPa, respectively. The mechanism of this phenomenon is that excavation led to rock low stability and stress redistribution. If the anchoring system is not applied, the strength of the rock decreases to the residual stress, instead, the stress of the surrounding rock redistributes again because of the anchoring force 5 , 0 (2019) MATEC Web of Conferences 295 SUS - Lille 2019 3002 , 0 (2019) MATEC Web of Conferences 295 SUS - Lille 2019 3002 https://doi.org/10.1051/matecconf /201929503002 which constitutes of the stress of the surrounding rock with initial stress. As a result, the maximum stress and residual stress increase as the strength of the anchoring system improves. 3.2 Stress field analysis On the other hand, due to the enhancement theory of rock-bolt, the physical and mechanical properties of the rock-bolt are improved and they improve a lot as the setting density is enhanced which also leads to the improving of the anchoring effect, suggesting that the radial stress improves with the setting density. which constitutes of the stress of the surrounding rock with initial stress. As a result, the maximum stress and residual stress increase as the strength of the anchoring system improves. On the other hand, due to the enhancement theory of rock-bolt, the physical and mechanical properties of the rock-bolt are improved and they improve a lot as the setting density is enhanced which also leads to the improving of the anchoring effect, suggesting that the radial stress improves with the setting density. As shown in Fig.4(b), no matter the rock-bolt exists or not, the regular of the tangent stress on the vault of the tunnel exists a concentration value in the anchoring area, and then decreases to a stable value. But the difference is that the maximum of the tangent stress anchored is much bigger than that without rock-bolt. The tangent stress and the maximum tangent value are both increase with the setting density increasing, and they tend to be the same value at last. For example, when the longitudinal and tangent interval are both 1.6m, 1.2m, 0.8m, 0.6m, and 0.4m, the tangent stress of the tunnel wall is 0.698MPa, 0.729MPa, 0.815MPa, 0.871MPa, 0.952MPa, respectively; the maximum value of the tunnel vault is 0.792MPa, 0.853MPa, 0.972MPa, 1.061MPa, 1.212MPa，which is much bigger than that without rock-bolt(0.60MPa, 0.61MPa). (a) (b) Fig. 4. The distribution of radial and tangent stress at various cases. (a) Radial stress, (b) Tangent stress. (a) (b) (a) (b) Fig. 4. The distribution of radial and tangent stress at various cases. (a) Radial stress, (b) Tangent stress. 3.3.1 Influence of quantity The effect of different setting density of rock-bolts on the radial convergence of the tunnel vault is shown in Fig.5. It can be seen that the stability of the surrounding rock has been greatly improved. Take the rock-bolts set as 1.6m × 1.6m for example, the vertical displacement of the vault is about -26mm, which reduces by 23.5% compared with -34mm without rock-bolt, and reduces by 27.6%, 35.3%, 39.7%, 44.1%, respectively, for rock-bolts set as 1.2m× 1.2m, 0.8m × 0.8m, 0.6m× 0.6m, 0.4m× 0.4m. With the longitudinal and tangent distance becoming small, the vertical displacement of the tunnel vault reduces significantly, the degree of which is in terms of the quantity of rock-bolt (Table 4). As shown in Fig.6, the deformation decreases as the quantity of rock-bolts increases, suggesting that they have obviously positive correlation with each other. When the quantity of rock-bolt is not very large, the deformation increases significantly with it and it does not increases obviously as the quantity increases, suggesting the property of convex function [9]. The characteristic is consistent with the results obtained by DEM[15]. It indexes that the regular of “the more, the better” is not reliable, we’d better choose the proper setting formation according to the vertical displacement. Table 4. The quantity of anchors of different setting formation Formation 1.6m×1.6m 1.2m×1.2m 0.8m×0.8m 0.6m×0.6m 0.4m×0.4m 1.0m×1.4m 0.6m×1.1m 6 , 0 (2019) MATEC Web of Conferences 295 SUS - Lille 2019 3002 https://doi.org/10.1051/matecconf /201929503002 Quantity 286 522 1276 1972 4472 525 522 The effect of the quantity of rock-bolts on the vertical displacement has been studied by the means of statistical method. The expression about the quantity of rock-bolts and the vertical displacement for tunnel vault and spandrel are listed in Eq.(1) and Eq.(2), respectively, and these equations can be translated into ordinary formation (Eq.(3)). Quantity 286 522 1276 1972 4472 525 522 The effect of the quantity of rock-bolts on the vertical displacement has been studied by the means of statistical method. The expression about the quantity of rock-bolts and the vertical displacement for tunnel vault and spandrel are listed in Eq.(1) and Eq.(2), respectively, and these equations can be translated into ordinary formation (Eq.(3)). yN=2.897×ln(xN)-42.7 (1) yN=3.307×ln(xN)-43.403 (2) yN=aN×ln(xN)+bN (3) (2) (3) where yN is the vertical displacement, xN is the quantity of rock-bolts. In these equations the R2 is 0.9882 and 0.9827, respectively, which means that the statistics fit well. 3.3.1 Influence of quantity Obviously, there is logarithmic relationship between the quantity of rock-bolts and the vertical displacement. In Eq.(3) aN and bN are parameters needed to be confirmed by the means of experiment or numerical simulation which are in terms of the lithology. As long as the expression is quantified, it is possible to get the quantity of rock-bolts according to the conditional parameters -- vertical displacement, then to confirm the setting formation. 0 5000 10000 15000 20000 -34 -32 -30 -28 -26 -24 -22 -20 -18 Vertical Displacement(mm) Distance along tunnel(mm) No rock-bolt 1.6m1.6m 1.2m1.2m 0.8m0.8m 0.6m0.6m 0.4m0.4m 0.6m1.1m 1.0m1.4m Fig. 5. The vertical displacement of the tunnel vault at different cases 0 1000 2000 3000 4000 5000 -28 -26 -24 -22 -20 -18 -16 Vault Spandrel Vertical Displacement(mm) The quantity of rock-bolt Vault Spandrel Fig. 6. Influence of the quantity of rock-bolts on the vertical displacement 0 5000 10000 15000 20000 -34 -32 -30 -28 -26 -24 -22 -20 -18 Vertical Displacement(mm) Distance along tunnel(mm) No rock-bolt 1.6m1.6m 1.2m1.2m 0.8m0.8m 0.6m0.6m 0.4m0.4m 0.6m1.1m 1.0m1.4m Fi 5 Th ti l di l t f th t l lt t diff t 0 5000 10000 15000 20000 -34 -32 -30 -28 -26 -24 -22 -20 -18 Vertical Displacement(mm) Distance along tunnel(mm) No rock-bolt 1.6m1.6m 1.2m1.2m 0.8m0.8m 0.6m0.6m 0.4m0.4m 0.6m1.1m 1.0m1.4m Fig. 5. The vertical displacement of the tunnel vault at different cases Fig. 5. The vertical displacement of the tunnel vault at different cases 0 1000 2000 3000 4000 5000 -28 -26 -24 -22 -20 -18 -16 Vault Spandrel Vertical Displacement(mm) The quantity of rock-bolt Vault Spandrel Fig. 6. Influence of the quantity of rock-bolts on the vertical displacement 0 1000 2000 3000 4000 5000 -28 -26 -24 -22 -20 -18 -16 Vault Spandrel Vertical Displacement(mm) The quantity of rock-bolt Vault Spandrel Fig. 6. Influence of the quantity of rock-bolts on the vertical displacement Fig. 6. Influence of the quantity of rock-bolts on the vertical displacement 7 7 , 0 (2019) MATEC Web of Conferences 295 SUS - Lille 2019 3002 https://doi.org/10.1051/matecconf /201929503002 3.2.2 Influence of diameter In this numerical model, the longitudinal and tangent distance of rock-bolts are both 0.6m, and the diameter is set to 18mm, 22mm, 25mm, 28mm, and 32mm, while other parameters are kept consistent with those in Table 1, 2. The results of the vertical displacement are shown in Table 5, and the features of the vertical displacement versus the diameter of rock- bolts are shown in Fig.7, which is similar with the effect caused by the quantity of rock- bolts. With the diameter of rock-bolt increasing, the vertical displacement does not reduce significantly. Similarly, we studied it with the method of statistical method, and the result is concluded with Eq.(4) and Eq.(5) for tunnel vault and spandrel, respectively. Like the phenomenon discussed above, the parameters called aD and bD are parameters which should be defined by laboratory test, field test or numerical simulation. So for the specifical project, the diameter of the rock-bolts can been chosen using Eq.(6). yD=4.8125×ln(xD)-36.011 (4) yD=4.77×ln(xD)-35.609 (5) yD=aD×ln(xD)+bD (6) (4) (5) (6) where yD is the vertical displacement, xD is the diameter of rock-bolts. In these equations the R2 is 0.9953 and 0.9945, which means that the statistics fit well. where yD is the vertical displacement, xD is the diameter of rock-bolts. In these equations the R2 is 0.9953 and 0.9945, which means that the statistics fit well. When the rock is unloaded, the surrounding rock deforms into blocks and the radial anchoring force is imposed on the rock body leading to rock compression. The cracks are closed, then the cohesion and frictional angle increase, the strength of the surrounding rock is enhanced[16]. If the quantity of the rock-bolts reaches to a critical value, the cracks are all closed and the rock body reaches to a limitation of the extrusion deformation. Beyond the critical, new cracks will appear which weaken the integrity and the stability of the rock. Table 5. The vertical displacement under various diameters of rock-bolts Diameter（mm） 15 20 25 30 35 Vertical displacement （mm） -23.325 -21.573 -20.455 -19.665 -19.069 18 20 22 24 26 28 30 32 34 -23 -22 -21 -20 -19 -18 Vertical Displacement(mm) The diameter of rock-bolt Vault Spandrel Fig.7. The vertical displacement with different diameters Table 5. The vertical displacement under various diameters of rock-bolts Table 5. 3.4 Axial stress analysis From the results discussed above, the effect on the stability of the surrounding rock of the longitudinal and tangent interval is due to the quantity of rock-bolts, suggesting that it is none of the interval’s business, but it will differ a lot for the axial stress of rock-bolt. When the quantity of rock-bolt is closed to each other, we need to study the axial stress distribution of rock-bolt. Take the cases in which rock-bolts are set as 1.0m×1.4m, 1.2m× 1.2m for example, there are 525 rock-bolts in the former case, and 522 in the latter one. The distribution of rock-bolt axial stress versus each excavation section is shown in Fig.8, and it can be seen that the axial force of the rock-bolt is a fluctuating distribution in each excavation section, with multiple extremums and one maximum. In order to do some research on the distribution of rock-bolt axial stress along the length, we studied the average axial stress of the tunnel vault and the hance in two cases. Fig.9(a) shows the characteristics of the axial stress versus the length of rock-bolt in the hance of the tunnel, showing a maximum at 601.92mm with value of 830MPa for the case of 1.0m×1.4m, and a maximum at 401.25mm with value of 828.6MPa for the case of 1.2m×1.2m. It can be seen that the maximums are similar in the two cases, but the maximum stress of the latter is closer to the tunnel wall than the former, and the maximum stress of the rock-bolt in both formations are offset the middle position and close to the inside part of the tunnel. 0 1000 2000 3000 4000 450 500 550 600 650 700 750 800 850 Rock-boltt axial stress(MPa) Distance along the rock-bolt(mm) 1.0m1.4m 1.2m1.2m (a) 0 1000 2000 3000 4000 334 336 338 340 342 344 346 348 Rock-bolt axial sress(MPa) Distance along the rock-bolt(mm) 1.0m1.4m 1.2m1.2m (b) Fig. 9. The axial stress distribution along the length of rock-bolt under different setting arrangements. (a) The hance of tunnel, (b) The tunnel vault. a) 0 1000 2000 3000 4000 334 336 338 340 342 344 346 348 Rock-bolt axial sress(MPa) Distance along the rock-bolt(mm) 1.0m1.4m 1.2m1.2m (b) 0 1000 2000 3000 4000 450 500 550 600 650 700 750 800 850 Rock-boltt axial stress(MPa) Distance along the rock-bolt(mm) 1.0m1.4m 1.2m1.2m (a) (a) (b) Fig. 9. 3.2.2 Influence of diameter The vertical displacement under various diameters of rock-bolts Diameter（mm） 15 20 25 30 35 Vertical displacement （mm） -23.325 -21.573 -20.455 -19.665 -19.069 Diameter（mm） 15 20 25 30 Vertical displacement （mm） -23.325 -21.573 -20.455 -19.665 18 20 22 24 26 28 30 32 34 -23 -22 -21 -20 -19 -18 Vertical Displacement(mm) The diameter of rock-bolt Vault Spandrel Fig.7. The vertical displacement with different diameters The diameter of rock-bolt Fig.7. The vertical displacement with different diameters 8 8 , 0 (2019) MATEC Web of Conferences 295 SUS - Lille 2019 3002 https://doi.org/10.1051/matecconf /201929503002 0 10000 20000 240 260 280 300 320 340 360 380 400 420 Rock-bolt axial stress(MPa) Distance along tunnel(mm) 0.8m0.8m 1.2m1.2m Section Fig. 8. Influence of excavation process on axial stress Fig. 8. Influence of excavation process on axial stress 3.4 Axial stress analysis The axial stress distribution along the length of rock-bolt under different setting arrangements. (a) The hance of tunnel, (b) The tunnel vault. 9 , 0 (2019) MATEC Web of Conferences 295 SUS - Lille 2019 3002 , 0 (2019) MATEC Web of Conferences 295 SUS - Lille 2019 3002 https://doi.org/10.1051/matecconf /201929503002 Fig.9(b) shows the distribution of axial stress at the tunnel vault in the two cases, presenting the similar features, but the axial stress of the former is slightly higher than that of the latter. By analyzing the distribution characteristics of the axial stress of the two cases, it can be seen that when the quantity of rock-bolts is similar, the effect they do on the stability of the surrounding rock is also the same and the vertical displacement can be calculated by Eq.(3) or Eq.(6). While, the plastic area of the former case is much larger than that of the latter and the axial stress of rock-bolts is also higher which means that the risk of instability increases. Therefore, we’d better choose the equivalent longitudinal and tangent intervals between rock-bolts when the quantity of rock-bolts is constant. 4 Conclusions In this study, an accurate model was built by CATIA, and was employed in the mechanical analysis of the bolt-reinforced tunnel which was excavated by full excavation for three sections. Besides, the factors and contributions of the parameters which have influence on the rock body have been studied by the means of numerical simulation. Primary conclusions are summarized as follows. (1) The increasing quantity of rock-bolts contributed to significantly reduce the plastic zone and the vertical displacement. While, it does not increase obviously any more with the number of rock-bolts increasing, which is the law of logarithm function, so as to the effect of the diameter of rock-bolt. (2) The process of excavation matters a lot to the rock-bolts axial force, and several extremum values and one maximum exist on the rock-bolt axial force along the length of tunnel. (2) The process of excavation matters a lot to the rock-bolts axial force, and several extremum values and one maximum exist on the rock-bolt axial force along the length of tunnel. (3) For the rectangular arrangement formation, the influence of the intervals between rock-bolts on the stability of the surrounding rock is due to the impact of the quantity of anchors, but the longitudinal and tangent rock-bolts with same intervals is stable than that of different one. References 1. S.R. Zhang, Y. Gu, Z.L. Zhang. The optimized design of rock-bolts supporting the large-scale underground cavities[J]. Journal of Hydroelectric Engineering, 26(5):47- 52(2007) 2. L.W Zhang, R. Wang. Research on status quo of anchorage theory of rock and soil[J]. Rock and Soil Mechanics, 23(5):627-631(2002) 3. Y. Cai, T. Esaki, Y.J. Jiang. An analytical model to predict axial load in grouted rock bolt for soft rock tunnelling[J]. Tunnelling and Underground Space Technology, 19(6) : 607-618(2004a) 4. Y. Cai, T. Esaki, Y.J. Jiang. A rock bolt and rock mass interaction model[J]. International Journal of Rock Mechanics & Mining Sciences, 41(7):1055-1067(2004b) 5. J.C. Gu, J. Shen, A.M. Chen, et al. Model testing study of strain distribution regularity in rock mass caused by prestressed anchorage cable[J]. Rock Mechanics and Rock Engineering, 19(Z1):917-921(2000) 6. A.T. Haile. A mechanistic evaluation and design of tunnel support systems for deep level South African mines. PhD thesis (unpublished), University of Natal, Durban, South Africa(1999a) 7. A.T. Haile. Observation of the dynamic support performance of South African tunnel support systems. In: Proceedings rock support and reinforcement practice in mining, 10 https://doi.org/10.1051/matecconf /201929503002 , 0 (2019) MATEC Web of Conferences 295 SUS - Lille 2019 3002 Kalgoorlie, Australia, 335–341(1999b) Kalgoorlie, Australia, 335–341(1999b) Kalgoorlie, Australia, 335–341(1999b) 8. L. Li. Parametric Study of Rockbolt Shear Behaviour by Double Shear Test[J]. Rock Mechanics and Rock Engineering, 49(12):4787-4797(2016) 9. Q.L. Dong, Research on deformation law of weak broken roadway and optimization of bolt support parameters under mining influence[D]. GuiZhou University(2018) 10. X.C. Han. Research on Application of BIM Concept Tunnel Portal Design Program[D]. Chongqing Jiaotong University(2018) 11. X. Qin. Research on Railway Geological Route Selection Method Based on BIM[D]. Shijiazhuang Tiedao University(2018) 12. G.L. Han. Analysis of Mechanism of Bolt-Anchor Cable Combined Support for Weak Rock Cavern by ABAQUS[J]. Metal Mine, 75-79(2008) 13. P.P. Chen, Q.W. Xu, H.H. Zhu, et al. Model experiment study on the failure characteristics of weak surrounding rock mass above tunnel arch and the anchoring effect of bolts[J]. Chinese Journal of Rock Mechanics & Engineering, 35(S2):3561- 3569(2016) 14. H. Chen, W.Z. Ren, D. Li, et al. Numerical simulation and model test study of mechanism of bolt in deep tunnel[J]. Rock and Soil Mechanics, 32(S1):719-724(2011) 15. H.N. Wang, X. Guo, M.J. Jiang, et al. Investigation of rock bolting for deeply buried tunnels via a new efficient hybrid DEM-Analytical model[J]. Tunnelling and Underground Space Technology, 82:366-379(2018) 16. H. Chen, W.Z. Ren, Z.G. Shu, et al. Model test study and numerical analysis of mechanism of anchor bolt under different supporting conditions[J]. Rock & Soil Mechanics, 33(S1):277-282(2012) 11
https://openalex.org/W4394941865	https://www.qeios.com/read/JRO84X/pdf	English	null	Review of: "A New Approach Towards Quantum Foundations and Some Consequences"	null	2,024	cc-by	518	Review of: "A New Approach Towards Quantum Foundations and Some Consequences" Jean Patrick Connerade1 1 Imperial College London Jean Patrick Connerade1 1 Imperial College London Potential competing interests: No potential competing interests to declare. None I hesitated to click on the box “I am knowledgeable about etc.” because I am no expert in this particular aspect of the field, but I do believe I can make a few comments which I hope are constructive. I found this to be a very interesting and (to me) novel approach. I fully agree that one must include in a quantum theory not only reality but also its perception by at least one observer (a minimum requirement), extendable to a wider group of different observers. Interestingly, this prerequisite to a physical theory can be traced back much further than QM. I found it in the aphorisms of Georg Christoph Lichtenberg, who was, of course, both a physicist and a philosopher. The existence of accessible and non-accessible states is the novelty here. Paul Dirac would no doubt have been against this idea (which smacks somewhat of hidden variables) because his approach was to allow in the theory only what is directly “measurable.” However, I do not think he really achieved that. Nor do I believe he gave a proper definition of what he termed “observability” other than by a semantic “pirouette” involving mathematical criteria, although I think (elsewhere than in his book) he suggested that what cannot be measured does not even exist. So, it seems to me that the field is indeed wide open for an alternative approach, as pursued in the present paper. I suppose it may be qualified as the development of a “quantum logic,” being a new branch of theoretical statistics imposed by, necessarily incomplete, knowledge of a physical system, along statistical lines Gibbs might have followed. Where I still find a problem is in the treatment of time, which does not appear as a proper variable in QM since (in contrast to space) it possesses no associated operator or quantum states. It seems to persist in QM as an entirely classical variable. This poses a real difficulty when attempting to harmonize QM with relativity, since the latter requires space and time to be treated on an equal footing to construct a continuous and differentiable four-dimensional space-time metric. Qeios, CC-BY 4.0 · Review, April 19, 2024 Qeios ID: JRO84X · https://doi.org/10.32388/JRO84X Review of: "A New Approach Towards Quantum Foundations and Some Consequences" I detect a similar difficulty here through the use of phrases like: “is the cat alive or dead at time t,” “at a fixed time,” “at the same time,” “if we let time vary,” which appear diversely in the text of this paper and seem to imply that time, once again, has been separated from space in its properties… y remarks are helpful. I found this an excellent paper, which is why I am happy to comment about it. I hope my remarks are helpful. I found this an excellent paper, which is why I am happy to comment about it. Qeios ID: JRO84X · https://doi.org/10.32388/JRO84X 1/1
https://openalex.org/W2016253265	https://asp-eurasipjournals.springeropen.com/counter/pdf/10.1155/2008/673040	English	null	A Minimax Mutual Information Scheme for Supervised Feature Extraction and Its Application to EEG-Based Brain-Computer Interfacing	EURASIP Journal on Advances in Signal Processing	2,008	cc-by	6,247	Hindawi Publishing Corporation EURASIP Journal on Advances in Signal Processing Volume 2008, Article ID 673040, 8 pages doi:10.1155/2008/673040 Hindawi Publishing Corporation EURASIP Journal on Advances in Signal Processing Volume 2008, Article ID 673040, 8 pages doi:10.1155/2008/673040 Hindawi Publishing Corporation EURASIP Journal on Advances in Signal Processing Volume 2008, Article ID 673040, 8 pages doi:10.1155/2008/673040 Recommended by Chein-I Chang This paper presents a novel approach for eﬃcient feature extraction using mutual information (MI). In terms of mutual information, the optimal feature extraction is creating a feature set from the data which jointly have the largest dependency on the target class. However, it is not always easy to get an accurate estimation for high-dimensional MI. In this paper, we propose an eﬃcient method for feature extraction which is based on two-dimensional MI estimates. At each step, a new feature is created that attempts to maximize the MI between the new feature and the target class and to minimize the redundancy. We will refer to this algorithm as Minimax-MIFX. The eﬀectiveness of the method is evaluated by using the classiﬁcation of electroencephalogram (EEG) signals during hand movement imagination. The results conﬁrm that the classiﬁcation accuracy obtained by Minimax- MIFX is higher than that achieved by existing feature extraction methods and by full feature set. Copyright © 2008 F. Oveisi and A. Erfanian. 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. Farid Oveisi and Abbas Erfanian Farid Oveisi and Abbas Erfanian Department of Biomedical Engineering, Faculty of Electrical Engineering, Iran University of Science and Technology, Narmak, Tehran 16844, Iran Department of Biomedical Engineering, Faculty of Electrical Engineering, Iran University of Science and Technolo Narmak, Tehran 16844, Iran Correspondence should be addressed to Abbas Erfanian, erfanian@iust.ac.ir Received 5 December 2007; Revised 29 May 2008; Accepted 3 July 2008 Received 5 December 2007; Revised 29 May 2008; Accepted 3 July 2008 Recommended by Chein-I Chang 1. INTRODUCTION training data through maximizing the variance of the projected data with aim of optimally representing the data in terms of minimal reconstruction error. However, in its feature extraction for classiﬁcation tasks, PCA does not suﬃciently use class information associated with patterns and its maximization to the variance of the projected patterns might not necessarily be in favor of discrimination among classes, thus naturally it likely loses some useful discriminating information for classiﬁcation. Classiﬁcation of the EEG signals associated with mental tasks plays an important role in the performance of the most EEG-based brain-computer interface (BCI) and reducing the dimensionality of the raw input variable space is an essential preprocessing step in the classiﬁcation process. There are two main reasons to keep the dimensionality of the input features as small as possible: computational cost and classiﬁcation accuracy. It has been observed that added irrelevant features may actually degrade the performance of classiﬁers if the number of training samples is small relative to the number of features [1]. These problems can be avoided by selecting relevant features (i.e., feature selection) or extracting new features containing maximal information about the class label from the original ones (i.e., feature extraction). g Linear discrimination analysis (LDA) is another popular linear dimensional reduction algorithm for supervised fea- ture extraction [3]. LDA computes a linear transformation by maximizing the ratio of between-class distance to within- class distance, thereby achieving maximal discrimination. In LDA, a transformation matrix from an n-dimensional feature space to a d-dimensional space is determined such that the Fisher criterion of between-class scatter over within- class scatter is maximized. LDA algorithm assumes the sample vectors of each class are generated from underlying multivariate normal distributions of common covariance matrix but diﬀerent means (i.e., homoscedastic data). Over A variety of linear feature extraction methods have been proposed. One well-known feature extraction methods may be principal component analysis (PCA) [2]. The purpose of PCA is to ﬁnd an orthogonal set of projection vectors or principal components for feature extraction from given EURASIP Journal on Advances in Signal Processing 2 the years, several extensions to the basic formulation of LDA have been proposed [4, 5]. Recently, a method based on discriminant analysis (DA) was proposed, known as subclass discriminant analysis (SDA), for describing a large number of data distributions [6]. In this approach, the underlying distribution of each class was approximated by a mixture of Gaussians. 2.1. Deﬁnition of mutual information Mutual information is a nonparametric measure of relevance between two variables. Shannon’s information theory pro- vides a suitable formalism for quantifying these concepts. Assume a random variable X representing continuous- valued random feature vector, and a discrete-valued random variable C representing the class labels. In accordance with Shannon’s information theory, the uncertainty of the class label C can be measured by entropy H(C) as p One of the most eﬀective approaches for optimal feature extraction is based on mutual information (MI). MI mea- sures the mutual dependence of two or more variables. In this context, the feature extraction process is creating a feature set from the data which jointly have largest dependency on the target class and minimal redundancy among themselves. However, it is almost impossible to get an accurate estimation for high-dimensional mutual information. In [11, 12], a method was proposed, known as MRMI, for learning linear discriminative feature transform using an approximation of the mutual information between transformed features and class labels as a criterion. The approximation is inspired by the quadratic Renyi entropy which provides a nonparametric estimate of the mutual information. However, there is no general guarantee that maximizing the approximation of mutual information using Renyi’s deﬁnition is equivalent to maximizing mutual information deﬁned by Shannon. Moreover, MRMI algorithm is subject to the curse of dimen- sionality [12]. To overcome the diﬃculties of MI estimation for feature extraction, Parzen window modeling was also employed to estimate the probability density function [13]. However, Parzen model may suﬀer from the “curse of dimensionality,” which refers to the overﬁtting of the training data when their dimension is high [14]. Due to this diﬃculty, some recent works on information-theoretic learning have proposed the use of alternative measures for MI [14], by means of an entropy estimation method that has succeeded in independent component analysis (ICA). The features are extracted one by one with maximal dependency to the target class. Although the mutual information between the features and the classes is maximized, but the proposed scheme does not produce minimal information redundancy between the extracted features. H(C) = − c∈C p(c) log p(c), (1) (1) where p(c) represents the probability of the discrete random variable C. 2.1. Deﬁnition of mutual information The uncertainty about C given a feature vector X is measured by the conditional entropy as H(C \| X) = − x p(x) c∈C p(c \| x) log p(c \| x) dx, (2) (2) where p(c \| x) is the conditional probability for the variable C given X. In general, the conditional entropy is less than or equal to the initial entropy. It is equal if and only if one has independence between two variables C and X. The amount by which the class uncertainty is decreased is, by deﬁnition, the mutual information, I(X; C) = H(C) −H(C \| X), and after applying the identities p(c, x) = p(c \| x)p(x) and p(c) = xp(c, x)dx can be expressed as I(X; C) = c∈C x p(c, x) log p(c, x) p(c)p(x)dx. (3) (3) If the mutual information between two random variables is large, it means two variables are closely related. Indeed, MI is zero if and only if the two random variables are strictly independent. 1. INTRODUCTION Then a generalized eigenvalue decomposition was used to ﬁnd the discriminant vectors that best (linearly) classify the data. All the above mentioned methods are based on the idea that a linear projection on the data is applied that maximizes the mutual information between the transformed features and the class labels. Finding the linear mapping was performed using standard gradient descent-ascent procedure which suﬀers from becoming stuck in local minima. The purpose of this paper is to introduce an eﬃcient method to extract feature with maximal dependency to the target class and minimal redundancy among themselves using two-dimensional MI estimates. The proposed method has been applied to the problem of the classiﬁcation of EEG signals during hand movement imagination. Moreover, the results of proposed method was compared to the results obtained using PCA, ICA, MRMI, and SDA. y Independent component analysis (ICA) has been also used for feature extraction. ICA is a signal processing technique in which observed random data are linearly trans- formed into components that are statistically independent from each other [7]. However, like PCA, the method is com- pletely unsupervised with regard to the class information of the data. A key question is which independent components (ICs) carry more information about the class label. In [8], a method was proposed for standard ICA to select a number of ICs (i.e., features) that carry information about the class label and a number of ICs that do not. It was shown that the proposed algorithm reduces the dimension of feature space while improving classiﬁcation performance. We have already used ICA-based feature extraction for classifying the EEG patterns associated with the resting state and the imagined hand movements [9, 10] and demonstrated the improvement of the performance. 2.2. Minimax mutual information approach to feature extraction The optimal feature extraction requires creating a new fea- ture set from the original features which jointly have largest 3 F. Oveisi and A. Erfanian features are created and included one by one such that the criteria (8) is maximized. Formally, the problem can be stated as dependency on the target class (i.e., maximal dependency). Let us denote by x the original feature set as the sample of continuous-valued random vector, and by discrete-valued random variable C the class labels. The problem is to ﬁnd a linear mapping W such that the transformed features features are created and included one by one such that the criteria (8) is maximized. Formally, the problem can be stated as wopt = argmax w I yi; c −β ys∈S I yi; ys ; yi = wTxi. (9) wopt = argmax w I yi; c −β ys∈S I yi; ys ; yi = wTxi. y = WTx (4) (4) (9) y (9) maximize the mutual information between the transformed features Y and the class labels C, I(Y, C). That is, we seek maximize the mutual information between the transformed features Y and the class labels C, I(Y, C). That is, we seek We use a genetic algorithm (GA) [18] for mutual informa- tion optimization and learning the linear mapping w. Unlike many classical optimization techniques, GA does not rely on computing local ﬁrst- or second-order derivatives to guide the search process; GA is a more general and ﬂexible method that is capable of searching wide solution spaces and avoiding local minima (i.e., it provides more possibilities of ﬁnding an optimal or near-optimal solution). To implement the GA, we use genetic algorithm and direct search toolbox for use in Matlab (The Mathworks, R2007b). The algorithm starts by generating an initial population of random candidate solutions. Each individual (chromosomes) in the population is then awarded a score based on its performance. The value of the ﬁtness function (i.e., the function to be optimize) for an individual is its score. The individuals with the best scores are chosen to be parents, which are cut and spliced together to make children. The genetic algorithm creates three types of children for the next generation: elite children, crossover children, and mutation children. Elite children are the individuals in the current generation with the best ﬁtness values. These individuals automatically survive to the next generation. 2.2. Minimax mutual information approach to feature extraction Crossover children are created by combining the genes of two chromosomes of a pair of parents in the current population. Mutation, on the other hand, arbitrarily alters one or more genes of a selected chromosome, by a random change with a probability equal to the mutation rate. These children are scored, with the best performers likely to be parents in the next generation. After some number of generations, it is hoped that the system converges with a near-optimal solution. Wopt = arg max W I(Y, C), (5) I(Y, C) = c∈C · · · p y1 · · · ym log p y1 · · · ym, c p y1 · · · ym p(c) × dy1 · · ·dym. (6) (5) (6) However, it is not always easy to get an accurate estimation for high-dimensional mutual information. It requires the knowledge on the underlying probability density functions (pdfs) of the data and the integration on these pdfs. Moreover, due to the enormous computational requirements of the method, the practical applicability of the above solution to complex classiﬁcation problems requiring a large number of features is limited. To overcome the abovementioned practical obstacle, we propose a heuristic method for feature extraction which is based on minimal-redundancy-maximal-relevance (min- imax) framework. The max-relevance and min-redundancy criterion has been already used for feature selection [15–17]. It was proved theoretically that minimax criteria is equivalent to maximal dependency (6) if one feature is added at one time [17]. This criterion is given by J = I xi; c −β xs∈S I xi; xs . (7) (7) (7) In this application, the genetic algorithm is run for 70 generations with population size of 20, crossover probability 0.8, and uniform mutation probability of 0.01. The number of individuals that automatically survive to the next genera- tion (i.e., elite individuals) is selected to be 2. The scattered function is used to create the crossover children by creating a random binary vector and selects the genes where the vector is a 1 from the ﬁrst parent, and the genes where the vector is a 0 from the second parent. According to this criteria, at each time, a new feature xi is selected with maximal dependency to the target class (i.e., maxiI(xi; c)) and minimal dependency among the new feature and already selected features (i.e., mini xs∈SI(xi; xs)). 2.2. Minimax mutual information approach to feature extraction The parameter β is the redundancy parameter which is used in considering the redundancy among input features and regulates the relative importance of the MI between the new extracted feature and the already extracted features with respect to the MI with the output class. According to this criteria, at each time, a new feature xi is selected with maximal dependency to the target class (i.e., maxiI(xi; c)) and minimal dependency among the new feature and already selected features (i.e., mini xs∈SI(xi; xs)). The parameter β is the redundancy parameter which is used in considering the redundancy among input features and regulates the relative importance of the MI between the new extracted feature and the already extracted features with respect to the MI with the output class. One is to implement MI-based feature extraction scheme, estimation of MI always poses a great diﬃculties as it requires the knowledge on the underlying probability density functions (pdfs) of the data and the integration on these pdfs. One of the most popular ways to estimate mutual information for low-dimensional data space is to use histograms as a pdf estimator. Histogram estimators can deliver satisfactory results under low-dimensional data spaces. Trappenberg et al. [19] have compared a number of MI estimation algorithms including standard histogram method, adaptive partitioning histogram method [20], and MI estimation based on the Gram-Charlier polynomial In this paper, we modiﬁed this criterion for purpose of feature extraction, namely minimax feature extraction, as follows: J = I yi; c −β ys∈S I yi; ys ; yi = wTxi, (8) (8) where yi and ys are the new and already extracted features, respectively. The parameter β was assigned the value 1/m, where m is the number of already extracted features. The proposed feature extraction method is an iterative process which begins with an empty feature set and additional EURASIP Journal on Advances in Signal Processing 4 expansion [19]. They have demonstrated that the adaptive partitioning histogram method showed superior perfor- mance in their examples. In this work, we used a two- dimensional mutual information estimation using adaptive partitioning histogram method. great number of data is lost. To overcome these problems and to shorten the experimental session, we have already developed an adaptive noise canceller (ANC) ﬁlter using artiﬁcial neural network for real-time removing the eye blinks interference from the EEG signals [21]. 3.3. Multiple classiﬁers (iii) output the set s containing the extracted features. (iii) output the set s containing the extracted features. Multiple classiﬁers are employed for classiﬁcation of extra- cted feature vectors. The Multiple Classiﬁer s are used if diﬀerent sensors are available to give information on one object. Each of the classiﬁers works independently on its own domain. The single classiﬁers are built and trained for their speciﬁc task. The ﬁnal decision is made on the results of the individual classiﬁers. In this work, for each EEG channel, separate classiﬁer is trained and the ﬁnal decision is implemented by a simple logical majority vote function. The desired output of each classiﬁer is −1 or +1. The output of classiﬁers is added and the signum function is used for computing the actual response of the classiﬁer. The block diagram of classiﬁcation process is shown in Figure 1. The diagonal linear discrimination analysis (DLDA) [23] is here considered as the classiﬁer. The classiﬁer is trained to distinguish between rest state and imaginative hand movement. 2.2. Minimax mutual information approach to feature extraction In this work, we use this method for real-time ocular artifact suppression without any visual inspection. The proposed MI-based feature extraction can be sum- marized by the following procedure: (i) initialization: 3.2. BCI competition 2003-data set III (a) set x to the initial feature set; To validate the proposed MI-based feature extraction and classiﬁcation methods for brain-computer Interfaces, the algorithms were also applied to the data set III of “BCI Competition 2003” which is obtained by Graz group [22]. This data set was recorded from a healthy subject during a feedback session. Three bipolar EEG channels were measured over C3, Cz, and C4. EEG signals were sampled with 128 Hz and was ﬁltered between 0.5 and 30 Hz. The task was to control a feedback bar in one dimension by imagination of left- or right-hand movements. The experiment included seven runs with 40 trials each. All runs were conducted on the same day with breaks of several minutes in between. The data set consists of 280 trials of 9 seconds length. The ﬁrst 2 seconds were quiet. At t = 2 seconds, an acoustic stimulus indicated the beginning of the trial, and a cross (“+”) was displayed for 1 seconds. Then, at t = 3 seconds, an arrow (left or right) was displayed as a cue stimulus. The subject was asked to use imagination as described above to move the feedback bar into the direction of the cue. (b) set s to the empty set; feature extraction (repeat until desired number of features are extracted): feature extraction (repeat until desired number of features are extracted): (ii) (a) set J = {I(wT i x, c) −β ys∈SI(wT i x, ys)} as the ﬁtness function; (b) initialize the GA; (1) specify type, size, and initial values of population; (2) specify the selection function (i.e., how the GA chooses parents for the next genera- tion); (3) specify the reproduction operators (i.e., how the genetic algorithm creates the next generation) (c) ﬁnd the weighting vector that maximizes the ﬁtness function and denote it as wopt; (d) extract the feature, y = wToptx; (e) put y into s; 3.1. Our experiments The EEG data of ﬁve healthy right-handed volunteer subjects were recorded at a sampling rate of 256 from positions Cz, T5, Pz, F3, F4, Fz, and C3 by Ag/AgCl scalp electrodes placed according to the International 10–20 system. The eye blinks were recorded by placing an electrode on the forehead above the left brow line. The signals were referenced to the right earlobe. Data were recorded for 5 seconds during each trial experiment and low-pass ﬁltered with a cutoﬀ45 Hz. Depending on the cue visual stimuli which was appeared on the monitor of computer at 2 seconds, the subject imagines either right-hand grasping or right-hand opening. If the visual stimuli was not appeared, the subject did not perform a speciﬁc task. In the present study, the tasks to be discriminated were the imagination of hand grasping and the idle state. The imaginative hand movement can be hand closing or hand opening. There were 200 trails acquired from each subject during each experiment day. 4.1. Our experiments Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 55 60 65 70 75 80 85 MRMI PCA ICA Minimax-MIFX SDA Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 55 60 65 70 75 80 85 MRMI ICA SDA PCA Minimax-MIFX Number of features (a) Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 55 60 65 70 75 80 85 MRMI PCA ICA Minimax-MIFX SDA (c) (a) (b) (b) Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 60 65 70 75 80 MRMI PCA ICA Minimax-MIFX SDA (d) (d) (c) Figure 2: Classiﬁcation accuracy for subject ST with diﬀerent sizes of feature set obtained by diﬀerent feature extraction methods: (a)–(c) diﬀerent experiment days. (d) Average classiﬁcation accuracy over diﬀerent days. MIFX with 5 features. During the second day, the best results obtained are 72.9% with 10 features using ICA, 72.3% using MRMI and 71.1% using Minimax-MIFX with 5 features, and 71.9% using full feature set. During the third experiment day, the best classiﬁcation accuracy obtained is 83.4% by using Minimax-MIFX with 5 features, while the rate is 74.0% with full feature set. Figure 2(d) shows the average classiﬁcation accuracies over three experiment days for the subject ST. It is observed that the Minimax-MIFX method provides a better performance compared to the other feature extraction methods. On average, the best rate for the subject ST is 76.5% which is obtained by Minimax-MIFX method with 5 extracted features. The average classiﬁcation performance of SDA for the subject ST is 73.96% which is poorer than that obtained by the Minimax-MIFX. The performance for full feature set is 72.43%. It is observed that the best performance of MRMI method takes place when the number of extracted parameters, variance, the mean absolute value (MAV), and 1 Hz frequency components between 1 and 35 Hz constitute the full set of features with size 44. In this application, the genetic algorithm was run for 70 generations with popu- lation size of 20, crossover probability 0.8, and mutation probability of.01. The classiﬁer is trained to distinguish between rest state and imaginative hand movement. The imaginative hand movement can be hand closing or hand opening. 4.1. Our experiments Original features are formed from 1second interval of EEG data of each channel, in the time period 2.3–3.3 seconds, during each trial of experiment. The window starting 0.3 seconds after cue presentation is used for classiﬁcation. The number of local extrema within interval, zero crossing, 5 AR One of the major problems in developing an EEG-based BCI is the eye blink artifact suppression. The traditional method of the eye blink suppression is the removal of the segment of EEG data in which eye blinks occur. This scheme is rigid and does not lend itself to adaptation. Moreover, a F. Oveisi and A. Erfanian 5 EEG Ch-1 EEG Ch-2 EEG Ch-n Original feature creation Original feature creation Original feature creation Feature extraction Feature extraction Feature extraction Classiﬁcation Classiﬁcation Classiﬁcation Σ + − ... ... ... ... Figure 1: The block diagram of classiﬁcation process. EEG Ch-1 EEG Ch-2 EEG Ch-n Original feature creation Original feature creation Original feature creation Feature extraction Feature extraction Feature extraction Classiﬁcation Classiﬁcation Classiﬁcation Σ + − ... ... ... ... Figure 1: The block diagram of classiﬁcation process. Original feature creation Feature extraction Figure 1: The block diagram of classiﬁcation process. Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 55 60 65 70 75 80 85 MRMI ICA SDA PCA Minimax-MIFX (a) Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 55 60 65 70 75 80 85 MRMI PCA ICA Minimax-MIFX SDA (b) Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 55 60 65 70 75 80 85 MRMI PCA ICA Minimax-MIFX SDA (c) Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 60 65 70 75 80 MRMI PCA ICA Minimax-MIFX SDA (d) Figure 2: Classiﬁcation accuracy for subject ST with diﬀerent sizes of feature set obtained by diﬀerent feature extraction methods: (a)–(c) diﬀerent experiment days. (d) Average classiﬁcation accuracy over diﬀerent days. 4.1. Our experiments From 200 data sets, 100 sets are randomly selected for training, while the rest is kept aside for validation purposes. Training and validating procedure is repeated 10 times and the results are averaged. Figure 2 shows the classiﬁcation accuracy for subject ST during diﬀerent experiment days for diﬀerent sizes of feature set obtained by Minimax-MIFX, PCA, MRMI, and ICA methods. During the ﬁrst day, the best classiﬁcation accuracy as high as 75.0% was obtained using Minimax- EURASIP Journal on Advances in Signal Processing 6 Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 55 60 65 70 75 80 85 MRMI ICA SDA PCA Minimax-MIFX (a) Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 55 60 65 70 75 80 85 MRMI ICA SDA PCA Minimax-MIFX (b) Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 55 60 65 70 75 80 85 MRMI ICA SDA PCA Minimax-MIFX (c) Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 55 60 65 70 75 80 85 MRMI ICA SDA PCA Minimax-MIFX (d) Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 55 60 65 70 75 80 85 MRMI ICA SDA PCA Minimax-MIFX (e) ure 3: The average of classiﬁcation accuracy over the three days for the subjects AE (a), ME (b), BM (c), and MM (d). Average iﬁcation accuracy over all days and all subjects (e). 4.1. Our experiments Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 55 60 65 70 75 80 85 MRMI ICA SDA PCA Minimax-MIFX (b) Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 55 60 65 70 75 80 85 MRMI ICA SDA PCA Minimax-MIFX (a) Minimax-MIFX Number of features Number of features (b) (a) Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 55 60 65 70 75 80 85 MRMI ICA SDA PCA Minimax-MIFX Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 55 60 65 70 75 80 85 MRMI ICA SDA PCA Minimax-MIFX Number of features Number of features (d) (c) Number of features 5 10 15 20 25 30 35 40 44 Classiﬁcation accuracy (%) 55 60 65 70 75 80 85 MRMI ICA SDA PCA Minimax-MIFX (e) (e) Figure 3: The average of classiﬁcation accuracy over the three days for the subjects AE (a), ME (b), BM (c), and MM (d). Average classiﬁcation accuracy over all days and all subjects (e). to be small. It should be noted that the MRMI method is subject to the curse of dimensionality as the number of extracted feature increases [12]. Due to this fact and low computation speed of MRMI, this method is performed for extraction of 5 and 10 features. degrades as the number of extracted features increases. The results indicate that classiﬁcation accuracy obtained by the Minimax-MIFX method is generally better than that obtained by other methods. The best classiﬁcation accuracy as high as 78.0% is obtained by Minimax-MIFX method only with 5 extracted features.The average performance of SDA is 77.85% which is identical to that obtained using Minimax- MIFX. Figure 3 shows the average of classiﬁcation accuracies over three days for all other subjects. The best classiﬁcation accuracy is obtained by the Minimax-MIFX in all subjects and is 78.4% with 5 features in AE, 80.0% with 10 features in ME, 78.37% with 20 features in BM, and 78.3% with 10 features in MM. Figure 3(e) shows the average of classiﬁcation accuracy over all subjects. The classiﬁcation performance obtained using ICA method is almost the same as that obtained using PCA. The best performance of MRMI method is achieved when ﬁve extracted features are used for classiﬁcation. However, the performance of MRMI REFERENCES [1] T. W. S. Chow and D. Huang, “Estimating optimal feature subsets using eﬃcient estimation of high-dimensional mutual information,” IEEE Transactions on Neural Networks, vol. 16, no. 1, pp. 213–224, 2005. [2] H. Li, T. Jiang, and K. Zhang, “Eﬃcient and robust feature extraction by maximum margin criterion,” IEEE Transactions on Neural Networks, vol. 17, no. 1, pp. 157–165, 2006. [3] R. O. Duda, P. E. Hart, and D. G. Stork, Pattern Classiﬁcation, Wiley-Interscience, New York, NY, USA, 2000. [4] H. Yu and J. Yang, “A direct LDA algorithm for high- dimensional data—with application to face recognition,” Pattern Recognition, vol. 34, no. 10, pp. 2067–2070, 2001. [5] R. P. W. Duin and M. Loog, “Linear dimensionality reduc- tion via a heteroscedastic extension of LDA: the Chernoﬀ criterion,” IEEE Transactions on Pattern Analysis and Machine Intelligence, vol. 26, no. 6, pp. 732–739, 2004. g p y Figure 4 shows the classiﬁcation accuracies obtained by diﬀerent feature extraction methods for diﬀerent number of extracted features. It is observed that the best classiﬁcation accuracy obtained is 90.0% using Minimax-MIFX with 7 extracted features, 87.85% using PCA with 8 features, 86.42% using ICA with 21 features, 75.71% using MRMI with 17 extracted features, and 87.14% using full feature set. It is observed that minimax-FX provides a robust performance against changes in the number of features extracted, while the performance of other feature extraction methods is sensitive with respect to the number of features. The performance of SDA for BCI competition data set is 83.57% with 15 extracted features. It is worthy to note that the best rate reported in the BCI competition 2003 for this data set is 89.3% [24]. [6] M. Zhu and A. M. Martinez, “Subclass discriminant analysis,” IEEE Transactions on Pattern Analysis and Machine Intelligence, vol. 28, no. 8, pp. 1274–1286, 2006. [7] A. Hyv¨arinen, J. Karhunen, and E. Oja, Independent Compo- nent Analysis, John Wiley & Sons, New York, NY, USA, 2001. [8] N. Kwak and C.-H. Choi, “Feature extraction based on ICA for binary classiﬁcation problems,” IEEE Transactions on Knowledge and Data Engineering, vol. 15, no. 6, pp. 1374–1388, 2003. [9] A. Erfanian and A. Erfani, “EEG-based brain-computer interface for hand grasp control: feature extraction by using ICA,” in Proceedings of the 9th Annual Conference of the Inter- national Functional Electrical Stimulation Society (IFESS ’04), Bournemouth, UK, September 2004. [10] A. Erfanian and A. 4.2. BCI competition 2003-data set III Six 0.7 second intervals of EEG data of each channel (i.e., C3 and C4) are considered during each trial of experiment. The ﬁrst window starts 0.5 seconds after cue stimulus and all 0.7 seconds windows overlap by 0.2 seconds. For each data window of each channel, one classiﬁer is designed. 7 F. Oveisi and A. Erfanian Number of features 2 4 6 8 10 12 14 16 18 20 22 Classiﬁcation accuracy (%) 60 65 70 75 80 85 90 95 MRMI ICA SDA PCA Minimax-MIFX Figure 4: Classiﬁcation accuracy obtained by using diﬀerent feature extraction methods for BCI competition 2003-data set III. Number of features 2 4 6 8 10 12 14 16 18 20 22 Classiﬁcation accuracy (%) 60 65 70 75 80 85 90 95 MRMI ICA SDA PCA Minimax-MIFX and the target class is maximized. However, the estimation of MI poses great diﬃculties as it requires estimating the multivariate probability density functions (pdfs) of the data space and the integration on these pdfs. The proposed MIFX method iteratively creates a new feature with maximal dependency to the target class and minimal redundancy among the new feature and previously extracted features. Our Minimax-MIFX scheme avoids the diﬃcult multivariate density estimation in maximizing dependency and mini- mizing redundancy. Only two-dimensional (2D) MIs are directly estimated, whereas the higher dimensional MIs are analyzed using the 2D MI estimates. The eﬀectiveness of the MIFX methods is evaluated by using the classiﬁcation of EEG signals during hand movement imagination. Our comprehensive experiments and BCI Competition 2003- Data Set III—demonstrate that the classiﬁcation accuracy can be improved by using the proposed feature extraction scheme. Figure 4: Classiﬁcation accuracy obtained by using diﬀerent feature extraction methods for BCI competition 2003-data set III. The ﬁnal decision is made on the results of the individual classiﬁers. The classiﬁers are trained to diﬀerentiate between EEG patterns associated with left- and right-hand movement imagery. The entire feature sets are formed from each data window, separately and consisted of 23 features including the number of local extrema within interval, zero crossing, energy of 8 wavelet packet nodes of a three-level decompo- sition, 5 AR parameters, variance, the mean absolute value (MAV), and the relative power in three common frequency bands of EEG spectral density—theta (4–8 Hz), alpha (9– 14 Hz), and beta (15–30 Hz). 4.2. BCI competition 2003-data set III Each classiﬁer is trained to diﬀerentiate between EEG patterns associated with left- and right-hand movement imagery. For each data window of each channel, one classiﬁer is designed. The ﬁnal decision is made on the results of the individual classiﬁers. From 280 data sets, 140 sets are assigned for training of each classiﬁer, while the rest is kept aside for validation purposes. 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https://openalex.org/W4230710683	https://hal.archives-ouvertes.fr/hal-00304259/document	English	null	Climate forcing and air quality change due to regional emissions reductions by economic sector	null	2,008	cc-by	20,409	ACPD ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 Climate and air quality by emission region and sector D. Shindell et al. Atmos. Chem. Phys. Discuss., 8, 11609–11642, 2008 www.atmos-chem-phys-discuss.net/8/11609/2008/ © Author(s) 2008. This work is distributed under the Creative Commons Attribution 3.0 License. Atmospheric Chemistry and Physics Discussions Climate forcing and air quality change due to regional emissions reductions by economic sector D. Shindell1, J.-F. Lamarque2, N. Unger1, D. Koch1, G. Faluveg1, S. Bauer1, and H. Teich1 1NASA Goddard Institute for Space Studies, New York, NY, USA 2National Center for Atmospheric Research, Boulder, CO, USA Received: 16 May 2008 – Accepted: 25 May 2008 – Published: 12 June 2008 Correspondence to: D. Shindell (dshindell@giss.nasa.gov) Published by Copernicus Publications on behalf of the European Geosciences Union. Atmos. Chem. Phys. Discuss., 8, 11609–11642, 2008 www.atmos-chem-phys-discuss.net/8/11609/2008/ © Author(s) 2008. This work is distributed under the Creative Commons Attribution 3.0 License. Atmos. Chem. Phys. Discuss., 8, 11609–11642, 2008 www.atmos-chem-phys-discuss.net/8/11609/2008/ © Author(s) 2008. This work is distributed under the Creative Commons Attribution 3.0 License. Atmospheric Chemistry and Physics Discussions ACPD ACPD We examine the air quality (AQ) and radiative forcing (RF) response to emissions re- ductions by economic sector for North America and developing Asia in the CAM and GISS composition/climate models. Decreases in annual average surface particulate 8, 11609–11642, 2008 8, 11609–11642, 2008 GISS composition/climate models. Decreases in annual avera are relatively robust, with intermodel variations in magnitude typically <30% and very 5 similar spatial structure. Surface ozone responses are small and highly model depen- dent. The largest net RF results from reductions in emissions from the North America industrial/power and developing Asia domestic fuel burning sectors. Sulfate reductions dominate the ﬁrst case, for which intermodel variations in the sulfate (or total) aerosol are relatively robust, with intermodel variations in magnitude typically <30% and very 5 similar spatial structure. Surface ozone responses are small and highly model depen- dent. The largest net RF results from reductions in emissions from the North America industrial/power and developing Asia domestic fuel burning sectors. Sulfate reductions dominate the ﬁrst case, for which intermodel variations in the sulfate (or total) aerosol Climate and air quality by emission region and sector D. Shindell et al. , ( ) optical depth (AOD) responses are ∼30% and the modeled spatial patterns of the AOD 10 reductions are highly correlated (R=0.9). Decreases in BC dominate the developing Asia domestic fuel burning case, and show substantially greater model-to-model dif- ferences. Intermodel variations in tropospheric ozone burdens are also large, though aerosol changes dominate those cases with substantial net climate forcing. The results optical depth (AOD) responses are ∼30% and the modeled spatial patterns of the AOD 10 reductions are highly correlated (R=0.9). Decreases in BC dominate the developing Asia domestic fuel burning case, and show substantially greater model-to-model dif- ferences. Intermodel variations in tropospheric ozone burdens are also large, though aerosol changes dominate those cases with substantial net climate forcing. The results Title Page Abstract Introduction Conclusions References Tables Figures ◭ ◮ ◭ ◮ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion g g indicate that across-the-board emissions reductions in domestic fuel burning in devel- 15 oping Asia and in surface transportation in North America are likely to oﬀer the greatest potential for substantial, simultaneous improvement in local air quality and near-term mitigation of global climate change via short-lived species. ACPD Conversely, reductions in industrial/power emissions have the potential to accelerate near-term warming, though indicate that across-the-board emissions reductions in domestic fuel burning in devel- 15 oping Asia and in surface transportation in North America are likely to oﬀer the greatest potential for substantial, simultaneous improvement in local air quality and near-term mitigation of global climate change via short-lived species. Conversely, reductions in industrial/power emissions have the potential to accelerate near-term warming, though p p g g they would improve AQ and have a long-term cooling eﬀect on climate. These broad 20 conclusions appear robust to intermodel diﬀerences. they would improve AQ and have a long-term cooling eﬀect on climate. These broad 20 conclusions appear robust to intermodel diﬀerences. Climate forcing and air quality change due to regional emissions reductions by economic sector 1NASA Goddard Institute for Space Studies, New York, NY, USA 2National Center for Atmospheric Research, Boulder, CO, USA Published by Copernicus Publications on behalf of the European Geosciences Union. 11609 ACPD To aid in formulation of policies to address both AQ and climate change we per- p y y g g y pp p both mitigate some of the eﬀects of global warming and improve AQ, while a poorly designed approach could improve AQ but accelerate warming. To aid in formulation of policies to address both AQ and climate change, we per- formed a series of simulations examining the impact of speciﬁc emission sectors on ra- g pp p g To aid in formulation of policies to address both AQ and climate change, we per- formed a series of simulations examining the impact of speciﬁc emission sectors on ra- To aid in formulation of policies to address both AQ and climate change, we per- formed a series of simulations examining the impact of speciﬁc emission sectors on ra- diatively active short-lived species using composition and climate models from the God- 20 dard Institute for Space Studies (GISS) and the Community Atmosphere Model (CAM) model, developed in collaboration with the National Center for Atmospheric Research (NCAR). In addition to calculating RF from short-lived species (in the GISS model), we also estimate the RF from long-lived species for comparison. These species were not simulated by the composition models, so their forcing estimates were instead based 25 on the carbon dioxide (CO2) and methane (CH4) emissions from each sector. We concentrate here on the near-term forcing over the next two decades from potential present-day emissions perturbations. This timescale is chosen to be long enough that the climate system would have suﬃcient time to respond to a substantial portion of formed a series of simulations examining the impact of speciﬁc emission sectors on ra diatively active short-lived species using composition and climate models from the God- 20 dard Institute for Space Studies (GISS) and the Community Atmosphere Model (CAM) model, developed in collaboration with the National Center for Atmospheric Research (NCAR). In addition to calculating RF from short-lived species (in the GISS model), we also estimate the RF from long-lived species for comparison. These species were not simulated by the composition models so their forcing estimates were instead based 25 diatively active short-lived species using composition and climate models from the God- 20 dard Institute for Space Studies (GISS) and the Community Atmosphere Model (CAM) model, developed in collaboration with the National Center for Atmospheric Research (NCAR). ACPD ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 change, and conversely are aﬀected by the impact of climate change on factors such as 5 temperature and humidity. Though emissions of long-lived greenhouse gases, espe- cially carbon dioxide, are the main driver of projected future climate change, AQ-related emissions have the potential to substantially aﬀect 21st Century climate as well (Shin- dell et al., 2008). In addition, AQ-related emissions reductions may lead to substantial i d h lth b ﬁt (W t t l 2006 W t t l 2007) change, and conversely are aﬀected by the impact of climate change on factors such as 5 temperature and humidity. Though emissions of long-lived greenhouse gases, espe- cially carbon dioxide, are the main driver of projected future climate change, AQ-related emissions have the potential to substantially aﬀect 21st Century climate as well (Shin- dell et al., 2008). In addition, AQ-related emissions reductions may lead to substantial economic and health beneﬁts (West et al., 2006; West et al., 2007). 10 5 Climate and air quality by emission region and sector D. Shindell et al. economic and health beneﬁts (West et al., 2006; West et al., 2007). 10 AQ regulations are typically made without consideration of their eﬀect on climate. Diﬀerent regulatory choices potentially aﬀect climate in opposite ways, however, as some AQ-related emissions lead to warming while others lead to cooling. Precipitation may also be either enhanced or suppressed by emissions changes resulting from AQ ( ) AQ regulations are typically made without consideration of their eﬀect on climate. Diﬀerent regulatory choices potentially aﬀect climate in opposite ways, however, as some AQ-related emissions lead to warming while others lead to cooling. Precipitation may also be either enhanced or suppressed by emissions changes resulting from AQ policy Hence a carefully designed regulatory approach for short lived pollutants could y y g g policy. Hence a carefully designed regulatory approach for short-lived pollutants could 15 both mitigate some of the eﬀects of global warming and improve AQ, while a poorly designed approach could improve AQ but accelerate warming. policy. Hence a carefully designed regulatory approach for short-lived pollutants could 15 both mitigate some of the eﬀects of global warming and improve AQ, while a poorly designed approach could improve AQ but accelerate warming. 1 Introduction Changes in AQ and climate typically occur on diﬀerent temporal and spatial scales. As climate change is by deﬁnition a long-term phenomenon, climate policy typically focuses on well-mixed greenhouse gases, as these have strong radiative forcing and 25 are long-lived (residence times of ∼10 years or longer) in the atmosphere. AQ, in con- 11610 trast, is usually concerned with short-lived pollutants (residence times of months or less) which do not currently play as prominent a role in climate policy. The two is- sues are linked in several ways, however. Tropospheric ozone and aerosols, among the primary short-lived pollutants regulated for AQ purposes, inﬂuence global climate trast, is usually concerned with short-lived pollutants (residence times of months or less) which do not currently play as prominent a role in climate policy. The two is- sues are linked in several ways, however. Tropospheric ozone and aerosols, among the primary short-lived pollutants regulated for AQ purposes, inﬂuence global climate trast, is usually concerned with short-lived pollutants (residence times of months or less) which do not currently play as prominent a role in climate policy. The two is- sues are linked in several ways, however. Tropospheric ozone and aerosols, among the primary short-lived pollutants regulated for AQ purposes, inﬂuence global climate ACPD ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 ) These experiments were done in support of the US Climate Change Science Pro- 5 gram (CCSP) Synthesis and Assessment Product 3.2: “Climate Projections Based on Emission Scenarios for Long-lived and Short-lived Radiatively Active Gases and Aerosols”. They were designed to examine the climate forcing due to short-lived species in a policy-relevant way by isolating the inﬂuence of economic activities subject These experiments were done in support of the US Climate Change Science Pro- 5 gram (CCSP) Synthesis and Assessment Product 3.2: “Climate Projections Based on Emission Scenarios for Long-lived and Short-lived Radiatively Active Gases and Aerosols”. They were designed to examine the climate forcing due to short-lived species in a policy-relevant way by isolating the inﬂuence of economic activities subject to regulation and/or reduction in usage (e.g. by improved eﬃciency). We look at 30% 10 reductions in total emissions from domestic fuel usage (residential and commercial fossil and biofuel burning), surface transportation, and from a combined industry and power generation sector. These three are the main anthropogenic sources for emis- sion of short-lived species and their precursors. Note that the combined industry/power sector can be largely separated into its two components for aerosols, as sulfur emis- 15 sions are largely from power generation while carbonaceous aerosol emissions are mostly from industrial processes (use of the combined sector saves computational re- sources). Since we consider across-the-board reductions in emissions from a given sector, our results are useful for assessing the potential impacts of reductions in total power/fuel usage rather than changes in the mix of power generation/transportation 20 types or in emissions control technologies targeted at speciﬁc pollutants. However, we examine both the net forcing from each sector and the contribution from individ- ual species, so that both the sectors and individual pollutants within sectors that are the most attractive climate mitigation targets can be determined. The responses to separate emissions perturbations in both North America (60–130 W, 25–60 N) and de- 25 veloping Asia (60–130 E, 0–50 N) are studied. These two regions were chosen based on their being the focus of CCSP interest and being the largest current emitter of many pollutants respectively 5 Climate and air quality by emission region and sector D. Shindell et al. to regulation and/or reduction in usage (e.g. by improved eﬃciency). ACPD In addition to calculating RF from short-lived species (in the GISS model), we also estimate the RF from long-lived species for comparison. These species were not g simulated by the composition models, so their forcing estimates were instead based 25 on the carbon dioxide (CO2) and methane (CH4) emissions from each sector. We concentrate here on the near-term forcing over the next two decades from potential present-day emissions perturbations. This timescale is chosen to be long enough that the climate system would have suﬃcient time to respond to a substantial portion of simulated by the composition models, so their forcing estimates were instead based 25 on the carbon dioxide (CO2) and methane (CH4) emissions from each sector. We concentrate here on the near-term forcing over the next two decades from potential present-day emissions perturbations. This timescale is chosen to be long enough that the climate system would have suﬃcient time to respond to a substantial portion of 11611 the forcing from short-lived species, which occurs virtually right away following emis- sions changes, but not to be so long that AQ-related emissions would likely diverge enormously from their current levels (the inﬂuence of the choice of time horizon is dis- cussed further in Sect. 5). the forcing from short-lived species, which occurs virtually right away following emis- sions changes, but not to be so long that AQ-related emissions would likely diverge enormously from their current levels (the inﬂuence of the choice of time horizon is dis- cussed further in Sect. 5). ACPD 8, 11609–11642, 2008 ACPD The components of the GISS model for Physical Understanding of Composition- Climate INteractions and Impacts (G-PUCCINI) version used here include photochem- istry from the surface to the mesosphere (Shindell et al., 2006), and sulfate, carbona- 8, 11609–11642, 2008 8, 11609–11642, 2008 ceous, sea salt, nitrate, and mineral dust aerosols (Koch et al., 2007; Bauer et al., 2006; 5 Miller et al., 2006). Dust and sea-salt are segregated into size bins, nitrate and sulfate aerosols have ﬁne and coarse modes, while only total mass is simulated for carbona- ceous aerosols. The components interact with one another, with linkages including oxidants aﬀecting sulfate, gas-phase nitrogen species aﬀecting nitrate, sulfate aﬀecting ceous, sea salt, nitrate, and mineral dust aerosols (Koch et al., 2007; Bauer et al., 2006; 5 Miller et al., 2006). Dust and sea-salt are segregated into size bins, nitrate and sulfate aerosols have ﬁne and coarse modes, while only total mass is simulated for carbona- ceous aerosols. The components interact with one another, with linkages including oxidants aﬀecting sulfate, gas-phase nitrogen species aﬀecting nitrate, sulfate aﬀecting Climate and air quality by emission region and sector D. Shindell et al. nitrogen heterogeneous chemistry, thermodynamic ammonium nitrate formation be- 10 ing aﬀected by sulfate, sea salt and mineral dust (Metzger et al., 2006), and sulfate and nitrate being absorbed onto mineral dust surfaces (Bauer et al., 2006; Bauer et al., 2007). As in our prior CCSP simulations (Shindell et al., 2007), the enhanced con- vective scavenging of aerosols in (Koch et al., 2007) was not included, leading to a g g ( ) g higher burden of these species relative to that study but values still roughly consistent 15 with observations. The simulations described here were run using a 4 by 5 degree hor- izontal resolution version of the ModelE climate model with 23-layers extending from the surface to ∼0.01 hPa (Schmidt et al., 2006). higher burden of these species relative to that study but values still roughly consistent 15 with observations. The simulations described here were run using a 4 by 5 degree hor- izontal resolution version of the ModelE climate model with 23-layers extending from the surface to ∼0.01 hPa (Schmidt et al., 2006). ACPD We look at 30% 10 reductions in total emissions from domestic fuel usage (residential and commercial fossil and biofuel burning), surface transportation, and from a combined industry and power generation sector. These three are the main anthropogenic sources for emis- sion of short-lived species and their precursors. Note that the combined industry/power p p y p sector can be largely separated into its two components for aerosols, as sulfur emis- 15 sions are largely from power generation while carbonaceous aerosol emissions are mostly from industrial processes (use of the combined sector saves computational re- sources). Since we consider across-the-board reductions in emissions from a given sector, our results are useful for assessing the potential impacts of reductions in total sector can be largely separated into its two components for aerosols, as sulfur emis- 15 sions are largely from power generation while carbonaceous aerosol emissions are mostly from industrial processes (use of the combined sector saves computational re- sources). Since we consider across-the-board reductions in emissions from a given sector, our results are useful for assessing the potential impacts of reductions in total power/fuel usage rather than changes in the mix of power generation/transportation 20 types or in emissions control technologies targeted at speciﬁc pollutants. However, we examine both the net forcing from each sector and the contribution from individ- ual species, so that both the sectors and individual pollutants within sectors that are the most attractive climate mitigation targets can be determined. The responses to g g p separate emissions perturbations in both North America (60–130 W, 25–60 N) and de- 25 veloping Asia (60–130 E, 0–50 N) are studied. These two regions were chosen based on their being the focus of CCSP interest and being the largest current emitter of many pollutants, respectively. 11612 2 Composition and climate models 2 Composition and climate models 2 ACPD ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 5 Climate and air quality by emission region and sector g Present-day simulations of trace gases and aerosols in both models agree fairly well with observations over North America for all species as in the model references given with observations over North America for all species as in the model references given previously, while sulfate, BC, OC and CO are all biased low over developing Asia. 10 Such biases are common among models, and have been linked to underestimates of developing Asian emissions in current inventories (e.g. Arellano et al., 2004; P´etron et al., 2004; Bergamaschi et al., 2000). Ratios of positive and negative forcing agents, e.g. BC/OC or SO4/BC, are fairly reasonable in both regions, however. Neither model included indirect aerosol eﬀects or radiative forcing from changes in black carbon de- 15 D. Shindell et al. previously, while sulfate, BC, OC and CO are all biased low over developing Asia. 10 Such biases are common among models, and have been linked to underestimates of developing Asian emissions in current inventories (e.g. Arellano et al., 2004; P´etron et al., 2004; Bergamaschi et al., 2000). Ratios of positive and negative forcing agents, e.g. BC/OC or SO4/BC, are fairly reasonable in both regions, however. Neither model Title Page Abstract Introduction Conclusions References Tables Figures ◭ ◮ ◭ ◮ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion 4 included indirect aerosol eﬀects or radiative forcing from changes in black carbon de- 15 position onto snow and ice surfaces. included indirect aerosol eﬀects or radiative forcing from changes in black carbon de- 15 position onto snow and ice surfaces. ACPD 8, 11609–11642, 2008 Simulations were also performed using the Community Atmosphere Model CAM3 Simulations were also performed using the Community Atmosphere Model CAM3 (Collins et al., 2006) coupled to the Model for Ozone and Related Tracers (MOZART) 20 chemistry (Horowitz et al., 2003). MOZART tropospheric chemistry is an extension of the mechanism presented in (Horowitz et al., 2003); changes include an updated terpene oxidation scheme and a better treatment of anthropogenic and natural non- methane hydrocarbons (NMHCs) treated up to isoprene, toluene and monoterpenes. (Collins et al., 2006) coupled to the Model for Ozone and Related Tracers (MOZART) 20 chemistry (Horowitz et al., 2003). MOZART tropospheric chemistry is an extension of the mechanism presented in (Horowitz et al., 2003); changes include an updated terpene oxidation scheme and a better treatment of anthropogenic and natural non- methane hydrocarbons (NMHCs) treated up to isoprene, toluene and monoterpenes. Aerosols have been extended from the work by (Lamarque et al., 2005) to include 25 actively simulated sea-salt and dust aerosols (each segregated into 4 bin sizes, from ﬁne mode to coarse mode) and a representation of ammonium nitrate that is depen- dent on sulfate following the parameterization of gas/aerosol partitioning by (Metzger Aerosols have been extended from the work by (Lamarque et al., 2005) to include 25 actively simulated sea-salt and dust aerosols (each segregated into 4 bin sizes, from ﬁne mode to coarse mode) and a representation of ammonium nitrate that is depen- dent on sulfate following the parameterization of gas/aerosol partitioning by (Metzger 11613 et al., 2002). Interactions are similar to those in the GISS model, with the excep- tion that there is no uptake of sulfate and nitrate onto mineral dust. CAM includes secondary organic aerosols, however, which GISS does not, and these are linked to the gas-phase chemistry through the oxidation of atmospheric NMHCs as in (Chung and Seinfeld, 2002). Only the bulk mass is calculated for aerosols other than sea- 5 salt and dust, and all aerosols are radiatively active. The horizontal resolution is 2 by 2.5 degrees, with 26 levels from the surface to ∼4 hPa. et al., 2002). Interactions are similar to those in the GISS model, with the excep- tion that there is no uptake of sulfate and nitrate onto mineral dust. ACPD 8, 11609–11642, 2008 CAM includes secondary organic aerosols, however, which GISS does not, and these are linked to the gas-phase chemistry through the oxidation of atmospheric NMHCs as in (Chung and Seinfeld, 2002). Only the bulk mass is calculated for aerosols other than sea- 5 salt and dust, and all aerosols are radiatively active. The horizontal resolution is 2 by 2.5 degrees, with 26 levels from the surface to ∼4 hPa. ACPD ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 , p y climate change). RF from changes in emissions of long-lived species based on such 5 estimates is discussed in Sect. 5. Our primary goal, however, is to use the composition- climate models to calculate the RF from all the short-lived species and hence to identify the relative contributions of the sectors and regions. As the forcings were expected to be small, we concentrate on the RF metric, which has little interannual variability, rather than the climate response (which would have a much lower signal to noise ratio) p y climate change). RF from changes in emissions of long-lived species based on such 5 estimates is discussed in Sect. 5. Our primary goal, however, is to use the composition- climate models to calculate the RF from all the short-lived species and hence to identify the relative contributions of the sectors and regions. As the forcings were expected to be small, we concentrate on the RF metric, which has little interannual variability, rather Climate and air quality by emission region and sector D. Shindell et al. than the climate response (which would have a much lower signal-to-noise ratio). 10 We ﬁrst compare changes in the surface concentrations relevant for AQ. We then examine burdens of radiatively active species, the optical depth for aerosols, and ﬁnally RF (though CAM did not calculate RF). We analyze the last 10 years of 11-year runs (use of the last 8 gives values with negligible diﬀerences) relative to an analogous control run. 15 3 Experimental setup Six simulations were performed, each reducing present-day emissions by 30% in one sector for one region. By using present-day emissions, the results are not tied to any Six simulations were performed, each reducing present-day emissions by 30% in one sector for one region. By using present-day emissions, the results are not tied to any particular future scenario. We use IIASA 2000 sectoral emissions (Table 1), based 20 on the 1995 EDGAR3.2 inventory extrapolated to 2000 using national and sector eco- nomic development data (Dentener et al., 2005). The exception is biomass burning emissions, which are averages over 1997–2002 from (Van der Werf et al., 2003) with emission factors from (Andreae and Merlet, 2001) for aerosols. The CAM model did sector for one region. By using present-day emissions, the results are not tied to any particular future scenario. We use IIASA 2000 sectoral emissions (Table 1), based 20 on the 1995 EDGAR3.2 inventory extrapolated to 2000 using national and sector eco- nomic development data (Dentener et al., 2005). The exception is biomass burning emissions, which are averages over 1997–2002 from (Van der Werf et al., 2003) with emission factors from (Andreae and Merlet, 2001) for aerosols. The CAM model did not perform the transportation sector simulations. 25 20 not perform the transportation sector simulations. 25 All simulations kept long-lived greenhouse gases ﬁxed at present day values as these were (generally) not part of the composition models that were used here, which instead All simulations kept long-lived greenhouse gases ﬁxed at present day values as these were (generally) not part of the composition models that were used here, which instead 11614 focused on shorter-lived pollutants. Changes in CO2 and CH4 in response to changes in anthropogenic emissions of those gases are relatively straightforward to estimate to ﬁrst-order, and do not require full 3-D chemical-transport models (though such estimate do not include all relevant feedbacks, such as the response of the carbon cycle to climate change). RF from changes in emissions of long-lived species based on such 5 estimates is discussed in Sect. 5. Our primary goal, however, is to use the composition- climate models to calculate the RF from all the short-lived species and hence to identify the relative contributions of the sectors and regions. 3 Experimental setup As the forcings were expected to be small, we concentrate on the RF metric, which has little interannual variability, rather than the climate response (which would have a much lower signal-to-noise ratio). 0 focused on shorter-lived pollutants. Changes in CO2 and CH4 in response to changes in anthropogenic emissions of those gases are relatively straightforward to estimate to ﬁrst-order, and do not require full 3-D chemical-transport models (though such estimate do not include all relevant feedbacks, such as the response of the carbon cycle to li t h ) RF f h i i i f l li d i b d h focused on shorter-lived pollutants. Changes in CO2 and CH4 in response to changes in anthropogenic emissions of those gases are relatively straightforward to estimate to ﬁrst-order, and do not require full 3-D chemical-transport models (though such estimate do not include all relevant feedbacks, such as the response of the carbon cycle to focused on shorter-lived pollutants. Changes in CO2 and CH4 in response to changes in anthropogenic emissions of those gases are relatively straightforward to estimate to ﬁrst-order, and do not require full 3-D chemical-transport models (though such estimate do not include all relevant feedbacks, such as the response of the carbon cycle to ACPD 8, 11609–11642, 2008 ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 may be related to variations in how the models simulate convective transport, planetary 5 boundary layer heights, or the transformation of BC from hydrophobic to hydrophilic. Reductions in surface ozone are generally small, with maxima of about 1–1.5 ppbv in both models but a response that extends over a larger, more spatially coherent area in the GISS model (Fig. 1). The surface ozone reduction in the GISS model also shows 5 Climate and air quality by emission region and sector D. Shindell et al. g a distinct local maximum over the Yellow Sea co-located with the largest decreases in 10 surface aerosols, while the CAM model does not. Local ozone reductions increase to levels of 1.5–3.5 ppbv during boreal summer over parts of India, China and Korea in the GISS model, but not in the CAM model. Both these annual and summer increases are statistically signiﬁcant in the GISS model (all signiﬁcance is based on the 95% con- ﬁdence level derived from the interannual variability in the simulations). Reductions 15 in surface pollution in areas far from the source region are not generally statistically signiﬁcant in these simulations. ﬁdence level derived from the interannual variability in the simulations). Reductions 15 in surface pollution in areas far from the source region are not generally statistically signiﬁcant in these simulations. Decreases in North American industrial/power emissions likewise lead to decreases in local pollutant levels (Fig 2) with the exception of BC for which no signiﬁcant ﬁdence level derived from the interannual variability in the simulations). Reductions 15 in surface pollution in areas far from the source region are not generally statistically signiﬁcant in these simulations. Decreases in North American industrial/power emissions likewise lead to decreases in local pollutant levels (Fig. 2), with the exception of BC for which no signiﬁcant changes are seen. Sulfate decreases exhibit very similar spatial structures in the two 20 models, with maxima over the Ohio River valley and reductions of more than 125 pptv extending over most of the Eastern US. In contrast to the response to reduced de- veloping Asia domestic emissions, in this case the GISS sulfate response is ∼20% larger than its CAM counterpart. 4.1 Surface pollutants Annual average local reductions take place for all the major pollutants in both mod- els in response to reductions in emissions from developing Asia domestic fuel burning p p g g (Fig. 1). The reductions have a similar spatial pattern in the two models for both BC 20 and sulfate, but the sulfate reductions are much larger in the CAM model than in the GISS model. The total change in particulate is dominated by BC, however, which shows decreases of similar magnitude (∼20% greater in the CAM model) and a nearly identical spatial structure in the two models. If we focus in on the ∼15 000 km2 with (Fig. 1). The reductions have a similar spatial pattern in the two models for both BC 20 and sulfate, but the sulfate reductions are much larger in the CAM model than in the GISS model. The total change in particulate is dominated by BC, however, which shows decreases of similar magnitude (∼20% greater in the CAM model) and a nearly identical spatial structure in the two models. If we focus in on the ∼15 000 km2 with the largest reductions (a scale more suitable for pollution studies, yet not so small as 25 to overstate the abilities of comparatively coarse-grid global models), we ﬁnd a similar 11615 result, with BC concentration reductions roughly an order of magnitude greater than those for sulfate, and an approximately 20% larger response in the CAM model (Ta- ble 2). Interestingly, the summer surface BC response is larger than the annual mean response in the GISS model, but smaller in the CAM model. Diﬀerences in seasonality result, with BC concentration reductions roughly an order of magnitude greater than those for sulfate, and an approximately 20% larger response in the CAM model (Ta- ble 2). Interestingly, the summer surface BC response is larger than the annual mean response in the GISS model, but smaller in the CAM model. Diﬀerences in seasonality ACPD ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 In all cases other than North America domestic fuel burning, substantial local re- 5 duction in both annual and summer surface particulate concentrations result from the regional sector emissions reductions (Table 2). They are especially large for the devel- oping Asia domestic case, but are also clear in the response to industrial/power and transportation emissions reductions in both regions. The annual average reductions In all cases other than North America domestic fuel burning, substantial local re- 5 duction in both annual and summer surface particulate concentrations result from the regional sector emissions reductions (Table 2). They are especially large for the devel- oping Asia domestic case, but are also clear in the response to industrial/power and transportation emissions reductions in both regions. The annual average reductions 5 Climate and air quality by emission region and sector D. Shindell et al. p g g are generally quite similar in the two models, with diﬀerences of <30% for all but one 10 of the statistically signiﬁcant local responses (Table 2) and in the large-scale aerosol responses (Figs. 1 and 2). Diﬀerences between the two models are much greater for seasonal pollutant responses (Table 2). Shifts in oxidants in response to ozone precur- sor emission decreases can sometimes outweigh eﬀects of reductions in sulfur-dioxide Title Page Abstract Introduction Conclusions References Tables Figures ◭ ◮ ◭ ◮ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion emissions, leading to increases in surface sulfate in these emission reduction experi- 15 ments (Table 2). Increases in surface aerosols are not statistically signiﬁcant, however. Nearly all the aerosols at the surface are in ﬁne mode, so that the PM2.5 (particulate matter with a mean radius of <2.5 microns) concentration changes are approximately the sum of the BC and sulfate concentration changes given in Table 2 (additional con- tributions from OC and nitrate are comparatively small). 20 4.2 Burdens and aerosol optical depths We now turn to global scale changes and their potential inﬂuence on climate. We begin by examining the global annual average change in the tropospheric burdens of radia- tively active species in the two models (Table 3). The models’ sulfate responses are We now turn to global scale changes and their potential inﬂuence on climate. ACPD 8, 11609–11642, 2008 Ozone reductions are stronger, and extend over a larger area, in the GISS model as well, as they were in the developing Asia domes- 25 tic case, with both models showing the greatest response near the western US Gulf Coast. Again the ozone reductions increase to 2–3.5 ppbv during summer over most of the eastern US in the GISS model, but not in the CAM results. Variations in the ozone response in the two models may be related to diﬀerences in incorporation of NMHCs in local pollutant levels (Fig. 2), with the exception of BC for which no signiﬁcant changes are seen. Sulfate decreases exhibit very similar spatial structures in the two 20 models, with maxima over the Ohio River valley and reductions of more than 125 pptv extending over most of the Eastern US. In contrast to the response to reduced de- veloping Asia domestic emissions, in this case the GISS sulfate response is ∼20% larger than its CAM counterpart. Ozone reductions are stronger, and extend over a changes are seen. Sulfate decreases exhibit very similar spatial structures in the two 20 models, with maxima over the Ohio River valley and reductions of more than 125 pptv extending over most of the Eastern US. In contrast to the response to reduced de- veloping Asia domestic emissions, in this case the GISS sulfate response is ∼20% larger than its CAM counterpart. Ozone reductions are stronger, and extend over a g p g larger area, in the GISS model as well, as they were in the developing Asia domes- 25 tic case, with both models showing the greatest response near the western US Gulf Coast. Again the ozone reductions increase to 2–3.5 ppbv during summer over most of the eastern US in the GISS model, but not in the CAM results. Variations in the ozone response in the two models may be related to diﬀerences in incorporation of NMHCs larger area, in the GISS model as well, as they were in the developing Asia domes- 25 tic case, with both models showing the greatest response near the western US Gulf Coast. Again the ozone reductions increase to 2–3.5 ppbv during summer over most of the eastern US in the GISS model, but not in the CAM results. Variations in the ozone response in the two models may be related to diﬀerences in incorporation of NMHCs 11616 and in gas-aerosol coupling (Sect. ACPD 8, 11609–11642, 2008 2). There is also a signiﬁcant remote response over Greenland in the GISS model (1.5–3.5 ppbv summertime decreases), consistent with the strong inﬂuence of North American emissions on surface pollution levels there (Stohl, 2006). and in gas-aerosol coupling (Sect. 2). There is also a signiﬁcant remote response over Greenland in the GISS model (1.5–3.5 ppbv summertime decreases), consistent with the strong inﬂuence of North American emissions on surface pollution levels there (Stohl, 2006). ACPD We begin by examining the global annual average change in the tropospheric burdens of radia- tively active species in the two models (Table 3). The models’ sulfate responses are quite consistent with one another. The largest changes are decreases of the global sul- 25 fate burden by ∼5.5% for reductions in developing Asia industrial/power emissions, by ∼3–4% for reductions in North American industrial/power emissions, and by 0.6–0.8% tively active species in the two models (Table 3). The models’ sulfate responses are quite consistent with one another. The largest changes are decreases of the global sul- 25 fate burden by ∼5.5% for reductions in developing Asia industrial/power emissions, by ∼3–4% for reductions in North American industrial/power emissions, and by 0.6–0.8% for reductions in developing Asia domestic fuel burning emissions. The two models’ quite consistent with one another. The largest changes are decreases of the global sul- 25 fate burden by ∼5.5% for reductions in developing Asia industrial/power emissions, by ∼3–4% for reductions in North American industrial/power emissions, and by 0.6–0.8% for reductions in developing Asia domestic fuel burning emissions. The two models’ 11617 changes in the sulfate burden in Tg agree within 5–12% (Table 3). Changes in BC and ozone burdens are less consistent between the two models. Responses are generally larger in the GISS model. This is especially so for BC burden changes in response to reductions in developing Asia emissions, in which cases the GISS model responses are about a factor of 2.5–3 times greater than the CAM model responses. 5 changes in the sulfate burden in Tg agree within 5–12% (Table 3). Changes in BC and ozone burdens are less consistent between the two models. Responses are generally larger in the GISS model. This is especially so for BC burden changes in response to reductions in developing Asia emissions, in which cases the GISS model responses are about a factor of 2.5–3 times greater than the CAM model responses. 5 ACPD ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 5 g p The climate inﬂuence of aerosols is not determined solely by the change in concen- tration or mass, but by the resulting change in optical properties. To examine this, we compare the AOD changes in the two models. The greatest reduction in AOD for the developing Asia perturbations is in the domestic case, while for North American it is in Climate and air quality by emission region and sector D. Shindell et al. the industrial/power case. The GISS and CAM models show similar AOD decreases 10 for these two perturbation experiments (Fig. 3). The two models’ responses are highly correlated spatially (R=0.8) and the global mean values diﬀer by only 20–30%. For North American industrial/power emissions, the main aerosol change is sulfate. The values of the global mean change in sulfate AOD in the two models are within 30%, the industrial/power case. The GISS and CAM models show similar AOD decreases 10 for these two perturbation experiments (Fig. 3). The two models’ responses are highly correlated spatially (R=0.8) and the global mean values diﬀer by only 20–30%. For North American industrial/power emissions, the main aerosol change is sulfate. The values of the global mean change in sulfate AOD in the two models are within 30%, and the spatial correlation of the AOD decreases is R=0.9 (the total AOD change is so 15 strongly dominated by sulfate in this case that the response pattern and magnitude are virtually identical to those shown in Fig. 3). The models are even consistent in showing a reduction in sulfate AOD over East Asia due to decreases in oxidants stemming from reduced emissions of ozone precursors in North America. Note that the ∼30% diﬀer- and the spatial correlation of the AOD decreases is R=0.9 (the total AOD change is so 15 strongly dominated by sulfate in this case that the response pattern and magnitude are virtually identical to those shown in Fig. 3). The models are even consistent in showing a reduction in sulfate AOD over East Asia due to decreases in oxidants stemming from reduced emissions of ozone precursors in North America. Note that the ∼30% diﬀer- ence in the AOD change between the models is larger than the ∼10% diﬀerence in the 20 burden change. For developing Asia domestic sector emission reductions, BC is the largest aerosol change (in absolute or percentage terms). ACPD ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 is consistent with the sulfate burden changes (Table 2), though AOD shows a larger 5 relative diﬀerence than burden. Conversely, the BC burden changes diﬀer much more than the AOD changes due to carbonaceous aerosols. This highlights how AOD (and RF) depend on several factors, including aerosol optical properties, water uptake, and vertical distribution, as well as on aerosol mass. is consistent with the sulfate burden changes (Table 2), though AOD shows a larger 5 relative diﬀerence than burden. Conversely, the BC burden changes diﬀer much more than the AOD changes due to carbonaceous aerosols. This highlights how AOD (and RF) depend on several factors, including aerosol optical properties, water uptake, and vertical distribution, as well as on aerosol mass. Climate and air quality by emission region and sector D. Shindell et al. Hence for industrial/power sector emissions reductions, where sulfate is the dom- 10 inant aerosol change, the total aerosol response appears to be reasonably robust across these two models in terms of the burden change and the magnitude and spatial pattern of AOD changes. For the domestic fuel burning sector emissions reductions, where BC has the greatest impact on climate, the aerosol changes are less robust be- Hence for industrial/power sector emissions reductions, where sulfate is the dom- 10 inant aerosol change, the total aerosol response appears to be reasonably robust across these two models in terms of the burden change and the magnitude and spatial pattern of AOD changes. For the domestic fuel burning sector emissions reductions, where BC has the greatest impact on climate, the aerosol changes are less robust be- 10 g p , g tween the two models for either the decrease in atmospheric mass of BC or the AOD 15 reductions due to individual aerosol species. Nevertheless, the total AOD change due to reductions in emissions from this sector is fairly similar in both magnitude and spatial pattern (Fig. 3). Although decreases in absorbing and reﬂective aerosols will clearly have opposing inﬂuences on climate, both models ﬁnd decreases in sulfate burdens of <1% and substantially greater percentage reductions in BC burdens. Combined with 20 tween the two models for either the decrease in atmospheric mass of BC or the AOD 15 reductions due to individual aerosol species. ACPD Nevertheless, the total AOD change due to reductions in emissions from this sector is fairly similar in both magnitude and spatial pattern (Fig. 3). Although decreases in absorbing and reﬂective aerosols will clearly have opposing inﬂuences on climate, both models ﬁnd decreases in sulfate burdens of <1% and substantially greater percentage reductions in BC burdens. Combined with 20 the larger forcing per Tg BC than sulfate, this indicates that RF will be dominated by BC changes and hence that the aerosol forcing is at least qualitatively consistent in these models, especially in its spatial structure (see also Sect. 4.3). 4.3 Radiative forcing ACPD As noted, the total AOD changes in the two models are similar (Fig. 3). In this case, the contribution to AOD from BC was not available for the CAM model, though total carbonaceous aerosol AOD changes 25 were recorded The AOD change from BC+OC is −0 0013 in the GISS model and p ence in the AOD change between the models is larger than the ∼10% diﬀerence in the 20 burden change. For developing Asia domestic sector emission reductions, BC is the largest aerosol change (in absolute or percentage terms). As noted, the total AOD changes in the two models are similar (Fig. 3). In this case, the contribution to AOD from BC was For developing Asia domestic sector emission reductions, BC is the largest aerosol change (in absolute or percentage terms). As noted, the total AOD changes in the two models are similar (Fig. 3). In this case, the contribution to AOD from BC was not available for the CAM model, though total carbonaceous aerosol AOD changes 25 were recorded. The AOD change from BC+OC is −0.0013 in the GISS model and −0.0008 in the CAM model. Much of the 47% greater response in the GISS model appears to be due to a larger eﬀect of OC on the AOD (the GISS AOD change due to OC is −0.0008, as large as the total AOD change from BC+OC in the CAM model). not available for the CAM model, though total carbonaceous aerosol AOD changes 25 were recorded. The AOD change from BC+OC is −0.0013 in the GISS model and −0.0008 in the CAM model. Much of the 47% greater response in the GISS model appears to be due to a larger eﬀect of OC on the AOD (the GISS AOD change due to OC is −0.0008, as large as the total AOD change from BC+OC in the CAM model). 11618 However, there may be important diﬀerences in the AOD change due to BC as well, to which the greater BC burden change in the GISS model certainly contributes. Also in the developing Asia domestic case, the AOD change from sulfate is twice as large in the CAM model as in the GISS model (−0.0004 and −0.0002, respectively). This ACPD ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 tor of RF, especially for regions where the forcing is greatest. In the industrial/power 5 cases, where AOD changes are largely from sulfate and are in reasonable agreement in the models, both the magnitude and spatial pattern of the RF are also likely to be quite consistent. The standard deviation in global mean RF per unit global mean AOD was 32% in a recent model intercomparison (Schulz et al., 2006), however, so that tor of RF, especially for regions where the forcing is greatest. In the industrial/power 5 cases, where AOD changes are largely from sulfate and are in reasonable agreement in the models, both the magnitude and spatial pattern of the RF are also likely to be quite consistent. The standard deviation in global mean RF per unit global mean AOD was 32% in a recent model intercomparison (Schulz et al., 2006), however, so that Climate and air quality by emission region and sector D. Shindell et al. p ( ) the total GISS/CAM intermodel variation in RF from aerosols may be slightly greater 10 than that for AOD. Part of the enhanced variability in RF versus AOD (and the discrep- ancy between contributions of individual species to AOD and RF) likely comes from the dependence of forcing on the vertical position of aerosols relative to clouds. As global AOD observations are largely clear-sky, it is diﬃcult to evaluate this aspect of the simulations. Note that the overall aerosol RF uncertainty in both models would be 15 substantially larger including aerosol indirect eﬀects. In the developing Asia domestic case, the total AOD changes are again compara- ble in magnitude and distribution. However, the contribution from sulfate (a cooling agent) diﬀeres substantially, and given the large diﬀerence in BC burden changes and in carbonaceous aerosol optical depths (from warming BC and cooling OC), the net 20 RF in the CAM model may have a substantial diﬀerent magnitude than that in the GISS model. Nonetheless, the relatively consistent response of total AOD in the GISS and CAM models (Fig. 3) suggests that the spatial pattern of RF will be fairly consistent. 4.3 Radiative forcing RF was calculated in the GISS model (Fig. 4), and is, unsurprisingly, closely related 25 to the AOD changes. The spatial pattern of the AOD changes is in fact highly corre- lated (R=0.7) with the areas of substantial (greater than ±100 mW/m2) RF in the GISS model in the cases where the net RF from short-lived species emissions is greatest: RF was calculated in the GISS model (Fig. 4), and is, unsurprisingly, closely related 25 to the AOD changes. The spatial pattern of the AOD changes is in fact highly corre- lated (R=0.7) with the areas of substantial (greater than ±100 mW/m2) RF in the GISS model in the cases where the net RF from short-lived species emissions is greatest: 11619 developing Asia domestic and North American industrial/power (Fig. 3 versus Fig. 4). Correlation values either with total RF, as in Fig. 4, or with aerosol RF only are within 0.1 of each other, emphasizing the dominance of aerosol forcing over ozone forcing in the largest local RF changes. Hence the AOD serves as a useful rough indica- developing Asia domestic and North American industrial/power (Fig. 3 versus Fig. 4). Correlation values either with total RF, as in Fig. 4, or with aerosol RF only are within 0.1 of each other, emphasizing the dominance of aerosol forcing over ozone forcing in the largest local RF changes. Hence the AOD serves as a useful rough indica- ACPD ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 (Kvalev˚ag and Myhre, 2007). 5 We also calculate the methane response to changes in oxidation for all three sets of sector experiments. With methane prescribed at present-day values, changes in mod- eled methane oxidation are due solely to changes in oxidizing agents. Note that the model’s oxidation rate fully captures spatial and seasonal variations. We also include 5 Climate and air quality by emission region and sector D. Shindell et al. the feedback of methane on its own lifetime (Prather et al., 2001). Finally, we calculate 10 the RF from these indirect methane changes as in (Ramaswamy et al., 2001). We con- sider the direct response of methane to methane emissions changes to be part of the response to emissions of long-lived species (Sect. 5), but include the indirect response of methane to oxidant changes as part of the RF due to short-lived species emissions the feedback of methane on its own lifetime (Prather et al., 2001). Finally, we calculate 10 the RF from these indirect methane changes as in (Ramaswamy et al., 2001). We con- sider the direct response of methane to methane emissions changes to be part of the response to emissions of long-lived species (Sect. 5), but include the indirect response of methane to oxidant changes as part of the RF due to short-lived species emissions (as it results from change in emissions of short-lived species and their precursors). 15 Tropospheric ozone changes take place with two distinct timescales. A nearly instan- taneous response, which we term O3 short-term (ST) occurs as a result of changes in emissions of NOx, CO, VOCs and SO2, while a slower response, O3 long-term (LT) occurs owing to the decadal timescale change in methane due to these same emission g g changes. The former is calculated by the composition model, while the latter is esti- 20 mated based on the methane response to oxidant changes. As both eﬀects result from change in emissions of short-lived species, both are included in the short-lived portion of Table 4. We can now examine the contribution of individual species to the RF from emissions We can now examine the contribution of individual species to the RF from emissions perturbations in the GISS model and compare the totals across sectors and regions. 25 The eﬀect of the perturbations is generally larger for developing Asian than North Amer- ican emissions. ACPD We have evaluated the RF due to reductions in the emissions of short-lived species in carbonaceous aerosol optical depths (from warming BC and cooling OC), the net 20 RF in the CAM model may have a substantial diﬀerent magnitude than that in the GISS model. Nonetheless, the relatively consistent response of total AOD in the GISS and CAM models (Fig. 3) suggests that the spatial pattern of RF will be fairly consistent. We have evaluated the RF due to reductions in the emissions of short-lived species We have evaluated the RF due to reductions in the emissions of short-lived species and their precursors in the GISS model for each individual forcing agent (Table 4). 25 In addition to the direct forcing due to ozone and aerosol changes, we include within the response to short-lived emissions several indirect eﬀects. The indirect eﬀects of aerosols on clouds are thought to be important, but the RF from these eﬀects is quite uncertain, with recent model estimates diﬀering by roughly a factor of ﬁve (Penner et and their precursors in the GISS model for each individual forcing agent (Table 4). 25 In addition to the direct forcing due to ozone and aerosol changes, we include within the response to short-lived emissions several indirect eﬀects. The indirect eﬀects of aerosols on clouds are thought to be important, but the RF from these eﬀects is quite uncertain, with recent model estimates diﬀering by roughly a factor of ﬁve (Penner et 11620 al., 2006). Hence rather than calculate these forcings directly in the models (which is computationally expensive as well as uncertain), we estimate their magnitude as in (Fuglestvedt et al., 2008) in which the indirect forcing is simply 1.5 times the direct sulfate forcing over land and 2 times the direct sulfate forcing over oceans, based on (Kvalev˚ag and Myhre, 2007). 5 al., 2006). Hence rather than calculate these forcings directly in the models (which is computationally expensive as well as uncertain), we estimate their magnitude as in (Fuglestvedt et al., 2008) in which the indirect forcing is simply 1.5 times the direct sulfate forcing over land and 2 times the direct sulfate forcing over oceans, based on ACPD ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 for both regions for the industry/power generation sector (∼30–40 mW/m2), a larger 5 response to surface transportation for North America, and larger response to domestic fuel burning emissions reductions from developing Asia. The largest positive forcing comes from the industrial/power sector, while the greatest negative forcing comes from developing Asia domestic fuel burning, whose net RF is almost exactly equal to the for both regions for the industry/power generation sector (∼30–40 mW/m2), a larger 5 response to surface transportation for North America, and larger response to domestic fuel burning emissions reductions from developing Asia. The largest positive forcing comes from the industrial/power sector, while the greatest negative forcing comes from developing Asia domestic fuel burning, whose net RF is almost exactly equal to the Climate and air quality by emission region and sector D. Shindell et al. RF from BC alone. For this sector, the net short-lived emission RF for developing Asia 10 is an order of magnitude larger than for North America, primarily owing to the much larger RF from BC decreases. Conversely, the net RF from short-lived emissions from the surface transportation sector is an order of magnitude larger for North America than for developing Asia, largely due to the diﬀerence in the sulfur content of fuels. Coupling between aerosols and oxidants is clear in the responses. Ozone increases 15 in response to reductions in North American emissions from the domestic or indus- trial/power sectors despite reduced emissions of ozone precursors, especially NOx for which the industrial/power sector in particular is a large source. This response is due to reductions in SO2 emissions, which lead to altered radiative ﬂuxes and to reduced Coupling between aerosols and oxidants is clear in the responses. Ozone increases 15 in response to reductions in North American emissions from the domestic or indus- trial/power sectors despite reduced emissions of ozone precursors, especially NOx for which the industrial/power sector in particular is a large source. This response is due to reductions in SO2 emissions, which lead to altered radiative ﬂuxes and to reduced Coupling between aerosols and oxidants is clear in the responses. Ozone increases 15 in response to reductions in North American emissions from the domestic or indus- trial/power sectors despite reduced emissions of ozone precursors, especially NOx for which the industrial/power sector in particular is a large source. ACPD The only RF from an individual species to reach 10 mW/m2 from a North American perturbation is the sulfate forcing from industrial/power emissions. In contrast, forcings from sulfate, BC, OC and ozone exceed 10 mW/m2 in response to perturbations in the GISS model and compare the totals across sectors and regions. 25 The eﬀect of the perturbations is generally larger for developing Asian than North Amer- ican emissions. The only RF from an individual species to reach 10 mW/m2 from a North American perturbation is the sulfate forcing from industrial/power emissions. In contrast, forcings from sulfate, BC, OC and ozone exceed 10 mW/m2 in response to 11621 perturbations in developing Asia, with the largest response due to decreased BC when domestic fuel burning emissions are reduced (−42 mW/m2). The total RF due to re- duced short-lived species emissions does not show such a simple relationship between the two regions. The net RF from short-lived species shows comparably large values 2 ACPD 8, 11609–11642, 2008 This will lead to additional uncertainty in the net RF, especially for the surface transportation sector for which the ozone response plays a substantial role, albeit still a small one compared to th i ﬂ f l Title Page Abstract Introduction Conclusions References Tables Figures ◭ ◮ ◭ ◮ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion ACPD 8, 11609–11642, 2008 ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 g y g Though forcings were calculated for the GISS model only, we have shown that values 5 for RF from sulfate are likely to be fairly consistent in the two models, indicating that the response to industrial/power sector perturbations is relatively robust. Uncertainties in the response of BC are larger, and could have a substantial eﬀect on RF. Given the dominance of BC in the RF from the domestic fuel burning sector, the net RF may be diﬀerent in magnitude than that shown here but is likely to be a substantial 10 negative value despite uncertainties. The RF from tropospheric ozone is also uncertain given the large diﬀerences in the burden changes in the two models. This will lead to additional uncertainty in the net RF, especially for the surface transportation sector for which the ozone response plays a substantial role albeit still a small one compared to g y g Though forcings were calculated for the GISS model only, we have shown that values 5 for RF from sulfate are likely to be fairly consistent in the two models, indicating that the response to industrial/power sector perturbations is relatively robust. Uncertainties in the response of BC are larger, and could have a substantial eﬀect on RF. Given the dominance of BC in the RF from the domestic fuel burning sector, the net RF 5 Climate and air quality by emission region and sector in the response of BC are larger, and could have a substantial eﬀect on RF. Given the dominance of BC in the RF from the domestic fuel burning sector, the net RF may be diﬀerent in magnitude than that shown here but is likely to be a substantial 10 negative value despite uncertainties. The RF from tropospheric ozone is also uncertain given the large diﬀerences in the burden changes in the two models. This will lead to additional uncertainty in the net RF, especially for the surface transportation sector for which the ozone response plays a substantial role, albeit still a small one compared to the inﬂuence of aerosols. 15 D. Shindell et al. may be diﬀerent in magnitude than that shown here but is likely to be a substantial 10 negative value despite uncertainties. The RF from tropospheric ozone is also uncertain given the large diﬀerences in the burden changes in the two models. ACPD This response is due to reductions in SO2 emissions, which lead to altered radiative ﬂuxes and to reduced 15 2 heterogeneous nitrogen chemistry on sulfate surfaces (Bell et al., 2005; Liao and Se- 20 infeld, 2005). Analogous results were seen previously for the response of ozone to emissions changes in these sectors in 2030 (Unger et al., 2008). Similarly, sulfate abundances are enhanced in response to reductions in North American surface trans- portation emissions. This occurs because reductions in CO and VOC emissions shift p the OH/HO2 ratio toward OH, enhancing gas-phase sulfate formation. This indirect in- 25 ﬂuence of oxidant changes outweighs the direct impact of reduced SO2 emissions for North American surface transportation, with low-sulfur fuels, but the opposite is true for developing Asia surface transportation emissions (Table 4). The spatial pattern of the RF reveals that aside from developing Asia surface trans- The spatial pattern of the RF reveals that aside from developing Asia surface trans- 11622 portation and North America domestic, the RF in response to emissions reduction from the other four region/sector combinations all show substantial forcing extending over large parts of the Northern Hemisphere (NH) (Fig. 4). This “remote” forcing is in addition to a strongly localized maximum near the source region. portation and North America domestic, the RF in response to emissions reduction from the other four region/sector combinations all show substantial forcing extending over large parts of the Northern Hemisphere (NH) (Fig. 4). This “remote” forcing is in addition to a strongly localized maximum near the source region. ACPD ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 reduction in emissions from power generation relative to continued use of the less ef- 5 ﬁcient technology. While the resulting decrease in CO2 emissions would indeed still aﬀect atmospheric concentrations a century later, short-lived species emissions result- ing from power generation to provide lightning at that time will depend upon the power consumption of 22nd Century lighting equipment, which is unlikely to have any clear re- 5 Climate and air quality by emission region and sector D. Shindell et al. g g lationship with a present-day mandate for CFL technology. Hence the 50 and 100 year 10 time horizons are not well-suited to short-lived species emissions. Conversely, using a very short timescale of only a few years or less would overemphasize the response to short-lived species emissions. This response is much faster than the response to long-lived species emissions and can give opposite forcing to the longer term response lationship with a present-day mandate for CFL technology. Hence the 50 and 100 year 10 time horizons are not well-suited to short-lived species emissions. Conversely, using a very short timescale of only a few years or less would overemphasize the response to short-lived species emissions. This response is much faster than the response to long-lived species emissions and can give opposite forcing to the longer term response when looking at all eﬀects (Table 4) or even when looking only at the eﬀect of short- 15 lived precursor emissions (Wild et al., 2001; Fuglestvedt et al., 1999). While the RF response to short-lived species emissions changes is nearly instantaneous (typically days to months for all but those forcing that take place via methane changes), we be- lieve the 20-year time horizon provides a reasonable balance between the eﬀects of when looking at all eﬀects (Table 4) or even when looking only at the eﬀect of short- 15 lived precursor emissions (Wild et al., 2001; Fuglestvedt et al., 1999). 5 Relative importance of long-lived and short-lived emissions To explore the total forcing in response to both short- and long-lived species emis- sions reductions from our three sectors, we calculate forcing based on the estimated atmospheric carbon dioxide response to 30% reductions in current emissions from each sector (Table 4). For these calculations, we use sectoral CO2 emissions from a 20 year 2000 inventory (updated from Olivier and Berdowski (2001)). Atmospheric con- To explore the total forcing in response to both short- and long-lived species emis- sions reductions from our three sectors, we calculate forcing based on the estimated atmospheric carbon dioxide response to 30% reductions in current emissions from each sector (Table 4). For these calculations, we use sectoral CO2 emissions from a 20 year 2000 inventory (updated from Olivier and Berdowski (2001)). Atmospheric con- centration changes and the resulting forcing are calculated for the 20-year time horizon, as in the Intergovernmental Panel on Climate Change Third Assessment Report (Ra- maswamy et al., 2001). We believe that using a longer time horizon, such as the 50 or 100 year values also used in that report, is not as useful for comparison with forc- 25 ing from short-lived emissions. As short-lived species responses times are less than a year, use of a many decades long timescale would in a sense be assuming that atmospheric carbon dioxide response to 30% reductions in current emissions from each sector (Table 4). For these calculations, we use sectoral CO2 emissions from a 20 year 2000 inventory (updated from Olivier and Berdowski (2001)). Atmospheric con- centration changes and the resulting forcing are calculated for the 20-year time horizon, as in the Intergovernmental Panel on Climate Change Third Assessment Report (Ra- maswamy et al., 2001). We believe that using a longer time horizon, such as the 50 20 or 100 year values also used in that report, is not as useful for comparison with forc- 25 ing from short-lived emissions. As short-lived species responses times are less than a year, use of a many decades long timescale would in a sense be assuming that 11623 changes in short-lived species precursor emissions took place and then were main- tained without further modiﬁcation for another 50 or 100 years, an implausible scenario for most emission changes. For example, mandating a shift from incandescent to more eﬃcient compact ﬂuorescent lighting (CFL) during the next 10 years would lead to a ACPD While the RF response to short-lived species emissions changes is nearly instantaneous (typically days to months for all but those forcing that take place via methane changes), we be- lieve the 20-year time horizon provides a reasonable balance between the eﬀects of long and short lived species is a widely used time horizon in the literature and is long- and short-lived species, is a widely used time horizon in the literature, and is 20 consistent with timescales for air quality policy. Using a longer time horizon would of course give larger relative importance to forcing from long-lived emissions, and vice- versa, and we stress that there is no “best” timescale. long- and short-lived species, is a widely used time horizon in the literature, and is 20 consistent with timescales for air quality policy. Using a longer time horizon would of course give larger relative importance to forcing from long-lived emissions, and vice- versa, and we stress that there is no “best” timescale. We also calculate the methane response to 30% reductions in regional fossil fuel methane emissions. Emissions are taken from (Fung et al., 1991), with the coal burning 25 source assigned to the power generation sector. As noted previously, simulations did not include interactive methane. We instead calculate the response of methane to the reduced emissions oﬄine, again including the feedback of methane on its own lifetime. We also include the longer-term response of ozone to the methane changes, 11624 the O3 LT response discussed previously. While strictly speaking this portion of the O3 LT response should be included under long-term emissions, the values are so small that we group all the O3 responses together in Table 4. As with the indirect methane response to short-lived precursor emissions, RF is calculated as in (Ramaswamy et al., 2001). 5 the O3 LT response discussed previously. While strictly speaking this portion of the O3 LT response should be included under long-term emissions, the values are so small that we group all the O3 responses together in Table 4. As with the indirect methane response to short-lived precursor emissions, RF is calculated as in (Ramaswamy et al., 2001). 5 ACPD ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 al., 2001). 5 Looking at the response to long-lived emissions alone, reductions in North Ameri- can industrial/power generation sector emissions are more than twice as eﬀective in mitigating RF as reductions in the surface transportation sector. The inclusion of the response to emissions of short-lived species alters the comparison markedly, with net Climate and air quality by emission region and sector D. Shindell et al. total RF from North American surface transportation emissions reductions now more 10 than double that for the North American industrial/power generation sector. For de- veloping Asia, the industrial/power generation sector is by far the most eﬀective for CO2, and long-lived emissions in general, but the inclusion of the response to short- lived species emissions makes the total net RF from the domestic fuel burning sector total RF from North American surface transportation emissions reductions now more 10 than double that for the North American industrial/power generation sector. For de- veloping Asia, the industrial/power generation sector is by far the most eﬀective for CO2, and long-lived emissions in general, but the inclusion of the response to short- lived species emissions makes the total net RF from the domestic fuel burning sector larger. Hence the inclusion of RF due to short-lived species emissions reductions 15 strongly shifts the relative importance of emissions from the diﬀerent economic sec- tors when looking at the 20-year time horizon. Relative to the values due to CO2 and CH4 (direct), adding the eﬀects of short-lived pollutants increases the forcing from the developing Asia domestic fuel burning sector several times over, enhances the forcing larger. Hence the inclusion of RF due to short-lived species emissions reductions 15 strongly shifts the relative importance of emissions from the diﬀerent economic sec- tors when looking at the 20-year time horizon. Relative to the values due to CO2 and CH4 (direct), adding the eﬀects of short-lived pollutants increases the forcing from the developing Asia domestic fuel burning sector several times over, enhances the forcing p g g g from North American transportation sector emissions by ∼2/3, and reduces the net 20 forcing from industrial/power emissions by ∼50–75%. We reiterate that the timescales for these species diﬀer dramatically, so that short-term and long-term total forcings can be substantially diﬀerent from one another. ACPD ACPD 6 Discussion and conclusion 8, 11609–11642, 2008 8, 11609–11642, 2008 The key results for radiative forcing can be summarized as follows. Reductions in surface transportation emissions in North America have a net cooling eﬀect (negative Climate and air quality by emission region and sector forcing). Negative forcing from long-lived emissions is compounded by negative forcing 5 from short-lived emissions, which results from the net eﬀect of several factors of com- parable magnitude: (1) reductions in BC (which is largely from diesel in this sector), (2) reduction in ozone precursors, which leads to direct reductions in ozone and indirect increases in methane (via reduced oxidation capacity), and (3) decreases in CO and forcing). Negative forcing from long-lived emissions is compounded by negative forcing 5 from short-lived emissions, which results from the net eﬀect of several factors of com- parable magnitude: (1) reductions in BC (which is largely from diesel in this sector), (2) reduction in ozone precursors, which leads to direct reductions in ozone and indirect increases in methane (via reduced oxidation capacity), and (3) decreases in CO and D. Shindell et al. ( p y) ( ) VOC emissions which shift the OH/HO2 ratio toward OH, thereby indirectly leading to 10 increased sulfate (Table 4), as noted previously. Note that the contrasting inﬂuence of OH changes on sulfate and methane reﬂects the diﬀerence between the more global methane oxidation and the more regional sulfate oxidation, and implies that these indi- rect eﬀects are quite sensitive to the NOx/(CO+VOCs) emission ratio. Direct impacts VOC emissions which shift the OH/HO2 ratio toward OH, thereby indirectly leading to 10 increased sulfate (Table 4), as noted previously. Note that the contrasting inﬂuence of OH changes on sulfate and methane reﬂects the diﬀerence between the more global methane oxidation and the more regional sulfate oxidation, and implies that these indi- rect eﬀects are quite sensitive to the NOx/(CO+VOCs) emission ratio. Direct impacts q x ( ) p on sulfate via SO2 emission changes are small owing to the low-sulfur fuel used in 15 North America. Reductions in developing Asia domestic emissions also have a sub- stantial cooling eﬀect via short-lived species emissions which results primarily from reductions in BC and ozone. There is a net decrease in surface particulate and ozone in both cases (Table 2), and hence reducing emissions from these regional sectors oﬀers a way to simultaneously improve human health and mitigate climate warming. to consideration of the eﬀects of long-lived emissions alone. to consideration of the eﬀects of long-lived emissions alone. ACPD This is especially important in the case of industrial/power generation emissions reductions, for which the short and long term forcings are of the opposite sign. They would hence lead to near-term warming 25 but long-term cooling. In the cases where emissions reductions lead to substantial from North American transportation sector emissions by ∼2/3, and reduces the net 20 forcing from industrial/power emissions by ∼50–75%. We reiterate that the timescales for these species diﬀer dramatically, so that short-term and long-term total forcings can be substantially diﬀerent from one another. This is especially important in the case of industrial/power generation emissions reductions, for which the short and long p g g term forcings are of the opposite sign. They would hence lead to near-term warming 25 but long-term cooling. In the cases where emissions reductions lead to substantial negative forcing via short-lived emissions, developing Asia domestic fuel burning and North America surface transportation, inclusion of the short-lived pollutants dramati- cally enhances near-term climate change mitigation potentials of these sectors relative 11625 ACPD ACPD tive impacts as incoming radiation is more abundant at lower latitudes. The GISS and CAM results diﬀer in the magnitude of the total AOD change resulting from the develop- ing Asia domestic sector perturbations by 22%, and this sector/region has the largest inﬂuence in both models for both AOD and surface pollution. However, substantial dif- ferences are seen in the two models’ BC change, which dominates the RF in this case, and further work to understand the diﬀerences in the aerosol simulations in these mod CAM results diﬀer in the magnitude of the total AOD change resulting from the develop- ing Asia domestic sector perturbations by 22%, and this sector/region has the largest 8, 11609–11642, 2008 8, 11609–11642, 2008 g p y g g inﬂuence in both models for both AOD and surface pollution. However, substantial dif- 5 ferences are seen in the two models’ BC change, which dominates the RF in this case, and further work to understand the diﬀerences in the aerosol simulations in these mod- els, which are substantial for present-day climatologies as well as for many aspects of the response to perturbations (Shindell et al., 2008), would clearly be useful. inﬂuence in both models for both AOD and surface pollution. However, substantial dif- 5 ferences are seen in the two models’ BC change, which dominates the RF in this case, and further work to understand the diﬀerences in the aerosol simulations in these mod- els, which are substantial for present-day climatologies as well as for many aspects of the response to perturbations (Shindell et al., 2008), would clearly be useful. Climate and air quality by emission region and sector D. Shindell et al. p p ( ) y Identical emissions inventories were used in the two models. Emissions can be 10 diﬃcult to quantify, however, especially for carbonaceous aerosols. The inventory of (Bond et al., 2004), for example, has anthropogenic non-biomass burning emissions that are 46% greater for BC and 57% greater for OC for North America than those used here. Similarly, for developing Asia it has 17% less BC and 19% less OC. The 1984 p p ( ) y Identical emissions inventories were used in the two models. Emissions can be 10 diﬃcult to quantify, however, especially for carbonaceous aerosols. 6 Discussion and conclusion In 20 contrast, industrial/power generation emissions have their largest eﬀect on climate via short-lived emissions through sulfate, and hence the net near-term eﬀect of reductions in short-lived species emissions is a positive forcing. O ll d l i A i d ti i i ﬀ th t t l Overall, developing Asia domestic emissions oﬀer the strongest leverage on near- term climate forcing via short-lived species emissions. This is partially a result of their 25 magnitude, and also because they yield a much greater eﬀect via BC changes than via sulfate changes. It also stems from their occurrence at lower latitudes than North Overall, developing Asia domestic emissions oﬀer the strongest leverage on near- term climate forcing via short-lived species emissions. This is partially a result of their 25 magnitude, and also because they yield a much greater eﬀect via BC changes than via sulfate changes. It also stems from their occurrence at lower latitudes than North 11626 American (or European) emissions, which enhances their photochemical and radia- tive impacts as incoming radiation is more abundant at lower latitudes. The GISS and CAM results diﬀer in the magnitude of the total AOD change resulting from the develop- ing Asia domestic sector perturbations by 22%, and this sector/region has the largest inﬂuence in both models for both AOD and surface pollution. However, substantial dif- 5 ferences are seen in the two models’ BC change, which dominates the RF in this case, and further work to understand the diﬀerences in the aerosol simulations in these mod- els, which are substantial for present-day climatologies as well as for many aspects of the response to perturbations (Shindell et al., 2008), would clearly be useful. American (or European) emissions, which enhances their photochemical and radia- tive impacts as incoming radiation is more abundant at lower latitudes. The GISS and ACPD The inventory of (Bond et al., 2004), for example, has anthropogenic non-biomass burning emissions that are 46% greater for BC and 57% greater for OC for North America than those used here. Similarly, for developing Asia it has 17% less BC and 19% less OC. The 1984 10 (Bond et al., 2004), for example, has anthropogenic non-biomass burning emissions that are 46% greater for BC and 57% greater for OC for North America than those used here. Similarly, for developing Asia it has 17% less BC and 19% less OC. The 1984 emissions inventory of (Cooke et al., 1999) has much larger (26–77%) carbonaceous 15 aerosol emissions for both regions from fossil fuels than (Bond et al., 2004). Thus the magnitude of the BC and OC forcings calculated here might be fairly diﬀerent using one of these other inventories. The sign of the net BC+OC eﬀect appears robust, however, as the ratio of emissions of warming BC to cooling OC is within 10% in the three inventories. The lone exception is that the BC/OC ratio is 40% more for North 20 America in (Cooke et al., 1999) compared with (Bond et al., 2004). Even this would emissions inventory of (Cooke et al., 1999) has much larger (26–77%) carbonaceous 15 aerosol emissions for both regions from fossil fuels than (Bond et al., 2004). Thus the magnitude of the BC and OC forcings calculated here might be fairly diﬀerent using one of these other inventories. The sign of the net BC+OC eﬀect appears robust, however, as the ratio of emissions of warming BC to cooling OC is within 10% in the three inventories. The lone exception is that the BC/OC ratio is 40% more for North 20 America in (Cooke et al., 1999) compared with (Bond et al., 2004). Even this would simply enhance the net cooling eﬀect seen here in response to reduced emissions from the surface transportation sector there, however, and would not dramatically alter our conclusions. Uncertainties in emissions of sulfate and ozone precursors are generally smaller 25 The spatial pattern of RF varies substantially between the regional sectors. The RF reduction from decreases in developing Asia domestic emissions extends over much of the NH (Fig. 4). ACPD Hence emission changes from this region could in some cases have a larger near-term eﬀect on the RF in other parts of the NH via short-lived 11627 species emissions than changes in those areas’ own local emissions. Additionally, re- ductions in transportation or domestic sector emissions cause zonal mean RF due to short-lived species emissions of the same sign throughout the NH, but reductions in industrial/power emissions induce a substantial negative RF in the Arctic while yielding positive RF at lower latitudes in the GISS model (Fig. 4). In this instance, sulfate re- ductions dominate the forcing closer to the emissions at lower latitudes, while BC and ozone are the most important (and roughly comparable) in the Arctic. species emissions than changes in those areas’ own local emissions. Additionally, re- ductions in transportation or domestic sector emissions cause zonal mean RF due to short-lived species emissions of the same sign throughout the NH, but reductions in industrial/power emissions induce a substantial negative RF in the Arctic while yielding positive RF at lower latitudes in the GISS model (Fig. 4). In this instance, sulfate re- ductions dominate the forcing closer to the emissions at lower latitudes, while BC and ozone are the most important (and roughly comparable) in the Arctic. species emissions than changes in those areas’ own local emissions. Additionally, re- ductions in transportation or domestic sector emissions cause zonal mean RF due to short-lived species emissions of the same sign throughout the NH, but reductions in industrial/power emissions induce a substantial negative RF in the Arctic while yielding positive RF at lower latitudes in the GISS model (Fig. 4). In this instance, sulfate re- ACPD ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 positive RF at lower latitudes in the GISS model (Fig. 4). In this instance, sulfate re- 5 ductions dominate the forcing closer to the emissions at lower latitudes, while BC and ozone are the most important (and roughly comparable) in the Arctic. 5 Climate and air quality by emission region and sector The response of AQ, especially particulate, appears to be more robust than the cli- mate forcing across models. This is perhaps not surprising given that RF depends not mate forcing across models. This is perhaps not surprising given that RF depends not only on concentrations, but also on such things as water uptake of aerosols, the verti- 10 cal distribution of aerosols and ozone, and the position of ozone and aerosols relative to clouds. Surface ozone amounts are determined by complex, non-linear photochem- istry involving an assortment of NMHCs, so that diﬀerences in the models’ chemical mechanisms may play a role in the greater uncertainties in surface ozone than in sur- D. Shindell et al. only on concentrations, but also on such things as water uptake of aerosols, the verti- 10 cal distribution of aerosols and ozone, and the position of ozone and aerosols relative to clouds. Surface ozone amounts are determined by complex, non-linear photochem- istry involving an assortment of NMHCs, so that diﬀerences in the models’ chemical mechanisms may play a role in the greater uncertainties in surface ozone than in sur- face aerosols. Additionally, surface pollution levels are particularly important to human 15 health in highly polluted areas, which often cover small areas and are diﬃcult to simu- late correctly in relatively low-resolution global models. Thus although the responses are similar in these two models, they may suﬀer from biases common to both. Hence our ability to predict both the AQ and RF responses to emissions perturbations has important limitations due to the relatively coarse resolution and simpliﬁed aerosol and 20 cloud physics used in current global models. ACPD This work complements prior regional and sectoral perturbations studies examining the response of gas-phase species only (Berntsen et al., 2005; Fuglestvedt et al., 1999; Derwent et al., 2001), aerosol species only (Koch et al., 2007), or both but with a sub- This work complements prior regional and sectoral perturbations studies examining the response of gas-phase species only (Berntsen et al., 2005; Fuglestvedt et al., 1999; Derwent et al., 2001), aerosol species only (Koch et al., 2007), or both but with a sub- set of the species included here (Unger et al., 2008) or for a single sector for the 25 entire globe (Fuglestvedt et al., 2008). Our analyses suggest that changes in aerosol emissions often have the largest potential leverage on near-term climate forcing via short-lived species emissions. Due to large intermodel diﬀerences, the role of ozone changes cannot yet be as clearly assessed, although it is likely that these will have a , ), p y ( , ), set of the species included here (Unger et al., 2008) or for a single sector for the 25 entire globe (Fuglestvedt et al., 2008). Our analyses suggest that changes in aerosol emissions often have the largest potential leverage on near-term climate forcing via short-lived species emissions. Due to large intermodel diﬀerences, the role of ozone changes cannot yet be as clearly assessed, although it is likely that these will have a ) p y ( ) set of the species included here (Unger et al., 2008) or for a single sector for the 25 entire globe (Fuglestvedt et al., 2008). Our analyses suggest that changes in aerosol emissions often have the largest potential leverage on near-term climate forcing via short-lived species emissions. Due to large intermodel diﬀerences, the role of ozone changes cannot yet be as clearly assessed, although it is likely that these will have a 11628 smaller but still important eﬀect both on air quality and climate. Further work is required to more thoroughly characterize the robustness of these conclusions across a larger number of models and emissions inventories, to explore the impact of aerosol mixing and aerosol indirect eﬀects on clouds more fully, and to examine alternative emissions smaller but still important eﬀect both on air quality and climate. Acknowledgements. We thank the NASA Atmospheric Chemistry Modeling and Analysis Pro- gram (ACMAP). JFL was supported by the SciDAC project from the Department of Energy. This research used resources of the National Energy Research Scientiﬁc Computing Center, which is supported by the Oﬃce of Science of the US Department of Energy under Contract No. DE-AC03-76SF00098. The National Center for Atmospheric Research is operated by the 25 University Corporation for Atmospheric Research under sponsorship of the National Science Foundation. ACPD ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 scenarios considering changes in the mix of sources constituting a given sector and 5 the inﬂuence of potential technological changes. The latter could be designed to re- duce emissions of particular pollutants, while not aﬀecting others. Our results for the RF from individual species provide a guide to the potential impact of such technolo- gies. However, we note that these technologies could also have eﬀects on overall fuel i b l i h ﬃi f i l scenarios considering changes in the mix of sources constituting a given sector and 5 the inﬂuence of potential technological changes. The latter could be designed to re- duce emissions of particular pollutants, while not aﬀecting others. Our results for the RF from individual species provide a guide to the potential impact of such technolo- gies. However, we note that these technologies could also have eﬀects on overall fuel consumption by altering the eﬃciency of a particular process. 10 5 Climate and air quality by emission region and sector D. Shindell et al. consumption by altering the eﬃciency of a particular process. 10 While there are many diﬃculties associated with inclusion of short-lived species emissions in international treaties (Rypdal et al., 2005; Bond and Sun, 2005), there are also many tangible near-term beneﬁts in human health, agriculture and forestry that result from improved AQ. Hence a strategy with a dual focus on both AQ and consumption by altering the eﬃciency of a particular process. 10 While there are many diﬃculties associated with inclusion of short-lived species emissions in international treaties (Rypdal et al., 2005; Bond and Sun, 2005), there are also many tangible near-term beneﬁts in human health, agriculture and forestry that result from improved AQ. Hence a strategy with a dual focus on both AQ and y p emissions in international treaties (Rypdal et al., 2005; Bond and Sun, 2005), there are also many tangible near-term beneﬁts in human health, agriculture and forestry that result from improved AQ. Hence a strategy with a dual focus on both AQ and climate change mitigation may present a unique opportunity to engage parties and na- 15 tions not yet fully commited to climate change mitigation for its own sake. ACPD Further work is required to more thoroughly characterize the robustness of these conclusions across a larger number of models and emissions inventories, to explore the impact of aerosol mixing and aerosol indirect eﬀects on clouds more fully, and to examine alternative emissions ACPD Although this is only an initial study, this work suggests that reducing emissions from the developing Asia domestic fuel burning and North America surface transportation sectors may be among the best options for pursuing such a strategy, providing substantial beneﬁts for air quality while simultaneously contributing to climate change mitigation. 20 p gy climate change mitigation may present a unique opportunity to engage parties and na- 15 tions not yet fully commited to climate change mitigation for its own sake. Although this is only an initial study, this work suggests that reducing emissions from the developing Asia domestic fuel burning and North America surface transportation sectors may be among the best options for pursuing such a strategy, providing substantial beneﬁts for air quality while simultaneously contributing to climate change mitigation. 20 climate change mitigation may present a unique opportunity to engage parties and na- 15 tions not yet fully commited to climate change mitigation for its own sake. Although this is only an initial study, this work suggests that reducing emissions from the developing Asia domestic fuel burning and North America surface transportation sectors may be among the best options for pursuing such a strategy, providing substantial beneﬁts for air quality while simultaneously contributing to climate change mitigation. 20 20 Acknowledgements. We thank the NASA Atmospheric Chemistry Modeling and Analysis Pro- gram (ACMAP). JFL was supported by the SciDAC project from the Department of Energy. This research used resources of the National Energy Research Scientiﬁc Computing Center, which is supported by the Oﬃce of Science of the US Department of Energy under Contract No DE AC03 76SF00098 The National Center for Atmospheric Research is operated by the 25 11629 ACPD ACPD Andreae, M. O. and Merlet, P.: Emission of trace gases and aerosols from biomass burning, Global Biogeochem. Cy., 15, 955–966, 2001. 8, 11609–11642, 2008 Arellano, A. F. J., Kasibhatla, P. S., Giglio, L., van der Werf, G. R., and Randerson, J. T.: Top- down estimates of global CO sources using mopitt measurements, Geophys. Res. Lett., 31, L01104, doi:10.1029/2003GL018609, 2004. 5 Climate and air quality by emission region and sector Bauer, S. E., Mishchenko, M. I., Lacis, A., Zhang, S., Perlwitz, J., and Metzger, S. M.: Do sulfate and nitrate coatings on mineral dust have important eﬀects on radiative properties and climate modeling?, J. 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J., and Giglio, L.: Carbon emissions from ﬁres in tropical and subtropical ecosystems, Global Change Biol., 9, 547–562., 2003. g West, J. J., Fiore, A. M., Horowitz, L. W., and Mauzerall, D. L.: Global health beneﬁts of miti- gating ozone pollution with methane emission controls, P. Natl. Acad. Sci., 103, 3988–3993, 30 2006. 30 West, J. J., Szopa, S., and Hauglustaine, D. A.: Human mortality eﬀects of future concentrations of tropospheric ozone, C. r. Geosci., doi:10.1016/j.crte.2007.08.005, 339, 775–783, 2007. 11633 ACPD 8, 11609–11642, 2008 ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 Climate and air quality by emission region and sector Table 1. Regional Annual Average Emissions by Sector (Tg/yr). Sector NOx CO SO2 BC OC CO2 CH4 Industrial/Power: North America Developing Asia 2.7 3.4 7 4 8.8 16.6 0.003 0.17 0.003 0.22 3890 5285 5.8 6.0 Domestic: North America Developing Asia 0.3 0.5 6 126 0.2 2.8 0.06 2.09 0.20 6.80 745 920 0 0 Surface North America Transportation: Developing Asia 3.5 3.4 70 38 0.5 1.1 0.18 0.43 0.24 0.60 1740 693 0 0 Emissions are in Tg N/yr for NOx, Tg S/yr for SO2, in Tg C/yr for BC and OC, and Tg/yr of the species for others. CO2 emissions are from the EDGAR 2000 inventory (updated from (Olivier and Berdowski, 2001)). The industry and power generation sector includes the EDGAR categories industrial, power generation, other transformation sector (reﬁneries, coke ovens, gas works, etc.), non-energy use and chemical feedstocks, oil production, transmission and handling, and cement manufacturing. Surface transportation includes on and oﬀroad transport and international shipping. Domestic includes residential and commercial fossil fuel usage and residential biofuel usage. Methane emissions are based on the coal burning source from (Fung et al., 1991). Sources for other emissions are given in Sect. 3. Table 1. Regional Annual Average Emissions by Sector (Tg/yr). D. Shindell et al. Emissions are in Tg N/yr for NOx, Tg S/yr for SO2, in Tg C/yr for BC and OC, and Tg/yr of the species for others. CO2 emissions are from the EDGAR 2000 inventory (updated from (Olivier and Berdowski, 2001)). The industry and power generation sector includes the EDGAR categories industrial, power generation, other transformation sector (reﬁneries, coke ovens, gas works, etc.), non-energy use and chemical feedstocks, oil production, transmission and handling, and cement manufacturing. Surface transportation includes on and oﬀroad transport and international shipping. Domestic includes residential and commercial fossil fuel usage and residential biofuel usage. Methane emissions are based on the coal burning source from (Fung et al., 1991). Sources for other emissions are given in Sect. 3. Emissions are in Tg N/yr for NOx, Tg S/yr for SO2, in Tg C/yr for BC and OC, and Tg/yr of the species for others. CO2 emissions are from the EDGAR 2000 inventory (updated from (Olivier and Berdowski, 2001)). ACPD 8, 11609–11642, 2008 ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 Climate and air quality by emission region and sector D. Shindell et al. 11634 ACPD 8, 11609–11642, 2008 The industry and power generation sector includes the EDGAR categories industrial, power generation, other transformation sector (reﬁneries, coke ovens, gas works, etc.), non-energy use and chemical feedstocks, oil production, transmission and handling, and cement manufacturing. Surface transportation includes on and oﬀroad transport and international shipping. Domestic includes residential and commercial fossil fuel usage and residential biofuel usage. Methane emissions are based on the coal burning source from (Fung et al., 1991). Sources for other emissions are given in Sect. 3. 11635 Values in brackets are not statistically signiﬁcant (as in Table 2). Values in parentheses are the percent change relative to each model’s own burden in its control run. ACPD 8, 11609–11642, 2008 ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 Table 2. Local change in surface pollutants (pptv) due to 30% emissions reductions in the given region and sector Region & sector summer SO4 summer BC annual SO4 annual BC North America domestic [+105] -198 [−62] [−70] [+18] [−44] [−53] [−14] North America surface transportation [+75] NA −235 NA [+36] NA −169 NA North America industry/power −383 −285 [−47] [21] −268 −164 [−14] [12] Developing Asia domestic −127 −264 −2430 −1815 [−64] −228 −1630 −2049 Developing Asia surface transportation [−119] NA −791 NA [−41] NA −436 NA Developing Asia industry/power −558 −264 −612 −178 −328 −242 −280 −309 Values are averages over comparable areas (∼15 000 km2) containing local maxima with the ﬁrst row giving results from the GISS PUCCINI model and the second from the CAM MOZART model for each sector. Values in brackets are not statistically signiﬁcant (signiﬁcance is based on the 95% conﬁdence level derived from the interannual variability). Table 2. Local change in surface pollutants (pptv) due to 30% emissions reductions in the given region and sector Climate and air quality by emission region and sector D. Shindell et al. Title Page Abstract Introduction Conclusions References Tables Figures ◭ ◮ ◭ ◮ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Values are averages over comparable areas (∼15 000 km2) containing local maxima with the ﬁrst row giving results from the GISS PUCCINI model and the second from the CAM MOZART model for each sector. Values in brackets are not statistically signiﬁcant (signiﬁcance is based on the 95% conﬁdence level derived from the interannual variability). 11636 ACPD 8, 11609–11642, 2008 ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 Climate and air quality by emission region and sector Table 3. Tropospheric burden changes due to 30% emissions reductions in the given region and sector (Tg and %). D. Shindell et al. Region & sector SO4 GISS SO4 CAM BC GISS BC CAM O3 GISS O3 CAM North America domestic [−0.0020] (−0.1%) [−0.0006] (−0.1%) −0.0029 (−1.1%) −0.0017 (−0.8%) [0.12] (−0.4%) −0.65 (−2.3%) North America surface transportation [−0.0029] (−0.2%) NA −0.0015 (−0.6%) NA −0.69 (−2.2%) NA North America industry/power −0.0460 (−2.9%) −0.0484 (−3.7%) [−0.0006] (−0.2%) [0.0004] (0.2%) −1.20 (−3.8%) −0.55 (−2.0%) Developing Asia domestic −0.0091 (−0.6%) −0.0101 (−0.8%) −0.0302 (−11.6%) −0.0093 (−4.4%) −1.14 (−3.6%) −0.65 (−2.3%) Developing Asia surface transportation −0.0068 (−0.4%) NA −0.0056 (−2.2%) NA −1.31 (−4.1%) NA Developing Asia industry/power −0.0834 (−5.3%) −0.0733 (−5.6%) −0.0027 (−1.0%) −0.0009 (−0.4%) −0.87 (−2.8%) −0.55 (−2.0%) Values in brackets are not statistically signiﬁcant (as in Table 2). Values in parentheses are the percent change relative to each model’s own burden in its control run. Values in brackets are not statistically signiﬁcant (as in Table 2). Values in parentheses are the percent change relative to each model’s own burden in its control run. 11637 ACPD 8, 11609–11642, 2008 ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 Table 4. Global mean radiative forcing due to 30% emissions reductions in the given region and sector in the GISS model (mW/m2). Climate and air quality by emission region and sector North America domestic North America transporta- tion North America industry & power Developing Asia domestic Developing Asia surface transportation Developing Asia industry & power Short-lived Emissions Sulfate, direct 0 −3 14 0 2 13 Sufate, indirect 0 −5 24 0 3 21 Black carbon −3 −5 −2 −42 −8 −4 Organic carbon 2 0 −1 13 1 0 Nitrate 1 1 0 1 2 −1 All aerosols 0 −12 35 −28 0 29 Ozone, ST 2 −5 5 −12 −5 −1 Ozone, LT 0 1 −2 −1 3 −1 CH4, indirect 1 4 2 −2 8 6 All short-lived 4 −14 42 −41 1 34 Long-lived Emissions CO2, 20 year −8 −21 −48 −9 −8 −60 CH4, direct 0 0 −8 0 0 −9 Total −4 −35 −14 −50 −6 −35 D. Shindell et al. Values are instantaneous tropopause forcings annually averaged and are grouped by the timescales for the emissions causing the indicated forcings. For methane, indirect forcing via changes in oxidants is included with ozone and aerosols in “all short-lived”, while direct forcing from reductions in methane emissions is given under long-lived. For ozone, short-term (ST) response is that due to changes in ozone precursor and aerosol emissions, while long-term (LT) is that due to changes in methane (see text for additional details). Signiﬁcance thresholds (90%) for individual forcings are ∼4 mW/m2. 11638 ACPD 8, 11609–11642, 2008 ACPD O3 (ppbv), Dev. Asia, Domestic, CAM -1.8 -1 .6 1 1.4 1.8 -.2 .2 -.6 O3 (ppbv), Dev. Asia, Domestic, GISS -1.8 -1 .6 1 1.4 1.8 -.2 .2 -.6 BC (pptv), Dev. Asia, Domestic, GISS -1800 -1000 1000 1800 -200 200 BC (pptv), Dev. Asia, Domestic, CAM -1800 -1000 1000 1800 -200 200 SO4 (pptv), Dev. Asia, Domestic, GISS -225 -125 125 225 -25 25 SO4 (pptv), Dev. Asia, Domestic, CAM -225 -125 125 225 -25 25 Fig. 1. Surface pollutant response to 30% reduction in Developing Asian domestic fu sector emissions in the GISS (left) and CAM (right) models for the indicated pollutan O3 (ppbv), Dev. Asia, Domestic, CAM -1.8 -1 .6 1 1.4 1.8 -.2 .2 -.6 O3 (ppbv), Dev. Asia, Domestic, GISS -1.8 -1 .6 1 1.4 1.8 -.2 .2 -.6 BC (pptv), Dev. Asia, Domestic, GISS -1800 -1000 1000 1800 -200 200 BC (pptv), Dev. Asia, Domestic, CAM -1800 -1000 1000 1800 -200 200 SO4 (pptv), Dev. Asia, Domestic, GISS -225 -125 125 225 -25 25 SO4 (pptv), Dev. Asia, Domestic, CAM -225 -125 125 225 -25 25 Fig. 1. Surface pollutant response to 30% reduction in Developing Asian domestic fuel burning sector emissions in the GISS (left) and CAM (right) models for the indicated pollutants. 8, 11609–11642, 2008 8, 11609–11642, 2008 BC (pptv), Dev. Asia, Domestic, GISS SO4 (pptv), Dev. Asia, Domestic, GISS -225 -125 125 225 -25 25 BC (pptv), Dev. Asia, Domestic, CAM SO4 (pptv), Dev. Asia, Domestic, CAM -225 -125 125 225 -25 25 Climate and air quality by emission region and sector D. Shindell et al. Title Page Abstract Introduction Conclusions References Tables Figures ◭ ◮ ◭ ◮ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Fig. 1. Surface pollutant response to 30% reduction in Developing Asian domestic fuel burning sector emissions in the GISS (left) and CAM (right) models for the indicated pollutants. 11639 ACPD 8, 11609–11642, 2008 ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 -1.8 -1 .6 1 1.4 1.8 -.2 .2 -.6 O3 (pptv), N. America, Ind/Pow, GISS O3 (pptv), N. America, Ind/Pow, CAM -1.8 -1 .6 1 1.4 1.8 -.2 .2 -.6 SO4 (pptv), N. America, Ind/Pow, GISS -225 -125 125 225 -25 25 SO4 (pptv), N. America, Ind/Pow, CAM -225 -125 125 225 -25 25 Fig. 2. As Fig. 1 but for 30% reduction in North American industrial and power generation emissions (decreases in BC are all less than 20 pptv, and hence are not shown). -1.8 -1 .6 1 1.4 1.8 -.2 .2 -.6 O3 (pptv), N. America, Ind/Pow, GISS O3 (pptv), N. America, Ind/Pow, CAM -1.8 -1 .6 1 1.4 1.8 -.2 .2 -.6 SO4 (pptv), N. America, Ind/Pow, GISS -225 -125 125 225 -25 25 SO4 (pptv), N. America, Ind/Pow, CAM -225 -125 125 225 -25 25 Fig. 2. As Fig. 1 but for 30% reduction in North American industrial and power gener emissions (decreases in BC are all less than 20 pptv and hence are not shown) rica, Ind/Pow, GISS SO4 (pptv), N. America, Ind/Pow, CAM Climate and air quality by emission region and sector O3 (pptv) N America Ind/Pow CAM SO4 (pptv), N. America, Ind/Pow, CAM -225 -125 125 225 -25 25 O3 (pptv), N. America, Ind/Pow, GISS SO4 (pptv), N. America, Ind/Pow, GISS -225 -125 125 225 -25 25 D. Shindell et al. Title Page Abstract Introduction Conclusions References Tables Figures ◭ ◮ ◭ ◮ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion O3 (pptv), N. America, Ind/Pow, CAM Fig. 2. As Fig. 1 but for 30% reduction in North American industrial and power generation emissions (decreases in BC are all less than 20 pptv, and hence are not shown). 11640 ACPD 8, 11609–11642, 2008 ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 x 10-2 -3 -2 -1.2 .8 1.2 2 3 -.4 .4 -.8 Developing Asia, Domestic, GISS -.15 Developing Asia, Domestic, CAM -.12 North America, Industrial/Power, GISS -.09 North America, Industrial/Power, CAM -.12 Fig. 3. Annual average total aerosol optical depth change due to 30% reductions in emis- sions from the given region and economic sector in the GISS (top) and CAM (bottom) models. Changes greater than ∼0.012 are statistically signiﬁcant (95%). Values in the upper right give the global annual mean. x 10-2 -3 -2 -1.2 .8 1.2 2 3 -.4 .4 -.8 Developing Asia, Domestic, GISS -.15 Developing Asia, Domestic, CAM -.12 North America, Industrial/Power, GISS -.09 North America, Industrial/Power, CAM -.12 -.15 Climate and air quality by emission region and sector North America, Industrial/Power, GISS -.09 North America, Industrial/Power, CAM -.12 D. Shindell et al. Title Page Abstract Introduction Conclusions References Tables Figures ◭ ◮ ◭ ◮ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Developing Asia, Domestic, CAM Fig. 3. Annual average total aerosol optical depth change due to 30% reductions in emis- sions from the given region and economic sector in the GISS (top) and CAM (bottom) models. Changes greater than ∼0.012 are statistically signiﬁcant (95%). Values in the upper right give the global annual mean. 11641 ACPD 8, 11609–11642, 2008 ACPD 8, 11609–11642, 2008 8, 11609–11642, 2008 Developing Asia, Domestic North America, Industrial/Power Developing Asia, Industrial/Power Developing Asia, Transportation North America, Transportation North America, Domestic -49 38 6 } } -45 -33 -11 } } } -1 -3 -2 } } } } -94 -108 -34 } } } -27 3 5 } } } -25 -28 -14 } } } Fig. 4. Annual average instantaneous tropopause radiative forcing (mW/m2) from short-lived species in the GISS model due to 30% reductions in emissions from the given region and eco- nomic sector. Values include the direct forcing from ozone and aerosols, but not indirect aerosol or chemical eﬀects (O3 LT, methane indirect, and sulfate indirect in Table 4). Area weighted av- erages over 60–90 N, 28–60 N, and 28 S to 28 N are given on the edges. Signiﬁcance thresh- olds (95%) are roughly 100 mW/m2, so that local responses are generally signiﬁcant while of the “remote” responses only the negative forcing covering much of the NH in the developing Asia domestic sector simulation is signiﬁcant. Developing Asia, Domestic North America, Industrial/Power Developing Asia, Industrial/Power Developing Asia, Transportation North America, Transportation North America, Domestic -49 38 6 } } -45 -33 -11 } } } -1 -3 -2 } } } } -94 -108 -34 } } } -27 3 5 } } } -25 -28 -14 } } } North America, Domestic Developing Asia, Domestic } Climate and air quality by emission region and sector D. Shindell et al. Title Page Abstract Introduction Conclusions References Tables Figures ◭ ◮ ◭ ◮ Back Close Full Screen / Esc Printer-friendly Version Interactive Discussion Fig. 4. Annual average instantaneous tropopause radiative forcing (mW/m2) from short-lived species in the GISS model due to 30% reductions in emissions from the given region and eco- nomic sector. Values include the direct forcing from ozone and aerosols, but not indirect aerosol or chemical eﬀects (O3 LT, methane indirect, and sulfate indirect in Table 4). Area weighted av- erages over 60–90 N, 28–60 N, and 28 S to 28 N are given on the edges. Signiﬁcance thresh- olds (95%) are roughly 100 mW/m2, so that local responses are generally signiﬁcant while of the “remote” responses only the negative forcing covering much of the NH in the developing Asia domestic sector simulation is signiﬁcant. 11642
https://openalex.org/W2808412012	https://figshare.com/articles/journal_contribution/Toll-like_receptors_signaling_pathways_as_a_potential_therapeutic_target_in_cardiovascular_disease/23458577/1/files/41289594.pdf	English	null	Toll-like Receptors Signaling Pathways as a Potential Therapeutic Target in Cardiovascular Disease	Current pharmaceutical design	2,018	cc-by	12,804	Published in Current Pharmaceutical Design Link to external publisher version https://doi.org/10.2174/1381612824666180614090224 Link to external publisher version https://doi.org/10.2174/1381612824666180614090224 Accepted version Citation for this work (American Psychological Association 7th edition) Parizadeh, S. M., Ghandehar, M., Heydari-Majd, M., Seifi, S., Mardani, R., Parizadeh, S. M., Ghayour- Mobarhan, M., Ferns, G., Hassanian, S. M., & Avan, A. (2018). Toll-like receptors signaling pathways as a potential therapeutic target in cardiovascular disease (Version 1). 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Toll-like receptors signaling pathways as a potential therapeutic target in cardiovascular disease Seyed Mostafa Parizadeh, Maryam Ghandehar, Motahareh Heydari-Majd, Sima Seifi, Ramin Mardani, Seyed Mohamahdreza Parizadeh, Majid Ghayour-Mobarhan, Gordon Ferns, Seyed Mahdi Hassanian, Amir Avan Document Version Accepted version Accepted version Tolllike receptors signaling pathways as a potential therapeutic target in cardiovascular disease Article (Accepted Version) Parizadeh, Seyed Mostafa, Ghandehar, Maryam, Heydari-Majd, Motahareh, Seifi, Sima, Mardani, Ramin, Parizadeh, Seyed Mohamahdreza, Ghayour-Mobarhan, Majid, Ferns, Gordon, Hassanian, Seyed Mahdi and Avan, Amir (2018) Toll-like receptors signaling pathways as a potential therapeutic target in cardiovascular disease. Current Pharmaceutical Design, 24 (17). pp. 1887-1898. 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Discover more of the University’s research at https://sussex.figshare.com/ Tolllike receptors signaling pathways as a potential therapeutic target in cardiovascular disease Copyright and reuse: py g Sussex Research Online is a digital repository of the research output of the University Copyright and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable, the material made available in SRO has been checked for eligibility before being made available. Copies of full text items generally can be reproduced, displayed or performed and given to third parties in any format or medium for personal research or study, educational, or not-for-profit purposes without prior permission or charge, provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. http://sro.sussex.ac.uk http://sro.sussex.ac.uk Affiliations 1) Metabolic syndrome Research center, Mashhad University of Medical Sciences, Mashhad, Iran 2) Department of Medical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. 3) Antimicrobial Resistance Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran; 4) Brighton & Sussex Medical School, Division of Medical Education, Falmer, Brighton, Sussex BN1 9PH, UK. 5) Department of Modern Sciences and Technologies; Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Toll-like receptors signaling pathways as a potential therapeutic target in cardiovascular disease Seyed Mostafa Parizadeh1,, Maryam Ghandehari1,, Motahareh Heydari-majd1, Sima Seifi1, Ramin Mardani1, Seyed Mohamahdreza Parizadeh1, Majid Ghayour-Mobarhan1, Gordon A. Ferns4, Seyed Mahdi Hassanian1,2,#, Amir Avan1,5,# # Corresponding Author: Amir Avan, Ph.D. Metabolic syndrome Research Center, Mashhad University of Medical Sciences, Mashhad, Iran. Tel:+9851138002298, Fax: +985118002287; E-mail: avana@mums.ac.ir & amir_avan@yahoo.com Seyed Mahdi Hassanian Ph.D. Department of Medical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Tel:+9851138002298, Fax: +985118002287; E-mail: hasanianmehrm@mums.ac.ir Seyed Mostafa Parizadeh1,, Maryam Ghandehari1,, Motahareh Heydari-majd1, Sima Seifi1, Ramin Mardani1, Seyed Mohamahdreza Parizadeh1, Majid Ghayour-Mobarhan1, Gordon A. Ferns4, Seyed Mahdi Hassanian1,2,#, Amir Avan1,5,# CAD in TLR title: Running r autho first as contributed Equally * Grant: This study was supported by a grant awarded to Dr Amir Avan (931753) by the Mashhad University of Medical Sciences. Grant: This study was supported by a grant awarded to Dr Amir Avan (931753) by the Mashhad University of Medical Sciences. Disclosure: The authors have no conflict of interest to disclose. Disclosure: The authors have no conflict of interest to disclose. 1 1 Abstract Cardiovascular disease (CVD) is one of the most important causes of morbidity and mortality, and associated with an important economic burden globally. Over the last decade, the prevalence of CVD has been rising globally, and is now associated with millions of death annually in both developed and developing countries. There is good evidence that the immune system is involved in the pathophysiology of CVD. Toll-like receptors (TLRs) and their down-stream signaling pathways play an important role in the immune system. Recent studies have suggested that the TLRs are involved in atherogenesis, including stroke, myocardial infarction, ischemia-reperfusion injury, cardiac remodeling and development of heart failure (HF). In this review we have summarized the recent studies investigating the role of TLRs in CVD and the potential for using TLRs signaling pathways as a therapeutic target in CVD. Key words: cardiovascular disease, immune system, Toll-like receptors, signaling pathway 2 Introduction Cardiovascular disease (CVD) is a major health problem with a high prevalence of morbidity and mortality worldwide, and particularly in developing countries. Over the last decade, the prevalence of CVD has risen alarmingly and it is now estimated that about 17 million deaths, and a very high rate of complications result from CVD and this imposes huge economic burden on healthcare system (1, 2). Ischemic heart disease and cerebrovascular disease are responsible for a large percentage of patients with CVD (3). Although a number of studies have been performed to clarify the mechanisms involved in the pathogenesis of CVD, it remains a complex disease that is incompletely understood. Recently molecular investigations of CVD pathophysiology have revealed some interesting new insights, that may have a bearing on its treatment and prevention. Toll-like receptors (TLRs) and their signaling pathways have emerged as being potentially important in the pathophysiology of CVD. The immune system has two main arms; the innate immune system (also called non-specific) and adaptive immune system (also called acquired or specific). The innate immune system plays a fundamental role as the first line of defensive mechanisms for sensing and eliminating invading pathogens (4). The Toll-like receptors (TLRs) are a group of receptors which related to a family of pattern recognition receptors (PRRs) that distinguish different molecular pattern of invading microbial pathogens called pathogen associated molecular patterns (PAMPs) (5). TLRs are expressed in various cell types, including immune cells and several non-immune cell types such as cardiovascular system cells (6, 7). After recognition of PAMPs the stimulation of TLRs results in multi- steps events which finally activates expression of a variety of genes related to 3 inflammation and immune response (8-10). There is increasing evidence that shows that there are many interactions between the immune system and various human disease (7, 11). There are associations between polymorphisms of the TLRs and signaling pathways which are related with different diseases such as autoimmune diseases, diabetes, cancers and also cardiovascular disease (7). The TLRs are involved in many pathophysiological processes suggests that with pharmacological manipulation of TLRs pathway as one of these signaling pathways can be a potential target for therapeutic management of cardiovascular disease. The aim of this review is to summarize recent studies in exciting field of role of TLRs in CVD for improvement of understandings pathophysiology and using TLRs signaling pathways as a therapeutic target in CVD (Table 1-2). Characteristics of Toll-like receptors The Toll-like receptors (TLRs) are type I transmembrane glycoproteins, that play a role as a part of immune system which related to a family of pattern recognition receptors (PRRs) that distinguish different molecular pattern of invading microbial pathogens called pathogen associated molecular patterns (PAMPs) such as lipid, protein, lipoprotein and nucleic acid (5). TLRs have two major domains: an intracellular portion that called the Toll/IL-1 receptor (TIR) domain and extracellular portion which contains leucine-rich repeats (LRRs) (5). Eleven types of TLRs (TLR1-TLR11) have been identified in human and for recognition of cytoplasmic and extracellular PAMPs, some of them are localized on the cell surface and others exist in intracellular compartments, localized in endosome, lysosome, endolysosome and endoplasmic reticulum (6, 12). Cell surface TLRs include type 1, 2, 4, 5, 6, 10 and 11 and in contrast 4 to these TLRs, intracellular TLRs that known as antiviral TLRs include type 3, 7, 8 and 9 (6). Stimulation of the TLR leads to initiating activation several signaling pathways which some of them are particular to specific type of TLR and some of them are common to all TLRs (6). TLRs are being expressed in various cell types, including immune cells in innate and adaptive system and several non-immune cell types such as fibroblast cells, endothelial cells and epithelial cells (13). The stimulation of TLRs results in a multi-step process which finally lads to the activation of regulatory pathways of the innate and adaptive immune system by producing inflammatory cytokines and other mediators (12). Toll-like receptors activation in immune and non-immune related conditions As mentioned above, TLRs can recognize conserved structural motifs of microbial pathogens known as PAMPs such as lipid, protein, lipoprotein and nucleic acid (5). For example, TLR2 recognizes specific components of cell wall in gram- negative or positive bacteria, viruses, mycobacteria, parasites and fungi, TLR4 recognizes lipopolysaccharide (LPS) of gram-negative bacteria and TLRs 3, 7, 8 and 9 (14) bind with viral PAMPs. Other TLRs bind to protein ligands (14). It was demonstrated that dendritic cells as a one type of immune system cells can be activated without existence of microbial pathogen and host tissue injury (15). There has been increasing evidence that in certain conditions TLRs can be also be activated by non- pathogen molecules such as host-derived molecule that could be release from tissues underwent physiological stress or any injury which is known as danger signal. These molecules are referred to as a danger-associated molecular pattern (DAMPs) include heat shock proteins (HSPs), fibronectin, fibrinogen, low-molecular-weight hyaluronic 5 acid, surfactant A protein, heparin sulfate, single- or double-strand RNA and high mobility group box-1 (HMGB-1) (16, 17). Alterations in the extracellular matrix could be a trigger for TLRs stimulation in the any presence of pathogens (6). Stimulation and activation of TLRs with host-derived molecules has risen attention to possible association between role of TLRs and CVD especially in development of atherosclerosis and heart failure. Toll-like receptors signaling and its association with microRNA TIR domain-containing adaptor molecules are different in TLRs signaling (18). At present, four major TIR domain-containing adaptors were identified: 1) myeloid differentiation factor 88 (MyD88), 2) TIR domain-containing adaptor protein (TIRAP), 3) TIR domain-containing adaptor inducing interferon β (TRIF), 4) TRIF-related adaptor molecule (TRAM) (6, 18). Following the recruitment of distinct adaptor molecules, downstream TLRs signaling can be separated into two different pathways: MyD88 dependent and MyD88-independent signaling pathways, or the so-called TRIF dependent pathway. MyD88 is bound by all TLRs except TLR3 (19). Each signaling pathway results in activation of inflammatory gene transcription factors (including nuclear factor-κB (NF-κB), interferon-regulatory factors (IRFs), activator protein 1 (AP1) (19). On the other hand, it has been observed that there is crosstalk between TLRs signaling pathways and the phosphoinositide 3 kinase (PI3K)/ Akt signaling pathway, so that activation of each one could be able to activate the other (18). TIRAP is an essential molecule for connecting of TLR2 and TLR4 to MyD88 (18). After connection of MyD88 and activated TLR, MyD88 communicates with IL-1 receptor- 6 associated kinases 4 (IRAK-4) and after that IRAK-4 stimulate IRAK-1 and IRAK-2. Following this, the IRAKs is separated from MyD88 and associate with tumor necrosis factor receptor-associated factor 6 (TRAF6). The complex of IRAKs and TRAF6 associate with another membrane complex, and activates the complex consisting TAK1 transforming growth factor-β-activated kinase 1 which also called mitogen-activated protein kinase kinase kinase 7 (MAP3K7), TAK1-binding protein 1 (TAB1), TAK1- binding protein 2 (TAB2) and TAK1-binding protein 3 (TAB3) which phosphorylates the inhibitor of nuclear factor kappa B (NF-ҡB) kinase (IKK) and mitogen-activated protein kinase kinase 6 (MAP2K6) and other downstream molecules such as JUN N-terminal kinase (JNK), P38 and ERK activate NF-κB and AP-1. Subsequently NF-ҡB and AP-1 translocate into the cellular nucleus and induce a number of gene transcription of proinflammatory cytokines and chemokines (16, 20, 21). In addition to MyD88-dependent signaling, there is another signaling pathway in which NF-ҡB activates in the absence of MyD88 association and known as MyD88- independent signaling that in this signaling pathways the main adaptor protein are TRIF and TRAM. The TLR3 transduces its signal mainly via MyD88-independent signaling pathways (22). In addition to activation of the MyD88-dependent pathways, TRIF- dependent pathways is another way that TLR7 and TLR9 could be able to transduce their signals and produce IFNs (10). Toll-like receptors signaling and its association with microRNA TLR4 is able to transduce its signals via TRIF- dependent pathways or MyD88 signaling pathways. In this pathway TRIF associates with TRAF family member-associated NF-kB activator (TANK)-binding kinase 1 (TBK1) and IKK-related kinases and IKK which make interferon regulatory factor 3 (IRF3) become active. IRF3 then transfer to the nucleus which leads to interferon (IFN-α and 7 IFN-β) production. The TRIF-dependent pathway may also lead to the activation of NF- ҡB (18). There is evidence of an association between the expression of microRNA (miRNA or miR) and regulation of TLRs-mediated signaling (18). miRNAs are short, non-coding RNA comprised of 19–22 nucleotides which play key roles in post- transcriptionally regulation of many biological activities in the cells such as gene expression in cardiovascular and immune system cells (1). In this regard, Taganov et al. observed that TLR4 ligand could be able to increase miR-132, miR-146a and miR-155. As well, elevation in miR-146a expression also observed by TLR2 and TLR5 ligands (23). They also reported that TRAF6 and IRAK-1 are two targets of miR-146 (23). Similarly, O’Connell et al. showed that TLR3 and TLR4 ligands and IFN, enhance miR- 155 expression (24). Tili et al. have suggested that the expression of miR-155 increases the expression of TNF-α (25).They also reported that TLR4 ligand down-regulates miR- 125b expression. Role of TLRs signaling in atherosclerosis and angiogenesis Although the inflammatory nature of atherosclerosis process is well established, the potential factors driving the pro-inflammatory process are not fully established (Table 1-2) (6). In this regard, infectious factors such as chlamydia pneumonia and cytomegalovirus have previously been suggested as being involved in the development of plaque formation and act as TLRs ligand (26). Several studies have evaluated polymorphisms of the genes encoding TLRs in CVD. The most evaluated polymorphisms are Asp299Gly and Thr399Ile (6). Kiechl et al. in a cohort study evaluated the effect of Asp299Gly and Thr399Ile polymorphisms of the TLR4 gene in 8 carotid artery atherosclerosis and found that subjects with specific alleles of these polymorphisms were associated with reduced risk of atherosclerosis and reduced intima-media thickness (27). However, later studies performed on larger sample size did not support this association (28-30). The relationship of these two polymorphisms and coronary artery disease (CAD) progression and risk of myocardial infarction (MI) have also been assessed. Several of these studies reported that the TLR4 Asp299Gly and Thr399Ile polymorphisms are associated with a reduced risk of MI (31-33). In contrast, Edfeldt et al. in an investigation of 2774 subjects reported that these polymorphisms were associated with an increased risk of MI in men, but not women (34). However Zee et al. and Koch et al. found no association between MI and these TLR4 polymorphisms (35, 36). Satoh and colleagues in 2005 evaluated serum proinflammatory cytokines and TLR4 expression on monocyte isolated from peripheral blood in patients with MI on admission and 14 days after MI and observed that baseline and after 14 days post MI onset; the expression of TLR4 and pro-inflammatory cytokines was higher in patients than healthy subjects. Also these levels were higher in patients with heart failure (HF) following a MI, than patients without HF (37). Methe et al. found that in patients with unstable angina (UA) and acute MI, levels of circulating TLR4-positive monocyte was significantly higher in UA than healthy subject and patients with stable angina (SA). In this study the increased level of TLR4 was associated with increased levels of IL-12 and B7-1 (38). Role of TLRs signaling in atherosclerosis and angiogenesis In addition to atherosclerosis progression, there is evidence that TLRs might also have role in angiogenesis so that, activation of TLRs particularly TLR2, TLR4, TLR7 and TLR9 with activating ligands such as LPS could activate endothelial cell even without presence of further cytokines with the mechanisms of increased adenosine and 9 adenine nucleotide concentration in involved tissue which makes releasing endothelial growth factors and induct angiogenesis (39, 40). Recently, Carnevale et al. has proposed that TLR4 as well as gut derived LPS can play an important role in atherosclerosis process in human. They have found that Escherichia coli LPS can be localized in carotid plaque and facilitate formation of atherosclerotic lesion (41). LPS can amplify platelet responses to common agonists upon binding to its receptor, TLR4. It has been proven that platelet activation and aggregation requires an active TLR4 pathway (42). Carnevale et al. study highlighted the possible role of bacterial LPS in atherosclerotic plaque formation via triggering the inflammation response through TLR4 (41). According to this study, dietary modifications can play an important role in reducing the risk of cardiovascular disease. Although still controversial, drugs and diets which are helpful in lowering the circulating LPS and bacteria can possibly modify the atherosclerosis development in susceptible patients. Buerger’s disease is characterized by peripheral arterial occlusive disease in young male smoker and is mostly presented with limb ischemia and pain, intermittent claudication and severe limb ulcers. The molecular pathogenesis of this vascular disease is not fully understood. Recently, the role of TLRs in Buerger’s disease is being better understood. A single neucleotide polymorphisms in the MyD88 gene has been reported to be associated with Buerger’s disease (43). Both myeloid MyD88-dependent and independent TLR signaling pathways can result in monocyte adhesion and therefore, inflammatory response in vessel walls. TLR activators are newly developed immunomodulators that are proposed as possible therapeutic approaches for Buerger’s disease and other vascular disease with similar adenine nucleotide concentration in involved tissue which makes releasing endothelial growth factors and induct angiogenesis (39, 40). Recently, Carnevale et al. has proposed that TLR4 as well as gut derived LPS can play an important role in atherosclerosis process in human. They have found that Escherichia coli LPS can be localized in carotid plaque and facilitate formation of atherosclerotic lesion (41). LPS can amplify platelet responses to common agonists upon binding to its receptor, TLR4. Role of TLRs signaling in myocardial ischemia-reperfusion injury Myocardial ischemia-reperfusion injury (IRI) is a condition that occurs after blood flow returns to myocardial tissue in an ischemic area after coronary artery occlusion (44). In this condition, neutrophil accumulation and excessive generation of oxygen radicals by ischemic myocardial and endothelial cells after restoration of blood supply result in further cellular damage and deleterious consequences (44). According to previous studies, TLRs are expressed in the myocardium are TLR2. TLR3. TLR4 and TLR6 and could be able to impact on myocardial disease (20, 45). Animal studies had shown that activation or conversely inhibition of activation of some of TLRs can cause adverse or favorable clinical effects, for example it was seen that stimulation of TLR4 leads to reduction in cardiac myocyte apoptosis (46). The immune system plays an important role in IRI and therefore contributes to cardiac remodeling and incidence of heart failure (HF) but the exact involved mechanisms have not been clarified (47, 48). Recently a numbers of studies have indicated that the TLRs signaling pathway could be a treatment target in patients with MI. in this regard, activation of NF-kB after stimulation of TLRs after myocardial reperfusion has shown that related to increase myocardial damage and inhibition this activation could be beneficial in reducing myocardial injury and improve cardiac function (49-51). In an animal study, Eritoran a TLR4 blocker was reported to reduce infarct size after myocardial infarction (MI) (52). Other animal studies conducted on TLR4-deficient mice represent similar results by reducing inflammatory cell infiltration and cytokine expression (53-55). Consistent with these observations, in another study evaluated transgenic mice results showed that infarct size in MyD88- deficient mice was significantly smaller. Furthermore, there was significantly better Myocardial ischemia-reperfusion injury (IRI) is a condition that occurs after blood flow returns to myocardial tissue in an ischemic area after coronary artery occlusion (44). In this condition, neutrophil accumulation and excessive generation of oxygen radicals by ischemic myocardial and endothelial cells after restoration of blood supply result in further cellular damage and deleterious consequences (44). According to previous studies, TLRs are expressed in the myocardium are TLR2. TLR3. TLR4 and TLR6 and could be able to impact on myocardial disease (20, 45). Animal studies had shown that activation or conversely inhibition of activation of some of TLRs can cause adverse or favorable clinical effects, for example it was seen that stimulation of TLR4 leads to reduction in cardiac myocyte apoptosis (46). Role of TLRs signaling in atherosclerosis and angiogenesis It has been proven that platelet activation and aggregation requires an active TLR4 pathway (42). Carnevale et al. study highlighted the possible role of bacterial LPS in atherosclerotic plaque formation via triggering the inflammation response through TLR4 (41). According to this study, dietary modifications can play an important role in reducing the risk of cardiovascular disease. Although still controversial, drugs and diets which are helpful in lowering the circulating LPS and bacteria can possibly modify the atherosclerosis development in susceptible patients. Buerger’s disease is characterized by peripheral arterial occlusive disease in young male smoker and is mostly presented with limb ischemia and pain, intermittent claudication and severe limb ulcers. The molecular pathogenesis of this vascular disease is not fully understood. Recently, the role of TLRs in Buerger’s disease is being better understood. A single neucleotide polymorphisms in the MyD88 gene has been reported to be associated with Buerger’s disease (43). Both myeloid MyD88-dependent and independent TLR signaling pathways can result in monocyte adhesion and therefore, inflammatory response in vessel walls. TLR activators are newly developed immunomodulators that are proposed as possible therapeutic approaches for Buerger’s disease and other vascular disease with similar h i 10 Role of TLRs signaling in myocardial ischemia-reperfusion injury Role of TLRs signaling in myocardial ischemia-reperfusion injury The immune system plays an important role in IRI and therefore contributes to cardiac remodeling and incidence of heart failure (HF) but the exact involved mechanisms have not been clarified (47, 48). Recently a numbers of studies have indicated that the TLRs signaling pathway could be a treatment target in patients with MI. in this regard, activation of NF-kB after stimulation of TLRs after myocardial reperfusion has shown that related to increase myocardial damage and inhibition this activation could be beneficial in reducing myocardial injury and improve cardiac function (49-51). In an animal study, Eritoran a TLR4 blocker was reported to reduce infarct size after myocardial infarction (MI) (52). Other animal studies conducted on TLR4-deficient mice represent similar results by reducing inflammatory cell infiltration and cytokine expression (53-55). Consistent with these observations, in another study evaluated transgenic mice results showed that infarct size in MyD88- deficient mice was significantly smaller. Furthermore, there was significantly better 11 11 cardiac function and less inflammatory cells infiltration to ischemic area after MI in these mice (56). Similarly, a study in IRAK4-deficient mice showed they were partially cardioprotected effects against IRI (57). Interestingly despite these results, in several animal studies it was reported that activation of TLR4 with administration of low dose of TLR4 agonists such as LPS 8-24 h before induction myocardial ischemia-reperfusion could be protective and reduced infarct size (58). This suggests that due to the existence of crosstalk between TLRs and PI3K/Akt signaling pathways activation of these signaling pathways modulated inflammatory responses and protect myocardial cells against apoptosis (59). It has been shown that PI3Kγ blockade is advantageous in cancer therapy. The blockade will result in both reduction of tumor growth and may also protect against anthracyclines cardiotoxicity (60). Moreover, TLR2 and TLR4 has been reported as an early markers of other drugs induced cardiomyopathy including doxorubicin. Patients who develop diastolic dysfunction will express higher rate of TLR2 and TLR4 (61). Ha et al. observed that administration of a PI3K inhibitor eliminated the cardioprotection of pretreating with low dose of LPS (62). Furthermore, TLR2 plays a role in IRI so that although in several studies it was observed that TLR2 deficiency or the administration of anti-TLR2 antibody, before myocardial reperfusion results in decreased infarct size and cytokine secretion and improved cardiac function (63, 64), but in a study of Ha et al. Role of TLRs signaling in myocardial ischemia-reperfusion injury administration of TLR2 agonist before reperfusion resulted in beneficial cardiac effects (18). Overall, cardiac protection induced by both TLR4 and TLR2 appear to be related to PI3K/Akt signaling and studies have revealed that inhibition of this pathway abolished cardiac protection of TLR2 and TLR4 (18, 65). 12 Role of TLRs signaling in post-MI cardiac remodeling and development of heart failure Role of TLRs signaling in post-MI cardiac remodeling and development of heart failure Due to negligible endogenous self-regenerative capacity of cardiomyocytes of human heart, elimination of necrotic cardiac tissue after MI and infiltration of immune cells ultimately leads to replacement with scar tissue (66). This is dependent of activation of immune system and production of cytokines which is a hallmark of MI (66). Although activation of TLRs-mediated signaling pathways as one of immune pathways in the early phase after MI, this may be beneficial but continuing this activation can accentuate detrimental remodeling in cardiac tissue injury and occurring heart failure (HF) which is defined as inability of pumping sufficient blood to different organs and tissues (67, 68). The two important factors in patient outcome following an MI are: infarct size and left ventricular (LV) remodeling. Although the exact role of activation of TLRs in cardiac pathology is not fully-understood, but there are evidences that TLR4 is highly expressed in heart and has a major role in response of immune system after incidence of MI (69). Oyama et al. observed smaller infarct size and suppressed inflammation in mice with TLR4 deficiency (70). Shishido et al. found that TLR4 deficiency was a protective condition in mice against adverse remodeling after MI.However, the infarct size was the same in mice with and without TLR2 deficiency, although TLR2-deficient mice had less TGF-β1 expression and type 1 collagen deposition, and also less fibrosis in histological examinations, ventricular remodeling and mortality (71). Furthermore, decreased LV remodeling and preserved systolic function in following MI was observed by Timmers et al. in TLR4-deficient mice. They also reported that although the collagen density was higher in the infarct area, 13 inflammatory cytokine expression was lower in TLR-4 deficient mice (72). On the other hand, Birks et al. showed that patients requiring left ventricular assist devices had higher levels of TLR4 and IL-1 receptor expression in their myocardium. They also observed significant association between expression of TLR4 and LI-1 receptor (49). Riad et al. reported that in mice with an induced MI, 6 days after MI those with TLR4 deficiency had improved LV function, lower LV remodeling and level of atrial natriuretic peptide and collagen density (73). Role of TLRs signaling in septic cardiomyopathy and myocarditis Septic cardiomyopathy is systolic and diastolic dysfunction of both sides of the heart following infection (74). As mentioned above, bacterial components such as LPS are recognized by surface TLRs and bacterial or viral nucleic acids are recognized by intracellular TLRs (19). Consequently, TLRs especially TLR4 may be involved in septic cardiomyopathy and cardiac dysfunction. In animal models it was shown that TLR4 or IRAK1 deficiency are protective against septic cardiomyopathy (6). Results of the study conducted by Tavener et al. showed that administration of LPS to mice with TLR4- deficient in myocardial cells but TLR4-positive in circulating leukocytes leads to impaired myocardial function. In contrast, giving LPS to TLR4-deficient in circulating leukocytes but positive in myocardial cells led to no change in cardiac function (75). However transplantation of TLR4-deficient bone marrow with the aim of cardioprotection did not lead to a positive effect (76). This could be because of roles of other TLRs such as TLR2 in myocardial cells or other tissues. In the studies investigated the role of TLR2 in bacteria-induced cardiomyopathy, results revealed the protective effect of TLR2- deficient condition during sepsis (77). Although the effect of Eritoran administration, a 14 14 synthetic TLR4 antagonist, in prevention of cardiomyopathy in severe septic patients are not fully demonstrated, Tidswell et al. in their clinical trial reported no significant reduction in all-causes mortality in patients with severe sepsis by administration of Eritoran (78). The most common cause of acute myocarditis is viral infection (19). The immune system and its responses are active in the subclinical phase of myocarditis (79). The definite diagnosis of myocarditis is made by endomyocardial biopsy and its histological evaluation (80). Activation of host immune response and TLRs signaling following viral infection and replication produce a variety of inflammatory cytokines that make infiltration of inflammatory cells found to be important factors with protective or deleterious effects on incidence of myocarditis and cardiomyopathy (81, 82). TLRs can be activated by double or single stranded RNA such as TLR3 have more correlation with viral myocarditis (19). Despite many animal studies on the roles of TLRs in pathogenesis of myocarditis, there is little clinical data available. In an animal model study, Negishi et al. Role of TLRs signaling in septic cardiomyopathy and myocarditis evaluated the role of TLR3 signaling pathway in infection with coxsackie virus, TLR3 deficient mice was more susceptible to be infected by some viral infection such as Coxsackie virus, whilst expression of TLR3 makes resistant against viruses (83). It was seen that TLR3 is important in controlling infection caused by Coxsackie virus due to subsequent activation of anti-viral infection (19). In viral myocarditis in mice deficient in TLR3 due to more viral replication in the primary phase, results in greater myocardial injury and increases risk of adverse clinical outcome (84). Also it was observed that in these mice infection with encephalomyocarditis virus resulted in more severe cardiac damage and earlier death (19). Gorbea et al. showed a 15 15 polymorphism of TLR3 gene in patients diagnosed with enteroviral myocarditis, reduce the ability to inhibit viral replication because of decrease in type I interferon (85). As mentioned previously, TLR3 transduces its signal through TRIF-dependent pathways. Although the presence of TLR3 inhibits viral replication, activation of TRIF which possesses pro-apoptotic activity, could give viruses, which are the obligatory intracellular pathogens, to replicate in infected cells. Therefore more studies should be undertaken to clarify its role in viral infection. On the other hand, it has been suggested that after viral infection, TLR4 deficient mice showed significantly lower viral replication in myocytes and reduced production of IL-1β and IL-18 and therefore less severe myocarditis (86). In this regard, Satoh et al. investigated 44 patients with myocarditis and found higher levels of TLR4 in these patients compared with control group. In addition, they found a positive association between the level of enteroviral RNA and TLR4 level (87). Pauschinger et al. observed that a greater enteroviral RNA level correlated with left ventricular dysfunction (88). Furthermore, Fuse et al. reported that Absence of MyD88 is associated with higher survival rate and lower viral load and reduction of production of interferon-γ (IFN- γ), IL-18, tumor necrosis factor-α (TNF-α) and cytokines of T-helper 1. However levels of IFN-β were significantly increased (89). Role of TLRs signaling in infective endocarditis Infective endocarditis (IE) is an important cardiac disease with a high mortality rate and in spite of available treatments its prognosis is poor (90). Several studies have reported the relationship between TLRs and pathogenesis of IE. Bustamante et al. investigated R753Q and R677W polymorphisms of TLR2 and D299G and T399I polymorphisms of TLR4 in 65 patients with IE and 66 healthy participants, and report that the TLR2 16 R735Q polymorphism was significantly associated with endocarditis and presence of this polymorphism makes individuals more susceptible to development of infective endocarditis (91). Golovkin et al. also investigated several polymorphisms of TLRs: rs5743551 and rs5743611 of TLR1, rs3804099 and rs5743708 of TLR2, rs4986790 and rs4986791 of TLR4 and rs3775073 and rs5743810 of TLR6. One hundred ten patients with infective endocarditis and 300 healthy matched participants were evaluated. They concluded that among mentioned polymorphisms, only TLR6 rs3775073 polymorphism was significantly correlated with decreased risk of IE (92). Single nucleotide variants of TLR4 were investigated by Weinstock et al. using blood that was obtained from 148 patients with IE and 185 controls. No significant difference in genetic variants of TLR4 was found between IE and control groups (93). Tsaganos et al. in their animal study induced bacterial endocarditis by two types of staphylococcus aureus, methicillin- susceptible (MSSA) and methicillin-resistant (MRSA), in rabbits. Results showed that after addition of TLR4 antagonist, infection with MSSA resulted in higher stimulation of TNF-α. This study revealed different pattern of TNF-α stimulation, independent to TLR4 mechanisms in MSSA and MRSA endocarditis (94). Banks et al. investigated Streptococcus sanguis as one of main causes of IE. They observed that the secreted components of this bacteria which was able to inhibit activation of monocytes by binding to a complex of TRL4 and CD14 (Table 1-2)(95). Role of TLRs signaling in atrial fibrillation Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia (96) and MI is an independent risk factor of new-onset AF (97). It has been reported that TLRs and its associated inflammation may be correlated with AF. Therefore they can be used as 17 potential predictor of new-onset AF after MI. In this regard, Zhang et al. investigated TLRs in peripheral blood mononuclear cell in 84 patients with MI and 85 patients with new-onset AF after MI. Eighty-two healthy individuals were selected as control group. Results showed significantly higher expression of TLR2 and TLR4 and their signaling proteins in group of patients with AF compared with MI patients without AF and also control group (97). Similarly, eighty six patients with AF and 86 participants with sinus rhythm were enrolled in a study conducted by Gurses and colleagues. The expression of TLR2 and TLR4 in peripheral blood monocyte were evaluated. At the end of the study, they found that AF patients had significantly higher TLR2 and TLR4. As well, after 17 months follow they found that the expression level of monocyte TLR4 was independent predictor of AF recurrence. These results suggest that TLR4 may be a potential therapeutic target in AF patients (96). Consistent with this result, Gurses et al. evaluation platelet TLR2, TLR4 and HMGB-1 in 53 AF patients and 22 controls. Left atrial and peripheral blood were obtained. This study showed significantly higher level of peripheral blood and left atrial TLR2, TLR4 and HMGB-1 in AF patients. Patients with persistent AF had higher serum HMGB-1 and also left atrial platelet TLR2 and TLR4 than patients with paroxysmal AF (98). Wang et al. found that patients with AF had higher level of TLR2 compared to controls and in contrast to a previous study comparison two group of AF patients revealed that TLR2 was significantly higher in paroxysmal AF than persistent AF patients (99). The most important complication of AF is atrial thrombi formation which can be resulted in ischemic accidents such as ischemic stroke (98). Association between TLRs signaling and thrombosis in patients with AF was investigated by Xu et al. with evaluation three groups: 15 AF patients without 18 18 thrombus, 15 AF patients with thrombus and 15 participants with sinus rhythm. Ultimately, they observed that in comparison to other groups, MyD88 and HMGB1 were significantly higher in AF patients with thrombus. Role of TLRs signaling in atrial fibrillation However expression level of TLR4 was not different between three groups (100). Role of TLRs signaling in cerebrovascular disease Cerebrovascular diseases refer to conditions in which the blood vessels supplying the brain are primarily involved (101). The most common manifestation of this group of disease is stroke that can be occurred as an ischemic or hemorrhagic which can cause major disability and mortality (101). Different endogenous ligands such as high-mobility group box 1 (HMGB1) protein, heat shock proteins (HSPs), hyaluronic acid and mRNA are released from damaged cells following to CNS injuries could be able to activate TLRs signaling pathways and result in triggers immune responses and infiltration of inflammatory elements and therefore further neuronal damage (102). Although the exact roles of TLRs in cerebrovascular disease is not fully-understood, two aspect of their effects have been suggested in stroke: on one hand the neuroprotective effects and neurodegenerative effects on the other hand. In this regard one of the important factors in outcomes resulted from activation of TLRs in nervous system depends on type of involved cells express TLR and another one is time course. It has been observed that TLRs activation could lead to the expression of anti-inflammatory cytokines such as IL-10 production that result in barricade neurodegenerative of several cytokines such as IFN-γ and TNF-α (103, 104). Samarasinghe et al. have reported that 12-24 h after activation of TLRs, the levels of serum IL-10 was raised (105). MyD88 deficient hematopoietic cells were investigated by Downes et al. who observed that 19 MyD88 deficient mice had a larger cerebral infarct size (106). Microglial cells have key role in stroke pathogenesis and study conducted by Jung et al. showed that the inhibition of activation of TLR4 leads to the release of IFN-β which contributes to induction of apoptosis in these cells (107). Aravalli et al. observed that blocking of TLR2 signaling pathway prevent progression of microglial cells towards apoptosis (108). Stevens et al. found that blockage of TLR9 also leads to neuroprotection (109). Tang et al. found that within 1 h after incidence of ischemic stroke the expression of TLR2 and TLR4 by neurons was elevated (110) and within 24 h microglial cells in ischemic area express high TLR2. This means activation of neuronal response prior to activation of microglial cells. Cao et al. showed that a reduced infarct size and lower levels of proinflammatory cytokines and overall better outcome in TLR2 and also TLR4 deficient mice with brain ischemia (111). Hyakkoku et al. confirmed these findings (112). Role of TLRs signaling in cerebrovascular disease Tang et al. concluded that activation of TLR 2 and TLR4 expressed by neurons with activation of caspase 3 can result in apoptosis (110). However, no significant difference was reported by Brea et al. in expression of TLR3 and TLR9 between good outcome and poor outcome in evaluation of 110 patients with ischemic stroke (113). Yang et al. found a correlation between higher TLR4 level in monocytes in peripheral blood and adverse neurological outcome (114). A positive association was also found by Ferronato et al. between TLR4 expression and expression of cyclooxygenase-2 (COX-2), an enzyme important in inflammation, after ischemic stroke which can cause more CNS damage (115). miRNAs act as key regulators of the expression of different genes post- transcriptionally (1). Xu et al. found high levels of miR-1906 in the ischemic zone and also in a peri-ischemic zone. They also found that after administration of exogenous 20 miR-1906, the expression of TLR4 and infarct zone was reduced and neurological functional outcomes were improved. However this protective effect was not seen in TLR4 knockout mice (116). Lin et al. investigated TLR4 gene polymorphisms among ethnic Chinese people and observed that Asp119Cys polymorphism significantly correlated with increased risk of ischemic stroke (117). approaches Rejection of cardiac stem cells is a major limitation of stem cell therapy that is mostly due to low tolerance of transplanted cells against hypoxia and inflammation which leads to apoptosis (Table 1-2). Disrupting the immune response is considered as a possible solution for improving the survival of transplanted stem cells. Modulation of toll like receptors is an interesting issue which under active research. TLR1-TLR6 and TLR9 which are expressed on outer surface or within intracellular organelles of mesenchymal stem cells (10, 118). TLRs signaling pathways can be activated by endogenous molecule secreted from damaged tissue. TLR4 is a well-known example of this issue. LPS which is a ligand for TLR4 will promote releasing of proinflammatory factors and prevent stem cell apoptosis (119, 120). Preconditioning of mesenchymal stem cells with LPS will results in better engraftment and greater survival rate of stem cells. Also it has been reported that angiogenesis will be enhanced by releasing of vascular endothelial growth factor (VEGF) (121). As well, activation of TLR3 will increase immunosuppressive capacity of transplanted stem cell by releasing immunosuppressive factors (120). 21 21 As mentioned earlier, TLRs play an important role in inflammation and inflammatory pathways. According the complexity of inflammatory pathways, there is still controversy about the exact therapeutic effect of TLR targeting. Activation of TLR will result in inflammatory response and this phenomenon has been reported with Escherichia coli and chlamydia infection (41, 122). Despite the established effect of gut microbiota on various metabolic and cardiovascular conditions, the role of antibiotic therapy on gut microbiota is not widely studied. Antibiotics can alter microbiota and induce or prevent some disease (123). By selective disruption of gut microbiota a certain bacteria can be targeted. Using highly specific antibiotics will eradicate a specific bacteria and therefore may interfere with specific pathways such as inflammation or atherogenesis. Another emerging issue about the therapeutic approaches for cardiovascular disease are probiotics. It has been suggested that use of different strains may interact with TLR and suppress inflammatory cytokine production (124). However, despite of recent trends toward new therapeutic approaches for cardiovascular disease, use of probiotics and selective antibiotics is not widely studied and the available data is controversial. Conclusion: Cardiovascular disease is among the leading cause death. Toll-like receptors and their down-stream signaling pathways play a crucial role in the immune system and its potential link with myocardial infarction, and heart failure, which is activated via endogenous molecule secreted from damaged tissue, indicating its value as a therapeutic target. Atherosclerosis is associated with inflammation and involvement of immune responses. TLR play an important role in macrophage activation within atherosclerotic lesions. 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(55) Mouse TLR4 Cardioprotective effect of myocardial TLR4 deficiency during MI Feng et al. (56) Mouse MyD88 Significantly smaller infarct size and better cardiac function after MI in MyD88-deficient Maekawa et al. (57) Mouse IRAK4 Cardioprotective effect of deficiency of IRAK4 against IRI Zacharowski et al. (58) Rat TLR4 Decrease infarct size with administration of TLR4 agonist before myocardial reperfusion Ha et al. (62) Mouse PI3K Cardioprotective effect with administration of PI3K inhibitor Ha et al. (18) Mouse TLR2 Cardioprotective effect with administration of TLR2 agonist Favre et al. (64) Mouse TLR2 Smaller infarct size after cardiac ischemia in deficiency of TLR2 Arslan et al. References (63) Mouse TLR2 Significantly improved cardiac function and smaller infarct size with administration of anti- TLR2 antibody Oyama et al. (70) Mouse TLR4 Decrease infarct size in deficiency of TLR4 Shishido et al. (71) Mouse TLR4, TLR2 Decreased ventricular remodeling after MI in deficiency of TLR4 and TLR2 No association between infarct size and deficiency of TLR2 Timmers et al. (72) Mouse TLR4 Decrease ventricular remodeling and better systolic function after MI and higher collagen density in infarct area in TLR4-deficient Riad et al. (73) Mouse TLR4 Decrease ventricular remodeling and better cardiac function after MI in TLR4 deficient Tavener et al. (75) Mouse TLR4 Administration of LPS to TLR4-deficient in myocardium but TLR4-positive in circulating leukocytes leaded to myocardial dysfunction Binck et al. (76) Mouse TLR4 No cardioprotective effect was observed with transplantation of TLR4-deficient bone marrow Zou et al. (77) Mouse TLR2 Cardioprotective effect in deficiency of TLR2 during sepsis Negishi et al. (83) Mouse TLR3 More susceptibility to viral infection in TLR3- deficient Richer et al. (84) Mouse TLR3 Important role of TLR3 in controlling coxsackie virus infection Increase myocardial injury and risk of adverse clinical outcome in TLR3-deficient mice Gorbea et al. (85) Mouse TLR3 Poor controlling of viral replication in TLR3- deficient mice with enteroviral myocarditis 32 Fairweather et al. (86) Mouse TLR3 Significantly lower viral replication and severity of myocarditis in TLR4 deficiency in myocytes Fuse et al. (89) Mouse MyD88 Association between absence of MyD88 and higher survival in viral myocarditis Downes et al. (106) Mouse MyD88 Larger infarct size after ischemic stroke in MyD88 deficient mice Jung et al. (107) Mouse TLR4 Blocking of TLR4 leaded to releasing IFN-β and cells apoptosis Aravalli et al. (108) Mouse cell line TLR2 Blocking of TLR2 prevented progression of microglial cells toward apoptosis Stevens et al. (109) Mouse TLR9 Blocking of TLR9 made neuroprotective Tang et al. (110) Mouse TLR2, TLR4 Elevation in expression of TLR2 and TLR4 within 1 h after ischemic stroke Cao et al. (111) Mouse TLR2, TLR4 Decrease in infarct size after stroke in TLR2 and TLR4-deficient mice Hyakkoku et al. (112) Mouse TLR3,TLR4, TLR9 Neuroprotective effect of TLR4 deficiency in brain ischemia but not with TLR3 and TLR9 Xu et al. References (116) Mouse TLR4 High level of miR-1906 in ischemic and peri- ischemic area Association between miR-1906 administration and decrease TLR4 level and size of infarct area and improvement in neurological function Tsaganos et al. (94) Rabbit TLR4 Different pattern of TNF-α stimulation, independent to TLR4 mechanisms in methicillin- susceptible and methicillin-resistant staphylococcus aureus endocarditis Banks et al. (95) Streptococcus sanguis TLR4 Inhibition activation of monocytes by binding to a complex of TRL4 and CD14 by secreted components of Streptococcus sanguis 33 Table2. Summary of the most relevant studies investigating TLRs signaling pathways in CVD patients Author (Reference) Population (number) Investigated TLR or other component Chief findings Kiechl et al. (27) Human (n=810) TLR4 Decreased risk of atherosclerosis in existence of Asp299Gly and Thr399Ile polymorphisms Labrum et al. (28) Human (n=3000) TLR4 No association between Asp299Gly and Thr399Ile polymorphisms and intima-media thickness Netea et al. (29) Human (n=493) TLR4 No association between Asp299Gly polymorphism and atherosclerosis Norata et al. (30) Human (n=1256) TLR4 No association between Asp299Gly and Thr399Ile polymorphisms and intima-media thickness Ameziane et al. (31) Human (n=399) TLR4 Decreased risk of MI in Asp299Gly polymorphism Boekholdt et al. (32) Human (n=885) TLR4 Association between Asp299Gly polymorphism and decreased risk of cardiovascular event in patients receiving statin treatment Holloway et al. (33) Human (n=166) TLR4 Association between Asp299Gly polymorphism and decreased risk of MI in patients receiving statin treatment Edfeldt et al. (34) Human (n=2774) TLR4 Increased risk of MI in men in existence of Asp299Gly and Thr399Ile polymorphisms Zee et al. (36) Human (n=1390) TLR4 No association between risk of MI and Asp299Gly polymorphism Koch et al. (35) Human (n=5264) TLR4 No association between Asp299Gly and Thr399Ile polymorphisms and risk of MI Satoh et al. (37) Human (n=85) TLR4 Increase level of TLR4 after MI and also higher TLR4 level in MI patients with HF compared with MI patients without HF Methe et al. (38) Human (n=118) TLR4 Higher TLR4 level in patients with UA Birks et al. (49) Human (n=36) TLR4 Higher TLR4 in patients with requiring ventricular assist devices Tidswell et al. (78) Human (n=300) TLR4 No significant reduction in all-cause mortality in severe septic patients received Eritoran Satoh et al. (87) Human (n=49) TLR4 Higher TLR4 level in patients with myocarditis Positive association between level of TLR4 and enteroviral RNA Brea et al. References (113) Human (n=110) TLR3, TLR9 No difference in expression of TLR3 and TLR9 between patients with good and poor outcome after ischemic stroke Yang et al. (114) Human (n=65) TLR4 Association between higher level of TLR4 and adverse neurological outcome Ferronato et al. (115) Human (n=60) TLR4 Positive association between TLR4 and expression of COX-2 enzyme which can cause more CNS damage after ischemic stroke Lin et al. (117) Human (n=457) TLR4 Increased risk of ischemic stroke in existence of Asp119Cys polymorphism Bustamante et al. (91) Human (n=131) TLR2, TLR4 Increased risk of infective endocarditis with presence of TLR2 R735Q polymorphism Golovkin et al. Human (n=410) TLR2, TLR4, Decreased risk of infective endocarditis with 34 (92) TLR6 presence of TLR6 rs3775073 polymorphism Weinstock et al. (93) Human (n=333) TLR4 No significant difference in genetic variants of TLR4 between patients with infective endocarditis and controls Zhang et al. (97) Human (n=251) TLR2, TLR4 Significantly higher expression of TLR2 and TLR4 and their signaling proteins in patients with AF compared with MI patients without AF Gurses et al. (96) Human (n=172) TLR2, TLR4 Significantly higher TLR2 and TLR4 in AF patients and expression level of monocyte TLR4 was associated with AF recurrence Gurses et al. (98) Human (n=75) TLR2, TLR4 Significantly higher level of peripheral blood and left atrial TLR2 and TLR4 in AF patients and also higher left atrial platelet TLR2 and TLR4 level in persistent AF than patients with paroxysmal AF Wang et al. (99) Human (n=48) TLR2 Higher level of TLR2 in AF patients and significantly higher of TLR2 level in paroxysmal AF compared to persistent AF patients Xu et al. (100) Human (n=45) TLR4, MyD88 Significantly higher MyD88 and HMGB1 in AF patients with thrombus and no difference in level of TLR4 between AF patients with and without thrombus Xu et al. (125) Human (n=163) TLR2 Significantly higher TLR2 level in patients with persistent AF compared to paroxysmal AF 35 Figure1. Schematic view of TLRs signaling pathways. Downstream TLRs signaling is divided into two different pathways: MyD88-dependent and -independent signaling pathways, which after several steps ultimately resulted in proinflammatory gene expression and production of various cytokines and chemokines 36
https://openalex.org/W2061380908	https://www.scielo.br/j/csp/a/3fQPZvKkfqt9LMXBZwYf7Sb/?lang=pt&format=pdf	Portuguese	null	As estatísticas de nascimento e os fatores maternos e da criança nas microrregiões do Nordeste brasileiro: uma investigação usando análise fatorial	Cadernos de Saúde Pública	2,010	cc-by	7,617	1 Centro de Ciências Exatas e da Natureza, Universidade Federal da Paraíba, João Pessoa, Brasil. 2 Programa de Pós-graduação em Biometria e Estatística Aplicada, Universidade Federal Rural de Pernambuco, Recife, Brasil. 311 ARTIGO ARTICLE 311 ARTIGO ARTICLE 311 ARTIGO ARTICLE As estatísticas de nascimento e os fatores maternos e da criança nas microrregiões do Nordeste brasileiro: uma investigação usando análise fatorial Birth statistics and maternal and infant risk factors in the micro-regions of Northeast Brazil: a factor analysis study Neir Antunes Paes 1 Carlos Sérgio Araújo dos Santos 2 Correspondência N. A. Paes Departamento de Estatística e Informática, Centro de Ciências Exatas e da Natureza, Universidade Federal da Paraíba. Campus Universitário, João Pessoa, PB 58000-000, Brasil. antunes@de.ufpb.br Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 Birth Certificates; Information Systems; Vital Statistics Neir Antunes Paes 1 Carlos Sérgio Araújo dos Santos 2 Abstract Historically, birth registration in Northeast Bra- zil has presented serious quality problems, with major regional variations and gaps in knowledge. The current study proposes to evaluate the quality of birth records and identify patterns of relation- ships between variables that reflect maternal and infant factors in the birth certificates and allow summarizing the data on live births in the 188 micro-regions of the Northeast in 2000, based on factor analysis. Data on live births were collected through the Information System on Live Births (SINASC). In general, regional distribution of birth coverage suggests an increase in the south- ern States of the Northeast. Quality of completion of variables in the micro-regions of the Northeast was considered satisfactory. In the factor analysis, data for the variables were reduced to two factors: favorable and unfavorable to delivery. O conhecimento da natalidade de um determina- do lugar se constitui em um fator determinante da dinâmica populacional, já que os nascimentos fazem parte da composição de inúmeros indi- cadores demográficos e epidemiológicos, como, por exemplo, taxas de mortalidade infantil, taxas de natalidade, fecundidade e de mortalidade ma- terna, os quais se constituem em informações preciosas no planejamento e na delimitação das políticas públicas nas áreas da saúde materna e infantil 1. Correspondência N. A. Paes Departamento de Estatística e Informática, Centro de Ciências Exatas e da Natureza, Universidade Federal da Paraíba. Campus Universitário, João Pessoa, PB 58000-000, Brasil. antunes@de.ufpb.br A maior parte do conhecimento sobre natali- dade no Brasil vem de informações geradas pelos censos e registros vitais. A qualidade das infor- mações e as condições em que os nascimentos ocorrem têm sido uma questão central e persis- tente. A falta de informação ou má declaração so- bre o nascido causa problemas na identificação correta de suas características e das condições que o levaram ao nascimento. Maior problema, no entanto, é a subnotificação dos registros de nascimentos, cuja estimação tem provocado dis- cussões entre estudiosos 1,2,3,4,5,6,7,8. Geralmente, os métodos de estimação tomam como base fa- tores da dinâmica populacional, como a fecundi- dade e a mortalidade, e outros propõem o uso de procedimentos baseados nos registros atrasados de nascimentos. Birth Certificates; Information Systems; Vital Statistics Unidades de análise para essa temática têm focado desde o país como um todo até unidades Cad. Abstract Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 Paes NA, Santos CSA 312 municipais, mas ainda são escassos os estudos que abordam agregados espaciais, particular- mente da Região Nordeste. Devido aos municí- pios variarem consideravelmente em tamanho, alguns deles são tão pequenos que impõem sé- rias restrições quanto à qualidade dos dados e informações declaradas sobre os nascimentos, além das variações no comportamento das va- riáveis relacionadas à natalidade. Dessa forma, agregações espaciais como as microrregiões, além de permitirem estimativas mais confiáveis de indicadores de natalidade preservam o má- ximo possível a homogeneidade das condições locais. A divisão político-administrativa do Nor- deste é formada por 188 microrregiões, as quais apresentam uma enorme diversidade e desigual- dades regionais. estimativas de indicadores da fecundidade e na- talidade no Nordeste 2. Assim, foi possível coletar informações agrupadas com respeito ao registro legal do recém-nascido, as quais possibilitaram avaliar a situação da mãe e da criança à época do nascimento. A população de mulheres por faixa etária qüinqüenal das microrregiões necessária para o cálculo da cobertura dos nascidos vivos foi extra- ída do banco de dados do Sistema IBGE de Recu- peração Automática (SIDRA; http://www.sidra. ibge.gov.br/bda/tabela/listabl.asp?z=t&c=200). Ademais, o ano 2000 foi escolhido por se tratar do último ano censitário disponível com infor- mações mais completas sobre as variáveis tra- tadas neste estudo, além de minimizar os erros de ajustamentos e projeções populacionais para anos não censitários, na construção de indica- dores que envolvem estes contingentes, parti- cularmente para as microrregiões. Além disso, até a confecção deste trabalho o ano de 2005 foi o último disponível referente aos dados de nascimentos. Contudo, importa conhecer as relações de similaridades ou padrões de relacionamento que as microrregiões guardam entre si quanto aos nascimentos. A utilização de ferramentas es- tatísticas de análise multivariada possibilita in- vestigar e identificar a existência desses padrões de relacionamento e resumir os dados sobre os nascidos vivos em um pequeno conjunto de fato- res ou componentes principais, não sacrificando boa parte das informações contidas nas variáveis originais. Os dados básicos sobre nascimentos para o ano 2000 foram coletados no banco de dados do SINASC. Esse sistema objetiva não somente levantar o número real dos nascidos vivos, mas também variáveis importantes para analisar as condições de nascimento. Abstract Conforme exposto, o principal propósito deste estudo consistiu em avaliar a qualidade dos dados e informações de- claradas nas variáveis constantes das DN, bem como investigar os padrões de relacionamentos entre as variáveis e não testar uma particular te- oria ou estudar os determinantes da natalidade. Pretendeu-se restringir o número de variáveis de modo a evitar instabilidades notadas em ex- plorações preliminares com modelos contendo um grande número de variáveis preditoras. Além do mais, o recurso metodológico utilizado neste trabalho – análise fatorial – busca simplificar a estrutura dos dados e sintetizar as informações quando o número de variáveis envolvidas é mui- to grande. Devido a esses motivos, o foco deste estudo foi direcionado às principais variáveis convencionais na expectativa de que elas possam atender aos objetivos propostos. Desse modo, as seguintes variáveis constantes nas DN foram uti- lizadas neste trabalho: peso ao nascer, duração da gestação, grau de instrução da mãe, idade da mãe, número de consultas durante o pré-natal, tipo de parto, raça/cor do recém-nascido e esta- do civil da mãe. Nesse contexto, este estudo se propõe a ava- liar a qualidade dos registros sobre os nascidos vivos e identificar padrões de similaridade en- tre variáveis que reflitam os fatores maternos e da criança constantes nas Declarações de Nas- cimentos (DN), que permitam resumir os da- dos sobre nascidos vivos das microrregiões do Nordeste em 2000 com base no uso da análise fatorial. Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 Fonte de dados As informações sobre os nascimentos são capta- das pelas secretarias de saúde ou órgãos de admi- nistração da saúde em cada estado, que por sua vez, recebem as DN dos cartórios ou hospitais. As fontes oficiais de dados contínuos são o Instituto Brasileiro de Geografia e Estatística (IBGE; http://www.ibge.gov.br) e o Sistema de Informações sobre Nascidos Vivos (SINASC. De- partamento de Informática do SUS. http://tab net.datasus.gov.br) do Ministério da Saúde. In- vestigações preliminares apontaram que no ano 2000 o SINASC captou um número maior de re- gistros, além de disponibilizar um número maior de variáveis. Essa fonte desde 2000 já se afirmava como muito útil em informações para permitir A lógica e justificativa para a escolha dessas variáveis são familiares, estão bem estabelecidas e são amplamente reconhecidas como fatores de risco para o nascimento, mesmo porque elas fazem parte de uma ampla discussão que resul- NASCIMENTOS E FATORES MATERNOS E DA CRIANÇA NO NORDESTE 313 culadas aqui servirão como proxy da verdadeira cobertura. tou na confecção e padronização de um único formulário oficializado para ser usado em todo o país 9. Para estimar a cobertura para os estados da Região Nordeste, extraiu-se a média das cober- turas das microrregiões pertencentes ao estado. Assim, a cobertura dos nascidos vivos para cada microrregião foi estabelecida da seguinte forma: Qualidade dos registros e declarações dos nascidos vivos Metodologias para estimação da cobertura de nascimentos foram apresentadas ou usa- das por diversos autores na literatura brasileira 2,10,11,12,13,14. Essas metodologias fazem uso de di- versos recursos: busca ativa dos registros de nas- cimentos; sistema de registro dual; registro atra- sado de nascimentos; ou fazem uso de estimati- vas indiretas. Essas últimas procuram estimar o número esperado de nascimentos, o qual é ob- tido por meio de projeções populacionais ou da distribuição específica da fecundidade por idade da mãe. É importante registrar que esses recursos metodológicos possuem restrições e produzem erros nas estimativas caso não sejam cumpridas as exigências impostas para seu adequado uso, o que é bastante usual. Neste estudo, por razões de disponibilidade dos dados para unidades ter- ritoriais desagregadas, como as microrregiões, foi usado o último procedimento mencionado, o que é descrito a seguir. Em que, Sendo, Sendo, = cobertura dos nascidos vivos para a micror- região i. = cobertura dos nascidos vivos para a micror- região i. = total de nascidos vivos observados da microrregião i. = total de nascidos vivos observados da microrregião i. = total de nascidos vivos estimados da microrregião i. = total de nascidos vivos estimados da microrregião i. = taxa específica de fecundidade do estado na faixa etária qüinqüenal j correspon- dente à microrregião i. = total de mulheres na faixa etária qüinqüe- nal j na microrregião i. O número de nascidos vivos para as micror- regiões foi estimado com base no produto do número de mulheres por grupos de idades qüin- qüenais de 15 a 49 anos da microrregião pelas correspondentes taxas específicas de fecundi- dade do estado, a qual a microrregião pertence. Essas taxas foram estimadas valendo-se da apli- cação da técnica indireta da razão de parturição desenvolvida por Brass 15. Admitiu-se, dessa ma- neira, que as taxas de fecundidades específicas dos estados do Nordeste em 2000 fossem válidas para cada microrregião pertencente ao mesmo estado, no mesmo ano. Esse suposto foi neces- sário em virtude das dificuldades de acesso aos dados sobre fecundidade neste nível de desagre- gação e à flutuação dos dados, o que significa não captar os possíveis diferenciais dos padrões de fecundidade intra-regionais. Nessa situação, os diferenciais dos níveis das coberturas ficarão expressos pelos pesos relativos da estrutura etá- ria censitária das mães. A implicação disso seria incorporar certo viés na estimativa dos níveis na cobertura dos nascimentos. No entanto, especu- la-se que não haja importantes diferenciais nos padrões de fecundidade intra-regionais, existen- tes, talvez, naquelas microrregiões que compor- tam grandes aglomerados urbanos, como as me- trópoles. Uma tendência à uniformização dos níveis e padrões da fecundidade no Brasil já vem sendo observada pelo menos desde os anos 90 4. Dessa forma, as estimativas das coberturas cal- Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 Medidas de adequação para o uso da análise fatorial A análise multivariada é uma ferramenta esta- tística que processa as informações de modo a simplificar a estrutura dos dados e a sintetizar as informações quando o número de variáveis envolvidas é muito grande, facilitando o enten- dimento do relacionamento existente entre as variáveis do processo. Os testes Kaiser-Meyer-Olkin (KMO) e de esfe- ricidade de Bartlett, indicam qual é o grau de suscetibilidade ou o ajuste dos dados à análise fatorial, isto é, qual é o nível de confiança que se pode esperar dos dados quando o seu tratamen- to pelo método multivariado de análise fatorial é empregado com sucesso 19. Dada uma matriz de correlação para um conjunto de variáveis, a análise fatorial permite investigar a existência de algum padrão de rela- cionamento e resumir os dados em um pequeno conjunto de fatores ou componentes principais, não sacrificando boa parte das informações (no sentido de variabilidade) contidas nas variáveis originais, sendo necessário a pressuposição da normalidade dos dados 19,20. O primeiro deles (KMO), apresenta valores normalizados (entre 0 e 1,0) e mostra qual é a proporção da variância que as variáveis (ques- tões do instrumento utilizado) apresentam em comum ou a proporção desta que é devida a fa- tores comuns. Para interpretação do resultado obtido, va- lores próximos de 1,0 indicam que o método de análise fatorial é perfeitamente adequado para o tratamento dos dados. Por outro lado, valo- res menores que 0,5 indicam a inadequação do método. Nessa técnica o que se faz é substituir um conjunto inicial de p variáveis, X1, X2, ..., Xp, correlacionadas, por um conjunto menor de fatores comuns (ou variáveis hipotéticas) que podem ser correlacionados (fatores ortogonais). Deseja-se determinar um número mínimo de fatores necessários para explicar a maior parte da variância do conjunto original de variáveis. O modelo de análise fatorial assume que cada variável observada é representada como uma função linear de um menor número de fatores comuns (por serem comuns as várias variáveis) mais uma componente de variação residual (fa- tores específicos). As variáveis usadas na análise fatorial foram aquelas especificadas na seção de fontes dos dados. j = faixa etária qüinqüenal de 15 a 49 anos. j = faixa etária qüinqüenal de 15 a 49 anos. Para a classificação das coberturas dos nas- cidos vivos foi estabelecido o seguinte critério: acima de 90%, como muito boa; de 81% a 90%, boa; de 71% a 80%, regular; abaixo de 71%, de- ficiente. Para efeito de comparação, os resultados en- contrados aqui são comparados com aqueles es- timados pelo SINASC e pelo SIDRA. A relação das microrregiões dos estados da Região Nordeste está apresentada na Tabela 1. Um outro problema diz respeito ao preenchi- mento do formulário da DN com relação às decla- rações errôneas ou omissões de informações re- ferentes às variáveis. Para dimensionar tal proble- ma, o grau de cobertura das declarações, ou seja, a completude é considerada um útil indicador para mensurar a qualidade dessas informações. Para tanto, a completude foi avaliada usando-se a pro- porção de ignorados em cada uma das variáveis. Alguns autores estabeleceram limites de tolerân- cia para a representatividade das variáveis. No caso das coberturas de óbitos Preston 16 estabe- leceu em 60%, a Organização das Nações Unidas (ONU) 17 55% e a Organization for Economic Co- operation and Development (OECD) 18 em 50%. Dessa forma, admitiu-se, aqui, um percentual igual ou inferior a 30% de ignorados, montante este considerado como razoável para expressar os padrões de representação das variáveis. Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 Paes NA, Santos CSA 314 Qualidade dos dados As coberturas dos nascidos vivos estimadas para as 188 microrregiões do Nordeste são ilustradas na Tabela 1. Boa parte das microrregiões da Bahia, Paraíba, Sergipe e Pernambuco apresentou co- berturas igual ou acima de 80%, cujas cobertu- ras estão classificadas entre boa e muito boa. As microrregiões de Alagoas, Ceará e Rio Grande do Norte registraram coberturas inferiores a 80%, configurando-se como coberturas regulares. Por sua vez, as microrregiões dos estados do Piauí e do Maranhão situaram-se em sua maioria em um patamar inferior a 70%, com coberturas conside- radas deficientes. Medidas de adequação para o uso da análise fatorial O segundo teste, o de esfericidade de Bartlett, é baseado na distribuição estatística de qui-qua- dradro e testa a hipótese (nula H0) de que a ma- triz de correlação é uma matriz identidade (cuja diagonal é 1,0 e todas as outras iguais a zero), isto é, que não há correlação entre as variáveis 6. Valores de significância maiores que 0,10 indicam que os dados não são adequados para o tratamento com o método em questão; que a hipótese nula não pode ser rejeitada. Já valores menores que o indicado permite rejeitar a hipó- tese nula 19. O teste de Shapiro & Wilk (W) 21 tem sido o mais utilizado para testar a normalidade dos da- dos, por causa de suas propriedades de poder quando comparado com uma larga faixa de tes- tes alternativos. Se a estatística W for significativa (para p = 0,05), então a hipótese de que a distri- buição dos dados em análise é normal deve ser rejeitada 21. Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 Método de rotação ortogonal varimax A rotação de fatores é uma técnica para girar os eixos de referência dos fatores, em torno da ori- gem, até alcançar uma posição ideal. Ela pode ser ortogonal ou oblíqua, caso os eixos se mante- nham ou não em 90 graus entre si durante o giro. O objetivo é facilitar a leitura dos fatores, pois a rotação deixa pesos fatoriais altos em um fator e baixos em outros, definindo mais claramente os grupos de variáveis que fazem parte de um fator estudado. O método de rotação mais utilizado é o varimax, o qual simplifica as colunas da matriz de fatores. Para os estados, os resultados são apresen- tados na Tabela 2, os quais foram confrontados com aqueles obtidos pelo Ministério da Saúde e pelo IBGE, ambos estimados por meio de méto- dos indiretos das projeções. Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 NASCIMENTOS E FATORES MATERNOS E DA CRIANÇA NO NORDESTE 315 Tabela 1 Estado/Microrregião Cobertura (%) Maranhão 63,40 Litoral Ocidental 49,85 São Luís 61,03 Rosário 71,20 Lençóis 49,37 Baixada 54,98 Itapecuru Mirim 91,77 Gurupi 46,11 Pindaré 61,66 Imperatriz 46,23 Médio Mearim 55,92 Alto Mearim/Grajaú 66,32 Presidente Dutra 68,85 Baixo Parnaíba 67,51 Chapadinha 85,06 Codó 72,67 Coelho Neto 71,46 Caxias 63,37 Chapada do Itapecuru 50,27 Porto Franco 66,57 Gerais Balsas 86,22 Chapada do Mangabeira 45,52 Piauí 61,40 Baixo Parnaíba 72,54 Litoral 66,92 Teresina 59,79 Campo Maior 60,39 Médio Parnaíba 56,57 Valença 58,53 Alto Parnaíba 65,96 Bertolínia 61,33 Floriano 57,79 Alto Médio Gurguéia 68,74 São Raimundo Nonato 50,71 Chapada do Extremo Sul Piauiense 62,17 Picos 59,47 Pio IX 64,04 Alto Médio Canindé 55,85 Ceará 76,50 Camocim/Acaraú 87,54 Ibiapaba 99,92 Coreaú 97,21 Meruoca 90,60 Sobral 88,67 Ipu 89,07 Santa Quitéria 79 44 Distribuição percentual da cobertura dos registros de nascimentos. Microrregiões e estados do Nordeste, Brasil, 2000. Método de rotação ortogonal varimax Ceará (cont.) 76,50 Itapipoca 82,83 Baixo Curu 65,92 Uruburetama 87,16 Médio Curu 69,35 Canindé 96,83 Baturité 76,97 Chorozinho 65,76 Cascavel 61,54 Fortaleza 57,12 Pacajus 70,04 Sertão Cratéus 80,67 Quixeramobim 76,59 Sertão Inhamuns 82,23 Senador Pompeu 76,65 Litoral Aracati 60,47 Baixo Jaguaribe 70,03 Médio Jaguaribe 78,60 Serra Pereiro 70,09 Iguatu 71,19 Várzea Alegre 78,26 Lavras da Mangabeira 75,50 Chapada do Araripe 95,29 Caririaçu 70,18 Barro 51,47 Cariri 69,31 Brejo Santo 52,56 Rio Grande do Norte 73,90 Mossoró 61,20 Chapada do Apodi 56,22 Vale do Açu 71,54 Serra de São Miguel 88,68 Pau dos Ferros 69,24 Umarizal 71,02 Macau 72,70 Angicos 63,94 Serra Santana 60,80 Seridó Ocidental 65,03 Seridó Oriental 68,03 Baixa Verde 91,35 Borborema 77,41 Agreste 86,22 Litoral Nordeste 82,58 Macaíba 83,28 Natal 73,87 Litoral Sul 99,53 Estado/Microrregião Cobertura (%) Paraíba 83,90 Catolé do Rocha 85,98 Cajazeiras 84,75 Sousa 72,11 Patos 91,83 Piancó 75,40 Itaporanga 77,19 Serra Teixeira 98,68 Médio Oeste 60,59 Seridó Ocidental 99,19 Seridó Oriental 97,98 Cariri Ocidental 88,92 Cariri Oriental 92,76 Curimataú Ocidental 79,60 Curimataú Oriental 99,72 Esperança 99,76 Brejo 97,05 Guarabira 73,30 Campina Grande 86,64 Itabaiana 63,97 Umbuzeiro 77,18 Litoral Norte 65,58 Sapé 56,92 João Pessoa 73,98 Litoral Sul 90,21 Pernambuco 79,90 Araripina 99,85 Salgueiro 89,83 Pajeú 69,94 Sertão Moxotó 82,49 Petrolina 86,67 Itaparica 98,43 Vale do Ipanema 99,98 Vale do Ipojuca 78,49 Médio Capibaribe 68,62 Garanhuns 80,88 Brejo 80,55 Mata Setentorial 71,64 Vitória de Santo Antão 74,34 Mata Meridional 80,63 Itamaracá 73,83 Recife 62,28 Suape 78,88 Fernando de Noronha 32,11 Estado/Microrregião Cobertura (%) Distribuição percentual da cobertura dos registros de nascimentos. Microrregiões e estados do Nordeste, Brasil, 2000. Cad. Método de rotação ortogonal varimax Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 Paes NA, Santos CSA 316 Tabela 1 (continuação) Alagoas 68,80 Serra do Sertão Alagoano 67,96 Alagoana do Sertão do São Francisco 76,98 Santana Ipanema 84,91 Batalha 73,18 Palmeira dos Índios 63,79 Arapiraca 70,16 Traipu 53,16 Serrana dos Quilombos 74,12 Mata 73,31 Litoral Norte 63,25 Maceió 53,38 São Miguel dos Campos 67,09 Penedo 73,35 Sergipe 81,70 Sergipana do Sertão do São Francisco 73,65 Carira 77,87 Nossa Senhora das Dores 77,73 Agreste de Itabaiana 71,73 Tobias Barreto 95,88 Estado/Microrregião Cobertura (%) Estado/Microrregião Cobertura (%) Estado/Microrregião Cobertura (%) Sergipe (cont.) 81,70 Agreste de Lagarto 89,49 Propriá 82,18 Cotinguiba 65,01 Japaratuba 89,81 Baixo Cotinguiba 84,34 Aracaju 72,22 Boquim 91,63 Estância 89,90 Bahia 87,00 Barreiras 99,93 Alto Capibaribe 61,09 Cotegipe 76,70 Santa Maria da Vitória 79,88 Juazeiro 99,97 Paulo Afonso 99,69 Barra 99,97 Bom Jesus da Lapa 93,13 Senhor do Bonfim 96,70 Irecê 99,98 Jacobina 99,38 Itaberaba 99,96 Bahia (cont.) 87,00 Feira de Santana 84,09 Jeremoabo 79,14 Euclides da Cunha 68,19 Ribeira do Pombal 85,48 Serrinha 91,41 Alagoinhas 87,02 Entre Rios 99,98 Catu 80,33 Santo Antônio de Jesus 54,51 Salvador 75,01 Boquira 95,69 Seabra 94,82 Jequié 92,85 Livramento do Brumado 82,79 Guanambi 79,11 Brumado 72,30 Vitória da Conquista 88,58 Itapetinga 81,80 Valença 97,86 Ilhéus-Itabuna 82,48 Porto Seguro 81,23 Fonte: Laboratório de Estudos Demográﬁ cos, Departamento de Estatística, Universidade Federal da Paraíba. Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 Rotulação ou classificação mo desejável. Como essa matriz tem dimensão 26 x 26, torna-se inviável sua apresentação neste trabalho. No fator 1 (condições favoráveis ao parto) en- contramos as variáveis: peso ao nascer (≤ 2.499g, 2.500-2.999g e 3.000-3.999g); raça do recém- nascido (branca e não-branca); parto Cesário; estado civil da mãe (separada, casada e viúva); 7 e mais consultas pré-natais; gestação em sema- nas (≤ 36; 37-41); idade da mãe de 20-29 anos e; instrução da mãe em anos de estudo (8-11; ≥ 12). Essas categorias apresentaram carregamentos elevados e positivos, podendo ser definidas por um fator que indica as microrregiões do Nordes- te, cujas mães possuíam no ano 2000 caracterís- ticas que favoreceram uma gestação com risco reduzido. O índice KMO (medida de adequação da amostra) apresentou um resultado de 0,938, in- dicando que a adequação do método de análise fatorial foi “muito boa” para o tratamento dos dados. Assim, comprovado que os dados foram adequados para realizar a análise fatorial e es- tabelecido o pressuposto da normalidade dos dados usando-se o teste de Shapiro & Wilk (valor de p = 0,514), aplicou-se tal procedimento. O mo- delo fatorial ajustado com 26 categorias explicou 92,05% da variação total (Tabela 3). Nesse mode- lo, nenhuma categoria apresentou comunalida- de baixa com dois fatores estimados pelo método das componentes principais. Na Tabela 3 encon- tram-se os percentuais da variância explicada por fator. A variância explicada no primeiro fator continuou sendo muito superior a dos demais fa- tores, pelo fato de que estas categorias estiveram muito correlacionadas, além do mais, são dados provenientes de uma mesma região geográfica, com características sócio-econômicas similares. No fator 2 (condições desfavoráveis ao parto) foram encontradas as seguintes variáveis: peso ao nascer (≥ 4.000g); consultas pré-natal (nenhu- ma, 1-6); estado civil da mãe (solteira); idade da mãe (10-19 anos, 30-39 anos e 40-49 anos); parto vaginal; semanas de gestação (≥ 42 semanas); e nível de instrução em anos de estudo (nenhuma, 1-7). Essas categorias apresentaram carregamen- tos elevados e positivos e foram classificadas com um fator que indica as microrregiões do Nordes- te, cujas mães possuíam características desfavo- ráveis a uma gestação “saudável”, ou seja, mais expostas a uma gestação com risco. Ainda na Tabela 3, encontram-se os carre- gamentos das 26 categorias nos dois fatores es- timados após ter sido feita a rotação de fatores pelo método varimax. Medidas de adequação para o uso da análise fatorial Estados da Região Nordeste, Distribuição percentual da cobertura dos registros de nascimentos, segundo diferentes fontes. Estados da Região Nordeste, Brasil 2000 Brasil, 2000. Estado Média das microrregiões * (%) Ministério da Saúde (%) IBGE * (%) Maranhão 63,4 60,5 38,4 Piauí 61,4 79,7 53,2 Ceará 76,5 78,0 58,3 Rio Grande do Norte 73,9 86,9 66,8 Paraíba 83,9 69,9 72,1 Pernambuco 79,9 88,6 69,4 Alagoas 68,8 78,9 47,4 Sergipe 81,7 90,6 75,4 Bahia 87,0 77,4 76,9 Nordeste 75,2 77,6 62,0 * Fonte: Laboratório de Estudos Demográﬁ cos, Departamento de Estatística, Universidade Federal da Paraíba; Fonte: Sistema de Informações sobre Nascidos Vivos (Departamento de Informática do SUS; http://tabnet.datasus.gov.br); * Fonte: Instituto Brasileiro de Geograﬁ a e Estatística (IBGE; http://www.ibge.gov.br). Medidas de adequação para o uso da análise fatorial As coberturas dos estados obtidas usando- se o método da razão de parturição (coluna 1) ficaram próximas daquelas obtidas pelo Minis- tério da Saúde. Por sua vez, as coberturas esti- madas pelo IBGE parecem subestimadas ao se distanciaram, em sua maioria, das coberturas produzidas pelas duas primeiras fontes. As es- tatísticas oficiais mostram que já no ano 2000 o IBGE captava menos óbitos que o Ministério da Saúde. Originalmente foram coletadas oito variáveis, conforme mencionadas anteriormente, as quais se reproduziram em 42 categorias. A maioria das categorias foi reagrupada, ficando reduzidas a 26. Na Tabela 3, são descritas essas variáveis com as respectivas categorias. Para saber se a matriz de correlações é uma matriz identidade, o teste de esfericidade Bar- tlett gerou a estatística qui-quadrado igual a 31.967,469 com grau de liberdade de 946, for- necendo uma significância com um valor de p = 0,000, cuja decisão foi rejeitar a hipótese nula de que a matriz de correlação é uma matriz iden- tidade. A matriz de correlações entre as catego- rias apresentou valores em sua maioria acima de 58,2%, indicando um alto grau de correlação entre as categorias. Valores altos nas correlações entre as categorias facilitam a utilização da aná- lise fatorial, pois este fato provoca uma diminui- ção no número de fatores gerados nesta técnica, tornando as interpretações mais simples. Por- tanto, a matriz de correlações revelou-se signifi- cativamente diferente da matriz identidade, co- Para o Nordeste, a diferença entre o resultado produzido aqui e a do Ministério da Saúde foi cerca de 2%, cuja média entre elas ficou em tor- no de 76%. As coberturas obtidas neste trabalho para os estados se diferenciaram no máximo em 10% quando comparadas com as do Ministério da Saúde, exceto para os estados do Piauí (18%), Rio Grande do Norte (13%) e Paraíba (14%). Nos dois primeiros uma cobertura mais elevada foi obtida pelo IBGE. Nas três situações, o Estado do Maranhão situou-se em um patamar inferior aos demais estados da região, ao contrário dos es- tados da Bahia, Sergipe, Paraíba e Pernambuco, que se destacaram com os graus mais elevados de coberturas dos registros de nascimentos. Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 NASCIMENTOS E FATORES MATERNOS E DA CRIANÇA NO NORDESTE 317 Tabela 2 Distribuição percentual da cobertura dos registros de nascimentos, segundo diferentes fontes. Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 Rotulação ou classificação Na última coluna dessa tabela pode-se observar a comunalidade de cada categoria. A matriz rotada possibilitou a interpre- tação dos fatores atribuindo-se uma rotulação de acordo com suas características diferenciadoras. Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 Paes NA, Santos CSA 318 Tabela 3 Matriz de carregamentos rotacionada pelo método varimax e número de fatores escolhidos: critério das raízes latentes (auto- valor > 1) e variância explicada e acumulativa nos fatores. Variáveis Fatores ou componentes * Comunalidades Fator 1 Fator 2 Inicial Extração * Peso 3.000-3.999g 0,955 0,207 1 0,956 Cor branca 0,941 0,245 1 0,946 Cor não-branca 0,857 0,438 1 0,927 Parto cesário 0,781 0,603 1 0,973 Estado civil separada 0,769 0,603 1 0,955 Estado civil casada 0,764 0,621 1 0,969 Estado civil viúva 0,762 0,584 1 0,921 Peso 2.500-2.999g 0,752 0,609 1 0,936 Peso ≤ 2.499g 0,729 0,635 1 0,935 ≥ 7 consultas pré-natal 0,728 0,648 1 0,951 ≤ 36 semanas gestação 0,726 0,661 1 0,964 8-11 anos instrução 0,720 0,670 1 0,967 37-41 semanas gestação 0,714 0,637 1 0,915 Idade 20-29 anos 0,713 0,697 1 0,994 ≥ 12 anos instrução 0,701 0,683 1 0,958 Peso ≥ 4000g 0,176 0,901 1 0,843 Nenhuma consulta pré-natal 0,242 0,883 1 0,837 Estado civil solteira 0,543 0,811 1 0,953 Idade 40-49 anos 0,543 0,765 1 0,880 Parto vaginal 0,637 0,762 1 0,987 ≥ 42 semanas gestação 0,584 0,760 1 0,920 1-6 consultas pré-natal 0,630 0,732 1 0,933 Idade 30-39 anos 0,659 0,722 1 0,955 Idade 10-19 anos 0,673 0,719 1 0,969 1-7 anos instrução 0,671 0,717 1 0,965 Nenhuma instrução 0,384 0,526 1 0,724 Autovalor (> 1) 22,68 1,25 - % da variância 87,23 4,82 - % acumulativo 87,23 92,05 - Fonte: Laboratório de Estudos Demográﬁ cos, Departamento de Estatística, Universidade Federal da Paraíba. Matriz de carregamentos rotacionada pelo método varimax e número de fatores escolhidos: critério das raízes latentes (auto- valor > 1) e variância explicada e acumulativa nos fatores. * Método de extração: componentes principais. Admite-se que as obtidas neste trabalho reflitam valores aproximados e plausíveis dos valores ver- dadeiros. Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 Fonte: Laboratório de Estudos Demográﬁ cos, Departamento de Estatística, Universidade Federal da Paraíba. Discussões As coberturas obtidas pelos métodos aplicados aqui e pelo Ministério da Saúde foram mais plau- síveis, uma vez que atendem às tendências e ex- pectativas históricas. Como as coberturas calcu- ladas neste trabalho foram baseadas na média daquelas obtidas para todas as microrregiões que compõem cada estado, parecem guardar mais coerência. É preciso considerar, no entanto, que como qualquer estimativa, deve-se atentar para os erros inerentes às estimativas pontuais. O comportamento geral das coberturas para as microrregiões do Nordeste (Tabela 1) revela que o Maranhão e o Piauí foram as microrregi- ões dominadas por baixas coberturas, enquanto que na Bahia predominaram coberturas acima dos 90%. Nos estados intermediários a esses, a composição das regiões foi predominante com coberturas acima dos 70%. Isolando-se as mi- crorregiões do extremo sul da Bahia, de forma Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 NASCIMENTOS E FATORES MATERNOS E DA CRIANÇA NO NORDESTE 319 refere à situação educacional, a população ne- gra tem apresentado desvantagem em relação à branca. Desde que a variável nível de instrução, com anos de escolaridade mais elevados, mos- trou-se associada ao fator 1, seria esperado que a variável raça/cor mostrasse algum diferencial. Segundo Perpétuo 26 , a influência dessa variável sobre a probabilidade de acesso às ações de saú- de desaparece apenas quando controlada pela classe social que pode ser considerada como um indicador do poder aquisitivo. Sugere que o principal problema não é ser negro, mas ser po- bre, mas que não se pode afastar a existência de discriminação racial no acesso às ações de saúde reprodutiva. Levanta que se pode argumentar que o poder aquisitivo, talvez mais que outras características sócio-econômicas, como a resi- dência e a escolaridade, estaria captando essa mesma discriminação. Existe uma outra dimen- são quando se discute a questão da discrimina- ção racial, seja ela a qualidade do atendimento à saúde disponível para brancas e negras ou não. Essa discussão merece mais investigações. No entanto, é preciso lembrar que a variável raça foi a que apresentou o maior percentual de igno- rados em todos os estados do Nordeste, poden- do desta maneira ter ocasionado algum tipo de “confundimento”. genérica, a distribuição regional da cobertura dos nascimentos sugere um aumento à medida que os estados se posicionam em direção ao sul do Nordeste. Discussões A qualidade do preenchimento das variá- veis cumpriu plenamente o requisito estabele- cido aqui como limite de aceitação mínimo de completude, 70%, à exceção da variável raça. No entanto, tomando como base o referencial adotado por instituições e pesquisadores im- portantes no cenário mundial (50% a 60% de completude), pode-se considerar que o sistema de registros dos nascimentos no Nordeste pro- duziu uma qualidade virtualmente satisfatória para se traçar perfis representativos das variá- veis investigadas. Foram identificadas em condições favorá- veis mães com considerável nível de instrução e número elevado de consultas pré-natais, o que sugere que essas mães possuíam um bom nível de informação sobre os cuidados com a gravidez e parto. O número de nascidos vivos de mães com idades entre 20-29 anos foi outra variável funda- mental, presente no fator 1, pois atribui-se nesta faixa etária um risco menor de ter um recém- nascido de baixo peso, por já possuir maturidade funcional do organismo, além do mais, é nesta faixa etária que as mulheres encontram-se no período mais fértil da vida. Menos de 42 semanas de gestação, incluída como favorável ao parto, é desejável para um parto saudável. O parto cesário encontra-se presente nesse fator, apesar de ser um tipo de parto que oferece certo risco à mãe. Leva-se em consideração que esse tipo de parto possui um custo mais elevado para a mãe do que o parto vaginal. A presença dessa variável, nesse fator, sugere que o parto cesário está restrito a mulheres com “maior” po- der aquisitivo e/ou com acesso aos serviços de atenção ao parto. Em estudo realizado por Carnel et al. 27 para Campinas, Estado de São Paulo, as chances para indicação de cesarianas foram mais elevadas para mulheres de melhor nível sócio- econômico e para as com pré-natal adequado, sugerindo que essa indicação não se baseia so- mente em normas técnicas, mas também em ra- zões não-médicas. Peso do recém-nascido entre 2.500-3.999g ex- pressa que a nutrição e cuidados da mãe durante o parto foram adequados e que comportaria me- nos riscos para os recém-nascidos 22. Estudo con- duzido no Município de Santo André no Estado de São Paulo, encontrou que os nascidos vivos de baixo peso ao nascer apresentaram um risco de morte 51,2 vezes superior ao daqueles que nas- ceram com peso igual ou superior a 2.500g 23. Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 Considerações finais financeiras nestes procedimentos, além de ou- tros fatores. Levando-se em consideração a distribuição das microrregiões do Nordeste brasileiro segundo as diferentes variáveis empregadas com relação às condições das mães e das crianças geradas, observou-se, em 2000, uma dicotomia sinteti- zada pelas “condições favoráveis” e “condições desfavoráveis” ao parto. Nessas regiões poder-se- ia traçar um perfil semelhante para o conjunto delas em cada fator. As características estariam ligadas a questões estruturais e diretamente rela- cionadas ao nível de desenvolvimento humano. Sugere-se como aprofundamento deste estudo a inclusão de variáveis econômicas associadas às condições que descrevam os nascidos vivos na Região Nordeste. A aplicação de técnicas de aná- lise multivariada, como a análise discriminan- te, para classificar as microrregiões com carac- terísticas similares em algum aspecto, segundo os fatores encontrados, poderia servir como um desdobramento deste trabalho. As microrregiões com valores altos nesse fa- tor, apresentaram características que descrevem condições favoráveis ao parto. As microrregiões com valores baixos nesse fator apresentaram ca- racterísticas que se aproximaram das caracterís- ticas do Fator 2. O número de nascidos vivos de mães anal- fabetas ou com baixo nível de instrução e baixo número de consultas pré-natal sugere que estas mães detêm um baixo nível de informação sobre os cuidados com a gravidez e o parto. Em estudo realizado por Morell & Melo 8, constatou-se pa- ra os municípios do Estado de São Paulo que as mulheres com menor nível de instrução tiveram seus filhos em idades mais precoces que aque- las que alcançaram maiores níveis de escolari- dade, o que elevaria o risco de um nascimento favorável. Nesse mesmo estudo, as autoras mostraram que uma idade materna precoce ou tardia está relacionada com o perfil dos nascidos vivos, co- mo o peso ao nascer. O número de nascimentos de mães na faixa etária de 10-19 anos (prema- turidade materna) representa um fator de risco elevado para o baixo peso ao nascer. As mães nas faixas etárias de 30-39 anos e de 40-49 são consi- deradas como gravidez de risco, pois estas faixas etárias são consideradas elevadas, podendo cau- sar risco durante a gravidez. Por sua vez, crianças com peso acima de 4.000g e mais de 42 semanas de gestação indicam complicações para um par- to saudável. Um outro aspecto que pode ser explorado é a questão do relacionamento entre qualidade dos registros de nascimentos com indicadores sócio- econômicos como nível de instrução e renda. Discussões O baixo peso reflete piores condições de vida, cuja maior concentração provém da clientela usuária do Sistema Único de Saúde (SUS), onde é maior a demanda por serviços de maior complexidade 24. A literatura é abundante em mostrar as ele- vadas proporções dos partos cesários no Brasil e no Nordeste, que facilmente têm ultrapassa- do 50% em muitas regiões do país 24,27. Segundo Almeida 24, do ponto de vista clínico, têm como fatores que concorrem para a realização de cesá- reas aqueles relacionados com a organização dos serviços de saúde e fatores que dizem respeito à parturiente. Destacam-se a redução do tempo do trabalho de parto, aumento da incorporação de tecnologia nos procedimentos relativo ao parto, agendamento prévio do parto como fator de oti- mização do trabalho médico, maior segurança do médico quanto ao procedimento e vantagens As variáveis como estado civil das mães nas categorias casada, separada e viúva presentes no fator 1, determinam que estas mães possuíam algum tipo de assistência, apoio do cônjuge ou do companheiro, ou mesmo uma rede de pro- teção familiar favorecendo um parto com con- dições mais estáveis, diferentemente das mães solteiras. A raça/cor do recém-nascido foi a única vari- ável que não se dividiu entre os fatores. Esse fato sugere que essa variável não se evidenciou como um diferenciador da natalidade para o Nordeste brasileiro. Simão et al. 25 sugerem que, no que se Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 Paes NA, Santos CSA 320 Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 Resumo N. A. Paes participou da redação e revisão do trabalho. C. S. A. Santos colaborou na análise e interpretação dos dados. Historicamente, o registro de nascimentos do Nordes- te brasileiro tem apresentado importantes problemas de qualidade, cujas variações regionais são muito grandes e pouco se conhece sobre elas. Este trabalho se propõe a avaliar a qualidade dos registros sobre os nascidos vivos e identificar padrões de relacionamento entre variáveis que reflitam os fatores maternos e da criança constantes nas declarações de nascimento, que permitam resumir os dados sobre nascidos vivos das 188 microrregiões do Nordeste em 2000, com base no uso da análise fatorial. Os dados sobre nascidos vivos foram coletados do Sistema de Informações sobre Nas- cidos Vivos (SINASC). De forma genérica, a distribui- ção regional da cobertura dos nascimentos sugere um aumento à medida que os estados se posicionam em direção ao sul do Nordeste. A qualidade no preenchi- mento das variáveis das microrregiões do Nordeste foi considerada satisfatória. Na análise fatorial, os dados das variáveis foram reduzidos em dois fatores: favorá- veis e desfavoráveis ao parto. Considerações finais Sugere-se uma não associação entre essas duas dimensões para o Nordeste em 2000. Especula-se que as pautas que nortearam a melhoria da qua- lidade das estatísticas vitais no Nordeste foram independentes daquelas que regularam os níveis da natalidade e da fecundidade. Investigações usando períodos mais recentes poderiam lançar mais luzes a essa questão. A variável número de nascidos vivos de mães solteiras também foi fundamental para caracte- rizar uma gestação com risco, pois os resultados da análise estatística sugerem que estas mães poderiam reunir um quadro mais desfavorável. Ou seja, essa condição civil poderia estar liga- da a alguns fatores sócio-econômicos e culturais que limitariam um acesso pleno à assistência na gravidez. Espera-se que este trabalho possa contribuir para a definição de estratégias que melhorem os registros de nascimentos, e que possa subsidiar o planejamento e delimitar políticas públicas nas áreas de saúde materna e infantil nos estados e suas respectivas microrregiões nordestinas. O parto vaginal, apesar de ser considerado “normal” ou mais recomendado às mães, carac- teriza-se por ser um tipo de parto de baixo custo, natural, mais acessível às mães. Mas, pode estar associado a menores cuidados, podendo suge- rir um maior risco. O diferencial com relação ao parto cesário pode também ser conseqüência de uma faceta da análise fatorial empregada neste trabalho. É possível que essa variável apresen- te um confundimento com outras variáveis ou mesmo que a qualidade desses dados tenha gera- do um viés na sua classificação junto com outras variáveis com um risco elevado. Essas especula- ções, no entanto, exigem maiores investigações que extrapolam o escopo deste trabalho. NASCIMENTOS E FATORES MATERNOS E DA CRIANÇA NO NORDESTE 321 Declaração de Nascimento; Sistemas de Informação; Estatísticas Vitais Agradecimentos Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) pelo financiamento. Declaração de Nascimento; Sistemas de Informação; Estatísticas Vitais Referências 1. Souza LM. Avaliação do Sistema de Informação sobre Nascidos Vivos. In: Anais do XIV Encontro Nacional de Estudos Populacionais [CD-ROM]. Campinas: Associação Brasileira de Estudos Popu- lacionais; 2004. 5. Szwarcwald CL, Leal MC, Andrade CLT, Souza Jr. PRB. Estimação da mortalidade infantil no Brasil: o que dizem as informações sobre óbitos e nasci- mentos do Ministério da Saúde? Cad Saúde Públi- ca 2002; 18:1725-36. 1. Souza LM. Avaliação do Sistema de Informação sobre Nascidos Vivos. In: Anais do XIV Encontro Nacional de Estudos Populacionais [CD-ROM]. Campinas: Associação Brasileira de Estudos Popu- lacionais; 2004. 5. Szwarcwald CL, Leal MC, Andrade CLT, Souza Jr. PRB. Estimação da mortalidade infantil no Brasil: o que dizem as informações sobre óbitos e nasci- mentos do Ministério da Saúde? Cad Saúde Públi- ca 2002; 18:1725-36. 2. Barbosa LM, Melo GHN. Avaliação da qualidade das informações sobre fecundidade provenientes do SINASC no Nordeste, 2000. Rev Bras Estud Po- pul 2005; 22:141-58. 6. Pereira JCR. Análise de dados qualitativos: estraté- gias metodológicas para as ciências da saúde, hu- manas e sociais. São Paulo: Edusp; 2001. 7. Paes NA. Um olhar sobre as estimativas da morta- lidade infantil do Nordeste provenientes de fontes tradicionais e do PACS. In: Teixeira P, organizador. Mortalidade infantil: fontes, metodologias e resul- tados. Recife: Fundação Joaquim Nabuco/Editora Massangana; 1998. p. 143-64. 7. Paes NA. Um olhar sobre as estimativas da morta- lidade infantil do Nordeste provenientes de fontes tradicionais e do PACS. In: Teixeira P, organizador. Mortalidade infantil: fontes, metodologias e resul- tados. Recife: Fundação Joaquim Nabuco/Editora Massangana; 1998. p. 143-64. 3. Scochi CGS, Costa IAR, Rocha SMM, Leite AM, Nascimento LC. Intervalo entre o nascimento e o registro civil: situação no Município de Ribeirão Preto, São Paulo, Brasil. Rev Bras Saúde Matern In- fant 2004; 4:171-8. 8. Morell MGG, Melo AV. A Declaração de Nascido Vivo no Estado de São Paulo: alguns resultados. Informe Demográfico 1995; 29:15-59. 4. Potter JE, Schmertmann CP, Cavenaghi SM. Fer- tility and development: evidence from Brazil. De- mography 2002; 39:739-61. Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 Paes NA, Santos CSA 322 9. Rede Interagencial de Informação para a Saúde. Indicadores básicos para a saúde no Brasil: concei- tos e aplicações. 2a Ed. Brasília: Organização Pan- Americana da Saúde; 2008. 18. Organization for Economic Cooperation and De- velopment. Mortality in developing countries. Tome III. Paris: Organization for Economic Coop- eration and Development; 1980. 10. Referências Albuquerque FRPC, Santos SR. Fatores de correção para o registro de nascimento utilizando registros tardios segundo os grupos de idades das mulhe- res Brasil e Unidades da Federação 1984/2001. In: Anais do XIV Encontro Nacional de Estudos Popu- lacionais [CD-ROM]. Campinas: Associação Brasi- leira de Estudos Populacionais; 2004. 19. Hair JF, Anderson RE, Tatham RL, Black WC. Aná- lise multivariada de dados. 5a Ed. São Paulo: Book- man Editora; 2005. 20. Johnson RA, Wichern DW. Applied multivariate statistical analysis. 5th Ed. New Jersey: Prentice- Hall; 2002. 21. Shapiro SS, Wilk MB. An analysis of variance test for normality (complete samples). Biometrika 1965; 52:591-611. 11. Wong L, Giraldelli B. Delayed Birth Registration (DBR) in São Paulo State: an attempt at correcting under-registration. Brazilian Journal for Popula- tion Studies 1997/1998; 1:84-104. 22. Maia MAC. Caracterização dos nascidos vivos hospitalares no primeiro ano de implantação do Subsistema de Informação sobre Nascidos vivos, em município de Minas Gerais, Brasil, 1996. Rev Saúde Pública 1997; 31:581-5. 12. Leal MC, Szwarcwald CL. Evolução da mortalidade neonatal no Estado do Rio de Janeiro, Brasil, de 1979 a 1993. 1 – Análise por grupo etário segun- do região de residência. Rev Saúde Pública 1996; 30:403-12. 23. Almeida MF. Fatores de risco da mortalidade ne- onatal em Santo André [Tese de Doutorado]. São Paulo: Faculdade de Saúde Pública, Universidade Estadual de São Paulo; 1994. 13. Frias LAM. Um modelo para estimar o sub-registro de nascimentos. Boletim Demográfico 1982; 13: 11-32. 24. Almeida MF. O uso da Declaração de Nascido Vivo na caracterização dos partos hospitalares. Informe Demográfico 1995; 29:107-22. 14. Altmann AMG, Ferreira CEC. A situação dos regis- tros dos fatos vitais no Brasil. Informe Demográfi- co 1982; 7:3-23. 25. Simão AB, Miranda-Ribeiro P, Caetano AJ, César CC. Comparando as idades à primeira relação se- xual, à primeira união e ao nascimento do primeiro filho de duas coortes de mulheres brancas e negras em Belo Horizonte: evidências quantitativas. Rev Bras Estud Popul 2006; 23:151-66. 15. Brass W. Methods for estimating fertility and mor- tality from limited and defective data. Chapel Hill: International Program of Laboratories for Popula- tions Statistics; 1975. 16. Preston SH. Use of direct and indirect techniques for estimating the completeness of death registra- tion systems. In: United Nations, editor. Data bases for mortality measurement. New York: United Na- tions; 1984. p. 66-76. 26. Perpétuo IHO. Raça e acesso às ações prioritárias na agenda da saúde reprodutiva. Cad. Saúde Pública, Rio de Janeiro, 26(2):311-322, fev, 2010 Referências In: Anais do XII Encontro Nacional de Estudos Populacionais [CD- ROM]. Campinas: Associação Brasileira de Estudos Populacionais; 2000. 17. United Nations. Model life tables for developing countries. New York: United Nations; 1982. (Popu- lation Studies, 77). 27. Carniel EF, Zanolli ML, Morcillo AM. Fatores de ris- co para indicação do parto cesário em Campinas (SP). Rev Bras Ginecol Obstet 2007; 29:34-40. Recebido em 18/Jul/2009 Versão final reapresentada em 30/Out/2009 Aprovado em 17/Nov/2009 Recebido em 18/Jul/2009 Versão final reapresentada em 30/Out/2009 Aprovado em 17/Nov/2009
https://openalex.org/W4394856717	https://annpublisher.org/ojs/index.php/toursci/article/download/249/259	English	null	Tourism Color Run Party Event Management in Ngawi	Jurnal Toursci	2,024	cc-by	1,669	Tourism Color Run Party Event Management in Ngawi Tourism Color Run Party Event Management in Ngawi Delfian Intan Nurmala Fadin Gadjah Mada University, Yogyakarta, Indonesia Gadjah Mada University, Yogyakarta, Indonesia Abstract. With every city, big or small, hosting a steady schedule of fresh events each year, events or festivals have become an integral element of Indonesian culture. A prime example is Ngawi City, East Java's Color Run Party. Even then, this event brings up several concerns, including the buildup of trash and the degradation of Indonesian culture due to the absence of regional components. The impact of the Color Run Party on society and culture, as well as destination and visitor management, are covered in this article. A qualitative technique is employed as the primary instrument in this research method, which describes the object's natural state. Findings indicate that although destination management and other event-related factors have been well-organized, some areas, such as trash management and the absence of regional cultural components, still need improvement. So that this event not only concentrates on quantity but also considers the effects on the environment and culture, more cautious management is required. Keywords: local culture, tourism destination management, Color Run Party, festival, and event management. Keywords: Festival, Party, Popular, Tourism Vol 1 No 5 April 2024 Vol 1 No 5 April 2024 https://annpublisher.org/ojs/index.php/toursci Journal of Tourism Sciences (Toursci) Tourism Color Run Party Event Management in Ngawi e-ISSN : 3046-7713 p-ISSN : 3032-7539 Journal of Tourism Sciences (Toursci) INTRODUCTION In Indonesia, celebrations and events are all the rage. Both large and small towns host recurring events or brand-new ones annually. As an illustration, consider the Colour Run event, which is held in numerous locations, including Ngawi City, East Java. The cleanup after the event featured much trash because so many people attended, which contributed to the event's holding; also, Indonesian culture was weakened because the Color Run Party was seen as being firmly at odds with it. Color Run Party is a 5 km event emphasizing pleasure and freedom. It originated in Arizona, United States, and then spread to Indonesia in 2014. Holi, an Indian festival, inspired the event. Pop culture and popular trends typically originate elsewhere and are seen as too easy for Indonesian society to embrace, which is sometimes seen as unprepared for globalization. As per Alastair M. Morrison's (2012) approach, which revolves around destination products comprising physical things, programs, people, and packages, the event organizer is hoping for many participants to ensure that the attempted Tourism Destination Management Fadin, Delfian Fadin, Delfian Fadin, Delfian \| 183 https://annpublisher.org/ojs/index.php/toursci Vol 1 No 5 April 2024 Vol 1 No 5 April 2024 feels comprehensive. Beginning with the theme, like the Color Run Party, the program includes music entertainment, a schedule of events from the opening to the closing, and amenities given to enthusiasts who purchase tickets for Rp. 50,000–65,000 (in the form of t-shirts and typical color- run merchandise). Additionally, the organizers worked with the government. They were eventually given full support because they were able to create an event that was seen as developing and having the potential to improve the image of Ngawi Regency. The event was implemented in the middle of the city, namely Ngawi City Square, and there were promotions through popular social media accounts about Ngawi, Ngawi tourism accounts, and promotional activities. Since the Ngawi Regency Government is supporting the Color Run Party, the management of tourist destinations has, in theory, been carried out accurately. This is because the event's target audience is students or those living far from the downtown square. News of the event is spreading, and an increasing number of people are interested in attending. It does not follow that there are still gaps from this event, especially with its excellent management as a tourist destination. Indeed, facility administration and visitor control could be more organized, positively impacting the surrounding area. Since guest control appears to be merely ceremonial and visitor limitations are not enforced, the organizer anticipates as many participants as possible, as was previously stated. Approximately 10,000 individuals took part in this event in 2019. Consequently, many crops were crushed, trash piled up, and traffic made the locals uncomfortable and polluted. The event's quality is neglected because it is exclusively focused on quantity, which needs to be addressed. Consideration should be given to visitor restrictions because, despite the event's large venue, a stampede is inevitable due to the large number of participants. Additionally, visitors' uncontrollable oxygen and carbon dioxide intake will cause breathing difficulties. If the organizers still desire many attendees, time division is required, such as using Color Run batch one and Color Run batch two participants. DISCUSSION The large number of motor vehicles causes significant air pollution. The district administration has completely backed this event. Therefore, waste management is also crucial because it needs to address any issues that may occur afterward. Since waste management is a significant issue today, enlisting volunteers to process waste and recycle it into more valuable materials is one way to address it. In exchange for their labor, these volunteers would receive pocket money and be guaranteed job or internship protection from the Ngawi Regency Government. Next is Event Management; as was previously noted, Color Run Parties are thought to have the potential to destroy Indonesian customs. This is due to the need for Indonesian culture during the event. Pop music abounds in the entertainment, and the concept of the American people is presented throughout the event. The organizer is forced to focus primarily on the interests of people in that age range rather than considering the value that should be placed on the intended target because the aim is a community of students. Even if the event is being held in conjunction with the promotion of Indonesian culture, such as through traditional dance performances as one of the entertainment options or the batik or puppet-themed key chain given out with tickets as ticket holders, the organizers did not include even the slightest hint of Indonesian culture in this event. Nevertheless, when the booth stand featuring traditional Indonesian or Ngawi food opens, it will make a significant impression, especially given that 10,000 people attended the Color Run Party. Attendees' perceptions of Color Run Party, which is thought to undermine Indonesian culture, are primarily influenced by event management because each event leaves a lasting impression on attendees. The impact that guests acquire is not just Color Run culture in general but also Indonesian culture, which is promoted thanks to the innovative administration of events that support Indonesian culture. Regarding other elements, like the Sunday event schedule, the colorful flour, booths, and stages, the square locations, the marketing that hits the mark, and the event schedule, everything seems appropriate and flawless if visitor control measures like time limits or restrictions are in place. Running 5 km with bursts of color flour every 1 km is one of the possible attractions for Tourism Attraction Management at this event. Other attractions include food stalls, door prizes, music, and games. METHOD With the researcher serving as the primary tool, this study employs a qualitative method to characterize the objects' natural state. Researchers opt for descriptive study to precisely and methodically describe the facts and features of the population or a specific field. Here, the researcher aims to present a synopsis of a specific currently occurring social phenomenon. Researchers may find it simpler to apply this strategy to examine, comprehend, and draw conclusions from each data set. \| 184 Fadin, Delfian Fadin, Delfian https://annpublisher.org/ojs/index.php/toursci https://annpublisher.org/ojs/index.php/toursci Vol 1 No 5 April 2024 DISCUSSION In light of the number of Color Run enthusiasts in Ngawi Regency, as previously mentioned, tourist attractions that concurrently promote Indonesian culture should be developed. Examples of such attractions include door prize distribution through the completion of nationality-related quizzes, stage entertainment featuring traditional dances and musical Fadin, Delfian Fadin, Delfian \| 185 https://annpublisher.org/ojs/index.php/toursci Vol 1 No 5 April 2024 instruments, painting exhibitions, batik, traditional Indonesian food, and miniature traditional houses. Suppose Generation Z is living in this era of technology and has easy access to everything. In that case, these measures are taken to prevent culture eroding in the memory of participants, the majority of whom are young. CONCLUSION As a result of globalization, several American customs, including the Color Run Party, made their way into Indonesia. The management of Ngawi City appears to be still focused on quantity, as seen by their counterarguments concerning the deterioration of Indonesian culture by the Colour Run Party. So that the execution of this event will not have a positive impact on the environment or culture, proper planning is required. The two most crucial management aspects are providing attractions that simultaneously connect visitors with Indonesian culture and managing garbage properly. This could lead to the Colour Run event being replaced by a cultural event that attracts just as many spectators. BIBLIOGRAPHY Muzha, V. K. (2015). Manajemen Strategi Pengembangan Pariwisata Dengan Pendekatan Blue Ocean Strategy. JISIP: Jurnal Ilmu Sosial dan Ilmu Politik, 43-52. Anonim. (2015, May). NGAWI COLOR RUN 2015. Retrieved from empatenamorganizer: https://empatenamorganizer.blogspot.com/ Armandhani, H. (2015). Colour Run : Menyehatkan atau Membahayakan? Retrieved from kompasiana:https://www.kompasiana.com/armandhani/54f36334745513a22b6c731 3/colour-run- menyehatkan-atau-membahayakan Armandhani, H. (2015). Colour Run : Menyehatkan atau Membahayakan? Retrieved from kompasiana:https://www.kompasiana.com/armandhani/54f36334745513a22b6c731 3/colour-run- menyehatkan-atau-membahayakan Dianisyani. (2015, March). Tingginya Animo Mayarakat dalam Event Color Run Party: Budaya Ikut-ikutan? Retrieved from dianisyani: https://dianisyani.wordpress.com/2015/03/19/tingginya-animo-mayarakat-dalam- event- color-run-party-budaya-ikut-ikutan/ Dianisyani. (2015, March). Tingginya Animo Mayarakat dalam Event Color Run Party: Budaya Ikut-ikutan? Retrieved from dianisyani: https://dianisyani.wordpress.com/2015/03/19/tingginya-animo-mayarakat-dalam- event- color-run-party-budaya-ikut-ikutan/ Harianto, S. (2019, February). Colour Run Millenial, Cara Polisi di Ngawi Minimalkan Kecelakaan. Retrieved from detikNews: https://news.detik.com/berita-jawa-timur/d- 4421728/colour-run-millenial-cara-polisi-di-ngawi-minimalkan-kecelakaan Harianto, S. (2019, February). Colour Run Millenial, Cara Polisi di Ngawi Minimalkan Kecelakaan. Retrieved from detikNews: https://news.detik.com/berita-jawa-timur/d- 4421728/colour-run-millenial-cara-polisi-di-ngawi-minimalkan-kecelakaan Marpaung, S. (2015). Mengapa harus ikut Color Run? Retrieved from subhievolution: https://subhievolution.blogspot.com/2015/01/mengapa-harus-ikut-color-run.html Marpaung, S. (2015). Mengapa harus ikut Color Run? Retrieved from subhievolution: https://subhievolution.blogspot.com/2015/01/mengapa-harus-ikut-color-run.html Unknown. (2014). Pengertian dari budaya populer atau Pop Culture. Retrieved from yupinter: https://yupinter.blogspot.com/2013/08/pengertian-dari-budaya-populer-atau- pop.html Unknown. (2014). Pengertian dari budaya populer atau Pop Culture. Retrieved from yupinter: https://yupinter.blogspot.com/2013/08/pengertian-dari-budaya-populer-atau- pop.html \| 186 Fadin, Delfian Fadin, Delfian
https://openalex.org/W2150694461	https://europepmc.org/articles/pmc1994061?pdf=render	English	null	A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities	Chemistry central journal	2,007	cc-by	4,944	Received: 10 April 2007 Accepted: 8 June 2007 J Chemistry Central Journal 2007, 1:16 doi:10.1186/1752-153X-1-16 p Accepted: 8 June 2007 This article is available from: http://journal.chemistrycentral.com/content/1/1/16 © 2007 Dalal 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/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2007 Dalal 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/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Chemistry Central Journal Open Access Abstract Background: The use of immobilized enzymes for catalyzing various biotransformations is now a widely used approach. In recent years, cross-linked enzyme aggregates (CLEAs) have emerged as a novel and versatile biocatalyst design. The present work deals with the preparation of a CLEA from a commercial preparation, Pectinex™ Ultra SP-L, which contains pectinase, xylanase and cellulase activities. The CLEA obtained could be used for any of the enzyme activities. The CLEA was characterized in terms of kinetic parameters, thermal stability and reusability in the context of all the three enzyme activities. Results: Complete precipitation of the three enzyme activities was obtained with n-propanol. When resulting precipitates were subjected to cross-linking with 5 mM glutaraldehyde, the three activities initially present (pectinase, xylanase and cellulase) were completely retained after cross- linking. The Vmax/Km values were increased from 11, 75 and 16 to 14, 80 and 19 in case of pectinase, xylanase and cellulase activities respectively. The thermal stability was studied at 50°C, 60°C and 70°C for pectinase, xylanase and cellulase respectively. Half-lives were improved from 17, 22 and 32 minutes to 180, 82 and 91 minutes for pectinase, xylanase and cellulase respectively. All three of the enzymes in CLEA could be reused three times without any loss of activity. Conclusion: A single multipurpose biocatalyst has been designed which can be used for carrying out three different and independent reactions; 1) hydrolysis of pectin, 2) hydrolysis of xylan and 3) hydrolysis of cellulose. The preparation is more stable at higher temperatures as compared to the free enzymes. ble enzyme aggregates produced by extensive chemical cross-linking of enzyme dissolved in a solution have been suggested as an alternative approach for obtaining stable and reusable enzyme preparations [5,6]. In recent years, it has been shown that chemical cross-linking of enzyme precipitates produces more robust biocatalysts. These have been called cross-linked enzyme aggregates (CLEAs) [7-9]. ble enzyme aggregates produced by extensive chemical cross-linking of enzyme dissolved in a solution have been suggested as an alternative approach for obtaining stable and reusable enzyme preparations [5,6]. In recent years, it has been shown that chemical cross-linking of enzyme precipitates produces more robust biocatalysts. These have been called cross-linked enzyme aggregates (CLEAs) [7-9]. Open Ac Research article A multipurpose immobilized biocatalyst with pectinase, xylanase and cellulase activities Sohel Dalal, Aparna Sharma and Munishwar Nath Gupta* Address: Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India Email: Sohel Dalal - sohel178@yahoo.co.uk; Aparna Sharma - ap19_12@yahoo.co.in; Munishwar Nath Gupta* - appliedbiocat@yahoo.co.in * Corresponding author Background g There is an increasing trend towards using enzymes for catalyzing biotransformations [1,2]. In view of their high costs (as compared to chemical catalysts), reusable forms of enzymes, called immobilized enzymes, are often used [3,4]. Immobilization is also sometimes accompanied by greater stability of enzymes during storage or operational stability. Immobilization generally consists of linking an enzyme to a solid matrix or entrapping it in a gel. Insolu- Page 1 of 6 (page number not for citation purposes) Page 1 of 6 (page number not for citation purposes) http://journal.chemistrycentral.com/content/1/1/16 Chemistry Central Journal 2007, 1:16 Precipitation of enzymatic activities with organic solvents Figure 1 Precipitation of enzymatic activities with organic sol- vents: Three different precipitants (acetone, dimethox- yethane (DME) and n-propanol, 5 ml each) were added to crude Pectinex™ Ultra SP-L (containing pectinase, xylanase and cellulase activity) (1 ml) for complete precipitation of proteins at 4°C as described in the experimental section. The percentage activities of the enzymes in the precipitate were calculated by taking the initial activities as 100%. The experi- ments were carried out in triplicate and error bars represent the percentage error in each set of readings. -■-: pectinase, - -: xylanase, - -: cellulase. 0 20 40 60 80 100 120 n- Propanol Acetone DME Solvents Precipitated activity (%) An interesting feature of the CLEAs is that these prepara- tions do not require extensive purification of the enzyme activities. In this respect, CLEAs differ from the CLEC™, another form of enzyme aggregates prepared by chemical cross-linking of enzyme crystals [10]. It has been shown that it is possible to form a CLEA that can catalyze more than one reaction. Thus, a CLEA may catalyze a sequence of reactions. Such CLEAs have been called Combi-CLEAs [8,11]. Recently, we suggested that it should be possible to extend this concept to Combi-CLEAs catalyzing non cas- cade reactions. Prepared from porcine pancreatic acetone powder, a CLEA was described which had lipase, alpha amylase and phospholipase A2 activities [11]. Such multi- purpose CLEAs would allow "one biocatalyst for many unrelated biological activities". In the present work, we show that this concept is especially useful in the context of crude/commercial preparations of microbial origin. In as much as enzymes are increasingly isolated from microbes, this work shows that a single biocatalyst preparation from microbial sources could catalyze several biotransforma- tions/bioconversions of industrial relevance. Precipitatio Figure 1 Precipitation of enzymatic activities with organic sol- vents: Three different precipitants (acetone, dimethox- yethane (DME) and n-propanol, 5 ml each) were added to crude Pectinex™ Ultra SP-L (containing pectinase, xylanase and cellulase activity) (1 ml) for complete precipitation of proteins at 4°C as described in the experimental section. The percentage activities of the enzymes in the precipitate were calculated by taking the initial activities as 100%. The experi- ments were carried out in triplicate and error bars represent the percentage error in each set of readings. -■-: pectinase, - -: xylanase, - -: cellulase. The system chosen is a well-known commercial prepara- tion called Pectinex™ Ultra SP-L that is used in the food processing industry for hydrolyzing pectin [12]. Our ear- lier work had shown that this preparation is also rich in xylanase and cellulase activities [13,14]. A multipurpose CLEA with substantial activities of pectinase, xylanase and cellulase was prepared and characterized. The other two activites, xylanase and cellulase, also have well known and extensively documented applications in biotechnology [15,16]. Results and discussion observed and reported for free enzyme (Data not shown as figure). observed and reported for free enzyme (Data not shown as figure). observed and reported for free enzyme (Data not shown as figure). The general protocol for the preparation of CLEAs consists of precipitating the enzyme activity by adding salt or an organic solvent [7,9]. This is followed by addition of cross-linking reagent, which is generally glutaraldehyde. Figure 1 shows that n-propanol precipitated all three of the enzymatic activities (viz. pectinase, xylanase and cellu- lase) completely. Figure 2 shows the effect of varying glu- taraldehyde concentration. Following the conditions described in the literature [9,11], 4 h of cross-linking time at 4°C was chosen for this study. As the results show, even a 5 mM glutaraldehyde concentration was good enough to result in CLEA, which retained 100% of all the three enzyme activities originally present in the solution. The temperature optima also did not reveal significant changes. The temperature optima observed for free enzymes agreed well with the reported value [17-19]. The pectinase temperature optima was 50°C for both free enzyme and CLEA. Free xylanase showed a temperature optimum of 50°C. CLEA showed broad temperature optima in the range of 50–55°C. Free cellulase has a tem- perature optimum of 50°C. CLEA showed a broad tem- perature optima in the range of 50–55°C (data not shown). The pH optima for the three activities in the CLEA were more or less same as those compared to free enzyme activ- ities. The pH optima were broad (at 4.5 for pectinase and 5.5 for xylanase and 5 for cellulase activities) and agreed with the literature value [17-19]. In the case of cellulase activity, CLEA showed pH optima at 5.5 as compared to 5 Table 1 summarizes the value for Michaelis-Menten parameters obtained with the three activities with free enzyme solution and CLEA. Values of Vmax were found to increase in the case of all three activities measured in CLEA, whereas Km values remained more or less unchanged upon CLEA formation. While not much data is available regarding these changes in the kinetic param- Table 1 summarizes the value for Michaelis-Menten parameters obtained with the three activities with free enzyme solution and CLEA. Values of Vmax were found to increase in the case of all three activities measured in CLEA, whereas Km values remained more or less unchanged upon CLEA formation. Background 0 20 40 60 80 100 120 n- Propanol Acetone DME Solvents Precipitated activity (%) Results and discussion While not much data is available regarding these changes in the kinetic param- Page 2 of 6 (page number not for citation purposes) http://journal.chemistrycentral.com/content/1/1/16 Chemistry Central Journal 2007, 1:16 Table 1: Comparison of kinetic parameters of pectinase, xylanase and cellulase in CLEA Enzyme Parameter Free enzyme CLEAs Pectinase Vmax (mgmin-1) 170 207 Km (mgml-1) 16 15 Vmax/Km (min-1) 11 14 Xylanase Vmax (mgmin-1) 300 400 Km (mgml-1) 4 5 Vmax/Km (min-1) 75 80 Cellulase Vmax (mgmin-1) 450 500 Km (mgml-1) 28 27 Vmax/Km (min-1) 16 19 The kinetic parameters were calculated by varying the substrate concentration. The data were fitted in Hanes-Woolf plot using Leonora software [26] as mentioned in the experimental section. The experiments were carried out in triplicate and the percentage error in each set of readings was within 3%. various CLEAs (and their clu in the range of 0.1 µ to 100 Figure 4 shows the reusabi enzyme catalyzed reactions could be reused three times enzyme activities. After this The Effect on CLEA activity of varying the amount of glutaral- dehyde Figure 2 The Effect on CLEA activity of varying the amount of glutaraldehyde: CLEA was prepared using varying concen- trations of glutaraldehyde (5–40 mM) as described in the experimental section. Experiments were carried out in tripli- cate and percentage error in each set of readings was within 3%. 5 10 20 40 Pectinase Xylanase Cellulase 0 20 40 60 80 100 Activity in CLEA (%) Glutaraldehyde conc ( mM) Enzyme Table 1: Comparison of kinetic xylanase and cellulase in CLEA Enzyme Parameter Pectinase Vmax (mgmin-1) Km (mgml-1) Vmax/Km (min-1 Xylanase Vmax (mgmin-1) Km (mgml-1) Vmax/Km (min-1 Cellulase Vmax (mgmin-1) Km (mgml-1) Vmax/Km (min-1 The kinetic parameters were calc concentration. The data were fitt Leonora software [26] as mention experiments were carried out in t each set of readings was within 3% Table 1: Comparison of kinetic parameters of pectinase, xylanase and cellulase in CLEA The Effect on CLEA activity of varying the amount of glutaral- dehyde Figure 2 The Effect on CLEA activity of varying the amount of glutaraldehyde: CLEA was prepared using varying concen- trations of glutaraldehyde (5–40 mM) as described in the experimental section. Experiments were carried out in tripli- cate and percentage error in each set of readings was within 3%. The Effect dehyde Figure 2 various CLEAs (and their clusters) has been reported to be in the range of 0.1 µ to 100 µm [7]. y y g g y g The Effect on CLEA activity of varying the amount of glutaraldehyde: CLEA was prepared using varying concen- trations of glutaraldehyde (5–40 mM) as described in the experimental section. Experiments were carried out in tripli- cate and percentage error in each set of readings was within 3%. y y g g y g The Effect on CLEA activity of varying the amount of glutaraldehyde: CLEA was prepared using varying concen- trations of glutaraldehyde (5–40 mM) as described in the experimental section. Experiments were carried out in tripli- cate and percentage error in each set of readings was within 3%. Figure 4 shows the reusability of CLEA for all the three enzyme catalyzed reactions. In all the three cases, CLEA could be reused three times without any loss of any of the enzyme activities. After this, slow decline in the activities in all three cases started. eters upon CLEA formation, the trends observed in the present case agree with those obtained with CLEA formed from pancreatic acetone powder [11]. It may be men- tioned that cross-linking is known to improve marginally the catalytic efficiency of enzyme [20]. Immobilized enzymes generally show increases in Km values especially when enzyme activities are assayed with high molecular weight substrates. In the present instance, Km values did not increase in a significant manner. Considering that CLEA was formed with just 5 mM glutaraldehyde, perhaps the enzyme aggregates had a very open structure and mass transfer remained as easy as with free enzyme. Conclusion To conclude, the present work outlines the preparation of a multipurpose robust biocatalyst. The biocatalyst prepa- ration is more thermostable and reusable. The multipur- pose biocatalyst can be used for bioconversions (such as hydrolysis of pectin, xylan and cellulose) of considerable biotechnological relevance. Results and discussion 5 10 20 40 Pectinase Xylanase Cellulase 0 20 40 60 80 100 Activity in CLEA (%) Glutaraldehyde conc ( mM) Enzyme 5 10 20 40 Pectinase Xylanase Cellulase 0 20 40 60 80 100 Activity in CLEA (%) Glutaraldehyde conc ( mM) Enzyme The kinetic parameters were calculated by varying the substrate concentration. The data were fitted in Hanes-Woolf plot using Leonora software [26] as mentioned in the experimental section. The experiments were carried out in triplicate and the percentage error in each set of readings was within 3%. Glutaraldehyde conc ( mM) Experimental The experiments were carried out in triplicate and error bars represent the percentage error in each set of readings.-■-: cellulase, --: xylanase, - -: pectinase. 0 20 40 60 80 100 120 1 2 3 4 5 No. of cycles Residual activity (%) 0 20 40 60 80 100 120 1 2 3 4 5 No. of cycles Residual activity (%) Scanning electron microscopy (SEM) image of CLEA Figure 3 Scanning electron microscopy (SEM) image of CLEA: SEM was carried out on Zeiss EVO50 scanning electron microscope, UK. Sample was dried by rinsing with anhydrous acetone, placed on a sample holder, coated with silver before being scanned under vacuum. The particle size was deter- mined from the micrograph with the scale of 20 µm unit. Scanning e Figure 3 g py ( ) g g Scanning electron microscopy (SEM) image of CLEA: SEM was carried out on Zeiss EVO50 scanning electron microscope, UK. Sample was dried by rinsing with anhydrous acetone, placed on a sample holder, coated with silver before being scanned under vacuum. The particle size was deter- mined from the micrograph with the scale of 20 µm unit. g py ( ) g g Scanning electron microscopy (SEM) image of CLEA: SEM was carried out on Zeiss EVO50 scanning electron microscope, UK. Sample was dried by rinsing with anhydrous acetone, placed on a sample holder, coated with silver before being scanned under vacuum. The particle size was deter- mined from the micrograph with the scale of 20 µm unit. Reusability of pectinase, xylanase and cellulase in CLEA Figure 4 Reusability of pectinase, xylanase and cellulase in y p , y g Reusability of pectinase, xylanase and cellulase in Reusability of pectinase, xylanase and cellulase in CLEA: The reusability of pectinase in CLEA was studied by carrying out the hydrolysis of polygalacturonic acid at 30°C for 30 minutes. The reusability of xylanse and cellulase was studied by carrying out hydrolysis of xylan and carboxyme- thyl cellulose at 50°C for 30 minutes respectively. After each cycle, the reaction mixture was centrifuged at 10, 000 × g and the reducing sugar in the supernatant was measured using 3,5-dinitro salicylic acid. The CLEA recovered after centrifugation was used for next cycle of hydrolysis. The experiments were carried out in triplicate and error bars represent the percentage error in each set of readings.-■-: cellulase, --: xylanase, - -: pectinase. Experimental Xylan and polygalacturonic acid were purchased from Sigma Chemical Co., St. Louis, USA. PectinexTM Ultra SP- L (a commercial preparation of pectolytic enzymes from a Table 2 shows the remarkable thermostabilization of the enzymes present in the preparation. In all three of the cases, half-lives have increased upon CLEA formation. Cellulase activity was most thermostable and its ther- moinactivation was measured at 70°C. Pectinase was least stable and its thermoiactivation was measured at 50°C. The largest increase in stability was in the case of pectinase in which the half-life increased from 17 to 180 minutes. Table 2: Half-life of pectinase, xylanase and cellulase in CLEA Enzyme Temperature (°C) Half-life (t1/2) (minutes) Free CLEAs Pectinase 50 17 180 Xylanase 60 22 82 Cellulase 70 32 91 The half-life values were calculated by plotting ln(residual activity of enzyme) vs time [19]. In all the three cases, the activity at 0 minute was taken as 100%. The experiments were carried out in triplicate and the percentage error in each set of readings was within 3%. Table 2: Half-life of pectinase, xylanase and cellulase in CLEA Enzyme Temperature (°C) Half-life (t1/2) (minutes) Free CLEAs Pectinase 50 17 180 Xylanase 60 22 82 Cellulase 70 32 91 Table 2: Half-life of pectinase, xylanase and cellulase in CLEA The Scanning electron micrograph (SEM) of this CLEA preparation is shown in Figure 3. The CLEAs were found to have diameter in the range of 15–30 µm. The size of The half-life values were calculated by plotting ln(residual activity of enzyme) vs time [19]. In all the three cases, the activity at 0 minute was taken as 100%. The experiments were carried out in triplicate and the percentage error in each set of readings was within 3%. http://journal.chemistrycentral.com/content/1/1/16 Chemistry Central Journal 2007, 1:16 Reusability of pectinase, xylanase and cellulase in CLEA Figure 4 Reusability of pectinase, xylanase and cellulase in CLEA: The reusability of pectinase in CLEA was studied by carrying out the hydrolysis of polygalacturonic acid at 30°C for 30 minutes. The reusability of xylanse and cellulase was studied by carrying out hydrolysis of xylan and carboxyme- thyl cellulose at 50°C for 30 minutes respectively. After each cycle, the reaction mixture was centrifuged at 10, 000 × g and the reducing sugar in the supernatant was measured using 3,5-dinitro salicylic acid. The CLEA recovered after centrifugation was used for next cycle of hydrolysis. Determination of pectinase activity The activity of pectinase was estimated using polygalac- turonic acid as the substrate according to the method described [21]. One unit of enzyme activity is defined as the amount of enzyme required to produce one µmol of galacturonic acid per minute under assay conditions. The amount of galacturonic acid was estimated using the din- itrosalicyclic acid method [22]. Determination of cellulase activity The activity of cellulase was estimated using carboxyme- thyl cellulose as a substrate [24]. One unit of enzyme activity is defined as the amount of enzyme required to produce one µmol of reducing sugar per minute under assay conditions. The amount of reducing sugar was esti- mated using the dinitrosalicyclic acid method [22]. Precipitation of enzymes by organic solvents p f y y g Chilled organic solvents (acetone, dimethoxyethane (DME) and n-propanol, 5 ml each) were added dropwise separately to 1 ml of commercial preparation Pectinex™ Ultra SP-L with shaking and kept for 15 minutes at 4°C for complete precipitation of enzymes and then centri- fuged for 5 minutes at 10,000 × g. The supernatant was discarded and the precipitate was redissolved in 0.05 M sodium acetate buffer pH 5. Pectinase, xylanase and cellu- lase activities were estimated in the solution. Experimental CLEA: The reusability of pectinase in CLEA was studied by carrying out the hydrolysis of polygalacturonic acid at 30°C for 30 minutes. The reusability of xylanse and cellulase was studied by carrying out hydrolysis of xylan and carboxyme- thyl cellulose at 50°C for 30 minutes respectively. After each cycle, the reaction mixture was centrifuged at 10, 000 × g and the reducing sugar in the supernatant was measured using 3,5-dinitro salicylic acid. The CLEA recovered after centrifugation was used for next cycle of hydrolysis. The experiments were carried out in triplicate and error bars represent the percentage error in each set of readings.-■-: cellulase, --: xylanase, - -: pectinase. selected strain of Aspergillus niger) was a kind gift from Dr. J.S.Rao, Novozymes, Bangalore, India. All other chem- icals used were of analytical grade. Protein estimation Protein concentration was determined according to the procedure described by Bradford [25] using bovine serum albumin as the standard. Determination of xylanase activity The activity of xylanase was estimated using xylan as the substrate [23]. One unit of enzyme activity is defined as the amount of enzyme required to produce one µmol of reducing sugar per minute under assay conditions. The amount of reducing sugar was estimated using the dinit- rosalicyclic acid method [22]. Thermal stability study gg g ( ) g 8. Sheldon RA, Schoevaart R, van Langen LM: Cross-linked enzyme aggregates (CLEAs): a novel and versatile method for enzyme immobilization. Biocatal Biotransform 2005, 23:141-147. Thermal stability was determined for pectinase, xylanase and cellulase present in CLEA. In the case of pectinase, the thermal stability of free enzyme and CLEA was studied at 50°C for 30 minutes and those of cellulase and xylanase were studied at 70°C and 60°C for 60 minutes respec- tively. In the three cases, the activity at 0 minute was taken as 100%. 9. Shah S, Sharma A, Gupta MN: Preparation of cross-linked enzyme aggregates by using bovine serum albumin as a proteic feeder. Anal Biochem 2006, 351:207-213. p 10. Persichetti RA, St Clair NL, Griffith JP, Navia MA, Margolin AL: Cross-linked enzyme crystals (CLECs) of thermolysin in the synthesis of peptides. J Am Chem Soc 1995, 117:2732-2737. the synthesis of peptides. J Am Chem Soc 1995, 117:2732-2737. 11. Dalal S, Kapoor M, Gupta MN: Preparation and characteriza- tion of combi-CLEAs catalyzing multiple non-cascade reactions. J Mol Catal B Enzyme 2006, 44:128-132. Reusability of pectinase, xylanase and cellulase To evaluate the reusability of pectinase, xylanase and cel- lulase in CLEA, the CLEA in each case was washed with the assay buffer after each use and then suspended again in a fresh reaction mixture to measure enzyme activity. The residual activity was calculated by taking the enzyme activity of the first cycle as 100%. 16. Subramaniyan S, Prema P: Biotechnology of microbial xyla- nases: enzymology, molecular biology and application. Crit Rev Biotechnol 2002, 22:33-64. , 17. Sreenath HK: Hydrolysis of carboxymethyl cellulose by cel- lulases. Lebensm Wiss Technol 1993, 26:224-228. 18. Gawande PV, Kamat MY: Preparation, characterization and application of Aspergillus sp. xylanase immobilized on Eudragit S-100. J Biotechnol 1998, 66:165-175. g J 19. Suh HJ, Noh DO, Choi YM: Solubilization of onion with polysaccharide- degrading enzymes. Int J Food Sci Tech 2002, 37:65-71. Determination of kinetic parameters 4. Gemeiner P, Stefuca V, Bales V: Biochemical engineering of biocatalysts immobilized on cellulosic materials. Enzyme Microb Technol 1993, 15:551-566. 4. Gemeiner P, Stefuca V, Bales V: Biochemical engineering of The kinetic parameters of free enzymes and CLEA were determined by measuring the initial rates of enzymes with varying amounts of respective substrate solutions under the assay conditions. The data were fitted in Hanes-Woolf equation using Leonora software to calculate the kinetic parameters [26]. biocatalysts immobilized on cellulosic materials. Enzyme Microb Technol 1993, 15:551-566. 5. Broun GH: Chemically aggregated enzymes. In Method in Enzymology Volume 44. Edited by: Mosbach K. Academic Press, New York; 1976:263-280. 6. Saleemuddin M: Bioaffinity based immobilization of enzymes. Adv Biochem Eng Biotechnol 1999, 64:203-226. y g , 7. Schoevaart R, Wolbers MW, Golubovic M, Ottens M, Kieboom APG, van Rantwijk F, van der Wielen LAM, Sheldon RA: Prepara- tion, optimization and structures of cross-linked enzyme aggregates (CLEAs). Biotechnol Bioeng 2004, 87:754-762. Preparation of CLEA Chilled n-propanol (5 ml) was added to the crude enzy- matic solution (1 ml) in capped centrifuge tubes. After Page 4 of 6 (page number not for citation purposes) http://journal.chemistrycentral.com/content/1/1/16 http://journal.chemistrycentral.com/content/1/1/16 http://journal.chemistrycentral.com/content/1/1/16 Chemistry Central Journal 2007, 1:16 pH and temperature optima of pectinase, xylanase and cellulase J y 12. Nikolic MV, Mojovic L: Hydrolysis of apple pectin by the coor- dinated activity of pectic enzymes. Food Chem 2007, 101:1-9. 13 Sh A M d l K G MN S i f b The pH optima of pectinase, xylanase and cellulase in CLEA were studied over the pH range of 3.5–9.5. Temper- ature optima of pectinase, xylanase and cellulase in free form and in CLEA were studied over the range of 25– 70°C. y p y 13. Sharma A, Mondal K, Gupta MN: Separation of enzymes by sequential macroaffinity ligand-facilitated three-phase par- titioning. J Chromatogr 2003, 995:127-134. g J g 14. Shah S, Sharma A, Gupta MN: Extraction of oil from Jatropha curcas L. seed kernels by combination of ultrasonication and aqueous enzymatic oil extraction. Bioresource Technol 2005, 96:121-123. 15. White AR, Brown RM Jr: Enzymatic hydrolysis of cellulose: visual characterization of the process. Proc Natl Acad Sci 1981, 78:1047-1051. Acknowledgements keeping the mixture for 15 minutes at 4°C for complete precipitation of enzymes, varying amounts of glutaralde- hyde were added. The tubes were shaken continuously during the addition. The mixture was kept at 4°C for 4 h with constant shaking at 300 rpm. At the end of the reac- tion time, the suspension was centrifuged at 10,000 × g for 5 minutes. The supernatant was decanted and the pellets were washed 3 times with 0.05 M sodium acetate buffer at pH 5 to remove unreacted glutaraldehyde. The final enzyme preparation was kept in the same buffer (1 ml) at 4°C. This work was supported by 'Core group grant for applied biocatalysis' by Department of Science & Technology, Government of India (DST). The financial support provided by All India Council for Technical Education (AICTE) to SD in the form of National Doctoral Fellowship is also gratefully acknowledged. This work was supported by 'Core group grant for applied biocatalysis' by Department of Science & Technology, Government of India (DST). The financial support provided by All India Council for Technical Education (AICTE) t SD i th f f N ti l D t l F ll hi i l t f ll (AICTE) to SD in the form of National Doctoral Fellowship is also gratefully acknowledged. (AICTE) to SD in the form of National Doctoral Fellowship is also gratefully acknowledged. References 1. Gupta MN: Enzyme functions in organic solvents. J Biochem 1992, 203:25-32. 2. Faber K, Kroutil W: New enzymes for biotransformations. Curr Opin Chem Biol 2005, 9:181-187. p 3. Mattiasson B: Affinity Immobilization. In Methods in Enzymology Volume 137. Edited by: Mosbach K. Academic Press, New York; 1988:647-656. Chemistry Central Journal 2007, 1:16 p p y g , 26. Cornish-Bowden A: Analysis of Enzyme Data Oxford: Oxford Uni- versity Press; 1995. 25. Bradford MM: A rapid and sensitive method for the quanti- tation of microgram quantities of protein utilizing the prin- ciple of protein-dye binding. Anal Biochem 1976, 72:248-254. 25. Bradford MM: A rapid and sensitive method for the quanti- tation of microgram quantities of protein utilizing the prin- ciple of protein-dye binding. Anal Biochem 1976, 72:248-254. 26. Cornish-Bowden A: Analysis of Enzyme Data Oxford: Oxford Uni- versity Press; 1995. Authors' contributions 20. Hartman FC, Wold F: Cross-linking of bovine pancreatic ribo- nuclease A with dimethyl adipimidate. Biochemistry 1967, 6:2439-2448. MNG suggested the idea about multipurpose CLEA, iden- tified the source of multiple enzyme activities and was actively involved in the discussions of the results. AS opti- mized the conditions for preparation of CLEA. SD carried out the characterization of CLEA, SEM studies of CLEA and enzyme assays. All authors jointly drafted, proof read and approved final manuscript. 21. Baily MJ, Pessa E: Strain and process for production of polyg- alacturonase. Enzyme Microb Technol 1990, 12:266-271. y 22. Miller GL: Use of dinitrosalicylic acid for the determination of reducing sugar. Anal Chem 1959, 31:426-428. g g 23. Baily MJ, Biely P, Poutanen K: Interlaboratory testing of meth- ods for assay of xylanase activity. J Biotechnol 1992, 23:257-270. 24. Ghose TK: Measurement of cellulase activities. Pure Appl Chem 1987, 59:257-268. Page 5 of 6 (page number not for citation purposes) (page number not for citation purposes) http://journal.chemistrycentral.com/content/1/1/16 http://journal.chemistrycentral.com/content/1/1/16 Chemistry Central Journal 2007, 1:16 Page 6 of 6 (page number not for citation purposes) Open access provides opportunities to our colleagues in other parts of the globe, by allowing anyone to view the content free of charge. Publish with ChemistryCentral and every scientist can read your work free of charge W. Jeffery Hurst, The Hershey Company. available free of charge to the entire scientific community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours you keep the copyright Submit your manuscript here: http://www.chemistrycentral.com/manuscript/ Page 6 of 6 Open access provides opportunities to our colleagues in other parts of the globe, by allowing anyone to view the content free of charge. Publish with ChemistryCentral and every scientist can read your work free of charge W. Jeffery Hurst, The Hershey Company. available free of charge to the entire scientific community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours you keep the copyright Submit your manuscript here: http://www.chemistrycentral.com/manuscript/ Open access provides opportunities to our colleagues in other parts of the globe, by allowing anyone to view the content free of charge. W Jeffery Hurst The Hershey Company
https://openalex.org/W1940526131	http://ainfo.cnptia.embrapa.br/digital/bitstream/item/119374/1/Distribuicao-dos-agregados-e-carbono-organico-influenciados.pdf	Portuguese	null	Aggregate and organic carbon distribution influenced by agroecological handling	null	2,009	cc-by	5,691	Arcângelo Loss1, Marcos Gervasio Pereira1, Nivaldo Schultz1, Edilene Pereira Ferreira1, Eliane Maria Ribeiro da Silva1 e Sidinei Julio Beutler2 1Departamento de Solos, Instituto de Agronomia, Universidade Federal Rural do Rio de Janeiro, BR 465, km 7, 23890-000, Seropédica, Rio de Janeiro, Brasil. 2Laboratório de Micorrizas, Centro Nacional de Pesquisa de Agrobiologia, Empresa Brasileira de Pesquisa Agropecuária, Seropédica, Rio de Janeiro, Brasil. Autor para correspondência. E-mail: arcangeloloss@yahoo.com.br RESUMO. O objetivo deste estudo foi verificar a influência do manejo agroecológico na distribuição dos agregados estáveis em água e no teor de carbono orgânico dos agregados em diferentes coberturas vegetais. Foram selecionadas cinco áreas, a saber: sistema agroflorestal; cultivo de figo (Ficus carica L.); consórcio maracujá (Passiflora edulis S.)–Desmodium sp.; cultivo de feijão (Phaseolus vulgaris L.), em manejo convencional, e cultivo de milho (Zea mays L.), em plantio direto. Em cada área, foram coletadas amostras de terra indeformadas, nas profundidades de 0 - 5 e 5 - 10 cm, e avaliada a estabilidade dos agregados e o teor de carbono orgânico nos agregados (COAGR). A maior massa de agregados encontra-se na classe de 2,00 mm, em ambas as profundidades, com exceção do cultivo de feijão. Nas duas profundidades, a classe de 2,00 mm apresentou os maiores valores de COAGR para a área do consórcio macaracujá - Desmodium sp. RESUMO. O objetivo deste estudo foi verificar a influência do manejo agroecológico na distribuição dos agregados estáveis em água e no teor de carbono orgânico dos agregados em diferentes coberturas vegetais. Foram selecionadas cinco áreas, a saber: sistema agroflorestal; cultivo de figo (Ficus carica L.); consórcio maracujá (Passiflora edulis S.)–Desmodium sp.; cultivo de feijão (Phaseolus vulgaris L.), em manejo convencional, e cultivo de milho (Zea mays L.), em plantio direto. Em cada área, foram coletadas amostras de terra indeformadas, nas profundidades de 0 - 5 e 5 - 10 cm, e avaliada a estabilidade dos agregados e o teor de carbono orgânico nos agregados (COAGR). A maior massa de agregados encontra-se na classe de 2,00 mm, em ambas as profundidades, com exceção do cultivo de feijão. Nas duas profundidades, a classe de 2,00 mm apresentou os maiores valores de COAGR para a área do consórcio macaracujá - Desmodium sp. Palavras-chave: agregação, resíduos vegetais, plantio direto, adubação verde. ABSTRACT. Aggregate and organic carbon distribution influenced by agroecological handling. The objective of this study was to verify the influence of agroecological handling in the distribution of stable aggregates in water and in the levels of aggregate organic carbon under different vegetable covers. Arcângelo Loss1*, Marcos Gervasio Pereira1, Nivaldo Schultz1, Edilene Pereira Ferreira1, Eliane Maria Ribeiro da Silva1 e Sidinei Julio Beutler2 Five areas were selected: agroflorestal system; fig cultivation (Ficus carica L.); passion fruit (Passiflora edulis S.) and Desmodium sp. consortium; bean cultivation (Phaseolus vulgaris L), under conventional system; and corn (Zea mays), in no-tillage system. In each area, undisturbed samples were collected, in 0-5 and 5-10 cm depths, and water aggregate distribution and organic carbon of aggregate (OCAGR) were quantified. The highest aggregate mass was observed in the 2.00 mm aggregate class, in both depths, except for the bean cultivation area. In both depths, the class with greater diameter showed the highest OCAGR values for the area of passion fruit – Desmodium sp. consortium. Key words: aggregation, vegetable residues, no-tillage system, green manuring. Acta Scientiarum. Agronomy DOI: 10.4025/actasciagron.v31i3.322 DOI: 10.4025/actasciagron.v31i3.322 Introdução Introdução Introdução Introdução Além disso, os solos de regiões tropicais são submetidos a altas temperaturas e maiores taxas de decomposição pelo aumento de disponibilidade de carbono orgânico ao ataque de microrganismos, diminuindo, dessa forma, o conteúdo de carbono orgânico do solo e, consequentemente, a estabilidade dos agregados (BRONICK; LAL, 2005). O SAF não recebe nenhum tipo de adubação complementar, somente resíduos culturais de sua própria vegetação. A área em cobertura de maracujá foi adubada nas covas com esterco de curral no momento do plantio, recebendo duas adubações de cobertura com cama de aviário por ano. As coberturas vegetais com figo, milho e feijão receberam uma adubação com cama de aviário no momento do plantio e depois, outra em cobertura, de acordo com as recomendações de cada cultura. Na área com plantio de figo, já foi utilizada adubação verde com crotalária (Crotalaria juncea) e siratro (Macroptilium artropurpureum); na cultura de feijão, mucuna cinza (Mucuna pruriens) e na cobertura com milho, mucuna cinza (Mucuna pruriens), mucuna anã (Mucuna deeringiana), Crotalaria spectabilis e Crotalaria juncea. A degradação dos agregados causa ao solo diminuição das condições favoráveis ao desenvolvimento vegetal e o predispõe ao aumento de erosão hídrica. A rotação de culturas e o manejo do solo amenizam esses problemas e agem com o intuito de restaurar-lhe a agregação. Diferentes práticas de manejo e sucessões de culturas induzem alterações nas propriedades físicas e químicas do solo. Neste contexto, a estabilidade dos agregados tem mostrado variação dependente do tipo de manejo do solo e das culturas (CAMPOS et al., 1995). O uso dessas leguminosas promoveu aporte de matéria seca de 1.612 kg ha-1 de Crotalaria spectabilis, 2.830 kg ha-1 de Crotalaria juncea, 5.560 kg ha-1 de mucuna cinza, 1.460 kg ha-1 de mucuna anã (SILVA et al., 2009) e 2.000 kg ha-1 de siratro (LOSS et al., 2009). O objetivo deste estudo foi verificar a influência do manejo agroecológico na distribuição dos agregados estáveis em água e no teor de carbono orgânico nas diferentes classes de agregados em diferentes coberturas vegetais no SIPA, Estado do Rio de Janeiro. A coleta das amostras foi realizada no início do inverno de 2006. Em cada uma das áreas, foram coletadas três amostras indeformadas, nas profundidades de 0-5 e 5-10 cm na entrelinha de cada cultura. Após esta etapa, as amostras foram transportadas para o laboratório e secadas ao ar. Introdução Introdução Introdução Introdução diversificação espacial e temporal do sistema de produção, subsidiando a fertilidade dos solos com menores aportes de insumos externos. Vários trabalhos desenvolvidos nas zonas intertropicais têm demonstrado o importante papel desempenhado pela matéria orgânica das camadas superficiais, principalmente sobre as propriedades edáficas que influenciam a fertilidade do solo (MOREAU, 1983; LEPSCH et al., 1994; FELLER et al., 1996; PINHEIRO et al., 2004; MERCANTE et al., 2008). Entretanto, em ambientes com manejo agroecológico, ainda são poucas as pesquisas que avaliam as modificações nas propriedades edáficas em função deste tipo de manejo (LOSS et al., 2009). A formação dos agregados do solo pode resultar da ação de união mecânica por células e hifas dos organismos, dos efeitos cimentantes dos produtos derivados da síntese microbiana ou da ação estabilizadora dos produtos de decomposição que agem individualmente ou em combinação (BAVER et al., 1973). Os preparos de solo conservacionistas, como a semeadura direta, com menor revolvimento, mantêm, parcial ou totalmente, os resíduos vegetais na superfície e aportam continuamente matéria orgânica ao solo, a qual é responsável pela manutenção e melhoria das propriedades físicas do O manejo agroecológico propicia ambiente favorável ao desenvolvimento de processos naturais e interações biológicas positivas no solo, por meio da Maringá, v. 31, n. 3, p. 523-528, 2009 Maringá, v. 31, n. 3, p. 523-528, 2009 Acta Scientiarum. Agronomy 524 Loss et al. solo (CASTRO FILHO et al., 1998). Na maioria dos sistemas de semeadura direta, a ausência quase que completa de preparo por longo tempo reduz o volume de macroporos e eleva a densidade do solo e a estabilidade dos agregados (BERTOL et al., 2001). gramíneas (Paspalum notatum); consórcio maracujá (Passiflora edulis S.)–Desmodium sp., sendo esta área cultivada com maracujá desde 1996; cultivo de feijão (Phaseolus vulgaris L.), em área que há oito anos vem sendo preparados experimentos com rotação de culturas em manejo convencional (aração e gradagem); e uma área com cultivo de milho (Zea mays L.), com a mesma rotação e idade da área com feijão, entretanto em sistema de plantio direto. A estabilização dos agregados depende do contínuo fornecimento de matéria orgânica de maneira suficiente para compensar a rápida perda de carbono orgânico do solo. Introdução Introdução Introdução Introdução Em seguida, foram passadas por peneiras de 8 e 4 mm de diâmetro de malha. A distribuição dos agregados, por classes de diâmetro médio (de 8,0 ≥ X > 2,0 cm, de 2,0 ≥ X > 1,0 cm, de 1,0 ≥ X > 0,5 cm, de 0,5 ≥ X > 0,25 cm e de 0,25 ≥ X > 0,105 cm), foi obtida submetendo-se as amostras de solo ao peneiramento via úmida (EMBRAPA, 1997). Para isso, foram pesadas amostras de 25 g que ficaram retidas na peneira de 4 mm, umedecidas com pulverizador, colocadas em um jogo de peneiras com malhas de 2,00; 1,00; 0,50; 0,25 e 0,105 mm, e submetidas à agitação vertical no aparelho de Yooder, durante 15 min. Após o tempo determinado, o material retido em cada peneira foi retirado, separadamente, com o auxílio de jato d'água, colocado em placas, previamente pesadas e Acta Scientiarum. Agronomy Distribuição dos agregados e carbono orgânico Distribuição dos agregados e carbono orgânico 525 no solo. Esse fato também foi verificado para cobertura com feijão na profundidade de 0 - 5 cm do presente trabalho (Figura 1), apresentando maior massa de agregados em todas as classes de diâmetro menores que 2,00 mm. identificadas, e levado à estufa até peso constante. Após a secagem, obteve-se a massa dos agregados retida em cada peneira. p Em seguida, foi determinado o teor de carbono orgânico nos agregados (COAGR) do material retido em cada peneira. Este foi seco em estufa a 65ºC, triturado em almofariz, peneirado em malha de 80 mesh e pesado 0,5 g. Em seguida, adicionaram-se 10 mL de dicromato de potássio 0,2 mol L-1 em meio sulfúrico, em cada amostra, que foi aquecida a 150°C em placa aquecedora até oxidação da matéria orgânica do solo. Posteriormente, adiciounaram-se 80 mL de água destilada, 3 mL de ácido fosfórico e três gotas de indicador difenilanima, sendo titulado com a solução de sulfato ferroso amoniacal 0,1 mol L-1 (EMBRAPA, 1997). A maior quantidade de agregados com diâmetro menor que 2,00 mm no solo é frequente em áreas em sistema convencional, concordando com os resultados encontrados por Silva et al. (2000), em que os autores estudaram a influência dos sistemas de manejo na agregação do solo, em cultivo convencional, plantio direto e floresta nativa. Segundo Tisdall e Oades (1982), o acúmulo dos agregados de diâmetro inferior a 1,00 mm em áreas cultivadas ocorre por estes agregados serem estáveis ao rápido umedecimento e não serem destruídos por práticas agrícolas, pois são constituídos, predominantemente, de partículas de 2 - 20 µm de diâmetro, unidas em cadeias por vários agentes cimentantes. Os resultados encontrados foram analisados quanto à normalidade e à homogeneidade dos dados, por meio dos testes de Lilliefors e Cochran e Barttlet, respectivamente. Depois, foram analisados como delineamento inteiramente casualizado. Os resultados obtidos foram submetidos à análise de variância com aplicação do teste F e os valores médios comparados entre si pelo teste de Scott- Knott a 5%. Na área com cobertura de feijão, o peso dos agregados da menor classe (0,105) diferiu estatisticamente das demais, apresentando maior massa de agregados, nas duas profundidades (Figuras 1 e 2). Este comportamento é resultado do maior revolvimento do solo, fragmentando os agregados de maior tamanho em unidades inferiores. Distribuição dos agregados e carbono orgânico Por essa ruptura, acelera-se a mineralização da matéria orgânica por meio do ataque de microrganismos e libera-se CO2 para atmosfera. Acta Scientiarum. Agronomy Resultados e discussão Resultados e discussão Resultados e discussão Resultados e discussão Observa-se, nas Figuras 1 e 2, que a maior massa média de agregados dos sistemas maracujá, SAF, figo e milho encontram-se na classe de diâmetro maior que 2,00 mm; entretanto, para o sistema feijão, esta observação foi apenas para a profundidade de 5 - 10 cm. Este comportamento pode ser pelo maior teor de carbono orgânico do solo verificado na área (feijão), quando comparada às demais (LOSS et al., 2009). Este maior teor de carbono, provavelmente, ocorreu pelo preparo do solo, pois antes da coleta das amostras, a área foi arada e gradeada, e os resíduos da cultura anterior (milho) foram incorporados e homogeneizados nos primeiros 10 cm de solo para plantio da cultura do feijão. Dessa maneira, o carbono atuou como agente cimentante, promovendo a formação de agregados mais estáveis, com maior massa de agregados para o sistema feijão na profundidade de 5-10 cm. Os resultados observados neste estudo, em relação à estabilidade dos agregados, são semelhantes àqueles encontrados por Lacerda et al. (2005) ao avaliarem o efeito do manejo do solo na estabilidade de agregados de um Nitossolo Vermelho, em três tipos de manejo: mata; preparo convencional por dez anos, seguido de semeadura direta por 12 anos; e preparo convencional por 22 anos. Para os três sistemas de manejo, os autores encontraram maior percentagem de agregados com diâmetro entre 7,93 e 2,00 mm. Pesos dos agregados (g) a a a a a c c b c c b b b b b b b b c b c d c a c b c d c a 0 2 4 6 8 10 12 14 > 2,00 2,00 - 1,00 1,00 - 0,50 0,50 - 0,25 0,25 - 0,105 < 0,105 Feijão Maracujá SAF Figo Milho Classes de agregados (mm) Figura 1. Distribuição dos agregados na profundidade de 0 - 5 cm com diferentes coberturas vegetais e em manejo orgânico no SIPA. Médias de três repetições seguidas de mesma letra, entre coberturas vegetais para cada classe de agregado, não diferem entre si pelo teste de Scott-Knott a 5%. Material e métodos Material e métodos Material e métodos Material e métodos O estudo foi realizado na área do Sistema Integrado de Produção Agroecológica - SIPA, denominado ‘Fazendinha Agroecológica do km 47’. O SIPA localiza-se na Embrapa Agrobiologia, em Seropédica, Rio de Janeiro, em área com 59 ha, e está situado na latitude 22º 45’S, longitude 43º 41’W Grw e altitude de 33 m, sendo o clima incluído na classificação de Köppen como do tipo Aw (NEVES et al., 2005). O solo da área experimental foi classificado como Argissolo Vermelho-Amarelo (EMBRAPA, 2006), sendo rotineiramente cultivado com oleráceas e frutíferas. Foram selecionadas cinco áreas de 0,12 ha, a saber: sistema agroflorestal (SAF) com cinco anos de implantação, sendo formado por banana (Musa sapientum), palmito jussara (Euterpe oleracea), cacau (Thebroma cacao), mamão (Carica papaya) e guapuruvu (Schizolobium parahyba); cultivo de figo (Ficus carica L.) com sete anos e as entrelinhas com Maringá, v. 31, n. 3, p. 523-528, 2009 Acta Scientiarum. Agronomy Resultados e discussão Resultados e discussão Resultados e discussão Resultados e discussão Em relação ao carbono orgânico dos agregados (COAGR), os maiores valores médios foram encontrados para a profundidade de 0 - 5 cm (Figura 3), decorrentes do maior aporte de resíduos vegetais nesta camada. De forma geral, nas duas profundidades, a classe de maior diâmetro (I) apresentou os maiores valores médios de COAGR (Figuras 3 e 4). Pinheiro et al. (2004), ao estudar as frações orgânicas e agregação do solo em função de diferentes sistemas de preparo do solo com oleráceas em Paty do Alferes, Estado do Rio de Janeiro, encontrou resultados similares aos observados neste estudo. Pesos de agregados (g) a a a a a c c b b a a a b b a a a b b b b b b a b a b b b a 0 2 4 6 8 10 12 14 > 2,00 2,00 - 1,00 1,00 - 0,50 0,50 - 0,25 0,25 - 0,105 < 0,105 Feijão Maracujá SAF Figo Milho Classes de agregados (mm) A área sob cobertura de feijão diferiu estatisticamente das demais, apresentando menor teor de COAGR na classe de 2,00 mm, na profundidade de 0 - 5 cm. Este fato pode resultar da menor massa de agregados retida nessa classe, em função do manejo convencional, favorecendo a quebra dos agregados e a consequente liberação do carbono orgânico protegido nos macroagregados à oxidação, assim como a atuação dos processos erosivos que promovem a remoção de partículas. Os valores médios de COAGR obtidos neste estudo são semelhantes aos encontrados por Beare et al. (1994), em um trabalho realizado em região de clima tropical, com plantio direto e convencional, onde se observou que os macroagregados (> 250 mm) do plantio direto continham 43% a mais de matéria orgânica total, quando comparados com os obtidos no plantio convencional, o que reflete maior teor de carbono orgânico nos macroagregados. Figura 2. Distribuição dos agregados na profundidade 5 - 10 cm com diferentes coberturas vegetais e em manejo orgânico no SIPA. Médias de três repetições seguidas de mesma letra, entre coberturas vegetais para cada classe de agregado, não diferem entre si pelo teste de Scott-Knott a 5%. No SAF, observaram-se, nas classes de menor diâmetro (0,50; 0,25 e < 0,105 mm), maiores massas de agregados que nas coberturas de maracujá, figo e berinjela, na profundidade de 0 - 5 cm (Figura 1). Resultados e discussão Resultados e discussão Resultados e discussão Resultados e discussão Pesos dos agregados (g) a a a a a c c b c c b b b b b b b b c b c d c a c b c d c a 0 2 4 6 8 10 12 14 > 2,00 2,00 - 1,00 1,00 - 0,50 0,50 - 0,25 0,25 - 0,105 < 0,105 Feijão Maracujá SAF Figo Milho Classes de agregados (mm) Feijão Maracujá SAF Figo Milho A redução da estabilidade dos agregados na cobertura com feijão pode ser pelas práticas de aração e gradagem realizadas na área. Segundo Tisdall e Oades (1982), este manejo provoca a quebra dos agregados de maior tamanho, interrompe o estabelecimento efetivo do sistema radicular das culturas e das hifas do solo e aumenta a proporção de agregados de menor tamanho > 2,00 2,00 - 1,00 1,00 - 0,50 0,50 - 0,25 0,25 - 0,105 < 0,105 Classes de agregados (mm) Figura 1. Distribuição dos agregados na profundidade de 0 - 5 cm com diferentes coberturas vegetais e em manejo orgânico no SIPA. Médias de três repetições seguidas de mesma letra, entre coberturas vegetais para cada classe de agregado, não diferem entre si pelo teste de Scott-Knott a 5%. Figura 1. Distribuição dos agregados na profundidade de 0 - 5 cm com diferentes coberturas vegetais e em manejo orgânico no SIPA. Médias de três repetições seguidas de mesma letra, entre coberturas vegetais para cada classe de agregado, não diferem entre si pelo teste de Scott-Knott a 5%. Maringá, v. 31, n. 3, p. 523-528, 2009 Acta Scientiarum. Agronomy Loss et al. 526 são os principais responsáveis pelos efeitos supracitados (WOHLENBERG et al., 2004). Pinheiro et al. (2004), avaliando o efeito de diferentes sistemas de preparo do solo e coberturas vegetais em Latossolo Vermelho, no município de Paty do Alferes, Estado do Rio de Janeiro, na distribuição dos agregados, também encontraram maiores valores de peso dos agregados na área com preparo convencional do solo, quando comparado ao cultivo mínimo e plantio em nível. Este comportamento, igualmente, foi atribuído às práticas de preparo da área, onde a aração e gradagem do solo ocasionaram a ruptura dos agregados de maior tamanho em unidades inferiores, culminando em maior peso de agregados de menor tamanho no sistema convencional. Acta Scientiarum. Agronomy Distribuição dos agregados e carbono orgânico 527 (2,00 mm). Neste estudo, observa-se, na Figura 3, variação de 9,28 g kg-1 (cultivo de feijão, classe < 0,105 mm) a 28,38 g kg-1 (cobertura de maracujá, classe de 2,00 mm) e, na Figura 4, de 7,12 g kg-1 (cultivo de feijão, classe < 0,105 mm) a 28,68 g kg-1 (cobertura de maracujá, classe de 2,00 mm). Este comportamento demonstra que o manejo adotado no SIPA está beneficiando o COAGR, já que nesta área observam-se maiores valores de carbono, quando comparados ao verificados por Cordeiro et al. (2004) em área com gramíneas. Para a camada de 5 - 10 cm, apenas a área com milho apresentou menores valores de COAGR que os verificados por Cordeiro et al. (2004), na classe de 2,00 mm. Estes resultados demonstram o efeito positivo do manejo agroecológico, propiciando maiores teores de carbono ao solo, que, por sua vez, poderá atuar como agente cimentante, promovendo a formação de agregados mais estáveis. Os maiores valores de COAGR encontrados na cobertura com maracujá para a classe I de agregados (Figuras 3 e 4) podem ser decorrentes do consórcio com a leguminosa Desmodium sp. Tarré et al. (2001), estudando o efeito da presença de Desmodium ovalifolium na pastagem de Brachiaria humidicola, em condições de clima tropical, verificaram que a taxa de acumulação de carbono no solo dobrou na área onde houve o consórcio com aquela espécie. Este comportamento ocorreu pela contribuição do N, por meio da leguminosa, e pela importância que este elemento possui na atividade biológica e na estabilização da matéria orgânica do solo (SISTI et al., 2004). Os valores de COAGR observados por Cordeiro et al. (2004) na classe de agregados de 2,00 mm (17,7; 18,6 e 15,9 g kg-1, respectivamente para braquiária, suázi e tifton) são menores que os teores de COAGR encontrados nas áreas do SIPA (maracujá, SAF, figo e milho), para a profundidade de 0 - 5 cm, e apenas a cobertura com feijão apresentou menores valores. Carbono orgânico (g kg-1) a c c c e a b c d a a a a a b a b b b c a b c c d 0,00 5,00 10,00 15,00 20,00 25,00 30,00 I II III IV V Feijão Maracujá SAF Figo Milho Classes de agregados (mm) Figura 3. Carbono orgânico dos agregados, na profundidade de 0 - 5 cm com diferentes coberturas vegetais e em manejo orgânico, nas classes de agregados. Resultados e discussão Resultados e discussão Resultados e discussão Resultados e discussão Este comportamento também foi observado para a profundidade de 5 - 10 cm (Figura 2), entretanto na classe de maior diâmetro (2,00 mm). Este resultado pode ser decorrente da utilização de adubação verde nas áreas de figo e berinjela e, na cultura do maracujá, do consórcio com Desmodium sp. Associadas à adubação orgânica, essas práticas podem propiciar melhor desenvolvimento radicular das culturas e, consequentemente, melhorar a agregação do solo, culminando na formação de agregados de maior tamanho. Dessa forma, tem-se ação direta das culturas na formação e estabilização dos agregados do solo. Estes resultados são corroborados por aqueles observados por Wohlenberg et al. (2004), estudando a dinâmica da agregação de um solo franco-arenoso em cinco sistemas de culturas em rotação e sucessão em Santa Maria, Estado do Rio Grande do Sul. Os autores verificaram que as sequências de culturas influenciam diferenciadamente na agregação do solo, dependendo da época do ano e do tempo de estabelecimento dos sistemas de culturas. Cordeiro et al. (2004), avaliando a distribuição do COAGR em solo sob pastagem em relevo movimentado, com as gramíneas braquiária (Brachiaria sp.), suázi (Digitaria swazilandensis) e tifton 85 (Cynodon spp.) da região Noroeste do Estado do Rio de Janeiro, observaram que os valores médios de COAGR (profundidade de 0 - 10 cm) foram 17,7; 18,6 e 15,9 g kg-1, respectivamente pra as três gramíneas avaliadas na classe de maior diâmetro Perin et al. (2002), avaliando o efeito da cobertura viva com leguminosas herbáceas perenes na agregação do mesmo solo, verificaram que as coberturas com as leguminosas amendoim forrageiro (Arachis pintoi) e cudzu tropical (Pueraria phaseoloides) propiciaram os maiores valores percentuais na classe de agregados > 2,00 mm, em média 38% superiores aos obtidos na área capinada. A matéria orgânica e o desenvolvimento de raízes Maringá, v. 31, n. 3, p. 523-528, 2009 Acta Scientiarum. Agronomy Distribuição dos agregados e carbono orgânico Médias de três repetições seguidas de mesma letra, para mesma classe de agregados, não diferem entre si pelo teste Scott-Knott a 5%. Legenda: I, II, III, IV e V correspondem à faixa de diâmetro de agregados de 8,0 ≥ X > 2,0 mm, de 2,0 ≥ X > 1,0 mm, de 1,0 ≥ X > 0,5 mm, de 0,5 ≥ X > 0,25 mm e de 0,25 ≥ X > 0,105 mm, respectivamente. Carbono orgânico (g kg-1) a c c c e a b c d a a a a a b a b b b c a b c c d 0,00 5,00 10,00 15,00 20,00 25,00 30,00 I II III IV V Feijão Maracujá SAF Figo Milho Classes de agregados (mm) Avaliando os índices de estabilidade de agregados estáveis em água, DMP (diâmetro médio ponderado) e DMG (diâmetro médio geométrico) nestas mesmas áreas do SIPA, Loss et al. (2009) encontraram maiores valores de DMP e DMG na área de maracujá. Esses resultados foram atribuídos ao consórcio com a leguminosa Desmodium sp., associado à adubação orgânica, que torna disponíveis elementos como N e C e propicia melhor desenvolvimento radicular das culturas e, consequentemente, melhora a agregação do solo, culminando na formação de agregados de maior tamanho. Dessa forma, pode apresentar também maiores teores de carbono orgânico nos agregados. I Figura 3. Carbono orgânico dos agregados, na profundidade de 0 - 5 cm com diferentes coberturas vegetais e em manejo orgânico, nas classes de agregados. Médias de três repetições seguidas de mesma letra, para mesma classe de agregados, não diferem entre si pelo teste Scott-Knott a 5%. Legenda: I, II, III, IV e V correspondem à faixa de diâmetro de agregados de 8,0 ≥ X > 2,0 mm, de 2,0 ≥ X > 1,0 mm, de 1,0 ≥ X > 0,5 mm, de 0,5 ≥ X > 0,25 mm e de 0,25 ≥ X > 0,105 mm, respectivamente. Conclusão Conclusão Conclusão Conclusão O manejo empregado na área sob cobertura de feijão desfavorece a formação de agregados mais estáveis. O manejo agroecológico propicia agregados de maior diâmetro, quando comparado a sistemas convencionais sem a utilização de práticas agroecológicas. A leguminosa Desmodium sp. consorciada com a cultura do maracujá está propiciando os maiores teores de carbono nos agregados com diâmetro > 2,00 mm. Carbono orgânico (g kg-1) b b b a b a a a a a b b a a b c b b a b c b a a c 0,00 5,00 10,00 15,00 20,00 25,00 30,00 I II III IV V Feijão Maracujá SAF Figo Milho Classes de agregados (mm) Figura 4: Carbono orgânico dos agregados, na profundidade de 5 - 10 cm com diferentes coberturas vegetais e em manejo orgânico, nas classes de agregados. Médias de três repetições seguidas de mesma letra, para mesma classe de agregados, não diferem entre si pelo teste Scott-Knott a 5%. Legenda: I, II, III, IV e V correspondem à faixa de diâmetro de agregados de 8,0 ≥ X > 2,0 mm, de 2,0 ≥ X > 1,0 mm, de 1,0 ≥ X > 0,5 mm, de 0,5 ≥ X > 0,25 mm e de 0,25 ≥ X > 0,105 mm, respectivamente. Carbono orgânico (g kg-1) b b b a b a a a a a b b a a b c b b a b c b a a c 0,00 5,00 10,00 15,00 20,00 25,00 30,00 I II III IV V Feijão Maracujá SAF Figo Milho Classes de agregados (mm) Acta Scientiarum. Agronomy Referências Referências Referências Referências um Argissolo. Revista Brasileira de Ciência do Solo, v. 26, n. 3, p. 713-720, 2002. CASTRO FILHO, C.; MUZILLI, O.; PODANOSCHI, A. L. Estabilidade dos agregados e sua relação com o teor de carbono orgânico num Latossolo Roxo distrófico, em função de sistemas de plantio, rotações de culturas e métodos de preparo das amostras. Revista Brasileira de Ciência do Solo, v. 22, n. 3, p. 527-538, 1998. PINHEIRO, E. F. M.; PEREIRA, M. G.; ANJOS, L. H. C. 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M.; FERREIRA, E.; ALVES, B. J. Referências Referências Referências Referências Figura 4: Carbono orgânico dos agregados, na profundidade de 5 - 10 cm com diferentes coberturas vegetais e em manejo orgânico, nas classes de agregados. Médias de três repetições seguidas de mesma letra, para mesma classe de agregados, não diferem entre si pelo teste Scott-Knott a 5%. Legenda: I, II, III, IV e V correspondem à faixa de diâmetro de agregados de 8,0 ≥ X > 2,0 mm, de 2,0 ≥ X > 1,0 mm, de 1,0 ≥ X > 0,5 mm, de 0,5 ≥ X > 0,25 mm e de 0,25 ≥ X > 0,105 mm, respectivamente. Maringá, v. 31, n. 3, p. 523-528, 2009 BAVER, L. D., GARDNER, W. H.; GARDNER, W. R. Soil structure: classification and genesis. In: BAVER, L. D.; GARDNER, W. H.; GARDNER, W. R. (Ed.). Soil physics. New York: John Wiley, 1973. p. 130-177. BEARE, M.; CABRERA, M.; HENDRIX, P.; COLEMAN, D. Aggregate-protected and unprotected Maringá, v. 31, n. 3, p. 523-528, 2009 Acta Scientiarum. Agronomy 528 Loss et al. MOREAU, R. 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Estabilidade estrutural de um Latossolo Vermelho-Escuro distrófico após sete anos de rotação de culturas e sistemas de manejo de solo. Revista Brasileira de Ciência do Solo v 19 n 1 PERIN, A.; GUERRA, J. G. M.; TEIXEIRA, M. G.; PEREIRA, M. G.; FONTANA, A. Efeito da cobertura viva com leguminosas herbáceas perenes na agregação de j Revista Brasileira de Ciência do Solo, v. 19, n. 1, p. 121-126, 1995. License information: 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 work is properly cited. Received on October 31, 2007. Accepted on April 17, 2008. Referências Referências Referências Referências R.; URQUIAGA, S.; BODDEY, R. M. The effects of the presence of a forage legume on nitrogen and carbon levels in soils under brachiaria pasture in the Atlantic Forest region of the south of Bahia, Brazil. Plant and Soil, v. 234, n. 1, p. 15-26, 2001. LACERDA, N. B.; ZERO, V. M.; BARILLI, J.; MORAES, M. H.; BICUDO, S. J. Efeito de sistemas de manejo na estabilidade de agregados de um NITOSSOLO VERMELHO. Engenharia Agrícola, v. 25, n. 3, p. 686-695, 2005. TISDALL, J. M.; OADES, J. M. Organic matter and water-stable aggregates in soils. Soil Science, v. 33, n. 2, p. 141-163, 1982. LEPSCH, I. F.; MENK, J. R. F.; OLIVEIRA, J. B. Carbon storage and other properties of soils under agriculture and natural vegetation in São Paulo State, Brazil. Soil Use and Management, v. 18, n. 10, p. 34-42, 1994. LOSS, A.; PEREIRA, M. G.; SCHULTZ, N.; ANJOS, L. H. C.; SILVA, E. M. R. Atributos químicos e físicos de um Argissolo Vermelho-Amarelo em sistema integrado de produção agroecológica. Pesquisa Agropecuária Brasileira, v. 44, n. 1, p. 68-75, 2009. LEPSCH, I. F.; MENK, J. R. F.; OLIVEIRA, J. B. Carbon storage and other properties of soils under agriculture and natural vegetation in São Paulo State, Brazil. Soil Use and Management, v. 18, n. 10, p. 34-42, 1994. WOHLENBERG, E. V. REICHERT, J. M. REINERT, D. J.; BLUME, E. Dinâmica da agregação de um solo franco arenoso em cinco sistemas de culturas em rotação em sucessão. Revista Brasileira de Ciência do Solo, v. 28, n. 5, p. 891-900, 2004. LOSS, A.; PEREIRA, M. G.; SCHULTZ, N.; ANJOS, L. H. C.; SILVA, E. M. R. Atributos químicos e físicos de um Argissolo Vermelho-Amarelo em sistema integrado de produção agroecológica. Pesquisa Agropecuária Brasileira, v. 44, n. 1, p. 68-75, 2009. MERCANTE, F. M.; SILVA, R.F.; FRANCELINO, C. S. F.; CAVALHEIRO, J. C. T.; OTSUBO, A. A. Biomassa microbiana, em um Argissolo Vermelho, em diferentes coberturas vegetais, em área cultivada com mandioca. Acta Scientiarum. Agronomy, v. 34, n. 4, p. 479-485, 2008. Maringá, v. 31, n. 3, p. 523-528, 2009 Acta Scientiarum. Agronomy
https://openalex.org/W4392344712	https://jurnal.ilmubersama.com/index.php/PubHealth/article/download/464/283	Indonesian	null	Asuhan Keperawatan pada Pasien Tuberkulosis Paru dengan Masalah Defisit Nutrisi di Rumah Sakit Tk. II Putri Hijau Medan	Pubhealth Jurnal Kesehatan Masyarakat	2,024	cc-by-sa	7,775	Asuhan Keperawatan pada Pasien Tuberkulosis Paru dengan Masalah Defisit Nutrisi di Rumah Sakit Tk. II Putri Hijau Medan Umi Febriwanti, Ade Irma Khairani, Rani Sartika Dewi Program Studi DIII Keperawatan, Akademi Keperawatan Kesdam I/Bukit Barisan Medan, Medan, Indonesia KATA KUNCI Tuberkulosis; Paru; Defisit Nutrisi; Asuhan Keperawatan KORESPONDENSI Phone: +62 812-6332-6429 E-mail: febriwantiumi@gmail.com INFORMASI ARTIKEL Penelitian ini menyoroti dua pasien dengan diagnosis tuberculosis (TB) yang mengalami keluhan batuk, sesak nafas, penurunan nafsu makan, serta riwayat penyakit paru. Melalui pendekatan sistem Gordon, perubahan pola kesehatan pasien diamati, termasuk nutrisi, eliminasi urine, alvi, istirahat-tidur, aktivitas, dan kebersihan diri. Kasus ini menunjukkan bahwa pasien dengan Riwayat penyakit paru dan penurunan nafsu makan didiagnosis dengan ketidakseimbangan nutrisi kurang dari kebutuhan tubuh. Intervensi keperawatan meliputi pemberian cairan, obat- obatan, monitoring tanda-tanda vital, dan pengajaran kepada pasien dan keluarganya. Evaluasi menunjukkan kemajuan dalam kondisi pasien, namun masalah belum sepenuhnya teratasi. Penelitian ini menekankan pentingnya perencanaan, tindakan, dan evaluasi yang sesuai dengan kondisi dan kebiasaan klien untuk mempercepat proses perawatan. Evaluasi juga harus mempertimbankan respons pasien terhadap intervensi yang dilakukan serta faktor-faktor lain yang dapat mempengaruhi hasil perawatan. Dengan demikian, perencanaan, tindakan, dan evaluasi yang komprehensif dan terkoordinasi dapat memastikan perawatan yang efektif dan efisien bagi pasien. Diterima Redaksi: 12 Februari 2024 Revisi Akhir: 28 Februari 2024 Diterbitkan Online: 29 Februari 2024 Diterima Redaksi: 12 Februari 2024 Revisi Akhir: 28 Februari 2024 Diterbitkan Online: 29 Februari 2024 https://doi.org/10.56211/pubhealth.v2i3.464 Attribution-ShareAlike 4.0 International Some rights reserved PENDAHULUAN Tindakan atau peran petugas rumah sakit selama memberikan pelayanan kesehatan kepada penderita Tubrkulosis paru yang mengalami defisit nutrisi adalah diet kalori tinggi protein (TKTP), cukup lemah, vitamin dan mineral. TKTP di berikan agar pasien mendapat cukup makanan untuk memenuhi kebutuhan kalori dan protein yang meningkat. Oleh karena itu rekomendasi kebutuhan energi total untuk pasien tuberkulosi paru di tinggkatkan menjadi 35-45 kkal/kgBB. Tuberkulosis sering dijuluki “the great iminator” yaitu suatu penyakit yang mempunyai banyak kemiripan dengan penyakit lain yang juga memberikan gejala umum seperti lemah dan demam. Pada sejumlah penderita gejala yang timbul tidak jelas sehingga sering diabaikan bahkan kadang-kadang asimmtomatik. Efek yang timbul jika tidak ditangani dengan baik maka akan menimbulkan komplikasi seperti: malnutrisi, empyema, efusi pelura, hepatitis, ketulian dan gangguan gastrointestinal (sebagai efek samping obat-obatan). Komplikasi Menurut Wahid & Imam (2013), dampak masalah yang sering terjadi pada TB paru adalah: Hemoptisis berat (pendarahan dari saluran nafas bawah) yang dapat mengakibatkan kematian karena syok hipovolemik atau tersumbatnya jalan nafas, kolaps dari lobus akibat retraksi bronchial, Bronkiektasis (peleburan bronkus setempat) dan fibrosis (pembentukan jaringan ikat pada proses pemulihan atau reaktif) pada-paru, pneumothorak (adanya udara dalam rongga pleura) spontan: koplas spontan karena kerusakan jaringan paru, Penyebaran infeksi ke organ lain seperti kardiopulmonar (Chardio Pulmonary Insuffciency). (Abd wahid; Imam Suprapto, 2013). Salah satu upaya untuk mencegah efek yang ditimbulkan maka diperlukan upaya pencegahan dan pemberantasan TB Paru yang dilakukan dengan pendekatan Directly Observe Treatment Shortcourse (DOTS) atau pengobatan TB paru dengan pengawasan langsung oleh Pengawas Menelan Obat (PMO) sehingga diharapkan dapat membantu penderita TB paru dalam pemenuhan kebutuhan keamana dan keselamatan agar terhindar dari risiko penyebaran infeksi dan dapat menurunkan resiko penyakit TB paru serta dapat mengurangi pasien yang mengalami penurunan kemampuan untuk melindungi dirinya dari penyakit, baik dari luar maupun dari dalam tubuh. Penanganan penyakit Tuberkulosis Paru pada kebutuhan keamanan dan keselamatan melibatkan banyak unsur termasuk didalamnya perawat yang berperan sebagai fasilitas perawatan kesehatan yang akan memberikan perawatan pada pasien pada pasien TB paru dalam pemenuhan kebutuhan keamanan dan keselamatan yang dapat mencegah timbulnya pertumbuhan bakteri dan penularan pada orang lain. Oleh karena itu, harapan dari peneliti adalah agar penderita Tuberkulosis Paru bisa sembuh dan mencegah penularan pada orang lain agar dapat menurunkan angka kematian dari pada pasien TB paru. Defisit nutrisi adalah suatu keadaan individu memiliki penurunan kemampuan mengonsumsi cairan dan makanan padat dari mulut ke lambung. PENDAHULUAN Defisit nutrisi adalah krtidak cukupan asupan zat gizi dalam memenuhi kebutuhan energi harian karena asupan makanan yang tidak memadai atau kareana gangguan pencernaan dan penyerapan makanan (Barbara et al., 2010). Defisit nutrisi adalah suatu keadaan dialami seseorang dalam keadaan tidak berpuasa atau resiko penurunan berat badan akibat krtidak cukupan asupan nutrisi untuk kebutuhan metabolism (Hidayat, 2009). Berdasarkan survey pendahuluan yang peneliti pada tanggal 03 Februari 2021 di Rumah Sakit TK II Putri Hijau Medan pada bulan September 2021 diperoleh data jumlah pasien rawat inap dengan diagnose tuberculosis paru di Rumah Sakit TK II Putri Hijau Medan 2021 sejak januari – desember tahun 2021 sebanyak 180 jiwa. yang terdiri dari pasien rawat jalan sebanyak 175 orang dan pasien rawat inap sebanyak 5 orang. PENDAHULUAN Tuberkulosis (TBC) merupakan penyakit lama yang masih menjadi pembunuh terbanyak diantara penyakit menular. Dunia pun masih belum bebas dari Tuberkulosis. Kerugian yang diakibatkannya sangat besar, bukan hanya dari aspek kesehatan semata tetapi juga dari aspek sosial maupun ekonomi. Dengan demikian Tuberkulosis merupakan ancaman terhadap cita-cita pembangunan dalam meningkat kesejahteraan rakyat secara menyeluruh, karenanya perang terhadap Tuberkulosis berarti pula perang terhadap kemiskinan, ketidak produktifan dan kelemahan akibat Tuberkulosis. (Kementrian Kesehatan RI, 2017). Tubercolusis merupakan penyakit infeksi menular yang disebabkan oleh mikrobakterium tubercolusis yang menyerang paru-paru dan hampir seluruh organ tubuh lainnya (Amin, 2015). Menurut data World Health Organization (WHO) tahun 2017 Prevalensi angka kejadi TBC Paru cukup tinggi mulai dari luar sampai dalam negri. Secara global pada tahun 2016 terdapat 10,4 juta kasus insiden Tuberkulosis (CI 8,8 juta – 12, juta) yang serta dengan 120 kasus per 100.000 penduduk. Lima Negara dengan insiden kasus tertinggi yaitu India, Indonesia, China, Philipina, dan Pakistan. Sebagian besar estimasi insiden Tuberkulosis pada tahun 2016 terjadi di Kawasan Asia Tenggara (45%) dimana Indonesia merupakan salah satu di dalamnya dan 25% nya terjadi di Kawasan Afrika (WHO, 2017). Menurut Kementrian RI (2017) jumlah kasus TB paru BTA (+) terbanyak di provinsi Jawa Barat sekitar 31.598, diikuti Jawa Timur 33.585, Jawa Tengah 18.248, DKI Jakarta 12.597, sumatera utara 11.897, banten 7.400, Sumatera barat 4.541, dan Sulawesi selatan 4.314, penderita TB paru disetiap provinsi mengalami penurunan yaitu papua barat sebanyak https://doi.org/10.56211/pubhealth.v2i3.464 UMI FEBRIWANTI / PUBHEALTH JURNAL KESEHATAN MASYARAKAT - VOL. 2 NO. 3 (2024) EDISI JANUARI ISSN: 2830-7224 (ONLINE) ISSN: 2830-7224 (ONLINE) 528% penderita TB paru. Adapun jumlah kasus tuberculosis paru di sulawesi selatan 8.508, penderita TB paru. Pada tahun 2017 provinsi gorontalo sebanyak 10%. (Profil Dinas Kesehatan, 2017). Menurut Data tuberkulosis di kota Medan saat ini cukup tinggi yakni di tahun 2021 mencapai 18.963 kasus. Namun yang terlapor baru 13,3 % perhari. Menurut Raharja, (2015). Tindakan atau peran petugas rumah sakit selama memberikan pelayanan kesehatan kepada penderita Tubrkulosis paru yang mengalami defisit nutrisi adalah diet kalori tinggi protein (TKTP), cukup lemah, vitamin dan mineral. TKTP di berikan agar pasien mendapat cukup makanan untuk memenuhi kebutuhan kalori dan protein yang meningkat. Oleh karena itu rekomendasi kebutuhan energi total untuk pasien tuberkulosi paru di tinggkatkan menjadi 35-45 kkal/kgBB. Menurut Data tuberkulosis di kota Medan saat ini cukup tinggi yakni di tahun 2021 mencapai 18.963 kasus. Namun yang terlapor baru 13,3 % perhari. Menurut Raharja, (2015). https://doi.org/10.56211/pubhealth.v2i3.464 Etiologi TB paru disebabkan oleh kuman Mycobacterium Tuberculosis yang dapat ditularkan ketika seseorang penderita penyakit paru aktif mengeluarkan organisme. Individu yang rentan menghirup droplet dan menjadi terinfeksi. Bakteria ditansmisikan ke alveoli dan memperbanyak diri. Reaksi inflamasi menghasilkan eksudat di alveoli dan bronkopnemonia, granuloma, dan jaringan fibrosa (Smaltzer & Bare, 2015) Menurut Smaltzer & Bare (2015) individu yang beresiko tinggi untuk tertular virus tuberculosis adalah mereka yang kontak dekat dengan seseorang yang mempunyai TB aktif, individu imunospresif (termasuk lansia, pasien dengan kanker, mereka yang dalam terapi kortikkosteroid, atau mereka yang terinfeksi dengan HIV), pengguna obat-obat IV dan alkhoholik, individu tanpa perawatan kesehatan yang adekuat (tunawisma; tahanan etnik dan ras minoritas, terutama anak- anak dibawah usia 15 tahun dan dewasa muda antara yang berusia 15-44 tahun), dengan gangguan medis yang sudah ada sebelumnya (misalkan diabetes, gagal ginjal kronis, silicosis, penyimpangan gizi), individu yang tinggal di daerah yang perumahan sub standar kumuh, pekerjaan (misalkan tenaga kesehatan, terutama yang melakukan aktifitas yang beresiko tinggi. Patofisiologi Seseorang yang dicurigai mengidap basil Mycobacterium tuberculosis akan menjadi terinfeksi. Bakteri menyebar melalui jalan napas ke alveoli, di mana pada derah tersebut bakteri menumpuk dan berkembang biak. Penyebaran basil ini bias juga melalui sistem limfe dan aliran darah ke bagian tubuh lain (ginjal, tulang, korteks serebri) dan area lain dari paru- paru (lobusats). Sistem kekebalan tubuh berespons dengan melakukan reaksi inflamasi. Neutropil dan magropag memfagositoris (menelan) bakteri. Limfosit yang spesifik terhadap tuberculosis menghancurkan (melisiskan) basil dan jaringan normal. Reaksi jaringan ini mengakibatkan terakumulasinya eksudat dalam alveoli dan terjadilah bronkopneumonia. Infeksi awal biasanya timbul dalam waktu 2-10 minggu setelah terpapar. Massa jaringan baru disebut granuloma, yang berisi gumpalan basil yang hidup dan yang sudah mati, dikelilingi oleh makrofag yang membentuk dinding. Granula berubah bentuk menjdi massa jaringan fibrosa. Bagian tengah dari massa tersebut disebut. Ghon Tubercle. Materi yang terdiri atas makrofag dan bakteri menjadi nekrotik, membentuk perkijuan (necrotizing caseosa). Setelah itu akan terbentuk kalfikasi, membetuk jaringan kolagen bakteri menjadi non-aktif. Penyakit akan berkembang menjadi aktif setelah infeksi awal, karena respons sistem imun yang tidak adekuat. Penyakit akan dapat juga timbul akibat infeksi ulang atau aktifnya kembali bakteri yang tidak aktif. Pada kasus ini, terjadi ulserasi pada ghon tubercle, dan akhirnya menjadi perkijuan. Tuberkel yang ulserasi mengalami proses penyembuhan membentuk jaringan parut. Paru- paru yang terinfeksikemudian meradang, mengakibatkan bronkopneumonia, pembentukan tuberkel, dan seterusnya. Pneumonia seluler ini dapat sembuh dengan sendirinya. Proses ini berjalan terus dan basil terus difagosit atau berkembang biak di dalam sel. Basil juga menyebar melalui kelenjar getah bening. Makrofag yang mengadakan infiltrasi menjadi lebih panjang dan sebagian bersatu membentuk sel tuberkel epiteloid yang dikelilingi oleh limfosit (membutuhkan 10-20 hari). Daerah yang mengalami nekrosis serta jaringan granulasi yang dikelilingi sel epiteloid dan fibroblast akan menimbulkan respons berbeda dan akhirnya membentuk suatu kapsul yang dikelilingi oleh tuberkel. Tuberkulosis Tuberkulosis atau TB Paru adalah suatu penyakit menular yang paling sering mengenai parenkim paru, biasanya disebabkan oleh Mycobacterium Tuberculosis. TB Paru dapat menyebar ke setiap bagian tubuh, termasuk maningen, ginjal, tulang dan nodus limfe (Smeltzter & Bare, 2015). Umi Febriwanti 113 https://doi.org/10.56211/pubhealth.v2i3.464 UMI FEBRIWANTI / PUBHEALTH JURNAL KESEHATAN MASYARAKAT - VOL. 2 NO. 3 (2024) EDISI JANUARI ISSN: 2830-7224 (ONLINE) ISSN: 2830-7224 (ONLINE) Tubercolusis merupakan penyakit infeksi menular yang disebsbkan oleh mikrobakterium tubercolusis yang menyerang paru-paru dan hampir seluruh organ tubuh lainnya (Amin, 2015). Tuberkulosis merupakan penyakit lama yang masih menjadi pembunuh terbanyak diantara penyakit menular. Dunia pun masih belum bebas dari TBC. Kerugian yang diakibatkannya sangat besar, bukan hanya dari aspek kesehatan semata tetapi juga dari aspek sosial maupun ekonomi. Dengan demikian TBC merupakan ancaman terhadap cita-cita pembangunan dalam meningkat kesejahteraan rakyat secara menyeluruh, karenanya perang terhadap TBC berarti pula perang terhadap kemiskinan, ketidak produktifan dan kelemahan akibat TBC. (Kementrian Kesehatan RI, 2017). Umi Febriwanti 114 Subjek Asuhan Subjek asuhan pada laporan tugas akhir ini adalah dua pasien terdiagnosa Tuberkulosis paru dengan masalah defisit nutrisi di Rumah Sakit Tk. II Putri Hijau Medan tahun 2022. Pengumpulan Data Pengumpulan data adalah berbagai cara yang digunakan untuk mengumpulkan data menghimpun, mengambil, atau menjaring data penelitian. Kita mengenal metode wawancara, pengamatan, angket, pengetesan, arsip, dan dokumen. Yang disebutkan dua terakhir lebih mengacu kepada sumber data. Cara-cara ini dipilih bukan tanpa alasan. Pertimbangan utama adalah kemampuan cara yang dipilih dalam menggali informasi. Kadang hanya diperlukan satu cara. Namun, kadang cara tunggal dinilai kurang mampu menjaring data secara lengkap, sehingga dibutuhkan metode lain sebagai metode sekunder (Suwartono, 2014). Asuhan keperawatan ini penulis menggunkan alat pengumpulan data dan berupa format pengkajian asuhan keperawatan. Selanjutnya menggunakan alat pemeriksaan fisik yang digunakan penulis antara lain: alat pemeriksaan tanda-tanda vital meliputi, thermometer, stetoskop, buku catatan dan pena. Kemudian hasil pegukuran di tulis dalam lembar format pengkajian. Teknik Pengumpulan Data Ada empat metode yang digunakan dalam pengumpulan data yang digunakan penulis dan termasuk dalam tahap pengkajian, yaitu anamnesi, observasi, pemeriksaan fisik, dan premeriksaan penunjang. 1. Anamnesis, mendapatkan informasi yang anda perlukan dalam mengidentifikasi dan merencanakan tindakan keperawatan. 1. Anamnesis, mendapatkan informasi yang anda perlukan dalam mengidentifikasi dan merencanakan tindakan keperawatan. 2. Meningkatkan hubungan anda dengan klien dalam berkomunikasi 3. Membantu klien memperoleh informasi dan berpartisipasi dalam indentifikasi masalah dan tuju 4. Membantu anda untuk menentukan investigasi lebih lanjut selama tahapan pengkajian Tempat dan Waktu Tempat penelitian pada kasus ini dilakukan di Rumah Sakit TK II Putri Hijau Medan. Waktu pelaksanaan penelitian direncanakan pada bulan Maret 2022. Fokus Asuhan Keperawatan Pada laporan tugas akhir ini penulis menggunakan pendekatan asuhan keperawatan yang berfokus pada pasien TB Paru dengan masalah defisit nutrisi untuk membantu pasien mengatasi tentang kesehatan yang dialami khususnya pasien TB Paru dengan masalah defisit nutrisi di Rumah Sakit TK II Putri Hijau Medan tahun 2022. Asuhan keperawatan merupakan proses atau rangkaian kegiatan praktik keperawatan langsung pada pasien atau diberbagai tatanan pelayanan kesehatan yang pelaksanaannya berdasarkan kaidah profesi keperawatan dan merupakan inti keperawatan. https://doi.org/10.56211/pubhealth.v2i3.464 Umi Febriwanti 114 UMI FEBRIWANTI / PUBHEALTH JURNAL KESEHATAN MASYARAKAT - VOL. 2 NO. 3 (2024) EDISI JANUARI ISSN: 2830-7224 (ONLINE) Sumber Data Sekunder Sumber data sekunder adalah data yang diperoleh selain klien, orang terdekat, teman, dan orang lain yang tahu tentang status kesehatan klien. Selain itu, tenang kesehatan lainnya seperti dokter, ahli gizi, ahli fisioterapi, laboraturium, juga termasuk sumber data sekunder. Sumber Data Primer Sumber data primer adalah klien. Sumber data primer bila klien dalam keadaan tidak sdar, mengalami gangguan bicara, atau pendengaran, klien masih bayi, karena beberapa sebab klien tidak dapat memberikan data subjektif untuk menegakkan diagnosis keperawatan. Namun, bila diperlukan klarifikasi data siubjektif, hendaknya perawat melakukan anamnesis kepada keluarga. 1. Riwayat kesehatan yang lalu Penyajian Data Penulis menyajikan data pada penelitian ini dengan cara tertular yaitu penyajian data penelitian dalam bentuk uraian kalimat dan juga dalam bentuk tabel. Penyajian textular adalah penyajian data hasil penelitian dalam bentuk uraian kalimat. Penyajian textular bias any digunakan untuk penelitian atau data kualitatif, penyajian textular disajikan dalam bentuk uraian kalimat. Penyajian data dalam bentuk tabel adalah suatu penyajian yang sistematik dari pada data numerik, yang tersusun dalam kolom atau jajaran penyajian adata dalam bentuk tabel digunakan untuk data yang sudah ditabulasi dan dikalsifikasikan. Umi Febriwanti 115 https://doi.org/10.56211/pubhealth.v2i3.464 UMI FEBRIWANTI / PUBHEALTH JURNAL KESEHATAN MASYARAKAT - VOL. 2 NO. 3 (2024) EDISI JANUARI ISSN: 2830-7224 (ONLINE) Pengkajian Pengkajian Identitas Pasien dan Hasil Anamnesa Tabel 1. Identitas Pasien dan Hasil Anamnesa Identitas pasien Kasus 1 Kasus 2 Nama Umur Jenis Kelamin Tn. I 50 Tahun Laki- laki Tn. T 49 Tahun Laki- laki Pendidikan SD SD Pekerjaan Petani Petani Status perkawinan Sudah Menikah Sudah Menikah Agama Islam Islam Alamat Suku/ bangsa Jawa / Indonesia Jawa/ Indoneia Tanggal masuk rumah sakit Jam masuk 23.50 WIB 10.30 WIB No. RM Diagnosa masuk TB TB Ditanggung oleh BPJS BPJS Tanggal dan Jam pengkajian Pukul: 08.00 WIB Pukul: 08.00 WIB Identitas Pasien dan Hasil Anamnesa Berdasarkan Tabel 1 didapatkan dari kedua pasien berjenis kelamin laki- laki mempunyai diagnosis yang sama yaitu Tuberkulosis. Pada kasus I dengan pasien berumur 52 tahun dan kasus II dengan pasien berumur 49 tahun. Umi Febriwanti htt //d i /10 56211/ bh 116 Tabel 2. Keluhan Utama dan Riwayat Sakit No Data Fokus Kasus I Kasus II 1. Keluhan utama saat masuk rumah sakit Klien mengatakan nafsu makan berkurang Keluarga klien mengatakan klien makannya sulit 2. Riwayat Penyakit Sekarang Keluarga klien mengatakan batuk lebih dari seminggu disertai dengan sesak nafas dan nafsu makannya menurun, sejak enam hari terakhir, Tn. I juga mengeluh badannya demam sejak hari jum’at, BAB cair lebih dari 5x/hari, nyeri perut mual, muntah 2x, pasien terlihat lemas, BB: 50Kg (Sekarang), BB:65Kg (Sebelum Sakit) kemudian keluarga membawanya ke RS TK II Putri Hijau Medan pada pukul 17.08 WIB untuk mendapatkan pengobatan dan selanjutnya Tn.I dirawat inap di RS TK II Putri Hijau Medan. Keluarga klien mengatakan batuk kurang lebih 2 bulan disertai sesak, nafsu makan menurun, mual, BAB jarang dan badannya bertambah kurus, Tn. T juga mengeluh demam, kurang lebih 6 hari, mual, muntah, pasien terlihat lemas, BB:35Kg (Sekarang), BB:45Kg (Sebelum Sakit) kemudian oleh keluarga dibawa ke Rumah Sakit TK II Putri Hijau Medan pada pukul 14.42 WIB untuk mendapatkan pengobatan dan selanjutnya Tn. T dirawat inap di Rumah Sakit TK II Putri .. 1. Riwayat kesehatan yang lalu Keluarga klien mengatakan Tn.I mempunyai Riwayat penyakit paru yaitu TB kurang lebih 1 tahun Keluarga klien mengatakan Tn.T tidak mempunyai riwayat penyakit sebelumnya 2. Riwayat keluarga Keluarga klien mengatakan keluarga klien tidak ada yang Keluarga klien mengatakan keluarga klien tidak ada yang Tabel 2. Keluhan Utama dan Riwayat Sakit Tabel 2. Pengkajian Perubahan Pola Kesehatan (Pendekatan Gordon / Pendekatan Sistem) Tabel 3. Perubahan Pola Kesehatan (Pendekatan Gordon / Pendekatan Sistem) Tabel 3. Perubahan Pola Kesehatan (Pendekatan Gordon / Pendekatan Sistem) Pola Kesehatan Pasien I Pasien II Pola Manajemen kesehatan Mengajarkan pasien dan keluarga merencanakan makanan. Mengajarkan pasien dan keluarga merencanakan makanan. Pola Nutrisi Ketika sehat Tn.I makan 3x/hari dengan jumlah yang banyak. Klien juga minum air 8x/hari jenis air putih 8gelas/hari. Ketika sakit Tn.I makan 3x/hari jenis diit bubur halus TKTP (Tinggi karbohidrat tinggi protein) dengan jumlah seperempat porsi, 4-5 sendok makan. Ketika sehat Tn.T makan 3x/hari dengan jumlah yang banyak, 2 bulan terakhir karena batuk napsu makan menurun 2x/hari jumlah sedikit. Ketika sakit Tn.T makan 3x/hari jenis diit bubur halus TKTP(tinggi karbohidrat tinggi protein) dengan jumlah 3-4 sendok, minum 3x/hari jumlah setengah gelas. Pola Eliminasi Urine Dirumah BAK 4x/hari warna kuningt keruh bdan berbau khas ,keika di rumah sakit BAK 3x/hari konsistensi sedang warna kuning keruh berbau khas. Dirumah BAK 3-4 x/hari konsistensi sedang warna kuning keruh dan berbau khas. Ketika di rumah sakit BAK 2-3 x/hari konsistensi sedang warna kuning keruh berbau khas. Alvi Dirumah Tn.I BAB 1x/hari jumlah sedang warna kuning kecoklatan bau khas, ketika di rumah sakit belum BAB sama sekali dari awal masuk rumah sakit sampai sekarang. Dirumah Tn.T jarang BAB setiap hari sekali dalam jumlah sedikit warna kuning kecoklatan berbau khas, ketika di rumah sakit Tn. T BAB baru sekali tadi dengan jumlah sedikit warna kuning kecoklatan dengan bau khas Pola istirahat – tidur Ketika Tn.I masih sehat, klien mengatakan waktu istrahat dan tidur kurang lebih 7 jam kalau siang tidur tidak menentu. Keika di rumah sakit waktu tidur 8 jam setiap hari dari terkadang tidur siang 1 jam dan tidak menentu. Ketika Tn.T masih sehat, klien mengatakan waktu istirahat dan tidur kurang lebih 8 jam kalau siang jarang tidur. Ketika di rumah sakit 8-9 jam setiap harinya dari dan tidur siang sekitar 1-2 jam. Pola Aktivitas Saat dirumah Tn.I selalu melakukan aktivitas sesuai dengan pekerjaannya yaitu petani. Tetapi waktu dirumah sakit semua Saat dirumah Tn.T selalu melakukan aktivitas sesuai dengan pekerjaanya yaitu petani. Tetapi waktu dirumah sakit semua Tabel 3. Perubahan Pola Kesehatan (Pendekatan Gordon / Pendekatan Sistem) Pola Kesehatan Pasien I Pasien II Pola Manajemen kesehatan Mengajarkan pasien dan keluarga merencanakan makanan. Mengajarkan pasien dan keluarga merencanakan makanan. Pola Nutrisi Ketika sehat Tn.I makan 3x/hari dengan jumlah yang banyak. Pengkajian Keluhan Utama dan Riwayat Sakit Kasus II Keluarga klien mengatakan klien makannya sulit Keluarga klien mengatakan batuk kurang lebih 2 bulan disertai sesak, nafsu makan menurun, mual, BAB jarang dan badannya bertambah kurus, Tn. T juga mengeluh demam, kurang lebih 6 hari, mual, muntah, pasien terlihat lemas, BB:35Kg (Sekarang), BB:45Kg (Sebelum Sakit) kemudian oleh keluarga dibawa ke Rumah Sakit TK II Putri Hijau Medan pada pukul 14.42 WIB untuk mendapatkan pengobatan dan selanjutnya Tn. T dirawat inap di Rumah Sakit TK II Putri .. (Sebelum Sakit) kemudian keluarga membawanya ke RS TK II Putri Hijau Medan pada pukul 17.08 WIB untuk mendapatkan pengobatan dan selanjutnya Tn.I dirawat inap di RS TK II Putri Hijau Medan. Keluarga klien mengatakan Tn.T tidak mempunyai riwayat penyakit sebelumnya Keluarga klien mengatakan Tn.I mempunyai Riwayat penyakit paru yaitu TB kurang lebih 1 tahun Keluarga klien mengatakan keluarga klien tidak ada yang Keluarga klien mengatakan keluarga klien tidak ada yang Umi Febriwanti 116 Umi Febriwanti 116 https://doi.org/10.56211/pubhealth.v2i3.464 UMI FEBRIWANTI / PUBHEALTH JURNAL KESEHATAN MASYARAKAT - VOL. 2 NO. 3 (2024) EDISI JANUARI ISSN: 2830-7224 (ONLINE) No Data Fokus Data Fokus No Kasus II mempunyai penyakit yang sama dengan yang diderita oleh klien. Kasus II mempunyai penyakit yang sama dengan yang diderita oleh klien. mempunyai penyakit yang sama dengan yang diderita oleh klien. mempunyai penyakit yang sama dengan yang diderita oleh klien. 3. Riwayat Psikososial 3. Riwayat Psikososial 1. Respon pasien terhadap penyakitnya : Tn.T menganggap penyakit nya ini adalah cobaan dari tuhan. 2. Pengaruh penyakit terhadap perannya di keluarga : Tn.T hanya bisa berbaring di tempat tidur, tidak dapat melakukan apa-apa dan tidak bisa bekerja. Pasien tidak bisa berkumpul dengan keluarganya dan masyarakat 1. Respon pasien terhadap penyakitnya : Tn.I menganggap penyakit nya ini adalah cobaan dari tuhan. 2. Pengaruh penyakit terhadap perannya di keluarga : Tn.I hanya bisa berbaring di tempat tidur, tidak dapat melakukan apa-apa dan tidak bisa bekerja. Pasien tidak bisa berkumpul dengan keluarganya dan masyarakat Berdasarkan Tabel 2 ditemukan keluhan utama dan riwayat penyakit kasus I yaitu klien mengatakan batuk lebih dari seminggu disertai dengan sesak nafas dan nafsu makannya menurun dan riwayat penyakit terdahulu adalah mempunyai Riwayat penyakit paru yaitu TB Paru kurang lebih 1 tahun. Sedangkan pasien dengan kasus II ditemukan keluhan klien mengatakan batuk kurang lebih 2 bulan disertai sesak, nafsu makan menurun, mual, BAB jarang dan badannya bertambah kurus, Tn.T tidak mempunyai riwayat penyakit sebelumnya. Tabel 3. Pengkajian Klien juga minum air 8x/hari jenis air putih 8gelas/hari. Ketika sakit Tn.I makan 3x/hari jenis diit bubur halus TKTP (Tinggi karbohidrat tinggi protein) dengan jumlah seperempat porsi, 4-5 sendok makan. Ketika sehat Tn.T makan 3x/hari dengan jumlah yang banyak, 2 bulan terakhir karena batuk napsu makan menurun 2x/hari jumlah sedikit. Ketika sakit Tn.T makan 3x/hari jenis diit bubur halus TKTP(tinggi karbohidrat tinggi protein) dengan jumlah 3-4 sendok, minum 3x/hari jumlah setengah gelas. Pola Eliminasi Urine Dirumah BAK 4x/hari warna kuningt keruh bdan berbau khas ,keika di rumah sakit BAK 3x/hari konsistensi sedang warna kuning keruh berbau khas. Dirumah BAK 3-4 x/hari konsistensi sedang warna kuning keruh dan berbau khas. Ketika di rumah sakit BAK 2-3 x/hari konsistensi sedang warna kuning keruh berbau khas. Alvi Dirumah Tn.I BAB 1x/hari jumlah sedang warna kuning kecoklatan bau khas, ketika di rumah sakit belum BAB sama sekali dari awal masuk rumah sakit sampai sekarang. Dirumah Tn.T jarang BAB setiap hari sekali dalam jumlah sedikit warna kuning kecoklatan berbau khas, ketika di rumah sakit Tn. T BAB baru sekali tadi dengan jumlah sedikit warna kuning kecoklatan dengan bau khas Pola istirahat – tidur Ketika Tn.I masih sehat, klien mengatakan waktu istrahat dan tidur kurang lebih 7 jam kalau siang tidur tidak menentu. Keika di rumah sakit waktu tidur 8 jam setiap hari dari terkadang tidur siang 1 jam dan tidak menentu. Ketika Tn.T masih sehat, klien mengatakan waktu istirahat dan tidur kurang lebih 8 jam kalau siang jarang tidur. Ketika di rumah sakit 8-9 jam setiap harinya dari dan tidur siang sekitar 1-2 jam. Pola Aktivitas Saat dirumah Tn.I selalu melakukan aktivitas sesuai dengan pekerjaannya yaitu petani. Tetapi waktu dirumah sakit semua Saat dirumah Tn.T selalu melakukan aktivitas sesuai dengan pekerjaanya yaitu petani. Tetapi waktu dirumah sakit semua Tabel 3. Perubahan Pola Kesehatan (Pendekatan Pola Kesehatan Pasien I Pola Manajemen kesehatan Mengajarkan pasien dan keluarga merencanakan makanan. Pola Nutrisi Ketika sehat Tn.I makan 3x/hari dengan jumlah yang banyak. Klien juga minum air 8x/hari jenis air putih 8gelas/hari. Ketika sakit Tn.I makan 3x/hari jenis diit bubur halus TKTP (Tinggi karbohidrat tinggi protein) dengan jumlah seperempat porsi, 4-5 sendok makan. Pola Eliminasi Urine Dirumah BAK 4x/hari warna kuningt keruh bdan berbau khas ,keika di rumah sakit BAK 3x/hari konsistensi sedang warna kuning keruh berbau khas. https://doi.org/10.56211/pubhealth.v2i3.464 Pengkajian Trakea di tengah, Cor ukuran bentuk dan letak jantung normal, Pulmo corakan bronkovaskuler kasar tidak tampak bercak pada kedua lapangan paru, Hilus tidak menebal, Diagfragma kanan dan kiri lancip, Soft tissue dan tulang yang tervisualisasi baik. f. Jantung Ada keluhan nyeri dada, irama jantung teratur, CRT < 3 detik, konjungtiva anemis dan JVP Ada kel uhan nyeri dada, irama jantung teratur, CRT > 3 detik, konjungtiva anemis dan JVP UMI FEBRIWANTI / PUBHEALTH JURNAL KESEHATAN MASYARAKAT - VOL. 2 NO. 3 (2024) EDISI JANUARI ISSN: 2830-7224 (ONLINE) kegiatan dibantu oleh keluarganya. kegi kelu Saat dirumah Tn.I mandi dan gosok gigi 2x/hari diwaktu pagi dan sore. Tetapi di rumah sakit Tn.I hanya diseka oleh keluarganya Saat goso dan Tn.T kelu Fisik Tabel 4. Observasi dan Pemeriksaan Fis Pasien I 37,6 ℃ 100 x/menit 120/80 mmHg 26 x/menit 160 cm 50 kg 4-5-6 Composmentis 3 9 1 2 1 3 4 C Toe) Bentuk kepala normal, rambut beruban, ubun-ubun tidak cekung, tidak ada benjolan dan lesi pada kepala, wajah simetris, tidak ada massa pada leher, tidak ada benjolan kelenjar tiroid dan tidak ada bendungan vena jugularis. B ti ce le si le k b Mata tidak strabismus (juling), alis mata simetris, tidak ada edema, ektropin, kalazion dan xantelesma, konjungtiva anemis, pupil isokor dan reflek cahaya kanan kiri positif. M al ed x an ca Hidung simetris, tidak terdapat perforasi, tidak ada situnisis, tidak ada nyeri tekan. Terpasang O2 nasal 4 lpm. H p ti T Mukosa bibir kering, tidak sianosis, pucat, tidak ada lesi, terdapat karie gigi, gigi sudah tidak lengkap, tidak ada gangguan pengecapan, tidak ada faringitis. M si te g ad Bentuk dada simetris, keluhan sesak, batuk kurang lebih satu minggu, irama nafas tidak teratur, adanya tambahan suara nafas ronchi. Trakhea di tengah Cor ukuran bentuk dan letak jantung normal, Pulmo corakan bronkovaskuler kasar tidak tampak bercak pada kedua lapangan paru, Hilus tidak menebal, Diafragma kanan dan kiri lancip, Soft tissue dan tulang yang tervisualisasi baik. B se b ad ro u ja b ta la m d tu Ada keluhan nyeri dada, irama jantung teratur, CRT < 3 detik, konjungtiva anemis dan JVP normal. A ja k n Hasil Observasi dan Pemeriksaan Fisik Bentuk kepala normal, rambut beruban, ubun-ubun tidak cekung, tidak ada benjolan dan lesi pada kepala, wajah simetris, tidak ada massa pada leher, tidak ada benjolan kelenjar tiroid dan tidak ada bendungan vena jugularis. f. Jantung Pengkajian Alvi Dirumah Tn.I BAB 1x/hari jumlah sedang warna kuning kecoklatan bau khas, ketika di rumah sakit belum BAB sama sekali dari awal masuk rumah sakit sampai sekarang. Pola istirahat – tidur Ketika Tn.I masih sehat, klien mengatakan waktu istrahat dan tidur kurang lebih 7 jam kalau siang tidur tidak menentu. Keika di rumah sakit waktu tidur 8 jam setiap hari dari terkadang tidur siang 1 jam dan tidak menentu. Pola Aktivitas Saat dirumah Tn.I selalu melakukan aktivitas sesuai dengan pekerjaannya yaitu petani. Tetapi waktu dirumah sakit semua Pola Kesehatan Pola Manajemen kesehatan Pola Nutrisi Pasien II Mengajarkan pasien dan keluarga merencanakan makanan. Ketika sehat Tn.I makan 3x/hari dengan jumlah yang banyak. Klien juga minum air 8x/hari jenis air putih 8gelas/hari. Ketika sakit Tn.I makan 3x/hari jenis diit bubur halus TKTP (Tinggi karbohidrat tinggi protein) dengan jumlah seperempat porsi, 4-5 sendok makan. Ketika sehat Tn.T makan 3x/hari dengan jumlah yang banyak, 2 bulan terakhir karena batuk napsu makan menurun 2x/hari jumlah sedikit. Ketika sakit Tn.T makan 3x/hari jenis diit bubur halus TKTP(tinggi karbohidrat tinggi protein) dengan jumlah 3-4 sendok, minum 3x/hari jumlah setengah gelas. Dirumah BAK 4x/hari warna kuningt keruh bdan berbau khas ,keika di rumah sakit BAK 3x/hari konsistensi sedang warna kuning keruh berbau khas. Dirumah BAK 3-4 x/hari konsistensi sedang warna kuning keruh dan berbau khas. Ketika di rumah sakit BAK 2-3 x/hari konsistensi sedang warna kuning keruh berbau khas. Dirumah Tn.I BAB 1x/hari jumlah sedang warna kuning kecoklatan bau khas, ketika di rumah sakit belum BAB sama sekali dari awal masuk rumah sakit sampai sekarang. Dirumah Tn.T jarang BAB setiap hari sekali dalam jumlah sedikit warna kuning kecoklatan berbau khas, ketika di rumah sakit Tn. T BAB baru sekali tadi dengan jumlah sedikit warna kuning kecoklatan dengan bau khas Ketika Tn.I masih sehat, klien mengatakan waktu istrahat dan tidur kurang lebih 7 jam kalau siang tidur tidak menentu. Keika di rumah sakit waktu tidur 8 jam setiap hari dari terkadang tidur siang 1 jam dan tidak menentu. g Ketika Tn.T masih sehat, klien mengatakan waktu istirahat dan tidur kurang lebih 8 jam kalau siang jarang tidur. Ketika di rumah sakit 8-9 jam setiap harinya dari dan tidur siang sekitar 1-2 jam. Saat dirumah Tn.T selalu melakukan aktivitas sesuai dengan pekerjaanya yaitu petani. Tetapi waktu dirumah sakit semua Umi Febriwanti 117 https://doi.org/10.56211/pubhealth.v2i3.464 https://doi.org/10.56211/pubhealth.v2i3.464 UMI FEBRIWANTI / PUBHEALTH JURNAL KESEHATAN MASYARAKAT - VOL. 2 NO. Pengkajian 3 (2024) EDISI JANUARI ISSN: 2830-7224 kegiatan dibantu oleh keluarganya. kegiatan dibantu oleh keluargannya. Pola kebersihan diri Saat dirumah Tn.I mandi dan gosok gigi 2x/hari diwaktu pagi dan sore. Tetapi di rumah sakit Tn.I hanya diseka oleh keluarganya Saat dirumah Tn.T mandi dan gosok gigi 2x/hari diwaktu pagi dan sore. Tetapi di rumah sakit Tn.T hanya di seka oleh keluarganya. Hasil Observasi dan Pemeriksaan Fisik Tabel 4. Observasi dan Pemeriksaan Fisik Observasi Pasien I Pasien II S N TD RR TB BB GCS Kesadaran 37,6 ℃ 100 x/menit 120/80 mmHg 26 x/menit 160 cm 50 kg 4-5-6 Composmentis 37 ℃ 90 x/menit 120/80 mmHg 24 x/menit 160 cm 35 kg 4-5-6 Composmentis (Pemeriksaan Head To Toe) a. Kepala Bentuk kepala normal, rambut beruban, ubun-ubun tidak cekung, tidak ada benjolan dan lesi pada kepala, wajah simetris, tidak ada massa pada leher, tidak ada benjolan kelenjar tiroid dan tidak ada bendungan vena jugularis. Bentuk kepala normal, rambut tipis kusam, ubunubun tidak cekung, tidak ada benjolan dan lesi pada kepala, wajah simetris, tidak ada massa pada leher, tidak ada benjolan kelenjar tiroid dan tidak ada bendungan vena jugularis b. Mata Mata tidak strabismus (juling), alis mata simetris, tidak ada edema, ektropin, kalazion dan xantelesma, konjungtiva anemis, pupil isokor dan reflek cahaya kanan kiri positif. Mata tidak strabismus (juling), alis mata simetris, tidak ada edema, ektropin, kalazion dan xantelesma, konjungtiva anemis, pupil isokor dan reflek cahaya kanan kiri positif. c. Hidung Hidung simetris, tidak terdapat perforasi, tidak ada situnisis, tidak ada nyeri tekan. Terpasang O2 nasal 4 lpm. Hidung simetris, tidak terdapat perforasi, tidak ada situnisis, tidak ada nyeri tekan. Terpasang O2 nasal 4 lpm. d. Mulut dan Faring Mukosa bibir kering, tidak sianosis, pucat, tidak ada lesi, terdapat karie gigi, gigi sudah tidak lengkap, tidak ada gangguan pengecapan, tidak ada faringitis. Mukosa bibir kering, tidak sianosis, pucat, tidak ada lesi, terdapat karie gigi, ada gangguan pengecapan, tidak ada faringitis. e. Thoraks dan Paru Bentuk dada simetris, keluhan sesak, batuk kurang lebih satu minggu, irama nafas tidak teratur, adanya tambahan suara nafas ronchi. Trakhea di tengah Cor ukuran bentuk dan letak jantung normal, Pulmo corakan bronkovaskuler kasar tidak tampak bercak pada kedua lapangan paru, Hilus tidak menebal, Diafragma kanan dan kiri lancip, Soft tissue dan tulang yang tervisualisasi baik. Bentuk dada simetris, keluhan sesak, batuk kurang lebih dua bulan, irama nafas tidak teratur, adanya tambahan suara nafas ronchi. e. Thoraks dan Paru Pengkajian 2 NO. 3 (2024) EDISI JANUARI ISSN: 2830-7224 (ONLINE) g. Ginjal Tidak ada perubahan dalam berkemih, tidak ada pembesaran dan tidak ada nyeri tekan pada kandung kencing, BAK kurang lebih 3-4 x/hari dengan warna kuning kerung dan bau khas. Tidak ada perubahan dalam berkemih, tidak ad pembesaran dan tidak ada nyer tekan pada kandung kencing BAK kurang lebih 2-3 x/har dengan warna kuning kerun dan bau khas. h. Abdomen Tidak ada luka operasi, tidak ada pembesaran hepar, tidak ada pembesaran lien, tidak mual dan muntah, anoreksia, tidak terpasang NGT dan bissing usus 7 x/menit. Tidak ada luka operasi, tidak ada pembesaran hepar, tidak ada pembesaran lien anoreksia, mual tidak muntah tidak terpasang NGT, bissing usus 6 x/menit. i. Ekstremitas dan persendian Pergerakan sendi bebas dan lemah, tidak ada kelainan ekstremitas, tidak ada kelainan tulang belakang, kulit ikterik, akral hangat, turgor kurang dan tidak ada luka. Terdapat infus NS di ekstremitas dextra. Pergerakan sendi bebas dan lemah, tidak ada kelainan ekstremitas, tidak ada kelainan tulang belakang, kulit ikterik akral dingin, turgor kurang dan tidak ada luka. Terdapat infu NS di ektremitas sinistra. j. Ingunial, genetalia, anus Tidak ada hernia, hemoroid, tidak ada nyeri tekan, tidak ada pendarahan, belum BAB selama di rumah sakit. Tidak ada hernia, hemoroid tidak ada nyeri tekan, tidak ad pendarahan, BAB sediki warna kuning kecoklatan dan bau khas Tidak ada perubahan dalam berkemih, tidak ada pembesaran dan tidak ada nyeri tekan pada kandung kencing, BAK kurang lebih 2-3 x/hari dengan warna kuning kerung dan bau khas. Tidak ada perubahan dalam berkemih, tidak ada pembesaran dan tidak ada nyeri tekan pada kandung kencing, BAK kurang lebih 3-4 x/hari dengan warna kuning kerung dan bau khas. g. Ginjal Tidak ada luka operasi, tidak ada pembesaran hepar, tidak ada pembesaran lien, tidak mual dan muntah, anoreksia, tidak terpasang NGT dan bissing usus 7 x/menit. Tidak ada luka operasi, tidak ada pembesaran hepar, tidak ada pembesaran lien, anoreksia, mual tidak muntah, tidak terpasang NGT, bissing usus 6 x/menit. Pergerakan sendi bebas dan lemah, tidak ada kelainan ekstremitas, tidak ada kelainan tulang belakang, kulit ikterik, akral dingin, turgor kurang dan tidak ada luka. Terdapat infus NS di ektremitas sinistra. Pergerakan sendi bebas dan lemah, tidak ada kelainan ekstremitas, tidak ada kelainan tulang belakang, kulit ikterik, akral hangat, turgor kurang dan tidak ada luka. Terdapat infus NS di ekstremitas dextra. Pengkajian Bentuk kepala normal, rambut tipis kusam, ubunubun tidak cekung, tidak ada benjolan dan lesi pada kepala, wajah simetris, tidak ada massa pada leher, tidak ada benjolan kelenjar tiroid dan tidak ada bendungan vena jugularis Mata tidak strabismus (juling), alis mata simetris, tidak ada edema, ektropin, kalazion dan xantelesma, konjungtiva Mata tidak strabismus (juling), alis mata simetris, tidak ada edema, ektropin, kalazion dan xantelesma, konjungtiva anemis, pupil isokor dan reflek cahaya kanan kiri positif. anemis, pupil isokor dan reflek cahaya kanan kiri positif. Hidung simetris, tidak terdapat perforasi, tidak ada situnisis, tidak ada nyeri tekan. Terpasang O2 nasal 4 lpm. Hidung simetris, tidak terdapat perforasi, tidak ada situnisis, tidak ada nyeri tekan. Terpasang O2 nasal 4 lpm. Hidung simetris, tidak terdapat perforasi, tidak ada situnisis, tidak ada nyeri tekan. Terpasang O2 nasal 4 lpm. Mukosa bibir kering, tidak sianosis, pucat, tidak ada lesi, terdapat karie gigi, gigi sudah tidak lengkap, tidak ada gangguan pengecapan, tidak ada faringitis. Hidung simetris, tidak terdapat perforasi, tidak ada situnisis, tidak ada nyeri tekan. Terpasang O2 nasal 4 lpm. Mukosa bibir kering, tidak sianosis, pucat, tidak ada lesi, terdapat karie gigi, ada gangguan pengecapan, tidak ada faringitis. d. Mulut dan Faring Mukosa bibir kering, tidak sianosis, pucat, tidak ada lesi, terdapat karie gigi, ada gangguan pengecapan, tidak ada faringitis. Mukosa bibir kering, tidak sianosis, pucat, tidak ada lesi, terdapat karie gigi, gigi sudah tidak lengkap, tidak ada gangguan pengecapan, tidak ada faringitis. e. Thoraks dan Paru Bentuk dada simetris, keluhan sesak, batuk kurang lebih satu minggu, irama nafas tidak teratur, adanya tambahan suara nafas ronchi. Trakhea di tengah Cor ukuran bentuk dan letak jantung normal, Pulmo corakan bronkovaskuler kasar tidak tampak bercak pada kedua lapangan paru, Hilus tidak menebal, Diafragma kanan dan kiri lancip, Soft tissue dan tulang yang tervisualisasi baik. Ada keluhan nyeri dada, irama jantung teratur, CRT < 3 detik, konjungtiva anemis dan JVP normal. Bentuk dada simetris, keluhan sesak, batuk kurang lebih dua bulan, irama nafas tidak teratur, adanya tambahan suara nafas ronchi. Trakea di tengah, Cor ukuran bentuk dan letak jantung normal, Pulmo corakan bronkovaskuler kasar tidak tampak bercak pada kedua lapangan paru, Hilus tidak menebal, Diagfragma kanan dan kiri lancip, Soft tissue dan tulang yang tervisualisasi baik. Ada kel uhan nyeri dada, irama jantung teratur, CRT > 3 detik, konjungtiva anemis dan JVP normal. https://doi.org/10.56211/pubhealth.v2i3.464 Umi Febriwanti 118 Umi Febriwanti 118 UMI FEBRIWANTI / PUBHEALTH JURNAL KESEHATAN MASYARAKAT - VOL. https://doi.org/10.56211/pubhealth.v2i3.464 Pengkajian Tidak ada hernia, hemoroid, tidak ada nyeri tekan, tidak ada pendarahan, BAB sedikit warna kuning kecoklatan dan bau khas. Tidak ada hernia, hemoroid, tidak ada nyeri tekan, tidak ada pendarahan, belum BAB selama di rumah sakit. Pemeriksaan Diagnostic Tabel 5. Hasil Pemeriksaan Diagnostik Klien 1 pada tanggal Pemeriksaan Hasil Nilai Normal HEMATOLOGI LED Darah lengkap Leukosit (WBC) Neutrofil Limfosit Monosit Eosinofil Basofil 37/51 18,1 16,7 0,6 0,8 0,0 0,0 0 / 10 mm/jam 3,70 - 10,1 Neutrofil % Limfosit % Monosit % Eosinofil % Basofil % Eritrosit (RBC) Hemoglobin(HGB) Hematrokit (HCT) MCV MCH MCHC RDW PLT MPV H 92,0 L 3,3 4,5 L 0,0 0,2 L 4,250 L 10,70 L 31,90 L 75,00 L 25,20 33,50 13,40 190 7,45 39,3 - 73,7 % 18,0 - 48,3 % 4,40 - 12,7 % 0,600 - 7,30 % 0,00 - 1,70 % 4,6 - 6,2 10̊̊ /𝜇𝐿 13,5 - 18,0 g/dL 40 - 54 % 81,1 - 96 𝜇𝑚ᶟ 27,0 - 31,2 pg 31,8 - 35,4 g/dL 11,5 - 14,5 % 155 - 366 10ᶟ/𝜇𝐿 6,90 - 10,6 Fl KIMIA KLINIK BUN Kreatinin Glukosa darah sewaktu H 39 1,154 115 7,80 - 20,23 mg/dL 0,8 - 1,3 mg/dL < 200 mg/Dl Pemeriksaan Radiologi : Foto Thorax Tampak berawan dan bercak- bercak di dinding paru sebelah kiri Pemeriksaan Diagnostic Tabel 5. Hasil Pemeriksaan Diagnostik Klien 1 pada tanggal Umi Febriwanti 119 https://doi.org/10.56211/pubhealth.v2i3.464 UMI FEBRIWANTI / PUBHEALTH JURNAL KESEHATAN MASYARAKAT - VOL. 2 NO. Pengkajian 3 (2024) EDISI JANUARI ISSN: 2830-7224 (ONLINE) mi Febriwanti https://doi.org/10.56211/pubhealth.v pada tanggal Pemeriksaan Hasil Nilai Normal HEMATOLOGI LED Darah Lengkap Leukosit (WBC) Neutrofil Limfosit Monosit Eosinofil Basofil 26/47 10,8 9,7 0,5 0,5 0,0 0,1 0 / 10 mm/jam 3,70 – 10,1 Neutrofil % Limfosit % Monosit % Eosinofil % Basofil % Eritrosit (RBC) Hemoglobin (HBG) Hematokrit (HTC) MCV MCH MCHC RDW PLT MPV H 89,7 L 4,8 4,8 L 0,1 0,5 6,070 L 11,40 L 38,50 L 63,50 L 18,70 L 29,50 12,90 H 532 6,43 39,3 – 73,7 % 18,0 – 48,3 % 4,40 – 12,7 % 0,600 – 7,30 % 0,00 – 1,70 % 4,6 – 6,2 10̊̊ /𝜇𝐿 13,5 – 18,0 g/dL 40 – 54 % 81,1 – 96,0 𝜇𝑚ᶟ 27,0 – 31,2 pg 31,8 – 35,4 g/dL 11,5 – 14,5 % 155 – 366 10ᶟ/𝜇𝐿 6,90 – 10,6 fL KIMIA KLINIK Faal Hati AST/SGOT ALT/SGPT Faal Ginjal BUN Kreatinin 24,99 19,16 H 38 1,085 < 35 U/L < 45 U/L 7,8 – 20,23 mg/dL 0,8 – 1,3 mg/dL Pemeriksaan Radiologi : Foto thorax Tampak berawan di dinding paru sebelah kiri Tabel 6. Terapi Terapi Pasien I Pasien II Infuse NS 1500 ml / 21 tpm NS 1500 ml / 21 tpm Injeksi Drip 1 aminophilin : Hydromal 2:1 1 aminophilin : Hydromal 2:1 Injeksi Via IV Ceftriaxone 2 x 1 ampl IV Ranitidine 1x50 mg IV Ranitidin 1 x 50 mg IV Per Oral Antaside 3 x 1 tab Isoniazid (INH) 1x200 mg Rifpasifin (R) 1x450 mg Pirasinamid (Z) 1x 750 mg Streptomisin (S) 1x250 mg Etabutol (E) 1x500 mg Antaside 3 x 1 tab Nebulizer Nebulizer ventolin 2,5 ml /8 jam Nebulizer ventolin 2,5 ml /5 jam a Data Tabel 7. Analisa Data Analisa Data Etiologi Masalah Pasien I Data subjektif : - Keluarga klien mengatakan nafsu makan berkurang Data Objektif : a. Keadaan umum : lemah, GCS 4-5-6 b. https://doi.org/10.56211/pubhealth.v2i3.464 Pengkajian TTV S : 37 ℃ N : 90 x/menit TD : 120/80 mmHg RR : 24 x/menit M.Tuberkulosis Menempel pada alveolus Proliferasi sel epitel disekeliling basil dan membentuk organ (tuberkel) Basil menyebar Inflamasi Anoreksia Perubahan nutrisi Ketidakseimbangan nutrisi kurang dari kebutuhan tubuh Ketidakseimbangan nutrisi kurang dari kebutuhan tubuh sa Keperawatan Tabel 8. Diagnosis Keperawatan Pasien I Pasien II Ketidakseimbangan nutrisi kurang dari kebutuhan tubuh berhubungan dengan penurunan nafsu makan ditandai dengan penurunan nafsu makan (anoreksia) , BB kurang dari normal Ketidakseimbangan nutrisi kurang dari kebutuhan tubuh berhubungan dengan penurunan nafsu makan ditandai dengan penurunan nafsu makan (anoreksia) , BB kurang dari normal Basil menyebar Inflamasi Anoreksia Perubahan nutrisi Ketidakseimbangan nutrisi kurang dari kebutuhan tubuh Ketidakseimbangan nutrisi kurang dari kebutuhan tubuh TD : 120/80 mmHg RR : 26 x/menit Pasien II Data Subjektif : - Keluarga klien mengatakan klien makannya sulit Data Objektif : a. Keadaan umum : lemah GCS 4-5-6 b. Rambut tipis dan kusam c. Wajah tampak lemas d. Makan hanya 2-3 sedok makan e. mual f. Mukosa bibir kering g. BAB 1x selama MRS h. Akral dingin i. Bissing usus 6 x/menit j. Turgor kulit jelek k. Mobilitas fisik lemah l. TB : 160 cm m. BB : 35 kg n. TTV S : 37 ℃ N : 90 x/menit TD : 120/80 mmHg RR : 24 x/menit M.Tuberkulosis Menempel pada alveolus Proliferasi sel epitel disekeliling basil dan membentuk organ (tuberkel) Basil menyebar Inflamasi Anoreksia Perubahan nutrisi Ketidakseimbangan nutrisi kurang dari kebutuhan tubuh Ketidakseimbangan nutrisi kurang dari kebutuhan tubuh Diagnosa Keperawatan Tabel 8. Diagnosis Keperawatan Pasien I Pasien II Ketidakseimbangan nutrisi kurang dari kebutuhan tubuh berhubungan dengan penurunan nafsu makan ditandai dengan penurunan nafsu makan (anoreksia) , BB kurang dari normal Ketidakseimbangan nutrisi kurang dari kebutuhan tubuh berhubungan dengan penurunan nafsu makan ditandai dengan penurunan nafsu makan (anoreksia) , BB kurang dari normal Berdasarkan Tabel 6 didapatkan kedua pasien mempunyai masalah yang sama dengan Ketidakseimbangan nutrisi dari kebutuhan tubuh berhubungan dengan penurunan nafsu makan ditandai dengan penurunan nafsu makan (anor BB kurang dari normal. M.Tuberkulosis Menempel pada alveolus Proliferasi sel epitel disekeliling basil dan membentuk organ (tuberkel) Basil menyebar Inflamasi Anoreksia Perubahan nutrisi Ketidakseimbangan nutrisi K k Ketidakseimbangan nutrisi kurang dari kebutuhan tubuh Berdasarkan Tabel 6 didapatkan kedua pasien mempunyai masalah yang sama dengan Ketidakseimbangan nutrisi kurang dari kebutuhan tubuh berhubungan dengan penurunan nafsu makan ditandai dengan penurunan nafsu makan (anoreksia), BB kurang dari normal. Pengkajian Wajah klien tampak lemas M.Tuberkulosis Menempel pada alveolus Proliferasi sel epitel disekeliling basil dan membentuk organ (tuberkel) Ketidakseimbangan nutrisi kurang dari kebutuhan tubuh Klien 2 pada tanggal Pemeriksaan Hasil Nilai Normal HEMATOLOGI LED Darah Lengkap Leukosit (WBC) Neutrofil Limfosit Monosit Eosinofil Basofil 26/47 10,8 9,7 0,5 0,5 0,0 0,1 0 / 10 mm/jam 3,70 – 10,1 Neutrofil % Limfosit % Monosit % Eosinofil % Basofil % Eritrosit (RBC) Hemoglobin (HBG) Hematokrit (HTC) MCV MCH MCHC RDW PLT MPV H 89,7 L 4,8 4,8 L 0,1 0,5 6,070 L 11,40 L 38,50 L 63,50 L 18,70 L 29,50 12,90 H 532 6,43 39,3 – 73,7 % 18,0 – 48,3 % 4,40 – 12,7 % 0,600 – 7,30 % 0,00 – 1,70 % 4,6 – 6,2 10̊̊ /𝜇𝐿 13,5 – 18,0 g/dL 40 – 54 % 81,1 – 96,0 𝜇𝑚ᶟ 27,0 – 31,2 pg 31,8 – 35,4 g/dL 11,5 – 14,5 % 155 – 366 10ᶟ/𝜇𝐿 6,90 – 10,6 fL KIMIA KLINIK Faal Hati AST/SGOT ALT/SGPT Faal Ginjal BUN Kreatinin 24,99 19,16 H 38 1,085 < 35 U/L < 45 U/L 7,8 – 20,23 mg/dL 0,8 – 1,3 mg/dL Pemeriksaan Radiologi : Foto thorax Tampak berawan di dinding paru sebelah kiri Tabel 6. Terapi Klien 2 pada tanggal Analisa Data UMI FEBRIWANTI / PUBHEALTH JURNAL KESEHATAN MASYARAKAT - VOL. 2 NO. 3 (2024) EDISI JANUARI ISSN: 2830-7224 (ONLINE) c. makan hanya 4-5 sedok makan d. Mukosa bibir kering, pucat e. Gigi sudah tidak lengkap f. Belum BAB sama sekali selama MRS g. Turgor kulit jelek h. Akral hangat i. Bissing usus 7 x/menit j. TB : 170 cm k. BB : 50 kg l. TTV S : 37,6 C N : 116 x/menit TD : 120/80 mmHg RR : 26 x/menit Basil menyebar Inflamasi Anoreksia Perubahan nutrisi Ketidakseimbangan nutrisi kurang dari kebutuhan tubuh Pasien II Data Subjektif : - Keluarga klien mengatakan klien makannya sulit Data Objektif : a. Keadaan umum : lemah GCS 4-5-6 b. Rambut tipis dan kusam c. Wajah tampak lemas d. Makan hanya 2-3 sedok makan e. mual f. Mukosa bibir kering g. BAB 1x selama MRS h. Akral dingin i. Bissing usus 6 x/menit j. Turgor kulit jelek k. Mobilitas fisik lemah l. TB : 160 cm m. BB : 35 kg n. Pengkajian Umi Febriwanti 121 https://doi.org/10.56211/pubhealth.v2i3.464 ISSN: 2830-7224 (ONLINE) UMI FEBRIWANTI / PUBHEALTH JURNAL KESEHATAN MASYARAKAT - VOL. 2 NO. 3 (2024) EDISI JANUARI UMI FEBRIWANTI / PUBHEALTH JURNAL KESEHATAN MASYARAKAT - VOL. 2 NO. 3 (2024) EDISI JANUARI Saran Bagi Klien dan Keluarga, sebagai tambahan pengetahuan bagi klien dan keluarga untuk mengambil keputusan yang sesuai dengan masalah serta ikut memperhatikan dan melaksanakan tindakan yang diberikan oleh perawat. Bagi Penulis, dasar pertimbangan dalam memberikan Asuhan keperawatan pada Klien Yang mengalami Tuberkulosis Dengan Ketidakseimbangan Nutrisi Kurang dari Kebutuhan tubuh. Bagi Pengembangan Ilmu dan Teknologi Keperawatan, hasil penelitian dapat digunakan untuk menambah referensi bagi mata kuliah kebutuhan dasar manusia tentang Ketidakseimbangan Nutrisi Kurang Dari kebutuhan Tubuh pada klien Tuberkulosis. Kesimpulan Berdasarkan pengkajian yang dilakukan pada Tn. I dan Tn. T dengan kasus tuberkulosis menunjukan bahwa Tn. I mempunyai riwayat penyakit TB mengalami penurunan nafsu makan kurang lebih satu minggu dan Berat badan kurang dari normal sedangkan Tn. I demam 6 hari yang lalu mengalami penurunan nafsu makan mulai 2 bulan yang lalu, klien merasa mual tapi tidak muntah, mobilitas fisik lemah dan berat badan kurang dari normal. Diagnosa keperawatan aktual yang muncul pada klien 1 dan klien 2 yaitu Ketidakseimbangan nutrisi kurang dari kebutuhan tubuh pada tuberculosis. Intervensi keperawatan yang diberikan kepada klien sesuai dengan NIC 2015 mengenai ketidakseimbangan nutrisi kurang dari kebutuhan tubuh meliputi : Identifikasi perubahan berat badan terakhir, Monitor turgor kulit dan mobilitas, Monitor tekanan darah, nada, suhu dan status pernafasan yang tepat, Monitor warna kulit, suhu dan kelembaban, Monitor mual dan muntah, Lakukan perawatan mulut sebelum makan , dan Ajarkan pasien dan keluarga merencanakan makanan. Hal tersebut sudah sesuai dengan keadaan dan kebiasaan klien, sehingga diharapkan pencapaian yang optimal. Implementasi Keperawatan yang dilakukan secara observasi, mandiri, edukapsi dan kolaborasi, disesuaikan dengan intervensi yang telah diambil dari NIC 2015 agar mencapai tujuan yang diharapkan. Peneliti melakukan implementasi sesuai dengan kondisi klien selama 3 hari. Setelah dilakukan implementasi, evaluasi yang di dapatkan pada klien dengan ketidakseimbangan nutrisi kurang dari kebutuhan tubuh menunjukan bahwa klien 2 lebih menunjukan kemajuan dibandingkan dengan klien 1. DAFTAR PUSTAKA Bulechek GM, Butcher HK, Dochterman JM, Wagner CM. (2012). Nursing Interventions Classification (NIC) - E-Book: Nursing Interventions Classification (NIC) - E-Book. Elsevier Health Sciences. Endalkachew K, Ferede YM, Derso T, Kebede A. (2022). Prevalence and associated factors of undernutrition among adult TB patients attending Amhara National Regional State hospitals, Northwest Ethiopia. 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https://openalex.org/W2613839215	https://europepmc.org/articles/pmc5422529?pdf=render	English	null	Reporting and Interpreting Task Performance in Go/No-Go Affective Shifting Tasks	Frontiers in psychology	2,017	cc-by	3,684	OPINION published: 09 May 2017 doi: 10.3389/fpsyg.2017.00701 OPINION published: 09 May 2017 doi: 10.3389/fpsyg.2017.00701 Reporting and Interpreting Task Performance in Go/No-Go Affective Shifting Tasks Adrian Meule 1, 2* Adrian Meule 1, 2* 1 Department of Psychology, University of Salzburg, Salzburg, Austria, 2 Center for Cognitive Neuroscience, University of Salzburg, Salzburg, Austria Keywords: motor response inhibition, inhibitory control, behavioral inhibition, impulsivity, go/no-go, food cues There is an increased interest in the study of impulsive reactions to food cues (Bartholdy et al., 2016). For this, computerized tasks that include lexical or pictorial food stimuli are often used. One of such tasks is the aﬀective shifting task, which is a type of go/no-go task. Unfortunately, it appears that reporting and interpreting performance in this task has been fairly inconsistent across studies. Therefore, the current article aims to highlight some of these issues in an eﬀort to provide some guidance for researchers who are interested in using this task. Response inhibition or inhibitory control is an executive function, which involves controlling one’s attention, behavior, thoughts, and/or emotions to override a strong internal predisposition or external lure (Diamond, 2013). Prominent psychological tasks that are used to measure inhibitory control include, for example, the Stroop task, Simon task, Flanker task, antisaccade tasks, or stop- signal tasks (Diamond, 2013). Another widely used measure is go/no-go tasks. In these tasks, participants are usually required to perform a quick motor response (e.g., pressing a button on a keyboard as fast as possible) when certain stimuli (i.e., targets) are displayed on a computer screen and to withhold this reaction for other stimuli (i.e., non-targets; also called distractors or lures). As this is an easy task, go/no-go paradigms usually involve a large number of trials of which only a small number of trials are no-go trials (e.g., Kaufman et al., 2003). This is necessary in order for the go-reaction to become pre-potent and, thus, to ensure that participants make enough commission errors (i.e., falsely pressing the button in no-go trials; also called false alarms). Reaction times in or the number of correct go-trials (i.e., hits) and omission errors (i.e., falsely not pressing the button in go trials; also called misses) can also be calculated, but they are usually not considered as indices of inhibitory control (or, speciﬁcally, lack thereof). Edited by: Peter Hall, University of Waterloo, Canada Edited by: Peter Hall, University of Waterloo, Canada Edited by: Peter Hall, University of Waterloo, Canada Reviewed by: Paschal Sheeran, University of North Carolina at Chapel Hill, USA Laura Nynke Van Der Laan, Utrecht University, Netherlands Correspondence: Adrian Meule adrian.meule@sbg.ac.at Reviewed by: Paschal Sheeran, University of North Carolina at Chapel Hill, USA Laura Nynke Van Der Laan, Utrecht University, Netherlands Reviewed by: Paschal Sheeran, University of North Carolina at Chapel Hill, USA Correspondence: Adrian Meule adrian.meule@sbg.ac.at Murphy et al. (1999) developed a modiﬁed version of such go/no-go tasks, which they termed aﬀective shifting task. In their task, words were presented on the screen one by one. Participants had to respond to targets by pressing the space bar as quickly as possible but withhold responses to distractors. The task comprised two practice blocks followed by eight test blocks, each including nine happy words and nine sad words (presented in randomized order within each block). Before each block, either happy or sad words were speciﬁed as targets. This means that when participants had to press the button in response to happy words, they were required to not press the button in response to sad words (or vice versa). The 10 blocks were presented either in the order happy-happy-sad-sad-happy-happy-sad-sad-happy-happy or sad-sad-happy-happy-sad- sad-happy-happy-sad-sad (with happy or sad indicating the respective target here). Thus, four test blocks were shift blocks, for which the instruction was reversed as compared to the previous block and four blocks were non-shift blocks, for which the instruction was the same as in the previous block. Specialty section: This article was submitted to Eating Behavior, a section of the journal Frontiers in Psychology Received: 16 February 2017 Accepted: 21 April 2017 Published: 09 May 2017 Citation: Meule A (2017) Reporting and Interpreting Task Performance in Go/No-Go Affective Shifting Tasks. Front. Psychol. 8:701. doi: 10.3389/fpsyg.2017.00701 Specialty section: This article was submitted to Eating Behavior, a section of the journal Frontiers in Psychology Received: 16 February 2017 Accepted: 21 April 2017 Published: 09 May 2017 Citation: Speciﬁcally, as opposed to traditional go/no-go tasks, there is an equal number of go and no-go trials and a low number of trials in total. Consider, for example, that the task employed by Murphy et al. (1999) merely contained 180 trials while other go/no-go tasks contain more than 1,000 trials (e.g., X-Y task, see Garavan et al., 2002; Kaufman et al., 2003; Meule et al., 2011). In addition, the several shifts make the single blocks very short und allow participants to take a break. Thus, the task is more comfortable (e.g., less boring) for participants and, thus, motivation may be higher to perform the task correctly than when using more exhausting go/no-go tasks. Murphy et al. (1999) and others used the aﬀective shifting task with emotional words (Rubinsztein et al., 2000; García-Blanco et al., 2013) or emotional pictures (Lange et al., 2012). However, the task can easily be adapted to other lexical or pictorial stimuli. For example, others have used versions with alcohol-related words (Noël et al., 2005, 2007) or pictures (Adams et al., 2013; Czapla et al., 2016) and food-related words (Mobbs et al., 2008, 2011; Loeber et al., 2012, 2013) or pictures (Meule and Kübler, 2014; Meule et al., 2014; Deux et al., 2017). To illustrate the importance of block type, I have combined data from two studies, in which food-related aﬀective shifting tasks and a short form of the Barratt Impulsiveness Scale (Spinella, 2007) were used in two samples of female students (Meule and Kübler, 2014; Meule et al., 2014, study 2). In shift blocks, commission errors (M = 11.4, SD = 4.76) were more common than in non-shift blocks (M = 7.12, SD = 3.85, t(156) = 12.2, p < 0.001). As depicted in Figure 1, higher attentional impulsivity related to more commission errors in shift blocks [r(n = 157) = 0.193, p = 0.015], but not in non-shift blocks [r(n = 157) = 0.108, p = 0.178]. Higher motor impulsivity also related to more commission errors in shift blocks [r(n = 157) = 0.264, p = 0.001], but not in non-shift blocks [r(n = 157) = 0.138, p = 0.086]. Non-planning impulsivity neither correlated with commission errors in shift blocks [r(n = 157) = 0.138, p = 0.086] nor in non-shift blocks [r(n = 157) = 0.076, p = 0.347]. Citation: Meule A (2017) Reporting and Interpreting Task Performance in Go/No-Go Affective Shifting Tasks. Front. Psychol. 8:701. doi: 10.3389/fpsyg.2017.00701 Meule A (2017) Reporting and Interpreting Task Performance in Go/No-Go Affective Shifting Tasks. Front. Psychol. 8:701. doi: 10.3389/fpsyg.2017.00701 It might be argued that the arrangement of the aﬀective shifting task demands mental ﬂexibility and, thus, it may not be a pure measure of inhibitory control. Although this might be a potential confound, the task also has a practical advantage over traditional go/no-go tasks. The instruction May 2017 \| Volume 8 \| Article 701 1 Frontiers in Psychology \| www.frontiersin.org Go/No-Go Affective Shifting Tasks Meule shifts increase task diﬃculty and, thus, a lower number of trials is suﬃcient for producing enough commission errors. Speciﬁcally, as opposed to traditional go/no-go tasks, there is an equal number of go and no-go trials and a low number of trials in total. Consider, for example, that the task employed by Murphy et al. (1999) merely contained 180 trials while other go/no-go tasks contain more than 1,000 trials (e.g., X-Y task, see Garavan et al., 2002; Kaufman et al., 2003; Meule et al., 2011). In addition, the several shifts make the single blocks very short und allow participants to take a break. Thus, the task is more comfortable (e.g., less boring) for participants and, thus, motivation may be higher to perform the task correctly than when using more exhausting go/no-go tasks. times have been interpreted as reﬂecting an “attention and/or response bias” (Murphy et al., 1999, p. 1314) or “an approach tendency” (Meule et al., 2014, p. 12). Yet, these interpretations lack a substantive empirical basis and are, therefore, speculative. While a high number of commission errors is widely accepted to index low inhibitory control (Schulz et al., 2007), interpretation is also not that trivial in the aﬀective shifting task. As would be expected, non-shift blocks are easier than shift blocks and, accordingly, participants commit more errors in shift blocks than in non-shift blocks. However, when examining the seven studies that used food-related aﬀective shifting tasks, only four of them (Mobbs et al., 2008, 2011; Meule and Kübler, 2014; Meule et al., 2014) reported results as a function of block type (i.e., shift vs. non-shift). shifts increase task diﬃculty and, thus, a lower number of trials is suﬃcient for producing enough commission errors. Citation: When using low-calorie food stimuli instead of neutral stimuli as control category, participants who frequently experienced cravings for high-calorie foods (Meule and Kübler, 2014) and non-overweight adolescents (Deux et al., 2017) committed more errors to low-calorie food distractors (i.e., when high-calorie foods were targets) than to high-calorie food distractors (i.e., when low-calorie foods were targets). In conclusion, it is my contention that is currently unclear in which type of condition [i.e., (high-calorie) food category as targets vs. (low-calorie or neutral) control category as targets] higher or lower inhibitory performance can be expected and how potential moderators (e.g., current hunger or body weight) may inﬂuence this inhibitory performance as a function of condition. of commission errors. Therefore, it appears that the construct that researchers actually want to measure (i.e., motor response inhibition/impulsivity) can primarily be found in shift blocks. Future studies are needed, however, in which other behavioral measures (e.g., stop-signal task) are used in order to provide further support for validity of commission errors in shift blocks as an index of motor response inhibition. Nevertheless, by not considering block type in analyses and interpretation of the data, researchers might miss important information and, therefore, may overlook interesting ﬁndings. y g g To complicate matters, researchers need to be aware that interpreting commission errors as a function of target type is diﬀerent from reaction time and omission errors in this task. As an example, consider that a researcher uses an aﬀective shifting task with food and neutral pictures. That is, participants have to either respond to food but not to neutral pictures in half of the blocks and to neutral but not to food pictures in the other half of the blocks. In food blocks (i.e., when food is the target category) reaction times represent reactions to food stimuli and omission errors represent missed reactions to food stimuli. Commission errors, however, represent false button presses in response to the distractors (i.e., neutral stimuli). While this may sound trivial, consider looking at the ﬁgures in Loeber et al. (2012) or Meule et al. (2014), where reaction times and commission errors are displayed in one ﬁgure with the conditions (i.e., target types) labeled as food and neutral/objects. As reaction time in food blocks refer to reactions to food, commission errors in food blocks refer to reactions to objects. Citation: When examining the relationships between all three impulsivity subscales and the number of commission errors together in one linear regression analysis, only motor impulsivity (β = 0.208, p = 0.026) but not attentional (β = 0.126, p = 0.122) or non-planning impulsivity (β = −0.002, p = 0.982) predicted the number Unfortunately, there seems to be quite a confusion about which task performance indices should be reported and how these can be interpreted. Some studies have reported indices based on signal detection theory such as discrimination index d’ or decision bias C and it has been argued that C is a better indicator of disinhibition than commission errors alone as it takes the number of both hits and false alarms into account (Noël et al., 2005; Mobbs et al., 2008). Yet, it is not clear if these should be actually preferred over simpler measures. For example, the same authors switched to reporting the more straightforward reaction times, omission errors and commission errors in their later works (Noël et al., 2007; Mobbs et al., 2011). While omission errors may indicate lapses of attention, reaction FIGURE 1 \| Scatterplots illustrating relationships between the number of commission errors in a go/no-go affective shifting task and subscales scores of a short form of the Barratt Impulsiveness Scale as a function of block type (shift vs. non-shift blocks). Higher attentional and motor impulsivity scores related to a higher number of commission errors in shift blocks (dashed line), but not in non-shift blocks (solid line). Non-planning impulsivity scores were not correlated with the number of commission errors. FIGURE 1 \| Scatterplots illustrating relationships between the number of commission errors in a go/no-go affective shifting task and subscales scores of a short form of the Barratt Impulsiveness Scale as a function of block type (shift vs. non-shift blocks). Higher attentional and motor impulsivity scores related to a higher number of commission errors in shift blocks (dashed line), but not in non-shift blocks (solid line). Non-planning impulsivity scores were not correlated with the number of commission errors. May 2017 \| Volume 8 \| Article 701 2 Frontiers in Psychology \| www.frontiersin.org Go/No-Go Affective Shifting Tasks Meule et al., 2014). Body weight did not interact with condition (i.e., food vs. neutral) regarding the number of commission errors (Mobbs et al., 2011; Loeber et al., 2012; Deux et al., 2017). Citation: This twist is a speciﬁc feature of the aﬀective shifting task as the meaning of the go and no- go stimuli is either not reversed (e.g., Batterink et al., 2010) or separate blocks with only food and only neutral stimuli are used (e.g., Houben et al., 2014) in other response inhibition tasks (but also see Teslovich et al., 2014). In conclusion, writing this opinion piece was motivated by increased interest in and inconsistent reporting and interpretation of the aﬀective shifting task as indicated by both the number of publications in recent years and personal requests that I received about this task. Because of its briefness and simplicity, I think that food-related aﬀective shifting tasks represent a promising tool for the investigation of impulsive reactions toward food cues. Although versions of the task have already been employed in students (Loeber et al., 2013; Meule and Kübler, 2014; Meule et al., 2014), individuals with bulimia (Mobbs et al., 2008), obese adults (Mobbs et al., 2011; Loeber et al., 2012), and adolescent, psychiatric inpatients (Deux et al., 2017), it appears that diﬀerent task designs and analyses lead to a rather confusing literature. Some of this confusion may be resolved by standardized reporting of task performance (e.g., reaction times, omission errors, and commission errors as a function of both block type and condition) and careful interpretation of this task performance (e.g., whether it is the distractors that lead participants to commit errors or whether it is the targets that lead participants to also respond to the distractors). I hope that this paper may provide a useful guidance for researchers who are interested in using this task, whether food-related or in other ﬁelds of research. Related to this issue is the question of whether more errors would be expected in blocks with food targets (i.e., neutral distractors) or in blocks with neutral targets (i.e., food distractors). It could be argued that having to respond to appealing food stimuli will lead participants to accidently press the button in response to neutral distractors as well. However, it may also be that food distractors will lure them to commit more errors when they actually are supposed to respond to neutral stimuli only. This second hypothesis was conﬁrmed in the studies by Loeber et al. (2012) and Meule et al. ACKNOWLEDGMENTS Publication of this article was supported by the Open Access Publication Fund of the University of Salzburg. Citation: (2014; study 2): participants committed more errors when neutral stimuli were targets than when food stimuli were targets (i.e., they committed more errors in response to food distractors than to neutral distractors). However, this diﬀerence was only found in hungry participants in one study (Loeber et al., 2013), but was unrelated to current hunger in two other studies (Meule AUTHOR CONTRIBUTIONS The author conﬁrms being the sole contributor of this work and approved it for publication. Adams, S., Ataya, A. F., Attwood, A. S., and Munafò, M. R. (2013). Eﬀects of alcohol on disinhibition towards alcohol-related cues. Drug Alcohol Depend. 127, 137–142. doi: 10.1016/j.drugalcdep.2012.06.025 REFERENCES Bartholdy, S., Dalton, B., O’Daly, O. G., Campbell, I. C., and Schmidt, U. (2016). A systematic review of the relationship between eating, weight and inhibitory control using the stop signal task. Neurosci. Biobehav. Rev. 64, 35–62. doi: 10.1016/j.neubiorev.2016. 02.010 Adams, S., Ataya, A. F., Attwood, A. S., and Munafò, M. R. (2013). Eﬀects of alcohol on disinhibition towards alcohol-related cues. 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Normative data and a short form of the Barratt Impulsiveness Scale. Int. J. Neurosci. 117, 359–368. doi: 10.1080/002074506005 88881 Loeber, S., Grosshans, M., Herpertz, S., Kiefer, F., and Herpertz, S. (2013). Hunger modulates behavioral disinhibition and attention allocation to food-associated cues in normal-weight controls. Appetite 71, 32–39. doi: 10.1016/j.appet.2013.07.008 Teslovich, T., Freidl, E. K., Kostro, K., Weigel, J., Davidow, J. Y., Riddle, M. C., et al. (2014). Probing behavioral responses to food: development of a food-speciﬁc go/no-go task. Psychiatry Res. 219, 166–170. doi: 10.1016/ j.psychres.2014.04.053 Loeber, S., Grosshans, M., Korucuoglu, O., Vollmert, C., Vollstädt-Klein, S., Schneider, S., et al. (2012). Impairment of inhibitory control in response to food-associated cues and attentional bias of obese participants and normal- weight controls. Int. J. Obes. 36, 1334–1339. doi: 10.1038/ijo.2011.184 Conﬂict of Interest Statement: The author declares that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Meule, A., and Kübler, A. (2014). Double trouble: trait food craving and impulsivity interactively predict food-cue aﬀected behavioral inhibition. Appetite 79, 174–182. doi: 10.1016/j.appet.2014.04.014 Meule, A., Lukito, S., Vögele, C., and Kübler, A. (2011). Enhanced behavioral inhibition in restrained eaters. Eat. Behav. 12, 152–155. Frontiers in Psychology \| www.frontiersin.org REFERENCES doi: 10.1016/j.eatbeh.2011.01.006 Copyright © 2017 Meule. 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) 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. Meule, A., Lutz, A. P. C., Krawietz, V., Stützer, J., Vögele, C., and Kübler, A. (2014). Food-cue aﬀected motor response inhibition and self-reported dieting success: a pictorial aﬀective shifting task. Front. Psychol. 5:216. doi: 10.3389/fpsyg.2014.00216 May 2017 \| Volume 8 \| Article 701 Frontiers in Psychology \| www.frontiersin.org

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